trichostatin a Abcam Search Results


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  • 99
    Thermo Fisher alexa fluor 488
    CFTR depletion impairs TfR recycling in bronchial epithelial cells. ( a – d ) 16HBE14o- cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides. ( a ) The cells were exposed to <t>Alexa-Fluor-488-Tf</t> for 1 h
    Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore triton x 100
    CFTR depletion impairs TfR recycling in bronchial epithelial cells. ( a – d ) 16HBE14o- cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides. ( a ) The cells were exposed to <t>Alexa-Fluor-488-Tf</t> for 1 h
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsa  (Abcam)
    99
    Abcam bsa
    CFTR depletion impairs TfR recycling in bronchial epithelial cells. ( a – d ) 16HBE14o- cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides. ( a ) The cells were exposed to <t>Alexa-Fluor-488-Tf</t> for 1 h
    Bsa, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 11355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dapi
    Treatment with Ola triggers nuclear translocation of endogenous HFn. Confocal microscopy images of MDA-MB 231, MDA-MB 468 and HCC1937 cells incubated for 1, 3 and 48 h at 37 °C with 1 µM Ola. Cells not previously treated with Ola were used as negative control (untreated). Nuclei were stained with <t>DAPI</t> (blue). Endogenous ferritin was recognized with anti-ferritin antibody and labeled with an anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (green; Thermo <t>Fischer</t> Scientific). Scale bar: 10 µm.
    Dapi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pbs
    Treatment with Ola triggers nuclear translocation of endogenous HFn. Confocal microscopy images of MDA-MB 231, MDA-MB 468 and HCC1937 cells incubated for 1, 3 and 48 h at 37 °C with 1 µM Ola. Cells not previously treated with Ola were used as negative control (untreated). Nuclei were stained with <t>DAPI</t> (blue). Endogenous ferritin was recognized with anti-ferritin antibody and labeled with an anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (green; Thermo <t>Fischer</t> Scientific). Scale bar: 10 µm.
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 45702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immobilon p membranes
    Drp1 SUMOylation selectively reduces binding to Mff. ( a ) Non-SUMOylatable HA-Drp1 shows enhanced association with GST-Mff. HA-Drp1 or HA-Drp1 4KR were transfected into HEK293 cells expressing GST-Mff, GST-Fis1, GST-MID49 or GST-MID51. GST-pull downs and lysates were immunoblotted with HA (for Drp1) and GST antibodies. The histogram shows ratio of Drp1 4KR to Drp1 binding to each receptor (For GST-Mff, n = 6; **P = 0.0042; for GST-Fis1, n = 5; P = 0.7749; For GST-MID49, n = 5; P = 0.4316; for GST-MID51, n = 5; P = 0.6926; Paired Student’s test). ( b ) In vitro , in the absence of SUMO, non-SUMOylatable Drp1-His and wild type Drp1-His show similar binding ability to GST-Mff. GST-PD was performed following incubation of His-tagged Drp1 or its 4KR mutant with either recombinant GST or GST-Mff. GST-pull downs and purified proteins were separated on different lanes in the same gels and immunoblotted with a Drp1 antibody. <t>PVDF</t> was stained with coomassie blue for GST or GST-Mff. ( c ) Cytosolic and mitochondrial localisations of Drp1, Mff and SENP3 in HEK293 cells. Drp1 and SENP3 are predominantly cytoplasmic under basal conditions and Mff is exclusively mitochondrial. GAPDH is a cytosolic marker and VDAC is a mitochondrial marker. ( d ) SENP3 knockdown, which promotes Drp1 SUMOylation, decreases the interaction between endogenous Drp1 and Mff. SENP3 siRNA (SENP3i) or non-specific siRNA (Nsi) was transfected in HEK293 cells followed by IP of Mff and SUMO-2/3. β-actin was used as a loading control. The histogram shows the degree of inhibition of Drp1 binding to Mff (n = 3; **P = 0.0091; Paired Student’s test).
    Immobilon P Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc cleaved caspase 3
    E-cadherin loss in Lgr5 + stem cells lead to their apoptosis. Co-IF for Lgr5-GFP (green), E-cadherin (white), and cleaved <t>caspase-3</t> (red), and quantification of cleaved caspase-3 + cells per gland in the gastric antrum of Lgr5-Cre;Cdh1 fl/+ ( n = 4) and Lgr5-Cre;Cdh1 fl/fl ( n = 6) mice after 7 weeks of Tam treatment. The data are presented as means ± SEM, * P
    Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 38170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam chicken anti gfp
    GFAP-YFP-expressing cells in the DG and population-level dynamics of hippocampal neural stem cells. (A,C) GFAP-YFP-positive cells in 8-week-old (A) and 56-week-old (C) GFAP-YFP reporter mice. Scale bars: 100 μm. (B,D) Representative confocal images of immunostaining for <t>GFP</t> (green) and S100β (red). Shown are examples of a GFAP + /S100β − neural stem cell (B) and a GFAP + /S100β + astrocyte (D). Scale bars: 20 μm. (E,F) Fit of the proposed model to the total number of NSCs (E) and the fraction of <t>BrdU-incorporating</t> NSCs (F). Estimated parameters are displayed in Table 1 .
    Chicken Anti Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse anti neun
    Progression of neurogenesis and gliogenesis in the tuberal hypothalamus of CD1 wildtype mice. a P0 brain sections of <t>BrdU</t> birthdating studies indicating the neurons, marked by <t>NeuN,</t> that were born at E11.5, E13.5 and E15.5 embryonic time points during neurogenesis in the tuberal hypothalamus. Yellow arrows indicate examples of NeuN+/BrdU+ co-labeled neurons, third ventricle location is highlighted with a white dotted line. b Sox9+ glioblasts in the tuberal hypothalamus at E13.5, E15.5 and E17.5. Scale bars equal 200 μm
    Mouse Anti Neun, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ki67  (Abcam)
    92
    Abcam ki67
    REG3B–GLP-1-treated pancreatic islets maintain insulin-positive cells after STZ induction. (A) Immunohistochemistry showing islet structure without STZ and REG3B–GLP-1 (left column), 1-week post STZ induction with or without REG3B–GLP-1 therapy (middle columns), and 8-weeks post STZ induction with or without REG3B–GLP-1 therapy (right columns). Insulin and glucagon (top row), glucagon and Nkx6.1 (middle row), and glucagon and <t>ki67</t> (bottom row) are shown. All images were taken with 40× objective. (B) Immunostaining of islets 1-week post STZ induction with AAV9 mRIP Reg3b–Glp-1 delivery. A small population of islets was found to be bi-hormonal for glucagon and insulin. The left panel is 10× objective of pancreas cross-section, with the islet with insulin and glucagon dual-positive cells indicated by a dashed box. Other panels show serial sections of a bi-hormonal islet, stained with combinations of anti-insulin, -glucagon or -Pdx1 antibodies, at higher magnifications (40× objective). Nuclei were counterstained by DAPI. Scale bars: 50 μm. (C) 8 weeks after STZ induction insulin-positive cell masses were determined as described in the Materials and Methods (left panel). Percent of total glucagon area to insulin area was determined from five random islets per individual (middle panel). Percent islet cell proliferation was determined by counting the number of ki67-positive cells within insulin- and glucagon-positive regions of five random islets (right panel).
    Ki67, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 8862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti alpha tubulin antibody
    MT EB3-tracking measurements reveal a significant decrease in spindle symmetry in the absence of Ccdc61. (A–C) HeLa cells stably expressing EGFP-EB3 were used for EB3-tracking experiments. Cells were either treated with control (ct), Ccdc61 O2 siRNA (O2), or a combination of Ccdc61 O2 siRNA together with FLAG-Ccdc61 siRNA-resistant construct (O2R). To study mitotic properties, like directionality and orientation, the individual EB3 tips were imaged every 0.13 s and analyzed via an automated process. (A) Example images of a single time frame (top row) and a time projection from 125 s recordings (bottom row) of HeLa cells treated as indicated. Western blot shows the efficiency of indicated Ccdc61 Oligo that was tested by its ability to deplete transiently transfected FLAG-Ccdc61 or siRNA-resistant FLAG-Ccdc61 (O2R). <t>α-tubulin</t> served as a loading control. (B) EGFP-EB3 tracks from HeLa cells were analyzed as described in the Materials and Methods section and as published in the preprint ( Jafarpour and Lorenz, 2017 ). The table represents velocity (µm/min), displacement (µm), duration (s) (mean ± SD.) and the total number of analyzed tracks (of 10 independent cells). The significance was calculated for the three conditions by a one-way ANOVA, and the <t>alpha</t> value at which there was no significant difference (n.s.d.) within the group as well as the p value is provided. (C) Shown are the results of calculated asymmetry and directionality indices of the indicated treated HeLa cells with 10 image series per group based on the histogram of calculated orientations (Supplemental Figure S4, B and C). Each mitosis event is characterized with two figures of merit, resulting in a single point. The y -axis represents the degree of asymmetry, whereby a low value indicates a symmetric direction of MT growth (low asymmetry) and a high value indicates an asymmetric direction of MT growth (reduced symmetry). The horizontal axis represents the directionality of MT growth, indicating different levels of point symmetry in MT growth paths.
    Monoclonal Anti Alpha Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dapi
    SMAD6 is Downstream of Notch Signaling in Homeostatic Endothelial Cell Flow-Mediated Alignment. A) Representative panels of HUVEC stained with <t>Phalloidin</t> (red, actin) and <t>DAPI</t> (white, nucleus) under flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001; ns, not significant. C) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. D) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. E) qPCR RNA levels (normalized to static control) in HUVEC treated with RPBJ siRNA. Statistical analysis, Student’s t-test; **, p≤0.01 F) Representative panels of HUVEC with additional RBPJ and DLL4 siRNAs (see Key Resources) stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions. White arrow, flow vector. Scale bar, 50μM. G) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. H) Representative panel of HAEC treated with RPBJ siRNA and stained with phalloidin (F-actin, white) and expression construct (SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive EC. Scale bar, 20μM. I) Quantification of cell axis ratio in indicated conditions. Statistical analysis, student’s t-test; ****, p≤0.0001. J) Diagram showing SMAD6 constructs. Numbers indicate amino acids in human SMAD6.
    Dapi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 87206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pbs
    SMAD6 is Downstream of Notch Signaling in Homeostatic Endothelial Cell Flow-Mediated Alignment. A) Representative panels of HUVEC stained with <t>Phalloidin</t> (red, actin) and <t>DAPI</t> (white, nucleus) under flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001; ns, not significant. C) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. D) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. E) qPCR RNA levels (normalized to static control) in HUVEC treated with RPBJ siRNA. Statistical analysis, Student’s t-test; **, p≤0.01 F) Representative panels of HUVEC with additional RBPJ and DLL4 siRNAs (see Key Resources) stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions. White arrow, flow vector. Scale bar, 50μM. G) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. H) Representative panel of HAEC treated with RPBJ siRNA and stained with phalloidin (F-actin, white) and expression construct (SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive EC. Scale bar, 20μM. I) Quantification of cell axis ratio in indicated conditions. Statistical analysis, student’s t-test; ****, p≤0.0001. J) Diagram showing SMAD6 constructs. Numbers indicate amino acids in human SMAD6.
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam total oxphos rodent wb antibody cocktail
    Loss of MTIF3 results in uncoordinated mitochondrial protein synthesis. ( A ) Levels of de novo protein synthesis were measured in heart mitochondria from control ( L/L ) and knockout ( L/L, cre ) 10-week-old mice by pulse and chase incorporation of 35 S-labeled cysteine and methionine. Mitochondrial protein was separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie, and visualized by autoradiography. Representative gels of five independent biological experiments are shown. ( B ) Levels of de novo protein synthesis in heart mitochondria from 25-week-old control ( L/L ) and knockout ( L/L, cre ) mice, determined as in (A). Mitochondrial proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( C ) and 25-week-old ( D ) mice were resolved on 4 to 20% SDS-PAGE gels and immunoblotted using antibodies to investigate the steady-state levels of <t>OXPHOS</t> proteins. <t>SDHA</t> was used as a loading control. Levels of mitoribosomal proteins, proteases, and MTIF2 proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( E ) and 25-week-old ( F ) mice. Porin was used as a loading control. All values are means ± SEM. * P
