Literature context: cam ab16667, RRID:AB_302459 Rat anti-Ki67 (clone SolA15) Th
Paneth cells are post-mitotic intestinal epithelial cells supporting the stem cell niche and mucosal immunity. Paneth cell pathologies are observed in various gastrointestinal diseases, but their plasticity and response to genomic and environmental challenges remain unclear. Using a knockin allele engineered at the mouse Lyz1 locus, we performed detailed Paneth cell-lineage tracing. Irradiation induced a subset of Paneth cells to proliferate and differentiate into villus epithelial cells. RNA sequencing (RNA-seq) revealed that Paneth cells sorted from irradiated mice acquired a stem cell-like transcriptome; when cultured in vitro, these individual Paneth cells formed organoids. Irradiation activated Notch signaling, and forced expression of Notch intracellular domain (NICD) in Paneth cells, but not Wnt/β-catenin pathway activation, induced their dedifferentiation. This study documents Paneth cell plasticity, particularly their ability to participate in epithelial replenishment following stem cell loss, adding to a growing body of knowledge detailing the molecular pathways controlling injury-induced regeneration.
Literature context: re as followed: Ki67 (#ab16667; RRID:AB_302459; PMID: 24659141; rabbit monoclo
An important feature of type 2 diabetes is a decrease in β-cell mass. Therefore, it is essential to find new approaches to stimulate β-cell proliferation. We have previously shown that heterozygous inactivation of the Na+/Ca2+ exchanger (isoform 1; NCX1), a protein responsible for Ca2+ extrusion from cells, increases β-cell proliferation, mass, and function in mice. Here, we show that Ncx1 inactivation also increases β-cell proliferation in 2-year-old mice and that NCX1 inhibition in adult mice by four small molecules of the benzoxyphenyl family stimulates β-cell proliferation both in vitro and in vivo. NCX1 inhibition by small interfering RNA or small molecules activates the calcineurin/nuclear factor of activated T cells (NFAT) pathway and inhibits apoptosis induced by the immunosuppressors cyclosporine A (CsA) and tacrolimus in insulin-producing cell. Moreover, NCX1 inhibition increases the expression of β-cell-specific genes, such as Ins1, Ins2, and Pdx1, and inactivates/downregulates the tumor suppressors retinoblastoma protein (pRb) and miR-193a and the cell cycle inhibitor p53. Our data show that Na+/Ca2+ exchange is a druggable target to stimulate β-cell function and proliferation. Specific β-cell inhibition of Na+/Ca2+ exchange by phenoxybenzamyl derivatives may represent an innovative approach to promote β-cell regeneration in diabetes and improve the efficiency of pancreatic islet transplantation for the treatment of the disease.
Literature context: anti-Ki67 Abcam Cat. # ab16667; RRID:AB_302459 Guinea pig anti-Insulin Dako Ca
Weight is defended so that increases or decreases in body mass elicit responses that favor restoration of one's previous weight. While much is known about the signals that respond to weight loss and the central role that leptin plays, the lack of experimental systems studying the overfed state has meant little is known about pathways defending against weight gain. We developed a system to study this physiology and found that overfed mice defend against increased weight gain with graded anorexia but, unlike weight loss, this response is independent of circulating leptin concentration. In overfed mice that are unresponsive to orexigenic stimuli, adipose tissue is transcriptionally and immunologically distinct from fat of ad libitum-fed obese animals. These findings provide evidence that overfeeding-induced obesity alters adipose tissue and central responses in ways that are distinct from ad libitum obesity and activates a non-leptin system to defend against weight gain.