    Total Oxphos Rodent Wb Antibody Cocktail, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 403 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam goat serum
    Loss of MTIF3 results in uncoordinated mitochondrial protein synthesis. ( A ) Levels of de novo protein synthesis were measured in heart mitochondria from control ( L/L ) and knockout ( L/L, cre ) 10-week-old mice by pulse and chase incorporation of 35 S-labeled cysteine and methionine. Mitochondrial protein was separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie, and visualized by autoradiography. Representative gels of five independent biological experiments are shown. ( B ) Levels of de novo protein synthesis in heart mitochondria from 25-week-old control ( L/L ) and knockout ( L/L, cre ) mice, determined as in (A). Mitochondrial proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( C ) and 25-week-old ( D ) mice were resolved on 4 to 20% SDS-PAGE gels and immunoblotted using antibodies to investigate the steady-state levels of <t>OXPHOS</t> proteins. <t>SDHA</t> was used as a loading control. Levels of mitoribosomal proteins, proteases, and MTIF2 proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( E ) and 25-week-old ( F ) mice. Porin was used as a loading control. All values are means ± SEM. * P
    Goat Serum, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 11031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti flag m2 antibody
    Banf1 binds to PARP1 following oxidative stress. a , b Banf1 and PARP1 are in a complex; immunoprecipitations from HEK293T cells expressing <t>Flag,</t> Flag-Banf1 or Flag-PARP1 using Flag <t>antibodies.</t> Immunoprecipitates were immunoblotted with the indicated antibodies. c The Banf1:PARP1 interaction after H 2 O 2 ; immunoprecipitations from HEK293T cells ectopically expressing Flag or Flag-Banf1 1 h after H 2 O 2 removal. Immunoprecipitates were immunoblotted with the indicated antibodies. d Banf1 and PARP1 directly interact. The indicated purified proteins (±NAD + /DNA/olaparib) were incubated together before immunoprecipitation with PARP1 antibodies and immunoblotting with the indicated antibodies. e Banf1 and PARP colocalise following oxidative stress induced by H 2 O 2 in U2OS cells. Representative cells fixed at the indicated times following removal of H 2 O 2 and stained with the indicated antibodies are shown, the colocalisation was analysed using ImageJ. The scale bar represents 10 μm. Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 37942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bsa
    <t>BHMT</t> is a potential direct oxidative-target of Nox4-generated ROS. A, Representative immunoblot showing decreasing labelling of BHMT after BIAM labelling of H 2 O 2 -oxidised purified recombinant BHMT and <t>BSA.</t> B, Graph of the densitometric analysis of labelled BHMT band intensities under the indicated H 2 O 2 concentrations. R 2 values indicate that there is a good correlation between the decreases in BHMT labelling and increasing H 2 O 2 concentration (i.e. increasingly oxidised BHMT), while there is a poor correlation between BSA labelling at the same H 2 O 2 concentrations. C, Extracellular catalase-inhibitable H2O2 generated by HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). D, Western blot and densitometric quanrification of BIAM-switch labelled (ie oxidised) BHMT levels in HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). Equivalent amounts (20μg) of “input” protein were blotted for βactin, to serve as a control. Above data are represented as mean +/-S.E.M. ⁎ : p=
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 42788 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher donkey anti rabbit igg h l highly cross adsorbed secondary antibody
    <t>BHMT</t> is a potential direct oxidative-target of Nox4-generated ROS. A, Representative immunoblot showing decreasing labelling of BHMT after BIAM labelling of H 2 O 2 -oxidised purified recombinant BHMT and <t>BSA.</t> B, Graph of the densitometric analysis of labelled BHMT band intensities under the indicated H 2 O 2 concentrations. R 2 values indicate that there is a good correlation between the decreases in BHMT labelling and increasing H 2 O 2 concentration (i.e. increasingly oxidised BHMT), while there is a poor correlation between BSA labelling at the same H 2 O 2 concentrations. C, Extracellular catalase-inhibitable H2O2 generated by HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). D, Western blot and densitometric quanrification of BIAM-switch labelled (ie oxidised) BHMT levels in HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). Equivalent amounts (20μg) of “input” protein were blotted for βactin, to serve as a control. Above data are represented as mean +/-S.E.M. ⁎ : p=
    Donkey Anti Rabbit Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Abcam)
    99
    Abcam gfp
    Ctdsp2 increases neural progenitor (NP) proliferation. (A–C) Overexpression of Ctdsp2 , but not the control construct pCAGIG, increased the proportion of cells expressing both proliferative marker bromodeoxyuridine + (BrdU + )/green fluorescence protein + <t>(GFP</t> + ) and radial glial cell (RGC) marker <t>Pax6</t> + /GFP + , but not intermediate progenitor (IP) marker Tbr2 + /GFP + , in GFP-positive cells. (D–F) shRNA-mediated knockdown (sh Ctdsp2 ) of Ctdsp2 decreased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P
    Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8593 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam triton x 100
    QIL1 localizes at cristae junctions and is present in the mature MICOS complex. ( A – C ) Immunogold labeling of 293T cells overexpressing C-terminally tagged QIL1 ( A ), MIC25 ( B ), or NDUFA13 ( C ) using an α-HA antibody coupled to 10-nm gold particles. Bars, 100 nm. Representative mitochondria are shown. The arrows point to the position of a gold particle. The distance in nanometers between the gold particles and the nearest CJ was measured using the ImageJ software. The histograms show the fraction of gold particles within the indicated distance to the crista junction in nanometers in QIL1-HA (n = 231 gold particles), MIC25-HA (n = 192 gold particles), and NDUFA13-HA (n = 309 gold particles) expressing cells. ( D ) Mitochondria isolated from stable 293T cell lines expressing C-terminally tagged QIL1, MIC60, MIC19, or MIC25 were lysed in 1% digitonin, subjected to BN-PAGE followed by immunotransfer to nitrocellulose membranes and probing with α-HA antibody. The mature ∼700 kDa MICOS complex is highlighted with an asterisk. MIC60, MIC19, and MIC25 were also detected in a sub-complex of ∼500 kDa (two asterisks). ( E ) Two-dimensional blue native electrophoresis of 293T mitochondrial lysates. Endogenous QIL1 is present in the mature ∼700 kDa MICOS complex (asterisk). MIC60, MIC19, and MIC25 were also present in a smaller sub-complex (two asterisks). ( F – G ) C-terminally tagged ( F ) or endogenous QIL ( G ) was immunopurified from mitochondria lysed in 1% digitonin or 1% Triton X-100 (TX100). Immunoblot analysis was performed to detect interaction with other MICOS subunits. DOI: http://dx.doi.org/10.7554/eLife.06265.006
    Triton X 100, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6600 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti beta actin antibody
    LRP6 and PIK3R2 are direct targets of miR-126-3p. (A) MiR-126-3p and its putative binding sequence in the 3′UTR of LRP6 and PIK3R2. The mutant miR-126-3p binding site was generated in the complementary site for the seed region of miR-126-3p. (B) Hep-G2 and BEL-7402 cells were co-transfected with miR-126-3p mimics or NC and wild-type or its mutant-type (either LRP6 or PIK3R2), and the luciferase activities were examined. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. (C) Western blot results of endogenous LRP6, PIK3R2, <t>beta-catenin</t> and p-AKT proteins in HCC cells transfected with miR-126-3p mimics or anti-mir-126-3p and its NC. (D) Analysis of LRP6 and PIK3R2 expression in subcutaneous tumors by immunohistochemistry. NC indicated negative control, **P
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    Abcam β actin
    LPS increased in P. gingivalis and thereby inhibited the functions of MSCs by activating NLRP3 inflammasome. a , b Western blot and real-time RT-PCR results revealed that NLRP3 expression was significantly increased in palatal tissues from the inoculation of P. gingivalis group compared to the mixture of L. reuteri and P. gingivalis group, and untreated group. <t>β-actin</t> was used as an internal control in western blot assay. c , d The cell migration assay results demonstrated that LPS significantly inhibited GMSC migration, and NLRP3 inhibitor CY-09 rescued the capacity of GMSC migration after LPS treatment. e – g Real-time RT-PCR results showed that LPS inhibited the expression of SOX2, OCT4, and NANOG, and CY-09 restored the capacities of MSCs. h , i ALP activity assay and alizarin red staining assay. j - m Real-time RT-PCR results further testified that NLRP3 activation inhibited the expression of the crucial transcription factors for modulating osteogenic differentiation: RUX2, OSX, OPN, and OCN, and CY-09 rescued the functions of osteogenic differentiation. n Counting kit-8 assay results showed that CY-09 restored the proliferation potential of GMSCs caused by LPS-induced NLRP3 activation. GAPDH was an internal control. Error bars represent SD ( n = 3). * P ≤ 0.05; ** P ≤ 0.01
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    Mobility of wild type and mutant <t>Cav3.1</t> and Cav3.2 channels in hippocampal neurons. ( a ) Fluorescence recovery after photobleaching (FRAP) assay of <t>Cav3.1-GFP</t> wild type and ∆CT (1851–1875) mutant channels or Cav3.2-GFP wild type and ∆CT (1860–1884) mutant channels transfected into mouse hippocampal neuron cultures. ( b ) Recovery (Fluorescence-Fluorescence bleach) of Cav3.1-GFP wild type and ∆CT (1851–1875) mutant channels . Recovery values are calculated as F(maximum after photobleach)-F(photobleach) ( n = 8 WT, n = 8 mutant, P
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    Abcam rat anti brdu
    Systemic LPS treatment increased the number of microglia and <t>BrdU-positive</t> proliferative cells. A : The lipopolysaccharide (LPS) treatment scheme. Intraperitoneal injection of LPS is performed at P3, and 5-bromo-2′-deoxyuridine (BrdU) is incorporated from P3 to P7. The retina was evaluated at P7. B , C : Immunostaining shows Iba-1-positive microglia (red). LPS treatment increases the number of microglia in the retina at P7. D : CD206, a marker of M2 microglia, is increased by LPS treatment. E : The number of cleaved <t>caspase-3-positive</t> cells is not changed by LPS treatment. F – H : Immunostaining shows BrdU-positive proliferative cells (red) in the peripheral retina. LPS treatment increases the number of BrdU-positive proliferative cells in the peripheral area. The increased BrdU-positive proliferative cells merge with the retinal precursor cell marker, Pax6. I , J : Pax6 (green) and Sox2 (magenta) double-positive cells are increased with LPS treatment. Data are the mean ± standard error of the mean (SEM; n=4 or 5). # ; p
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    Abcam immunocytochemistry
    Systemic LPS treatment increased the number of microglia and <t>BrdU-positive</t> proliferative cells. A : The lipopolysaccharide (LPS) treatment scheme. Intraperitoneal injection of LPS is performed at P3, and 5-bromo-2′-deoxyuridine (BrdU) is incorporated from P3 to P7. The retina was evaluated at P7. B , C : Immunostaining shows Iba-1-positive microglia (red). LPS treatment increases the number of microglia in the retina at P7. D : CD206, a marker of M2 microglia, is increased by LPS treatment. E : The number of cleaved <t>caspase-3-positive</t> cells is not changed by LPS treatment. F – H : Immunostaining shows BrdU-positive proliferative cells (red) in the peripheral retina. LPS treatment increases the number of BrdU-positive proliferative cells in the peripheral area. The increased BrdU-positive proliferative cells merge with the retinal precursor cell marker, Pax6. I , J : Pax6 (green) and Sox2 (magenta) double-positive cells are increased with LPS treatment. Data are the mean ± standard error of the mean (SEM; n=4 or 5). # ; p
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    Image Search Results