Literature context: nti-Ki67 antibody (IFC)AbcamCat#ab16667Anti-Cytokeratin 14 antibody (IF
Genetic mutations of CARD14 (encoding CARMA2) are observed in psoriasis patients. Here we showed that Card14E138A/+ and Card14ΔQ136/+ mice developed spontaneous psoriasis-like skin inflammation, which resulted from constitutively activated CARMA2 via self-aggregation leading to the enhanced activation of the IL-23-IL-17A cytokine axis. Card14-/- mice displayed attenuated skin inflammation in the imiquimod-induced psoriasis model due to impaired IL-17A signaling in keratinocytes. CARMA2, mainly expressed in keratinocytes, associates with the ACT1-TRAF6 signaling complex and mediates IL-17A-induced NF-κB and MAPK signaling pathway activation, which leads to expression of pro-inflammatory factors. Thus, CARMA2 serves as a key mediator of IL-17A signaling and its constitutive activation in keratinocytes leads to the onset of psoriasis, which indicates an important role of NF-κB activation in keratinocytes in psoriatic initiation.
Literature context: am Cat# ab16667 RRID:AB_302459 Rabbit polyclonal anti-Calnexin
A primary cause of disease progression in type 2 diabetes (T2D) is β cell dysfunction due to inflammatory stress and insulin resistance. However, preventing β cell exhaustion under diabetic conditions is a major therapeutic challenge. Here, we identify the vitamin D receptor (VDR) as a key modulator of inflammation and β cell survival. Alternative recognition of an acetylated lysine in VDR by bromodomain proteins BRD7 and BRD9 directs association to PBAF and BAF chromatin remodeling complexes, respectively. Mechanistically, ligand promotes VDR association with PBAF to effect genome-wide changes in chromatin accessibility and enhancer landscape, resulting in an anti-inflammatory response. Importantly, pharmacological inhibition of BRD9 promotes PBAF-VDR association to restore β cell function and ameliorate hyperglycemia in murine T2D models. These studies reveal an unrecognized VDR-dependent transcriptional program underpinning β cell survival and identifies the VDR:PBAF/BAF association as a potential therapeutic target for T2D.
Literature context: D:RRID:AB_302459), LOX (IMGENEX Cat# IMG-6442A R
The potential of altering the tumor ECM to improve drug response remains fairly unexplored. To identify targets for modification of the ECM aiming to improve drug response and overcome resistance, we analyzed expression data sets from pre-treatment patient cohorts. Cross-evaluation identified a subset of chemoresistant tumors characterized by increased expression of collagens and collagen-stabilizing enzymes. We demonstrate that strong collagen expression and stabilization sets off a vicious circle of self-propagating hypoxia, malignant signaling, and aberrant angiogenesis that can be broken by an appropriate auxiliary intervention: Interfering with collagen stabilization by inhibition of lysyl oxidases significantly enhanced response to chemotherapy in various tumor models, even in metastatic disease. Inhibition of collagen stabilization by itself can reduce or enhance tumor growth depending on the tumor type. The mechanistical basis for this behavior is the dependence of the individual tumor on nutritional supply on one hand and on high tissue stiffness for FAK signaling on the other.
Literature context: RRID:AB_302459 Anti-mouse BrdU Sigma Cat#B8434
Psoriasis is a chronic autoinflammatory skin disease. Although interleukin-17, derived from lymphocytes, has been shown to be critical in psoriasis, the initiation and maintenance of chronic skin inflammation has not been well understood. IL-25 (also called IL-17E), another IL-17 family cytokine, is well known to regulate allergic responses and type 2 immunity. Here we have shown that IL-25, also highly expressed in the lesional skin of psoriasis patients, was regulated by IL-17 in murine skin of a imiquimod (IMQ)-induced psoriasis model. IL-25 injection induced skin inflammation, whereas germline or keratinocyte-specific deletion of IL-25 caused resistance to IMQ-induced psoriasis. Via IL-17RB expression in keratinocytes, IL-25 stimulated the proliferation of keratinocytes and induced the production of inflammatory cytokines and chemokines, via activation of the STAT3 transcription factor. Thus, our data demonstrate that an IL-17-induced autoregulatory circuit in keratinocytes is mediated by IL-25 and suggest that this circuit could be targeted in the treatment of psoriasis patients.