    CFTR depletion impairs TfR recycling in bronchial epithelial cells. ( a – d ) 16HBE14o- cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides. ( a ) The cells were exposed to Alexa-Fluor-488-Tf for 1 h

    Journal: Cell Death and Differentiation

    Article Title: Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator

    doi: 10.1038/cdd.2013.46

    Figure Lengend Snippet: CFTR depletion impairs TfR recycling in bronchial epithelial cells. ( a – d ) 16HBE14o- cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides. ( a ) The cells were exposed to Alexa-Fluor-488-Tf for 1 h

    Article Snippet: The Alexa-Fluor-488, Alexa-Fluor-546 secondary antibodies were obtained from Molecular Probes (Invitrogen).

    Techniques: Transfection

    Treatment with Ola triggers nuclear translocation of endogenous HFn. Confocal microscopy images of MDA-MB 231, MDA-MB 468 and HCC1937 cells incubated for 1, 3 and 48 h at 37 °C with 1 µM Ola. Cells not previously treated with Ola were used as negative control (untreated). Nuclei were stained with DAPI (blue). Endogenous ferritin was recognized with anti-ferritin antibody and labeled with an anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (green; Thermo Fischer Scientific). Scale bar: 10 µm.

    Journal: Scientific Reports

    Article Title: H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer

    doi: 10.1038/s41598-017-07617-7

    Figure Lengend Snippet: Treatment with Ola triggers nuclear translocation of endogenous HFn. Confocal microscopy images of MDA-MB 231, MDA-MB 468 and HCC1937 cells incubated for 1, 3 and 48 h at 37 °C with 1 µM Ola. Cells not previously treated with Ola were used as negative control (untreated). Nuclei were stained with DAPI (blue). Endogenous ferritin was recognized with anti-ferritin antibody and labeled with an anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (green; Thermo Fischer Scientific). Scale bar: 10 µm.

    Article Snippet: A blocking step was performed for 1 h at RT with a solution containing 2% BSA (Sigma), 2% goat serum (Euroclone) and 0.2 µg/mL DAPI (4’,6-diamino-2-phenylindole; Thermo Fischer Scientific) in PBS.

    Techniques: Translocation Assay, Confocal Microscopy, Multiple Displacement Amplification, Incubation, Negative Control, Staining, Labeling

    HFn recognition of Triple-Negative Breast Cancer cells. ( a ) MDA-MB 231, MDA-MB 468, HCC1937 (for TNBC) and HUVEC cells were incubated 2 h at 4 °C in PBS buffer and 0.3% BSA with different amounts of FITC-labelled HFn nanoparticles (20 and 100 µg/mL). Then, cells were processed for flow cytometry, using untreated cells to set the positive region and the singlet gate. Reported values are the mean ± s.e. (n = 3). ( b ) MDA-MB 231, MDA-MB 468, HCC1937 (for TNBC) and HUVEC cells seeded in a multiwell plate were incubated 1 h at 37 °C in complete cell culture medium with different amounts of FITC-labeled HFn nanoparticles (20 and 100 µg/mL). Then, cells were detached, washed and processed for flow cytometry, using untreated cells to set the positive region and the singlet gate. Reported values are the mean ± s.e. (n = 3). ( c ) Competition assay. HCC1937 cells were incubated 1 h at 37 °C with 100 µg/mL of FITC-labelled HFn with or without an excess of unlabeled HFn ( i.e ., 5 mg) as competitor. Cells were then detached and treated for flow cytometry. Untreated cells have been used to set the singlet gate and the positive region. Reported values are the mean ± s.e. (n = 3). ( d ) Colocalization of HFn and TfR1. Confocal images of MDA-MB 231, MDA-MB 468, HCC1937 (for TNBC) and HUVEC cells incubated 15 min or 1 h at 37 °C in complete cell culture medium with FITC-labeled HFn nanoparticles (green; 100 µg/mL). Nuclei were stained with DAPI (blue). TfR1 was recognized with anti-TfR1 antibody (Abcam) and labeled with an anti-rabbit secondary antibody conjugated with Alexa Fluor 546 (magenta; Thermo Fischer Scientific). Scale bar: 10 µm.

    Journal: Scientific Reports

    Article Title: H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer

    doi: 10.1038/s41598-017-07617-7

    Figure Lengend Snippet: HFn recognition of Triple-Negative Breast Cancer cells. ( a ) MDA-MB 231, MDA-MB 468, HCC1937 (for TNBC) and HUVEC cells were incubated 2 h at 4 °C in PBS buffer and 0.3% BSA with different amounts of FITC-labelled HFn nanoparticles (20 and 100 µg/mL). Then, cells were processed for flow cytometry, using untreated cells to set the positive region and the singlet gate. Reported values are the mean ± s.e. (n = 3). ( b ) MDA-MB 231, MDA-MB 468, HCC1937 (for TNBC) and HUVEC cells seeded in a multiwell plate were incubated 1 h at 37 °C in complete cell culture medium with different amounts of FITC-labeled HFn nanoparticles (20 and 100 µg/mL). Then, cells were detached, washed and processed for flow cytometry, using untreated cells to set the positive region and the singlet gate. Reported values are the mean ± s.e. (n = 3). ( c ) Competition assay. HCC1937 cells were incubated 1 h at 37 °C with 100 µg/mL of FITC-labelled HFn with or without an excess of unlabeled HFn ( i.e ., 5 mg) as competitor. Cells were then detached and treated for flow cytometry. Untreated cells have been used to set the singlet gate and the positive region. Reported values are the mean ± s.e. (n = 3). ( d ) Colocalization of HFn and TfR1. Confocal images of MDA-MB 231, MDA-MB 468, HCC1937 (for TNBC) and HUVEC cells incubated 15 min or 1 h at 37 °C in complete cell culture medium with FITC-labeled HFn nanoparticles (green; 100 µg/mL). Nuclei were stained with DAPI (blue). TfR1 was recognized with anti-TfR1 antibody (Abcam) and labeled with an anti-rabbit secondary antibody conjugated with Alexa Fluor 546 (magenta; Thermo Fischer Scientific). Scale bar: 10 µm.

    Article Snippet: A blocking step was performed for 1 h at RT with a solution containing 2% BSA (Sigma), 2% goat serum (Euroclone) and 0.2 µg/mL DAPI (4’,6-diamino-2-phenylindole; Thermo Fischer Scientific) in PBS.

    Techniques: Multiple Displacement Amplification, Incubation, Flow Cytometry, Cytometry, Cell Culture, Labeling, Competitive Binding Assay, Staining

    Drp1 SUMOylation selectively reduces binding to Mff. ( a ) Non-SUMOylatable HA-Drp1 shows enhanced association with GST-Mff. HA-Drp1 or HA-Drp1 4KR were transfected into HEK293 cells expressing GST-Mff, GST-Fis1, GST-MID49 or GST-MID51. GST-pull downs and lysates were immunoblotted with HA (for Drp1) and GST antibodies. The histogram shows ratio of Drp1 4KR to Drp1 binding to each receptor (For GST-Mff, n = 6; **P = 0.0042; for GST-Fis1, n = 5; P = 0.7749; For GST-MID49, n = 5; P = 0.4316; for GST-MID51, n = 5; P = 0.6926; Paired Student’s test). ( b ) In vitro , in the absence of SUMO, non-SUMOylatable Drp1-His and wild type Drp1-His show similar binding ability to GST-Mff. GST-PD was performed following incubation of His-tagged Drp1 or its 4KR mutant with either recombinant GST or GST-Mff. GST-pull downs and purified proteins were separated on different lanes in the same gels and immunoblotted with a Drp1 antibody. PVDF was stained with coomassie blue for GST or GST-Mff. ( c ) Cytosolic and mitochondrial localisations of Drp1, Mff and SENP3 in HEK293 cells. Drp1 and SENP3 are predominantly cytoplasmic under basal conditions and Mff is exclusively mitochondrial. GAPDH is a cytosolic marker and VDAC is a mitochondrial marker. ( d ) SENP3 knockdown, which promotes Drp1 SUMOylation, decreases the interaction between endogenous Drp1 and Mff. SENP3 siRNA (SENP3i) or non-specific siRNA (Nsi) was transfected in HEK293 cells followed by IP of Mff and SUMO-2/3. β-actin was used as a loading control. The histogram shows the degree of inhibition of Drp1 binding to Mff (n = 3; **P = 0.0091; Paired Student’s test).

    Journal: Scientific Reports

    Article Title: SENP3-mediated deSUMOylation of Drp1 facilitates interaction with Mff to promote cell death

    doi: 10.1038/srep43811

    Figure Lengend Snippet: Drp1 SUMOylation selectively reduces binding to Mff. ( a ) Non-SUMOylatable HA-Drp1 shows enhanced association with GST-Mff. HA-Drp1 or HA-Drp1 4KR were transfected into HEK293 cells expressing GST-Mff, GST-Fis1, GST-MID49 or GST-MID51. GST-pull downs and lysates were immunoblotted with HA (for Drp1) and GST antibodies. The histogram shows ratio of Drp1 4KR to Drp1 binding to each receptor (For GST-Mff, n = 6; **P = 0.0042; for GST-Fis1, n = 5; P = 0.7749; For GST-MID49, n = 5; P = 0.4316; for GST-MID51, n = 5; P = 0.6926; Paired Student’s test). ( b ) In vitro , in the absence of SUMO, non-SUMOylatable Drp1-His and wild type Drp1-His show similar binding ability to GST-Mff. GST-PD was performed following incubation of His-tagged Drp1 or its 4KR mutant with either recombinant GST or GST-Mff. GST-pull downs and purified proteins were separated on different lanes in the same gels and immunoblotted with a Drp1 antibody. PVDF was stained with coomassie blue for GST or GST-Mff. ( c ) Cytosolic and mitochondrial localisations of Drp1, Mff and SENP3 in HEK293 cells. Drp1 and SENP3 are predominantly cytoplasmic under basal conditions and Mff is exclusively mitochondrial. GAPDH is a cytosolic marker and VDAC is a mitochondrial marker. ( d ) SENP3 knockdown, which promotes Drp1 SUMOylation, decreases the interaction between endogenous Drp1 and Mff. SENP3 siRNA (SENP3i) or non-specific siRNA (Nsi) was transfected in HEK293 cells followed by IP of Mff and SUMO-2/3. β-actin was used as a loading control. The histogram shows the degree of inhibition of Drp1 binding to Mff (n = 3; **P = 0.0091; Paired Student’s test).

    Article Snippet: Immunoblotting Samples were resolved by SDS-PAGE (7.5–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated.

    Techniques: Binding Assay, Transfection, Expressing, In Vitro, Incubation, Mutagenesis, Recombinant, Purification, Staining, Marker, Inhibition

    E-cadherin loss in Lgr5 + stem cells lead to their apoptosis. Co-IF for Lgr5-GFP (green), E-cadherin (white), and cleaved caspase-3 (red), and quantification of cleaved caspase-3 + cells per gland in the gastric antrum of Lgr5-Cre;Cdh1 fl/+ ( n = 4) and Lgr5-Cre;Cdh1 fl/fl ( n = 6) mice after 7 weeks of Tam treatment. The data are presented as means ± SEM, * P

    Journal: International Journal of Biological Sciences

    Article Title: E-cadherin is Required for the Homeostasis of Lgr5+ Gastric Antral Stem Cells

    doi: 10.7150/ijbs.28879

    Figure Lengend Snippet: E-cadherin loss in Lgr5 + stem cells lead to their apoptosis. Co-IF for Lgr5-GFP (green), E-cadherin (white), and cleaved caspase-3 (red), and quantification of cleaved caspase-3 + cells per gland in the gastric antrum of Lgr5-Cre;Cdh1 fl/+ ( n = 4) and Lgr5-Cre;Cdh1 fl/fl ( n = 6) mice after 7 weeks of Tam treatment. The data are presented as means ± SEM, * P

    Article Snippet: Antibodies were as follows: GFP (1:200, Cat. no. 2956, CST, Danvers, MS), BrdU (1:300, Cat. no. ab6326, Abcam, Cambridge, UK), Ki67 (1:1000, Cat. no. ab15580, Abcam), E-cadherin (1:300, Cat. no. 610181, BD Biosciences, Franklin Lakes, NJ), cleaved caspase-3 (1:200, Cat. no. 9661S, CST), TRITC-labelled UEA (1:100; Cat. no. L4889, Sigma), ChrgA (1:100, Cat. no. ZA-0066, Zhongshanjinqiao, Beijing, China), Tff2 (1:500, R33452, Sigma), FITC-labelled DBA (1:100, L9142, Sigma), RFP (1:500, Cat. no. 600-401-379, Rockland, Limerick, PA; tdTomato can be recognized by the RFP antibody), p53 (1:100, Cat. no. 2524S, CST,).

    Techniques: Mouse Assay

    GFAP-YFP-expressing cells in the DG and population-level dynamics of hippocampal neural stem cells. (A,C) GFAP-YFP-positive cells in 8-week-old (A) and 56-week-old (C) GFAP-YFP reporter mice. Scale bars: 100 μm. (B,D) Representative confocal images of immunostaining for GFP (green) and S100β (red). Shown are examples of a GFAP + /S100β − neural stem cell (B) and a GFAP + /S100β + astrocyte (D). Scale bars: 20 μm. (E,F) Fit of the proposed model to the total number of NSCs (E) and the fraction of BrdU-incorporating NSCs (F). Estimated parameters are displayed in Table 1 .