Literature context: AB_16667; Abcam; Cambridge, MA RRID:AB_302459 Goat anti-rabbit (Hâ€¯+â€¯L) IgG (b
Multiple sclerosis is a demyelinating disease in which neurological deficits result from damage to myelin, axons, and neuron cell bodies. Prolonged or repeated episodes of demyelination impair remyelination. We hypothesized that augmenting Sonic hedgehog (Shh) signaling in chronically demyelinated lesions could enhance oligodendrogenesis and remyelination. Shh regulates oligodendrocyte development during postnatal myelination, and maintains adult neural stem cells. We used genetic approaches to detect Shh expression and Shh responding cells in vivo. ShhCreERT2 or Gli1CreERT2 mice were crossed to reporter mice for genetic fate-labeling of cells actively transcribing Shh or Gli1, an effective readout of canonical Shh signaling. Tamoxifen induction enabled temporal control of recombination at distinct stages of acute and chronic cuprizone demyelination of the corpus callosum. Gli1 fate-labeled cells were rarely found in the corpus callosum with tamoxifen given during acute demyelination stages to examine activated microglia, reactive astrocytes, or remyelinating cells. Gli1 fate-labeled cells, mainly reactive astrocytes, were observed in the corpus callosum with tamoxifen given after chronic demyelination. However, Shh expressing cells were not detected in the corpus callosum during acute or chronic demyelination. Finally, SAG, an agonist of both canonical and type II non-canonical Hedgehog signaling pathways, was microinjected into the corpus callosum after chronic demyelination. Significantly, SAG delivery increased proliferation and enhanced remyelination. SAG did not increase Gli1 fate-labeled cells in the corpus callosum, which may indicate signaling through the non-canonical Hedgehog pathway. These studies demonstrate that Hedgehog pathway interventions may have therapeutic potential to modulate astrogliosis and to promote remyelination after chronic demyelination.
Literature context: 6667; RRID:AB_302459 Mouse anti-E-Cadherin BD Biosci
The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells.
Literature context: (1 : 500, Abcam, Cat# ab16667, RRID:AB_302459) a general cell proliferation m
Ischemic postconditioning is increasingly being investigated as a therapeutic approach for cerebral ischemia. However, the majority of studies are focused on the acute protection of neurons per se. Whether and how postconditioning affects multiple cells in the recovering neurovascular unit remains to be fully elucidated. Here, we asked whether postconditioning may modulate help-me signaling between injured neurons and reactive microglia. Rats were subjected to 100 min of focal cerebral ischemia, then randomized into a control versus postconditioning group. After 3 days of reperfusion, infarct volumes were significantly reduced in animals treated with postconditioning, along with better neurologic outcomes. Immunostaining revealed that ischemic postconditioning increased expression of vascular endothelial growth factor (VEGF) in neurons within peri-infarct regions. Correspondingly, we confirmed that VEGFR2 was expressed on Iba1-positive microglia/macrophages, and confocal microscopy showed that in postconditioned rats, these cells were polarized to a ramified morphology with higher expression of M2-like markers. Treating rats with a VEGF receptor 2 kinase inhibitor negated these effects of postconditioning on microglia/macrophage polarization. In vitro, postconditoning after oxygen-glucose deprivation up-regulated VEGF release in primary neuron cultures, and adding VEGF to microglial cultures partly shifted their M2-like markers. Altogether, our findings support the idea that after postconditioning, injured neurons may release VEGF as a 'help-me' signal that promotes microglia/macrophage polarization into potentially beneficial phenotypes.
Literature context: 67) Rabbit; monoclonal 1:200 RRID:AB_302459
Prenatal dexamethasone exposure (PDE) induces multiorgan developmental toxicities in offspring. Here we verified the transgenerational inheritance effect of ovarian developmental toxicity by PDE and explored its intrauterine programming mechanism. Pregnant rats subcutaneously received 0.2 mg/kg/d dexamethasone from gestational day (GD) 9 to GD20. A subgroup was euthanized for fetuses on GD20, and the other group went on to spontaneous labor to produce F1 offspring. The adult F1 females were mated with normal males to produce the F2 and F3 generations. The PDE fetal rats exhibited ovarian mitochondrial structural abnormalities, decreased serum estradiol (E2) levels, and lower expression levels of ovarian steroidogenic factor 1 (SF1), steroidal synthetases, and insulinlike growth factor 1 (IGF1). On postnatal week (PW) 6 and PW12, the PDE F1 offspring showed altered reproductive behavior and ovarian morphology. The serum E2 level and ovarian expression of SF1, steroidal synthetases, and IGF1 were also decreased. The adult F3 offspring showed alterations in reproductive phenotype and ovarian IGF1, SF1, and steroidal synthetase expression similar to those of F1. PDE induces ovarian developmental toxicity and transgenerational inheritance effects. The mechanism by which this toxicity occurs may be related to PDE-induced low-functional programming of fetal ovarian IGF1/SF1 and steroidal synthetases.
Literature context: o CHOP (RRID:AB_2245733), Ki67 (RRID:AB_302459), and BrdU (RRID:AB_298940) wer
Adult hippocampal neurogenesis has been shown to be required for certain types of cognitive function. For example, studies have shown that these neurons are critical for pattern separation, the ability to store similar experiences as distinct memories. Although traumatic brain injury (TBI) has been shown to cause the loss of newborn hippocampal neurons, the signaling pathway(s) that triggers their death is unknown. Endoplasmic reticulum (ER) stress activates the PERK-eIF2α pathway that acts to restore ER function and improve cell survival. However, unresolved/intense ER stress activates C/EBP homologous protein (CHOP), leading to cell death. We show that TBI causes the death of hippocampal newborn neurons via CHOP. Using CHOP KO mice, we show that loss of CHOP markedly reduces newborn neuron loss after TBI. Injured CHOP mice performed significantly better in a context fear discrimination task compared with injured wild-type mice. In contrast, the PERK inhibitor GSK2606414 exacerbated doublecortin cell loss and worsened contextual discrimination. Administration of guanabenz (which reduces ER stress) to injured male rats reduced the loss of newborn neurons and improved one-trial contextual fear memory. Interestingly, we also found that the surviving newborn neurons in brain-injured animals had dendritic loss, which was not observed in injured CHOP KO mice or in animals treated with guanabenz. These results indicate that ER stress plays a key role in the death of newborn neurons after TBI. Further, these findings indicate that ER stress can alter dendritic arbors, suggesting a role for ER stress in neuroplasticity and dendritic pathologies.SIGNIFICANCE STATEMENT The hippocampus, a structure in the temporal lobe, is critical for learning and memory. The hippocampus is one of only two areas in which neurons are generated in the adult brain. These newborn neurons are required for certain types of memory, and are particularly vulnerable to traumatic brain injury (TBI). However, the mechanism(s) that causes the loss of these cells after TBI is poorly understood. We show that endoplasmic reticulum (ER) stress pathways are activated in newborn neurons after TBI, and that manipulation of the CHOP cascade improves newborn neuron survival and cognitive outcome. These results suggest that treatments that prevent/resolve ER stress may be beneficial in treating TBI-triggered memory dysfunction.
Literature context: clonal anti-Ki67 Abcam ab16667, RRID:AB_302459 Mouse monoclonal anti-Î± smooth
Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.
Literature context: RRID:AB_302459 Rabbit anti-mouse/human pRaf1 C
Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and NrasG12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.