    Journal: Development (Cambridge, England)

    Article Title: Revealing age-related changes of adult hippocampal neurogenesis using mathematical models

    doi: 10.1242/dev.153544

    Figure Lengend Snippet: GFAP-YFP-expressing cells in the DG and population-level dynamics of hippocampal neural stem cells. (A,C) GFAP-YFP-positive cells in 8-week-old (A) and 56-week-old (C) GFAP-YFP reporter mice. Scale bars: 100 μm. (B,D) Representative confocal images of immunostaining for GFP (green) and S100β (red). Shown are examples of a GFAP + /S100β − neural stem cell (B) and a GFAP + /S100β + astrocyte (D). Scale bars: 20 μm. (E,F) Fit of the proposed model to the total number of NSCs (E) and the fraction of BrdU-incorporating NSCs (F). Estimated parameters are displayed in Table 1 .

    Article Snippet: Immunohistochemistry For each animal, six 50 μm coronal brain sections (250 μm between sections) were washed four times, for 10 min in Tris-buffered saline pH 7.4 (TBS) at room temperature, blocked for 1 h in TBS containing 3% horse serum (Millipore; S9135) and 0.3% Triton X-100 (Sigma; 9002-93-1) at room temperature, followed by an overnight staining at 4°C in TBS containing 3% horse serum and 0.3% Triton X-100 with the following primary antibodies: rat anti-BrdU (Abcam, ab6326; 1/200), chicken anti-GFP (Aves, GFP-1020; 1/500), mouse anti-S100β (Abcam, ab66028; 1/100) and rabbit anti-Tbr2 (Abcam, ab23345; 1/300).

    Techniques: Expressing, Mouse Assay, Immunostaining

    Progression of neurogenesis and gliogenesis in the tuberal hypothalamus of CD1 wildtype mice. a P0 brain sections of BrdU birthdating studies indicating the neurons, marked by NeuN, that were born at E11.5, E13.5 and E15.5 embryonic time points during neurogenesis in the tuberal hypothalamus. Yellow arrows indicate examples of NeuN+/BrdU+ co-labeled neurons, third ventricle location is highlighted with a white dotted line. b Sox9+ glioblasts in the tuberal hypothalamus at E13.5, E15.5 and E17.5. Scale bars equal 200 μm

    Journal: Neural Development

    Article Title: Oligodendrocyte development in the embryonic tuberal hypothalamus and the influence of Ascl1

    doi: 10.1186/s13064-016-0075-9

    Figure Lengend Snippet: Progression of neurogenesis and gliogenesis in the tuberal hypothalamus of CD1 wildtype mice. a P0 brain sections of BrdU birthdating studies indicating the neurons, marked by NeuN, that were born at E11.5, E13.5 and E15.5 embryonic time points during neurogenesis in the tuberal hypothalamus. Yellow arrows indicate examples of NeuN+/BrdU+ co-labeled neurons, third ventricle location is highlighted with a white dotted line. b Sox9+ glioblasts in the tuberal hypothalamus at E13.5, E15.5 and E17.5. Scale bars equal 200 μm

    Article Snippet: Primary antibodies were as follows: Mouse anti-NeuN (Millipore; 1:400), Rat anti-BrdU (Cedar Lane; 1:300), Goat anti-Ki67 (Santa Cruz; 1:300), Rabbit anti-Ki67 (Abcam; 1:100), Goat anti-Sox9 (R & D systems; 1:40); Rabbit anti-Olig2 (Millipore; 1:500); Mouse anti-Olig2 (Millipore; 1:300), Goat anti-PdgfRα (R & D Systems; 1:150); Rat anti-SF-1 (graciously provided by Dr Taro Tachibana, Osaka City University JAPAN, 1:800); Rabbit anti-TTF-1 (alternatively Nkx2.1; Santa Cruz; 1:500), Goat anti-Sox10 (Santa Cruz, 1:500), Rabbit anti-pHH3 (Millipore; 1:500), Rabbit anti-Cyclin E (Santa Cruz; 1:200), Rabbit anti-Cyclin B1 (Santa Cruz; 1:200), Mouse anti-Cyclin D2 (ThermoFisher Scientific; 1:200), Rabbit anti-p57kip (Sigma; 1:200), Rabbit anti-Aldh1L1 (Abcam; 1:500), Rat anti-MBP (Millipore; 1:50), and Rabbit anti-Cleaved Caspase 3 (Abcam; 1:800).

    Techniques: Mouse Assay, Labeling

    REG3B–GLP-1-treated pancreatic islets maintain insulin-positive cells after STZ induction. (A) Immunohistochemistry showing islet structure without STZ and REG3B–GLP-1 (left column), 1-week post STZ induction with or without REG3B–GLP-1 therapy (middle columns), and 8-weeks post STZ induction with or without REG3B–GLP-1 therapy (right columns). Insulin and glucagon (top row), glucagon and Nkx6.1 (middle row), and glucagon and ki67 (bottom row) are shown. All images were taken with 40× objective. (B) Immunostaining of islets 1-week post STZ induction with AAV9 mRIP Reg3b–Glp-1 delivery. A small population of islets was found to be bi-hormonal for glucagon and insulin. The left panel is 10× objective of pancreas cross-section, with the islet with insulin and glucagon dual-positive cells indicated by a dashed box. Other panels show serial sections of a bi-hormonal islet, stained with combinations of anti-insulin, -glucagon or -Pdx1 antibodies, at higher magnifications (40× objective). Nuclei were counterstained by DAPI. Scale bars: 50 μm. (C) 8 weeks after STZ induction insulin-positive cell masses were determined as described in the Materials and Methods (left panel). Percent of total glucagon area to insulin area was determined from five random islets per individual (middle panel). Percent islet cell proliferation was determined by counting the number of ki67-positive cells within insulin- and glucagon-positive regions of five random islets (right panel).

    Journal: Disease Models & Mechanisms

    Article Title: Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced ?-cell damage and pancreatic Glp-1 gene therapy

    doi: 10.1242/dmm.012591

    Figure Lengend Snippet: REG3B–GLP-1-treated pancreatic islets maintain insulin-positive cells after STZ induction. (A) Immunohistochemistry showing islet structure without STZ and REG3B–GLP-1 (left column), 1-week post STZ induction with or without REG3B–GLP-1 therapy (middle columns), and 8-weeks post STZ induction with or without REG3B–GLP-1 therapy (right columns). Insulin and glucagon (top row), glucagon and Nkx6.1 (middle row), and glucagon and ki67 (bottom row) are shown. All images were taken with 40× objective. (B) Immunostaining of islets 1-week post STZ induction with AAV9 mRIP Reg3b–Glp-1 delivery. A small population of islets was found to be bi-hormonal for glucagon and insulin. The left panel is 10× objective of pancreas cross-section, with the islet with insulin and glucagon dual-positive cells indicated by a dashed box. Other panels show serial sections of a bi-hormonal islet, stained with combinations of anti-insulin, -glucagon or -Pdx1 antibodies, at higher magnifications (40× objective). Nuclei were counterstained by DAPI. Scale bars: 50 μm. (C) 8 weeks after STZ induction insulin-positive cell masses were determined as described in the Materials and Methods (left panel). Percent of total glucagon area to insulin area was determined from five random islets per individual (middle panel). Percent islet cell proliferation was determined by counting the number of ki67-positive cells within insulin- and glucagon-positive regions of five random islets (right panel).

    Article Snippet: Antibodies and concentrations used for immunocytochemistry include: guinea pig anti-insulin (1:400, Dako Corporation, Carpinteria, CA), mouse anti-insulin (1:300, Sigma-Aldrich, St Louis, MO), mouse monoclonal to glucagon (1:300, Abcam, Cambridge, MA), rabbit polyclonal to Pdx-1 (1:100, Abcam, Cambridge, MA), goat polyclonal against Nkx6.1 (1:200, R & D Systems, Minneapolis, MN), rabbit polyclonal to ki67 (1:100, Abcam, Cambridge, MA), and goat polyclonal to Cxcl13 (1:200, R & D Systems, Minneapolis, MN).

    Techniques: Immunohistochemistry, Immunostaining, Staining

    MT EB3-tracking measurements reveal a significant decrease in spindle symmetry in the absence of Ccdc61. (A–C) HeLa cells stably expressing EGFP-EB3 were used for EB3-tracking experiments. Cells were either treated with control (ct), Ccdc61 O2 siRNA (O2), or a combination of Ccdc61 O2 siRNA together with FLAG-Ccdc61 siRNA-resistant construct (O2R). To study mitotic properties, like directionality and orientation, the individual EB3 tips were imaged every 0.13 s and analyzed via an automated process. (A) Example images of a single time frame (top row) and a time projection from 125 s recordings (bottom row) of HeLa cells treated as indicated. Western blot shows the efficiency of indicated Ccdc61 Oligo that was tested by its ability to deplete transiently transfected FLAG-Ccdc61 or siRNA-resistant FLAG-Ccdc61 (O2R). α-tubulin served as a loading control. (B) EGFP-EB3 tracks from HeLa cells were analyzed as described in the Materials and Methods section and as published in the preprint ( Jafarpour and Lorenz, 2017 ). The table represents velocity (µm/min), displacement (µm), duration (s) (mean ± SD.) and the total number of analyzed tracks (of 10 independent cells). The significance was calculated for the three conditions by a one-way ANOVA, and the alpha value at which there was no significant difference (n.s.d.) within the group as well as the p value is provided. (C) Shown are the results of calculated asymmetry and directionality indices of the indicated treated HeLa cells with 10 image series per group based on the histogram of calculated orientations (Supplemental Figure S4, B and C). Each mitosis event is characterized with two figures of merit, resulting in a single point. The y -axis represents the degree of asymmetry, whereby a low value indicates a symmetric direction of MT growth (low asymmetry) and a high value indicates an asymmetric direction of MT growth (reduced symmetry). The horizontal axis represents the directionality of MT growth, indicating different levels of point symmetry in MT growth paths.

    Journal: Molecular Biology of the Cell

    Article Title: Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry

    doi: 10.1091/mbc.E18-02-0115

    Figure Lengend Snippet: MT EB3-tracking measurements reveal a significant decrease in spindle symmetry in the absence of Ccdc61. (A–C) HeLa cells stably expressing EGFP-EB3 were used for EB3-tracking experiments. Cells were either treated with control (ct), Ccdc61 O2 siRNA (O2), or a combination of Ccdc61 O2 siRNA together with FLAG-Ccdc61 siRNA-resistant construct (O2R). To study mitotic properties, like directionality and orientation, the individual EB3 tips were imaged every 0.13 s and analyzed via an automated process. (A) Example images of a single time frame (top row) and a time projection from 125 s recordings (bottom row) of HeLa cells treated as indicated. Western blot shows the efficiency of indicated Ccdc61 Oligo that was tested by its ability to deplete transiently transfected FLAG-Ccdc61 or siRNA-resistant FLAG-Ccdc61 (O2R). α-tubulin served as a loading control. (B) EGFP-EB3 tracks from HeLa cells were analyzed as described in the Materials and Methods section and as published in the preprint ( Jafarpour and Lorenz, 2017 ). The table represents velocity (µm/min), displacement (µm), duration (s) (mean ± SD.) and the total number of analyzed tracks (of 10 independent cells). The significance was calculated for the three conditions by a one-way ANOVA, and the alpha value at which there was no significant difference (n.s.d.) within the group as well as the p value is provided. (C) Shown are the results of calculated asymmetry and directionality indices of the indicated treated HeLa cells with 10 image series per group based on the histogram of calculated orientations (Supplemental Figure S4, B and C). Each mitosis event is characterized with two figures of merit, resulting in a single point. The y -axis represents the degree of asymmetry, whereby a low value indicates a symmetric direction of MT growth (low asymmetry) and a high value indicates an asymmetric direction of MT growth (reduced symmetry). The horizontal axis represents the directionality of MT growth, indicating different levels of point symmetry in MT growth paths.

    Article Snippet: Mouse anti-FLAG M2 (1:5000; F3165), mouse anti-α-tubulin (1:1000; T5168) and mouse anti-γ-tubulin (1:500; T6557) were obtained from Sigma, goat anti-γ-tubulin C-20 (1:50, sc-7396) from Santa Cruz, rabbit anti-GFP (1:500; NB600-303) from Novus, rabbit anti-pericentrin (1:3000; ab4448) and rabbit anti-Cep170 (1:500; ab72505) from Abcam, mouse anti-Cep170 72-413-1 (1:1000 or 1 µg per 1 mg total cell lysate; 41-3200) from Thermo Scientific, rabbit anti-Cep290 (1:500; A301-659A-2) from Bethyl and rabbit anti-Mad2 (1:100; PRB-452C) from Covance.

    Techniques: Stable Transfection, Expressing, Construct, Western Blot, Transfection

    SMAD6 is Downstream of Notch Signaling in Homeostatic Endothelial Cell Flow-Mediated Alignment. A) Representative panels of HUVEC stained with Phalloidin (red, actin) and DAPI (white, nucleus) under flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001; ns, not significant. C) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. D) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. E) qPCR RNA levels (normalized to static control) in HUVEC treated with RPBJ siRNA. Statistical analysis, Student’s t-test; **, p≤0.01 F) Representative panels of HUVEC with additional RBPJ and DLL4 siRNAs (see Key Resources) stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions. White arrow, flow vector. Scale bar, 50μM. G) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. H) Representative panel of HAEC treated with RPBJ siRNA and stained with phalloidin (F-actin, white) and expression construct (SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive EC. Scale bar, 20μM. I) Quantification of cell axis ratio in indicated conditions. Statistical analysis, student’s t-test; ****, p≤0.0001. J) Diagram showing SMAD6 constructs. Numbers indicate amino acids in human SMAD6.

    Journal: bioRxiv

    Article Title: SMAD6 Integrates Endothelial Cell Homeostatic Flow Responses Downstream of Notch

    doi: 10.1101/2020.07.02.184820

    Figure Lengend Snippet: SMAD6 is Downstream of Notch Signaling in Homeostatic Endothelial Cell Flow-Mediated Alignment. A) Representative panels of HUVEC stained with Phalloidin (red, actin) and DAPI (white, nucleus) under flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001; ns, not significant. C) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. D) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. E) qPCR RNA levels (normalized to static control) in HUVEC treated with RPBJ siRNA. Statistical analysis, Student’s t-test; **, p≤0.01 F) Representative panels of HUVEC with additional RBPJ and DLL4 siRNAs (see Key Resources) stained with VE-cadherin (green, junctions) and DAPI (white, nucleus) under control (static) or flow conditions. White arrow, flow vector. Scale bar, 50μM. G) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. H) Representative panel of HAEC treated with RPBJ siRNA and stained with phalloidin (F-actin, white) and expression construct (SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive EC. Scale bar, 20μM. I) Quantification of cell axis ratio in indicated conditions. Statistical analysis, student’s t-test; ****, p≤0.0001. J) Diagram showing SMAD6 constructs. Numbers indicate amino acids in human SMAD6.

    Article Snippet: After washing 5X with 1% BSA, samples were incubated with Alexa-fluor-conjugated anti-species secondary antibodies (1:250—see Key Resources) plus Alexa-fluor-conjugated phalloidin (1:50— Invitrogen, #A12379 or #A12381) and DAPI or DRAQ7 (1:300—Sigma #10236276001 or Abcam #ab109202, respectively) in 1% BSA for 30 min at RT.

    Techniques: Staining, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Construct

    SMAD6 is Downstream of Notch Signaling in Homeostatic Endothelial Cell Flow-Mediated Alignment. A ) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DAPI (blue, nucleus) under control (static) or flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio. Statistical analysis, One-way ANOVA; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001. C) qPCR RNA levels (normalized to vehicle control under flow) of SMAD6 in HUVEC treated with DAPT under flow. Statistical analysis, Student’s t-test; *, p≤0.05. D ) Representative panels of HUVEC stained with Phalloidin (F-actin, white) with indicated treatments and expression constructs (Empty Vector (EV) or SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive HUVEC. Scale bar, 50μM. E) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ***, p≤0.001; ****, p≤0.0001. F) qPCR RNA levels (normalized to vehicle control under flow) in in HUVEC treated with Notch1 siRNA under flow. Statistical analysis, Student’s t-test; *, p≤0.05. G) Representative panels of HUVEC stained with Phalloidin (F-actin, white) with indicated treatments and expression construct (SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive EC. Scale bar, 20μM. H) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; **, p≤0.01.

    Journal: bioRxiv

    Article Title: SMAD6 Integrates Endothelial Cell Homeostatic Flow Responses Downstream of Notch

    doi: 10.1101/2020.07.02.184820

    Figure Lengend Snippet: SMAD6 is Downstream of Notch Signaling in Homeostatic Endothelial Cell Flow-Mediated Alignment. A ) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DAPI (blue, nucleus) under control (static) or flow conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio. Statistical analysis, One-way ANOVA; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001. C) qPCR RNA levels (normalized to vehicle control under flow) of SMAD6 in HUVEC treated with DAPT under flow. Statistical analysis, Student’s t-test; *, p≤0.05. D ) Representative panels of HUVEC stained with Phalloidin (F-actin, white) with indicated treatments and expression constructs (Empty Vector (EV) or SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive HUVEC. Scale bar, 50μM. E) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ***, p≤0.001; ****, p≤0.0001. F) qPCR RNA levels (normalized to vehicle control under flow) in in HUVEC treated with Notch1 siRNA under flow. Statistical analysis, Student’s t-test; *, p≤0.05. G) Representative panels of HUVEC stained with Phalloidin (F-actin, white) with indicated treatments and expression construct (SMAD6) under flow conditions. White arrow, flow vector. Red arrowhead, positive EC. Scale bar, 20μM. H) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; **, p≤0.01.

    Article Snippet: After washing 5X with 1% BSA, samples were incubated with Alexa-fluor-conjugated anti-species secondary antibodies (1:250—see Key Resources) plus Alexa-fluor-conjugated phalloidin (1:50— Invitrogen, #A12379 or #A12381) and DAPI or DRAQ7 (1:300—Sigma #10236276001 or Abcam #ab109202, respectively) in 1% BSA for 30 min at RT.

    Techniques: Staining, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Construct

    SMAD6 is Required for Homeostatic Endothelial Cell Flow-Mediated Alignment and Polarization. A) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DRAQ7 (white, nucleus) under static (no flow) or flow (laminar, 72h, 15 d/cm 2 for all panels) conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. C) Representative panels of HUVEC stained with GM130 (red, Golgi) and DAPI (white, nucleus) under indicated conditions with indicated treatments. White arrow, flow vector. Scale bar, 50μM. D ) Quantification of Golgi localization relative to nucleus in indicated conditions. ≥30 cells/condition were analyzed. Statistical analysis, One-way ANOVA; **, p≤0.01; ***, p≤0.001. NT, non-targeting (control) siRNA.

    Journal: bioRxiv

    Article Title: SMAD6 Integrates Endothelial Cell Homeostatic Flow Responses Downstream of Notch

    doi: 10.1101/2020.07.02.184820

    Figure Lengend Snippet: SMAD6 is Required for Homeostatic Endothelial Cell Flow-Mediated Alignment and Polarization. A) Representative panels of HUVEC stained with VE-cadherin (green, junctions) and DRAQ7 (white, nucleus) under static (no flow) or flow (laminar, 72h, 15 d/cm 2 for all panels) conditions with indicated treatments. White arrow, flow vector. Scale bar, 20μM. B) Quantification of cell axis ratio in indicated conditions. Statistical analysis, One-way ANOVA; ****, p≤0.0001. C) Representative panels of HUVEC stained with GM130 (red, Golgi) and DAPI (white, nucleus) under indicated conditions with indicated treatments. White arrow, flow vector. Scale bar, 50μM. D ) Quantification of Golgi localization relative to nucleus in indicated conditions. ≥30 cells/condition were analyzed. Statistical analysis, One-way ANOVA; **, p≤0.01; ***, p≤0.001. NT, non-targeting (control) siRNA.

    Article Snippet: After washing 5X with 1% BSA, samples were incubated with Alexa-fluor-conjugated anti-species secondary antibodies (1:250—see Key Resources) plus Alexa-fluor-conjugated phalloidin (1:50— Invitrogen, #A12379 or #A12381) and DAPI or DRAQ7 (1:300—Sigma #10236276001 or Abcam #ab109202, respectively) in 1% BSA for 30 min at RT.

    Techniques: Staining, Plasmid Preparation

    Loss of MTIF3 results in uncoordinated mitochondrial protein synthesis. ( A ) Levels of de novo protein synthesis were measured in heart mitochondria from control ( L/L ) and knockout ( L/L, cre ) 10-week-old mice by pulse and chase incorporation of 35 S-labeled cysteine and methionine. Mitochondrial protein was separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie, and visualized by autoradiography. Representative gels of five independent biological experiments are shown. ( B ) Levels of de novo protein synthesis in heart mitochondria from 25-week-old control ( L/L ) and knockout ( L/L, cre ) mice, determined as in (A). Mitochondrial proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( C ) and 25-week-old ( D ) mice were resolved on 4 to 20% SDS-PAGE gels and immunoblotted using antibodies to investigate the steady-state levels of OXPHOS proteins. SDHA was used as a loading control. Levels of mitoribosomal proteins, proteases, and MTIF2 proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( E ) and 25-week-old ( F ) mice. Porin was used as a loading control. All values are means ± SEM. * P

    Journal: Science Advances

    Article Title: Fidelity of translation initiation is required for coordinated respiratory complex assembly

    doi: 10.1126/sciadv.aay2118

    Figure Lengend Snippet: Loss of MTIF3 results in uncoordinated mitochondrial protein synthesis. ( A ) Levels of de novo protein synthesis were measured in heart mitochondria from control ( L/L ) and knockout ( L/L, cre ) 10-week-old mice by pulse and chase incorporation of 35 S-labeled cysteine and methionine. Mitochondrial protein was separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie, and visualized by autoradiography. Representative gels of five independent biological experiments are shown. ( B ) Levels of de novo protein synthesis in heart mitochondria from 25-week-old control ( L/L ) and knockout ( L/L, cre ) mice, determined as in (A). Mitochondrial proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( C ) and 25-week-old ( D ) mice were resolved on 4 to 20% SDS-PAGE gels and immunoblotted using antibodies to investigate the steady-state levels of OXPHOS proteins. SDHA was used as a loading control. Levels of mitoribosomal proteins, proteases, and MTIF2 proteins from isolated heart mitochondria of control ( L/L ) and knockout ( L/L, cre ) 10-week-old ( E ) and 25-week-old ( F ) mice. Porin was used as a loading control. All values are means ± SEM. * P

    Article Snippet: Immunoblotting Specific proteins were detected using rabbit antibodies against Afg3l2 (14631-1-AP, Proteintech, diluted 1:500), CCDC44 (TACO1) (21147-1-AP, Proteintech, diluted 1:500), LONP1 (15440-1-AP, Proteintech, diluted 1:500), HSPA9 (mtHSP70) (PA5-48035, Thermo Fisher Scientific, diluted 1:1000), LRPPRC (sc66844, Santa Cruz Biotechnology; diluted 1:1000), MRPL45 (15682-1-AP, Proteintech, diluted 1:1000), MRPL44 (16394-1-AP, Proteintech, diluted 1:500), MRPL37 (15190-1-AP, Proteintech, diluted 1:500), MRPS35 (16457-1-AP, Proteintech, diluted 1:500), MRPS34 (HPA042112-100 μl, Sigma, diluted 1:500), MRPS16 (16735-1-AP, Proteintech, diluted 1:1000), MTIF3 (14219-1-AP, Proteintech, diluted 1:500), and MTIF2 (LS-C164664/56970, LSBio, diluted 1:500) and mouse antibodies against ATP5a (ab14748, Abcam, diluted 1:1000), COXI (ab14705, Abcam, diluted 1:1000), COXIV (ab14744, Abcam, diluted 1:500), NDUFA9 (ab14713, Abcam, diluted 1:1000), SDHA (ab14715, Abcam, diluted 1:1000), Total OXPHOS Antibody Cocktail (ab110413, Abcam, diluted 1:5000), Total OXPHOS Blue Native Antibody Cocktail (ab110412, Abcam, diluted 1:5000), UQCRC2 (ab14745, Abcam, diluted 1:1000), and VDAC1/Porin (ab14734, Abcam, diluted 1:1000) in Odyssey Blocking Buffer (Li-Cor).