Literature context: Ab16667, RRID:AB_302459), Nestin (
The bcl-2 family of survival and death promoting proteins play a key role in regulating cell numbers during nervous system development. Bcl-xL, an anti-apoptotic bcl-2 family member is highly expressed in the developing nervous system. However; the early embryonic lethality of the bcl-x germline null mouse precluded an investigation into its role in nervous system development. To identify the role of bcl-x in spinal cord neurogenesis, we generated a central nervous system-specific bcl-x conditional knockout (BKO) mouse. Apoptotic cell death in the BKO embryo was initially detected at embryonic day 11 (E11) in the ventrolateral aspect of the spinal cord corresponding to the location of motor neurons. Apoptosis reached its peak at E13 having spread across the ventral and into the dorsal spinal cord. By E18, the wave of apoptosis had passed and only a few apoptotic cells were observed. The duration and direction of spread of apoptosis across the spinal cord is consistent with the spatial and temporal sequence of neuronal differentiation. Motor neurons, the first neurons to become post mitotic in the spinal cord, were also the first apoptotic cells. As neurogenesis spread across the spinal cord, later born neuronal populations such as Lim2+ interneurons were also affected. The onset of apoptosis occurred in cells that had exited the cell cycle within the previous 24h and initiated neural differentiation as demonstrated by BrdU birthdating and βIII tubulin immunohistochemistry. This data demonstrates that spinal cord neurons become Bcl-xL dependent at an early post mitotic stage in developmental neurogenesis.
Literature context: 7 (clone SP6)AbcamCat# ab16667; RRID:AB_302459Rabbit monoclonal anti mTOR (clo
Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling-driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is inherently resistant to existing SMO inhibitors, prolonging the survival of the mice. Our study reveals that mTORC1-mediated translation is a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.
Literature context: ab16667 RRID:AB_302459 Anti-GFP antibody Abcam Cat#: a
Myeloid-biased hematopoietic stem cells (MB-HSCs) play critical roles in recovery from injury, but little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc+ myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H2 receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine-producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H2 agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MB-HSCs to enforce homeostasis.
Literature context: RRID:AB_302459 Rabbit polyclonal anti-E-cadher
Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous carcinoma in which various tumor-suppressor genes are lost by mutation, deletion, or silencing. Here we report a tumor-suppressive mode of action for growth-differentiation factor 11 (GDF11) and an unusual mechanism of its inactivation in TNBC. GDF11 promotes an epithelial, anti-invasive phenotype in 3D triple-negative cultures and intraductal xenografts by sustaining expression of E-cadherin and inhibitor of differentiation 2 (ID2). Surprisingly, clinical TNBCs retain the GDF11 locus and expression of the protein itself. GDF11 bioactivity is instead lost because of deficiencies in its convertase, proprotein convertase subtilisin/kexin type 5 (PCSK5), causing inactive GDF11 precursor to accumulate intracellularly. PCSK5 reconstitution mobilizes the latent TNBC reservoir of GDF11 in vitro and suppresses triple-negative mammary cancer metastasis to the lung of syngeneic hosts. Intracellular GDF11 retention adds to the concept of tumor-suppressor inactivation and reveals a cell-biological vulnerability for TNBCs lacking therapeutically actionable mutations.
Literature context: am Cat# ab16667; RRID:AB_302459 Mouse monoclonal anti-Nestin EM
Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.
Literature context: Ki67 Abcam ab16667, RRID:AB_302459 Î±/Î² Tubulin Cell Signaling Cat.
Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21Cip. CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.