    Techniques: Knock-Out, Mouse Assay, Labeling, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Autoradiography, Isolation

    Banf1 binds to PARP1 following oxidative stress. a , b Banf1 and PARP1 are in a complex; immunoprecipitations from HEK293T cells expressing Flag, Flag-Banf1 or Flag-PARP1 using Flag antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies. c The Banf1:PARP1 interaction after H 2 O 2 ; immunoprecipitations from HEK293T cells ectopically expressing Flag or Flag-Banf1 1 h after H 2 O 2 removal. Immunoprecipitates were immunoblotted with the indicated antibodies. d Banf1 and PARP1 directly interact. The indicated purified proteins (±NAD + /DNA/olaparib) were incubated together before immunoprecipitation with PARP1 antibodies and immunoblotting with the indicated antibodies. e Banf1 and PARP colocalise following oxidative stress induced by H 2 O 2 in U2OS cells. Representative cells fixed at the indicated times following removal of H 2 O 2 and stained with the indicated antibodies are shown, the colocalisation was analysed using ImageJ. The scale bar represents 10 μm. Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Barrier-to-autointegration factor 1 (Banf1) regulates poly [ADP-ribose] polymerase 1 (PARP1) activity following oxidative DNA damage

    doi: 10.1038/s41467-019-13167-5

    Figure Lengend Snippet: Banf1 binds to PARP1 following oxidative stress. a , b Banf1 and PARP1 are in a complex; immunoprecipitations from HEK293T cells expressing Flag, Flag-Banf1 or Flag-PARP1 using Flag antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies. c The Banf1:PARP1 interaction after H 2 O 2 ; immunoprecipitations from HEK293T cells ectopically expressing Flag or Flag-Banf1 1 h after H 2 O 2 removal. Immunoprecipitates were immunoblotted with the indicated antibodies. d Banf1 and PARP1 directly interact. The indicated purified proteins (±NAD + /DNA/olaparib) were incubated together before immunoprecipitation with PARP1 antibodies and immunoblotting with the indicated antibodies. e Banf1 and PARP colocalise following oxidative stress induced by H 2 O 2 in U2OS cells. Representative cells fixed at the indicated times following removal of H 2 O 2 and stained with the indicated antibodies are shown, the colocalisation was analysed using ImageJ. The scale bar represents 10 μm. Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Antibodies The antibodies used were as follows: anti-Banf1 N-terminus (SAB1409950, Sigma-Aldrich and ab88464, Abcam, 1:1000 for WB and 1:500 for IF), anti-Banf1 C-terminus (PRS40170604, Sigma-Aldrich, 1:1000 for WB and 1:500 for IF), PARP1 (9532, Cell Signalling Technology 1:1000 for WB and 1:500 for IF), PARP1 (ab191217, Abcam Fig. 1:500 for IF), anti-Emerin (5430, Cell Signalling Technology, 1:500 for IF), anti-Flag M2 Antibody (F3165, Sigma-Aldrich, 1:1000 for WB and 1:300 for IF), anti-PAR (ab14459, Abcam, 1:1000 for WB), anti-γ-Tubulin (T6557, Sigma-Aldrich, 1:2000 for WB), anti-H3 (4499, Cell Signalling Technology, 1:2000 for WB), anti-H4 (2935 Cell Signalling Technology 1:1000 for WB), anti-SP1 (9389, Cell Signalling Technology, 1:1000 for WB), anti-EGFR (sc-03, Santa-cruz, 1:500 for WB), anti-LC3B (2775, Cell Signalling Technologies, 1:1000 for WB), anti-phospho-p53 ser15 (2984, Cell Signalling Technology, 1:1000 for WB), anti-β-actin (612656, BD Biosciences, 1:2000 for WB).

    Techniques: Expressing, Purification, Incubation, Immunoprecipitation, Staining, Western Blot

    Banf1 A12T inhibits repair of oxidative lesions in NGPS patient cells. a Interactions of PARP1 with Flag-Banf1 mutants. Flag immunoprecipitations from HEK293T cells ectopically expressing the Flag-Banf1 WT or A12T proteins following H 2 O 2 . b Purified Banf1 WT or A12T proteins were incubated with PARP1, immunopreciptated with PARP1 antibodies and immunoblotted with the indicated antibodies. c Inhibition of auto-poly-ADP-ribose activity of PARP1 in U2OS expressing ectopic Flag-Banf1 WT or A12T following H 2 O 2 . d The PAR bands in ( c ), were analysed via densitometry and normalised to γ-Tubulin. Histogram data shown in ( d ), represent the mean and S.D. of three independent experiments. e In vitro inhibition of PARP1 poly-ADP-ribose activity on histones by purified Banf1 WT or A12T. f The PAR bands on ( e ), were analysed via densitometry. g Banf1 inhibits repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in control cells and cells ectopically expressing Flag-Banf1 WT or A12T following H 2 O 2 treatment and recovery. Paired t test was used for statistical analysis. h Inhibition of Poly-ADP-ribose activity of PARP1 purified from NGPS patient cells on an immobilised histone substrate HEK293T following H 2 O 2 . i NGPS patient cells exhibit defective repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in control cells and NGPS cells after H 2 O 2 treatment and recovery. Paired t test was used for statistical analysis. Immunoblots are representative of n = 3 independent experiments. Unless otherwise stated, histogram data shown represent the mean and S.D. of n = 4 independent experiments and statistical significance was defined via ANOVA. * P

    Journal: Nature Communications

    Article Title: Barrier-to-autointegration factor 1 (Banf1) regulates poly [ADP-ribose] polymerase 1 (PARP1) activity following oxidative DNA damage

    doi: 10.1038/s41467-019-13167-5

    Figure Lengend Snippet: Banf1 A12T inhibits repair of oxidative lesions in NGPS patient cells. a Interactions of PARP1 with Flag-Banf1 mutants. Flag immunoprecipitations from HEK293T cells ectopically expressing the Flag-Banf1 WT or A12T proteins following H 2 O 2 . b Purified Banf1 WT or A12T proteins were incubated with PARP1, immunopreciptated with PARP1 antibodies and immunoblotted with the indicated antibodies. c Inhibition of auto-poly-ADP-ribose activity of PARP1 in U2OS expressing ectopic Flag-Banf1 WT or A12T following H 2 O 2 . d The PAR bands in ( c ), were analysed via densitometry and normalised to γ-Tubulin. Histogram data shown in ( d ), represent the mean and S.D. of three independent experiments. e In vitro inhibition of PARP1 poly-ADP-ribose activity on histones by purified Banf1 WT or A12T. f The PAR bands on ( e ), were analysed via densitometry. g Banf1 inhibits repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in control cells and cells ectopically expressing Flag-Banf1 WT or A12T following H 2 O 2 treatment and recovery. Paired t test was used for statistical analysis. h Inhibition of Poly-ADP-ribose activity of PARP1 purified from NGPS patient cells on an immobilised histone substrate HEK293T following H 2 O 2 . i NGPS patient cells exhibit defective repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in control cells and NGPS cells after H 2 O 2 treatment and recovery. Paired t test was used for statistical analysis. Immunoblots are representative of n = 3 independent experiments. Unless otherwise stated, histogram data shown represent the mean and S.D. of n = 4 independent experiments and statistical significance was defined via ANOVA. * P

    Article Snippet: Antibodies The antibodies used were as follows: anti-Banf1 N-terminus (SAB1409950, Sigma-Aldrich and ab88464, Abcam, 1:1000 for WB and 1:500 for IF), anti-Banf1 C-terminus (PRS40170604, Sigma-Aldrich, 1:1000 for WB and 1:500 for IF), PARP1 (9532, Cell Signalling Technology 1:1000 for WB and 1:500 for IF), PARP1 (ab191217, Abcam Fig. 1:500 for IF), anti-Emerin (5430, Cell Signalling Technology, 1:500 for IF), anti-Flag M2 Antibody (F3165, Sigma-Aldrich, 1:1000 for WB and 1:300 for IF), anti-PAR (ab14459, Abcam, 1:1000 for WB), anti-γ-Tubulin (T6557, Sigma-Aldrich, 1:2000 for WB), anti-H3 (4499, Cell Signalling Technology, 1:2000 for WB), anti-H4 (2935 Cell Signalling Technology 1:1000 for WB), anti-SP1 (9389, Cell Signalling Technology, 1:1000 for WB), anti-EGFR (sc-03, Santa-cruz, 1:500 for WB), anti-LC3B (2775, Cell Signalling Technologies, 1:1000 for WB), anti-phospho-p53 ser15 (2984, Cell Signalling Technology, 1:1000 for WB), anti-β-actin (612656, BD Biosciences, 1:2000 for WB).

    Techniques: Expressing, Purification, Incubation, Inhibition, Activity Assay, In Vitro, Alkaline Single Cell Gel Electrophoresis, Western Blot

    BHMT is a potential direct oxidative-target of Nox4-generated ROS. A, Representative immunoblot showing decreasing labelling of BHMT after BIAM labelling of H 2 O 2 -oxidised purified recombinant BHMT and BSA. B, Graph of the densitometric analysis of labelled BHMT band intensities under the indicated H 2 O 2 concentrations. R 2 values indicate that there is a good correlation between the decreases in BHMT labelling and increasing H 2 O 2 concentration (i.e. increasingly oxidised BHMT), while there is a poor correlation between BSA labelling at the same H 2 O 2 concentrations. C, Extracellular catalase-inhibitable H2O2 generated by HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). D, Western blot and densitometric quanrification of BIAM-switch labelled (ie oxidised) BHMT levels in HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). Equivalent amounts (20μg) of “input” protein were blotted for βactin, to serve as a control. Above data are represented as mean +/-S.E.M. ⁎ : p=

    Journal: Free Radical Biology & Medicine

    Article Title: NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice

    doi: 10.1016/j.freeradbiomed.2015.09.015

    Figure Lengend Snippet: BHMT is a potential direct oxidative-target of Nox4-generated ROS. A, Representative immunoblot showing decreasing labelling of BHMT after BIAM labelling of H 2 O 2 -oxidised purified recombinant BHMT and BSA. B, Graph of the densitometric analysis of labelled BHMT band intensities under the indicated H 2 O 2 concentrations. R 2 values indicate that there is a good correlation between the decreases in BHMT labelling and increasing H 2 O 2 concentration (i.e. increasingly oxidised BHMT), while there is a poor correlation between BSA labelling at the same H 2 O 2 concentrations. C, Extracellular catalase-inhibitable H2O2 generated by HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). D, Western blot and densitometric quanrification of BIAM-switch labelled (ie oxidised) BHMT levels in HepG2 cells transduced with AdNox4 or control, AdβGal (n=3). Equivalent amounts (20μg) of “input” protein were blotted for βactin, to serve as a control. Above data are represented as mean +/-S.E.M. ⁎ : p=

    Article Snippet: 300 ng of purified BHMT (Abcam) or BSA (Sigma) were used per reaction, in labelling buffer (100 mM phosphate buffer, pH 6.0, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA).

    Techniques: Generated, Purification, Recombinant, Concentration Assay, Transduction, Western Blot

    Ctdsp2 increases neural progenitor (NP) proliferation. (A–C) Overexpression of Ctdsp2 , but not the control construct pCAGIG, increased the proportion of cells expressing both proliferative marker bromodeoxyuridine + (BrdU + )/green fluorescence protein + (GFP + ) and radial glial cell (RGC) marker Pax6 + /GFP + , but not intermediate progenitor (IP) marker Tbr2 + /GFP + , in GFP-positive cells. (D–F) shRNA-mediated knockdown (sh Ctdsp2 ) of Ctdsp2 decreased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

    doi: 10.3389/fnmol.2018.00044

    Figure Lengend Snippet: Ctdsp2 increases neural progenitor (NP) proliferation. (A–C) Overexpression of Ctdsp2 , but not the control construct pCAGIG, increased the proportion of cells expressing both proliferative marker bromodeoxyuridine + (BrdU + )/green fluorescence protein + (GFP + ) and radial glial cell (RGC) marker Pax6 + /GFP + , but not intermediate progenitor (IP) marker Tbr2 + /GFP + , in GFP-positive cells. (D–F) shRNA-mediated knockdown (sh Ctdsp2 ) of Ctdsp2 decreased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P

    Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

    Techniques: Over Expression, Construct, Expressing, Marker, Fluorescence, shRNA

    miR-26 promotes NP proliferation. (A,D,G) Overexpression of miR-26a, but not the control construct pCAGIG, proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + , but not IP marker Tbr2 + /GFP + , in GFP-positive cells. (B,E,H) miRNA sponge-mediated knockdown (miR-26-SP), but not the mutated sponge (miR-26-SPmut), decreased proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + in GFP-positive cells. (C,F,I) Ratio of BrdU + /GFP + , Pax6 + /GFP + or Tbr2 + /GFP + cells vs. GFP + cells in the electroporated cortex. Values represent mean ± SEM. n = 9 sections from at least three brains. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

    doi: 10.3389/fnmol.2018.00044

    Figure Lengend Snippet: miR-26 promotes NP proliferation. (A,D,G) Overexpression of miR-26a, but not the control construct pCAGIG, proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + , but not IP marker Tbr2 + /GFP + , in GFP-positive cells. (B,E,H) miRNA sponge-mediated knockdown (miR-26-SP), but not the mutated sponge (miR-26-SPmut), decreased proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + in GFP-positive cells. (C,F,I) Ratio of BrdU + /GFP + , Pax6 + /GFP + or Tbr2 + /GFP + cells vs. GFP + cells in the electroporated cortex. Values represent mean ± SEM. n = 9 sections from at least three brains. * P

    Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

    Techniques: Over Expression, Construct, Expressing, Marker

    miR-26a regulates cell cycle progression in NPs. (A) Illustration of in utero electroporation and BrdU incorporation. Electroporation was performed at E13.5, followed by BrdU incorporation 23 hours (23 h) later, and tissues were analyzed at E14.5. BrdU + , Ki67 + and GFP + cells were labeled and counted. (B) miR-26a overexpression increased the proportion of cells entering the S phase of the cell cycle, while miR-26a-SP decreased the proportion, by measuring the BrdU labeling index. (C) Electroporation was performed at E13.5, followed by BrdU incorporation 1 day (1d) later, and tissues were analyzed at E15.5. BrdU + , Ki67 + and GFP + cells were labeled and counted. (D) Neither miR-26a nor miR-26-SP altered the proportion of cells exiting the cell cycle. Values represent mean ± SEM. n = 9 sections from at least three brains. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

    doi: 10.3389/fnmol.2018.00044

    Figure Lengend Snippet: miR-26a regulates cell cycle progression in NPs. (A) Illustration of in utero electroporation and BrdU incorporation. Electroporation was performed at E13.5, followed by BrdU incorporation 23 hours (23 h) later, and tissues were analyzed at E14.5. BrdU + , Ki67 + and GFP + cells were labeled and counted. (B) miR-26a overexpression increased the proportion of cells entering the S phase of the cell cycle, while miR-26a-SP decreased the proportion, by measuring the BrdU labeling index. (C) Electroporation was performed at E13.5, followed by BrdU incorporation 1 day (1d) later, and tissues were analyzed at E15.5. BrdU + , Ki67 + and GFP + cells were labeled and counted. (D) Neither miR-26a nor miR-26-SP altered the proportion of cells exiting the cell cycle. Values represent mean ± SEM. n = 9 sections from at least three brains. * P

    Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

    Techniques: In Utero, Electroporation, BrdU Incorporation Assay, Labeling, Over Expression

    Emx2 is functionally inhibited by miR-26 in regulating NP proliferation. (A–F) Overexpression of Emx2 , but not the control construct pCAGIG, decreased the proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + , but not IP marker Tbr2 + /GFP + , in GFP-positive cells. short hairpin RNA (shRNA)-mediated knockdown (sh Emx2 ) of Emx2 increased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. (G–J) Emx2 expression suppressed the proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + in GFP-positive cells. Co-expressing Emx2 with miR-26, but not miR-26-mut, dramatically reversed the suppression. (K,L) Emx2 expression did not alter the proportion of cells expressing IP marker Tbr2 + /GFP + , in GFP-positive cells. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

    doi: 10.3389/fnmol.2018.00044

    Figure Lengend Snippet: Emx2 is functionally inhibited by miR-26 in regulating NP proliferation. (A–F) Overexpression of Emx2 , but not the control construct pCAGIG, decreased the proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + , but not IP marker Tbr2 + /GFP + , in GFP-positive cells. short hairpin RNA (shRNA)-mediated knockdown (sh Emx2 ) of Emx2 increased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. (G–J) Emx2 expression suppressed the proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + in GFP-positive cells. Co-expressing Emx2 with miR-26, but not miR-26-mut, dramatically reversed the suppression. (K,L) Emx2 expression did not alter the proportion of cells expressing IP marker Tbr2 + /GFP + , in GFP-positive cells. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P

    Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

    Techniques: Over Expression, Construct, Expressing, Marker, shRNA

    QIL1 localizes at cristae junctions and is present in the mature MICOS complex. ( A – C ) Immunogold labeling of 293T cells overexpressing C-terminally tagged QIL1 ( A ), MIC25 ( B ), or NDUFA13 ( C ) using an α-HA antibody coupled to 10-nm gold particles. Bars, 100 nm. Representative mitochondria are shown. The arrows point to the position of a gold particle. The distance in nanometers between the gold particles and the nearest CJ was measured using the ImageJ software. The histograms show the fraction of gold particles within the indicated distance to the crista junction in nanometers in QIL1-HA (n = 231 gold particles), MIC25-HA (n = 192 gold particles), and NDUFA13-HA (n = 309 gold particles) expressing cells. ( D ) Mitochondria isolated from stable 293T cell lines expressing C-terminally tagged QIL1, MIC60, MIC19, or MIC25 were lysed in 1% digitonin, subjected to BN-PAGE followed by immunotransfer to nitrocellulose membranes and probing with α-HA antibody. The mature ∼700 kDa MICOS complex is highlighted with an asterisk. MIC60, MIC19, and MIC25 were also detected in a sub-complex of ∼500 kDa (two asterisks). ( E ) Two-dimensional blue native electrophoresis of 293T mitochondrial lysates. Endogenous QIL1 is present in the mature ∼700 kDa MICOS complex (asterisk). MIC60, MIC19, and MIC25 were also present in a smaller sub-complex (two asterisks). ( F – G ) C-terminally tagged ( F ) or endogenous QIL ( G ) was immunopurified from mitochondria lysed in 1% digitonin or 1% Triton X-100 (TX100). Immunoblot analysis was performed to detect interaction with other MICOS subunits. DOI: http://dx.doi.org/10.7554/eLife.06265.006

    Journal: eLife

    Article Title: QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology

    doi: 10.7554/eLife.06265

    Figure Lengend Snippet: QIL1 localizes at cristae junctions and is present in the mature MICOS complex. ( A – C ) Immunogold labeling of 293T cells overexpressing C-terminally tagged QIL1 ( A ), MIC25 ( B ), or NDUFA13 ( C ) using an α-HA antibody coupled to 10-nm gold particles. Bars, 100 nm. Representative mitochondria are shown. The arrows point to the position of a gold particle. The distance in nanometers between the gold particles and the nearest CJ was measured using the ImageJ software. The histograms show the fraction of gold particles within the indicated distance to the crista junction in nanometers in QIL1-HA (n = 231 gold particles), MIC25-HA (n = 192 gold particles), and NDUFA13-HA (n = 309 gold particles) expressing cells. ( D ) Mitochondria isolated from stable 293T cell lines expressing C-terminally tagged QIL1, MIC60, MIC19, or MIC25 were lysed in 1% digitonin, subjected to BN-PAGE followed by immunotransfer to nitrocellulose membranes and probing with α-HA antibody. The mature ∼700 kDa MICOS complex is highlighted with an asterisk. MIC60, MIC19, and MIC25 were also detected in a sub-complex of ∼500 kDa (two asterisks). ( E ) Two-dimensional blue native electrophoresis of 293T mitochondrial lysates. Endogenous QIL1 is present in the mature ∼700 kDa MICOS complex (asterisk). MIC60, MIC19, and MIC25 were also present in a smaller sub-complex (two asterisks). ( F – G ) C-terminally tagged ( F ) or endogenous QIL ( G ) was immunopurified from mitochondria lysed in 1% digitonin or 1% Triton X-100 (TX100). Immunoblot analysis was performed to detect interaction with other MICOS subunits. DOI: http://dx.doi.org/10.7554/eLife.06265.006

    Article Snippet: For the in vivo analysis, Drosophila muscle tissue was dissected in PBS, fixed for 30 min in 4% paraformaldehyde, washed, permeabilized for 2 hr in 0.5% Triton X-100 in PBS, and blocked with normal goat serum in 0.1% Tween in PBS (PBST). α-ATP5A primary antibody (1:300; Abcam) was diluted in blocking buffer and incubated overnight at 4°C.

    Techniques: Labeling, Software, Expressing, Isolation, Polyacrylamide Gel Electrophoresis, Electrophoresis

    LRP6 and PIK3R2 are direct targets of miR-126-3p. (A) MiR-126-3p and its putative binding sequence in the 3′UTR of LRP6 and PIK3R2. The mutant miR-126-3p binding site was generated in the complementary site for the seed region of miR-126-3p. (B) Hep-G2 and BEL-7402 cells were co-transfected with miR-126-3p mimics or NC and wild-type or its mutant-type (either LRP6 or PIK3R2), and the luciferase activities were examined. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. (C) Western blot results of endogenous LRP6, PIK3R2, beta-catenin and p-AKT proteins in HCC cells transfected with miR-126-3p mimics or anti-mir-126-3p and its NC. (D) Analysis of LRP6 and PIK3R2 expression in subcutaneous tumors by immunohistochemistry. NC indicated negative control, **P

    Journal: Journal of Translational Medicine

    Article Title: MiR-126-3p suppresses tumor metastasis and angiogenesis of hepatocellular carcinoma by targeting LRP6 and PIK3R2

    doi: 10.1186/s12967-014-0259-1

    Figure Lengend Snippet: LRP6 and PIK3R2 are direct targets of miR-126-3p. (A) MiR-126-3p and its putative binding sequence in the 3′UTR of LRP6 and PIK3R2. The mutant miR-126-3p binding site was generated in the complementary site for the seed region of miR-126-3p. (B) Hep-G2 and BEL-7402 cells were co-transfected with miR-126-3p mimics or NC and wild-type or its mutant-type (either LRP6 or PIK3R2), and the luciferase activities were examined. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. (C) Western blot results of endogenous LRP6, PIK3R2, beta-catenin and p-AKT proteins in HCC cells transfected with miR-126-3p mimics or anti-mir-126-3p and its NC. (D) Analysis of LRP6 and PIK3R2 expression in subcutaneous tumors by immunohistochemistry. NC indicated negative control, **P

    Article Snippet: The primary antibodies used for western blot and IHC: LRP6 (abcam, ab75358, 1:1000, ab24386, 1:300), beta-catenin (epitomics, 1247–1, 1:1000), PIK3R2 (abcam, ab28356, 1:1000), phospho-ser473-AKT (epitomics, 2118–1, 1:1000), CD34 (epitomics, 2749–1, 1:500), β-actin (A5441, Sigma-Aldrich, 1:2000).

    Techniques: Binding Assay, Sequencing, Mutagenesis, Generated, Transfection, Luciferase, Activity Assay, Western Blot, Expressing, Immunohistochemistry, Negative Control

    LPS increased in P. gingivalis and thereby inhibited the functions of MSCs by activating NLRP3 inflammasome. a , b Western blot and real-time RT-PCR results revealed that NLRP3 expression was significantly increased in palatal tissues from the inoculation of P. gingivalis group compared to the mixture of L. reuteri and P. gingivalis group, and untreated group. β-actin was used as an internal control in western blot assay. c , d The cell migration assay results demonstrated that LPS significantly inhibited GMSC migration, and NLRP3 inhibitor CY-09 rescued the capacity of GMSC migration after LPS treatment. e – g Real-time RT-PCR results showed that LPS inhibited the expression of SOX2, OCT4, and NANOG, and CY-09 restored the capacities of MSCs. h , i ALP activity assay and alizarin red staining assay. j - m Real-time RT-PCR results further testified that NLRP3 activation inhibited the expression of the crucial transcription factors for modulating osteogenic differentiation: RUX2, OSX, OPN, and OCN, and CY-09 rescued the functions of osteogenic differentiation. n Counting kit-8 assay results showed that CY-09 restored the proliferation potential of GMSCs caused by LPS-induced NLRP3 activation. GAPDH was an internal control. Error bars represent SD ( n = 3). * P ≤ 0.05; ** P ≤ 0.01

    Journal: Stem Cell Research & Therapy

    Article Title: Balanced oral pathogenic bacteria and probiotics promoted wound healing via maintaining mesenchymal stem cell homeostasis

    doi: 10.1186/s13287-020-1569-2

    Figure Lengend Snippet: LPS increased in P. gingivalis and thereby inhibited the functions of MSCs by activating NLRP3 inflammasome. a , b Western blot and real-time RT-PCR results revealed that NLRP3 expression was significantly increased in palatal tissues from the inoculation of P. gingivalis group compared to the mixture of L. reuteri and P. gingivalis group, and untreated group. β-actin was used as an internal control in western blot assay. c , d The cell migration assay results demonstrated that LPS significantly inhibited GMSC migration, and NLRP3 inhibitor CY-09 rescued the capacity of GMSC migration after LPS treatment. e – g Real-time RT-PCR results showed that LPS inhibited the expression of SOX2, OCT4, and NANOG, and CY-09 restored the capacities of MSCs. h , i ALP activity assay and alizarin red staining assay. j - m Real-time RT-PCR results further testified that NLRP3 activation inhibited the expression of the crucial transcription factors for modulating osteogenic differentiation: RUX2, OSX, OPN, and OCN, and CY-09 rescued the functions of osteogenic differentiation. n Counting kit-8 assay results showed that CY-09 restored the proliferation potential of GMSCs caused by LPS-induced NLRP3 activation. GAPDH was an internal control. Error bars represent SD ( n = 3). * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The expressions of protein were detected using anti-NLRP3 (1:300, Abcam), and β-actin (1:2000, Abcam) was used as an internal control.