Literature context: m Cat# ab16667, RRID:AB_302459), mouse anti-FLAG M2 (1:1,000;
Production of an appropriate number of distinct cell types in precise locations during embryonic development is critical for proper tissue function. Homeostatic renewal or repair of damaged tissues in adults also requires cell expansion and transdifferentiation to replenish lost cells. However, the responses of diverse cell types to tissue injury are not fully elucidated. Moreover, the molecular mechanisms underlying transdifferentiation remain poorly understood. This knowledge is essential for harnessing the regenerative potential of individual cell types. This study investigated the fate of pulmonary neuroendocrine cells (PNECs) following lung damage to understand their plasticity and potential. PNECs are proposed to carry out diverse physiological functions in the lung and can also be the cells of origin of human small cell lung cancer. We found that Notch signaling is activated in proliferating PNECs in response to epithelial injury. Forced induction of high levels of Notch signaling in PNECs in conjunction with lung injury results in extensive proliferation and transdifferentiation of PNECs toward the fate of club cells, ciliated cells and goblet cells. Conversely, inactivating Notch signaling in PNECs abolishes their ability to switch cell fate following lung insult. We also established a connection between PNEC transdifferentiation and epigenetic modification mediated by the polycomb repressive complex 2 and inflammatory responses that involve the IL6-STAT3 pathway. These studies not only reveal a major pathway that controls PNEC fate change following lung injury but also provide tools to uncover the molecular basis of cell proliferation and fate determination in response to lung injury. Stem Cells 2018;36:377-391.
Literature context: 667 Rabbit; monoclonal 1:200 RRID:AB_302459 Ki67, rabbit, Abcam
In utero exposure to the endocrine disrupting compound bisphenol A (BPA) is known to disrupt mammary gland development and increase tumor susceptibility in rodents. It is unclear whether different periods of in utero development might be more susceptible to BPA exposure. We exposed pregnant CD-1 mice to BPA at different times during gestation that correspond to specific milestones of in utero mammary gland development. The mammary glands of early-life and adult female mice, exposed in utero to BPA, were morphologically and molecularly (estrogen receptor-α and Ki67) evaluated for developmental abnormalities. We found that BPA treatment occurring before mammary bud invasion into the mesenchyme [embryonic day (E)12.5] incompletely resulted in the measured phenotypes of mammary gland defects. Exposing mice up to the point at which the epithelium extends into the precursor fat pad (E16.5) resulted in a nearly complete BPA phenotype and exposure during epithelial extension (E15.5 to E18.5) resulted in a partial phenotype. Furthermore, the relative differences in phenotypes between exposure windows highlight the substantial correlations between early-life molecular changes (estrogen receptor-α and Ki67) in the stroma and the epithelial elongation defects in mammary development. These data further implicate BPA action in the stroma as a critical mediator of epithelial phenotypes.
Literature context: 7; RRID:AB_302459 Ki67 (mouse) BD Biosciences Cat
The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.
Literature context: ID: RRID:AB_302459 Anti-PD-L1 abcam Cat#ab205921
Glioblastoma is an immunosuppressive, fatal brain cancer that contains glioblastoma stem-like cells (GSCs). Oncolytic herpes simplex virus (oHSV) selectively replicates in cancer cells while inducing anti-tumor immunity. oHSV G47Δ expressing murine IL-12 (G47Δ-mIL12), antibodies to immune checkpoints (CTLA-4, PD-1, PD-L1), or dual combinations modestly extended survival of a mouse glioma model. However, the triple combination of anti-CTLA-4, anti-PD-1, and G47Δ-mIL12 cured most mice in two glioma models. This treatment was associated with macrophage influx and M1-like polarization, along with increased T effector to T regulatory cell ratios. Immune cell depletion studies demonstrated that CD4+ and CD8+ T cells as well as macrophages are required for synergistic curative activity. This combination should be translatable to the clinic and other immunosuppressive cancers.
Literature context: ab16667; RRID:AB_302459) (1:200),
The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.
Literature context: Kingdom; RRID:AB_302459) recognize
According to previous opinion, the derivation of neurons and glia from the central canal (CC) lining of the spinal cord in rodents should occur in the embryonic period. Reports of the mitotic activity observed in the lining during postnatal development have often been contradictory, and proliferation was ascribed to the generation of ependymocytes, which are necessary for the elongation of CC walls. Our study quantifies the intensity of proliferation and determines the cellularity of the CC lining in reference to lumbar spinal segment L4 during the postnatal development of rats. The presence of dividing cells peaks in the CC lining on postnatal day 8 (P8), with division occurring in 19.2% ± 3.2% of cells. In adult rats, 3.6% ± 0.9% of cells still proliferate, whereas, in mice, 10.3% ± 2.3% of cells at P8 and only 0.6% ± 0.2% of cells in the CC lining in adulthood are proliferating. In the rat, the length of the cell cycle increases from 100.3 ± 35.7 hours at P1 to 401.4 ± 80.6 hours at P43, with a sudden extension between P15 and P22. Despite the intensive proliferation, the total cellularity of the CC lining at the L4 spinal segment significantly descended in from P8 to P15. According to our calculations, the estimated cellularity was significantly higher compared with the measured cellularity of the CC lining at P15. Our results indicate that CC lining serves as a source of cells beyond ependymal cells during the first postnatal weeks of the rat. J. Comp. Neurol. 525:693-707, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: lone SP6, RRID:AB_302459, 1:200), a
TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.
Literature context: :100, Ki67 (Abcam cat# ab16667, RRID: AB_302459) diluted at 1:100, and B220 (BD
During the humoral immune response, B cells undergo a dramatic change in phenotype to enable antibody affinity maturation in germinal centers (GCs). Using genome-wide chromosomal conformation capture (Hi-C), we found that GC B cells undergo massive reorganization of the genomic architecture that encodes the GC B cell transcriptome. Coordinate expression of genes that specify the GC B cell phenotype-most prominently BCL6-was achieved through a multilayered chromatin reorganization process involving (1) increased promoter connectivity, (2) formation of enhancer networks, (3) 5' to 3' gene looping, and (4) merging of gene neighborhoods that share active epigenetic marks. BCL6 was an anchor point for the formation of GC-specific gene and enhancer loops on chromosome 3. Deletion of a GC-specific, highly interactive locus control region upstream of Bcl6 abrogated GC formation in mice. Thus, large-scale and multi-tiered genomic three-dimensional reorganization is required for coordinate expression of phenotype-driving gene sets that determine the unique characteristics of GC B cells.
Glucagon-like peptide-2 (GLP-2) is an enteroendocrine hormone that stimulates the growth of the intestinal epithelium. We have previously demonstrated that GLP-2 exerts its intestinotropic effect through an indirect mechanism that requires both IGF-1 and the intestinal epithelial IGF-1 receptor. However, the biological activity of IGF-1 is modulated by IGF binding proteins (IGFBPs), including IGFBP-4, which is highly expressed in the intestine. To determine the role of IGFBP-4 in the tropic effects of GLP-2, IGFBP-4 knockout (KO) and control mice were treated with degradation-resistant GLP-2 or vehicle for 10 days. Comparable levels of IGFBP-1-3/5-7 mRNAs were observed in the intestinal mucosa of all animals. IGFBP-4 KO mice had greater small intestinal weight and length, and deeper crypts (P < .05) as compared with controls, suggesting that IGFBP-4 has an inhibitory role in basal intestinal growth. However, small intestinal weight, crypt-villus height and crypt cell proliferation increased in response to GLP-2 in control mice (P < .05), and these changes were abrogated with IGFBP-4 KO. In contrast, pregnancy-associated plasma protein-A KO mice, which have increased levels of circulating IGFBP-4, demonstrated a normal intestinotropic response to GLP-2. Finally, GLP-2 treatment of control mice significantly increased IGFBP-4 mRNA expression in the jejunal mucosa (P < .05), a finding that was recapitulated by GLP-2 treatment of fetal rat intestinal cells in culture (10(-8)M for 2 h; P < .05). Collectively, these results indicate that the IGF-I-modulating protein, IGFBP-4, exerts a negative effect on basal intestinal growth but plays a positive regulatory role in the intestinotropic actions of GLP-2.
Literature context: tic Ki67 proteinAbcam, ab16667, AB_302459, rabbit, monoclonal1:500Anti-Br
Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC-VZ) during rat development, which take place between embryonic days 14 and 18 (E14-E18) and E20-E21, have been described. In the VZ of the brain, unlike the SC-VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2(+) /Sox-10(+) cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2(+) /Sox-10(+) cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40-μm section during the second postnatal week. After the latter development, the number of Olig2(+) /Sox-10(+) cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2(+) /Sox-10(+) cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2(+) /Sox-10(+) cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5-bromo-2-deoxyuridine administration at P2, P11, P22, or P120-P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats.
Vasomotor thermo-dysregulation (hot flashes) are an often debilitating symptom of menopause. Effective treatment is achieved primarily through activation of the estrogen receptor (ER)α with estrogens but is also associated with increased risk for breast and uterine cancer. In this study, we have tested novel compounds lacking the B ring of 17-hydroxy-β-estradiol (E2) (A-CD compounds) with differing ratios of ERα:ERβ binding affinities for the ability to reduce diurnal/nocturnal tail-skin temperatures (TSTs) in the ovariectomized female rat menopausal hot flash model. Normal mammary tissue expresses the predominantly antiproliferative ERβ. Therefore, we hypothesized that a preferential ERβ agonist with fractional ERα activity would safely reduce TSTs. The A-CD compound, L17, is a preferential ERβ agonist that has a ratio of ERβ:ERα binding affinity relative to E2 of 9.3 (where ERβ:ERα for E2, 1.0). In the ovariectomized rat, daily administration of low doses (1 mg/kg) of the A-CD compound TD81 (ERα:ERβ relative affinity, 15.2) was ineffective in temperature regulation, whereas L17 showed a trend toward TST reduction. Both E2 and the A-CD compound, TD3 (ERβ:ERα relative affinity, 5.0), also reduced TSTs but had marked proliferative effects on mammary and uterine tissues. At 2 mg/kg, L17 strongly reduced TSTs even more effectively than E2 but, importantly, had only minimal effect on uterine weight and mammary tissues. Both E2- and L17-treated rats showed similar weight reduction over the treatment period. E2 is rapidly metabolized to highly reactive quinones, and we show that L17 has 2-fold greater metabolic stability than E2. Finally, L17 and E2 similarly mediated induction of c-fos expression in neurons within the rat thermoregulatory hypothalamic median preoptic nucleus. Thus, the A-CD compound, L17, may represent a safe and effective approach to the treatment of menopausal hot flashes.
Sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor-based incretin therapy intended for the treatment of type 2 diabetes mellitus (T2DM), has not been linked to adverse effects on the pancreas in prospective clinical trials or in nonclinical toxicology studies. To further assess potential pancreatic effects, sitagliptin was studied in the male Zucker diabetic fatty (ZDF) rat model of T2DM. Following 3 months of oral dosing with vehicle, or sitagliptin at doses 3- to 19-fold above the clinically therapeutic plasma concentration, which increased active plasma glucagon-like peptide-1 levels up to approximately 3-fold, or following 3 months of oral dosing with metformin, a non-incretin-based reference T2DM treatment, the pancreas of male ZDF rats was evaluated using qualitative and quantitative histopathology techniques. In the quantitative evaluation, proliferative index was calculated in exocrine pancreatic ducts and ductules using computer-based image analysis on sections stained by immunohistochemistry for cytokeratin (a cytoplasmic epithelial cell marker) and Ki-67 (a nuclear marker of recent cell division). Relative to controls, sitagliptin treatment did not alter disease progression based on detailed clinical signs and clinical pathology assessments. Sitagliptin treatment did not result in pancreatitis or any adverse effect on the pancreas based on a qualitative histopathology evaluation. Proliferative index did not increase with sitagliptin treatment based on quantitative assessment of more than 5000 sections of pancreas, where control group means ranged from 0.698-0.845% and sitagliptin-treated group means ranged from 0.679-0.701% (P = .874). Metformin treatment was similarly evaluated and found not to have adverse effects on pancreas.