    Techniques: Western Blot, Quantitative RT-PCR, Expressing, Cell Migration Assay, Migration, ALP Activity Assay, Staining, Activation Assay

    Mobility of wild type and mutant Cav3.1 and Cav3.2 channels in hippocampal neurons. ( a ) Fluorescence recovery after photobleaching (FRAP) assay of Cav3.1-GFP wild type and ∆CT (1851–1875) mutant channels or Cav3.2-GFP wild type and ∆CT (1860–1884) mutant channels transfected into mouse hippocampal neuron cultures. ( b ) Recovery (Fluorescence-Fluorescence bleach) of Cav3.1-GFP wild type and ∆CT (1851–1875) mutant channels . Recovery values are calculated as F(maximum after photobleach)-F(photobleach) ( n = 8 WT, n = 8 mutant, P

    Journal: Molecular Brain

    Article Title: T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B

    doi: 10.1186/s13041-018-0368-5

    Figure Lengend Snippet: Mobility of wild type and mutant Cav3.1 and Cav3.2 channels in hippocampal neurons. ( a ) Fluorescence recovery after photobleaching (FRAP) assay of Cav3.1-GFP wild type and ∆CT (1851–1875) mutant channels or Cav3.2-GFP wild type and ∆CT (1860–1884) mutant channels transfected into mouse hippocampal neuron cultures. ( b ) Recovery (Fluorescence-Fluorescence bleach) of Cav3.1-GFP wild type and ∆CT (1851–1875) mutant channels . Recovery values are calculated as F(maximum after photobleach)-F(photobleach) ( n = 8 WT, n = 8 mutant, P

    Article Snippet: Supernatants were transferred to new tubes and solubilized proteins were incubated with 50 μl of Protein G/A beads (Piercenet) and 2 μg of anti-Cav3.2 (H-300, Santa Cruz Biotechnologies, Inc) antibody or anti-GFP antibody (Abcam) overnight while tumbling at 4 °C.

    Techniques: Mutagenesis, Fluorescence, Photobleaching Assay, Transfection

    Effect of Cav3.1-GFP and Cav3.2-GFP C-terminal deletions on SPTAN1 and SPTBN2 binding in tsA-201 cells. ( a ) Cav3.1-GFP ∆CT (1851–1875) and wild type channel immunoprecipitates probed with anti-Spectrin αII (SPTAN1) polyclonal antibody. Densitometry analysis of SPTAN1 bound to Cav3.1-GFP immunoprecipitates is shown. ( P = 0.0051, n = 3). ( b ) Cav3.2-GFP ∆CT (1860–1884) and wild type channel immunoprecipitates probed with anti-Spectrin αII (SPTAN1) polyclonal antibody. Densitometry analysis of SPTAN1 bound to Cav3.2-GFP immunoprecipitates is shown ( P = 0.0005, n = 3). ( c ) Cav3.1-GFP ∆CT (1851–1875) and wild type channel immunoprecipitates probed with anti-Spectrin βII (SPTBN2) polyclonal antibody. Densitometry analysis of SPTBN2 bound to Cav3.1-GFP immunoprecipitates is shown ( P = 0.0085, n = 3). ( d ) Cav3.2-GFP ∆CT (1860–1884) and wild type channel immunoprecipitates probed with anti-Spectrin βII (SPTBN2) polyclonal antibody. Densitometry analysis of SPTBN2 bound to Cav3.2-GFP immunoprecipitates is shown. ( P = 0.0047, n = 3)

    Journal: Molecular Brain

    Article Title: T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B

    doi: 10.1186/s13041-018-0368-5

    Figure Lengend Snippet: Effect of Cav3.1-GFP and Cav3.2-GFP C-terminal deletions on SPTAN1 and SPTBN2 binding in tsA-201 cells. ( a ) Cav3.1-GFP ∆CT (1851–1875) and wild type channel immunoprecipitates probed with anti-Spectrin αII (SPTAN1) polyclonal antibody. Densitometry analysis of SPTAN1 bound to Cav3.1-GFP immunoprecipitates is shown. ( P = 0.0051, n = 3). ( b ) Cav3.2-GFP ∆CT (1860–1884) and wild type channel immunoprecipitates probed with anti-Spectrin αII (SPTAN1) polyclonal antibody. Densitometry analysis of SPTAN1 bound to Cav3.2-GFP immunoprecipitates is shown ( P = 0.0005, n = 3). ( c ) Cav3.1-GFP ∆CT (1851–1875) and wild type channel immunoprecipitates probed with anti-Spectrin βII (SPTBN2) polyclonal antibody. Densitometry analysis of SPTBN2 bound to Cav3.1-GFP immunoprecipitates is shown ( P = 0.0085, n = 3). ( d ) Cav3.2-GFP ∆CT (1860–1884) and wild type channel immunoprecipitates probed with anti-Spectrin βII (SPTBN2) polyclonal antibody. Densitometry analysis of SPTBN2 bound to Cav3.2-GFP immunoprecipitates is shown. ( P = 0.0047, n = 3)

    Article Snippet: Supernatants were transferred to new tubes and solubilized proteins were incubated with 50 μl of Protein G/A beads (Piercenet) and 2 μg of anti-Cav3.2 (H-300, Santa Cruz Biotechnologies, Inc) antibody or anti-GFP antibody (Abcam) overnight while tumbling at 4 °C.

    Techniques: Binding Assay

    Calcium currents evoked by wild type and mutant Cav3.1 and Cav3.2 channels in tsA-201 cells. ( a ) Current-voltage (I/V) relationship of wild type or mutant Cav3.1-GFP ∆CT (1851–1875) mutant channels ( n = 10). Values are represented as means +/− S.E. The solid lines are fits with the Boltzmann equation. ( b ) Mean peak current density representation from wild type or mutant Cav3.1-GFP ∆CT (1851–1875) channels ( P

    Journal: Molecular Brain

    Article Title: T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B

    doi: 10.1186/s13041-018-0368-5

    Figure Lengend Snippet: Calcium currents evoked by wild type and mutant Cav3.1 and Cav3.2 channels in tsA-201 cells. ( a ) Current-voltage (I/V) relationship of wild type or mutant Cav3.1-GFP ∆CT (1851–1875) mutant channels ( n = 10). Values are represented as means +/− S.E. The solid lines are fits with the Boltzmann equation. ( b ) Mean peak current density representation from wild type or mutant Cav3.1-GFP ∆CT (1851–1875) channels ( P

    Article Snippet: Supernatants were transferred to new tubes and solubilized proteins were incubated with 50 μl of Protein G/A beads (Piercenet) and 2 μg of anti-Cav3.2 (H-300, Santa Cruz Biotechnologies, Inc) antibody or anti-GFP antibody (Abcam) overnight while tumbling at 4 °C.

    Techniques: Mutagenesis

    Effect of Cav3.1-GFP and Cav3.2-GFP C-terminal deletions on ankyrin B binding in tsA-201 cells. ( a ) Cav3.1-GFP ∆CT (1851–1875) and wild type channel immunoprecipitates probed with anti-ankyrin B polyclonal antibody. Densitometry analysis of ankyrin B bound to Cav3.1-GFP immunoprecipitates is shown ( P = 0.0005, n = 4). ( b ) Cav3.2-GFP ∆CT (1860–1884) and wild type channel immunoprecipitates probed with anti-ankyrin B polyclonal antibody. Densitometry analysis of ankyrin B bound to Cav3.2-GFP immunoprecipitates is shown ( P = 0.015, n = 4)

    Journal: Molecular Brain

    Article Title: T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B

    doi: 10.1186/s13041-018-0368-5

    Figure Lengend Snippet: Effect of Cav3.1-GFP and Cav3.2-GFP C-terminal deletions on ankyrin B binding in tsA-201 cells. ( a ) Cav3.1-GFP ∆CT (1851–1875) and wild type channel immunoprecipitates probed with anti-ankyrin B polyclonal antibody. Densitometry analysis of ankyrin B bound to Cav3.1-GFP immunoprecipitates is shown ( P = 0.0005, n = 4). ( b ) Cav3.2-GFP ∆CT (1860–1884) and wild type channel immunoprecipitates probed with anti-ankyrin B polyclonal antibody. Densitometry analysis of ankyrin B bound to Cav3.2-GFP immunoprecipitates is shown ( P = 0.015, n = 4)

    Article Snippet: Supernatants were transferred to new tubes and solubilized proteins were incubated with 50 μl of Protein G/A beads (Piercenet) and 2 μg of anti-Cav3.2 (H-300, Santa Cruz Biotechnologies, Inc) antibody or anti-GFP antibody (Abcam) overnight while tumbling at 4 °C.

    Techniques: Binding Assay

    Systemic LPS treatment increased the number of microglia and BrdU-positive proliferative cells. A : The lipopolysaccharide (LPS) treatment scheme. Intraperitoneal injection of LPS is performed at P3, and 5-bromo-2′-deoxyuridine (BrdU) is incorporated from P3 to P7. The retina was evaluated at P7. B , C : Immunostaining shows Iba-1-positive microglia (red). LPS treatment increases the number of microglia in the retina at P7. D : CD206, a marker of M2 microglia, is increased by LPS treatment. E : The number of cleaved caspase-3-positive cells is not changed by LPS treatment. F – H : Immunostaining shows BrdU-positive proliferative cells (red) in the peripheral retina. LPS treatment increases the number of BrdU-positive proliferative cells in the peripheral area. The increased BrdU-positive proliferative cells merge with the retinal precursor cell marker, Pax6. I , J : Pax6 (green) and Sox2 (magenta) double-positive cells are increased with LPS treatment. Data are the mean ± standard error of the mean (SEM; n=4 or 5). # ; p

    Journal: Molecular Vision

    Article Title: Microglia increases the proliferation of retinal precursor cells during postnatal development

    doi:

    Figure Lengend Snippet: Systemic LPS treatment increased the number of microglia and BrdU-positive proliferative cells. A : The lipopolysaccharide (LPS) treatment scheme. Intraperitoneal injection of LPS is performed at P3, and 5-bromo-2′-deoxyuridine (BrdU) is incorporated from P3 to P7. The retina was evaluated at P7. B , C : Immunostaining shows Iba-1-positive microglia (red). LPS treatment increases the number of microglia in the retina at P7. D : CD206, a marker of M2 microglia, is increased by LPS treatment. E : The number of cleaved caspase-3-positive cells is not changed by LPS treatment. F – H : Immunostaining shows BrdU-positive proliferative cells (red) in the peripheral retina. LPS treatment increases the number of BrdU-positive proliferative cells in the peripheral area. The increased BrdU-positive proliferative cells merge with the retinal precursor cell marker, Pax6. I , J : Pax6 (green) and Sox2 (magenta) double-positive cells are increased with LPS treatment. Data are the mean ± standard error of the mean (SEM; n=4 or 5). # ; p

    Article Snippet: The following primary antibodies were used: mouse anti-Pax6 (1:300 dilution; Abcam, Cambridge, MA), mouse anti-Chx10 (1:200 dilution; SantaCruz, Dallas, TX), rabbit anti-Iba-1 (1:50 dilution; Wako Pure Chemical Industries, Ltd.), rabbit anti-CRX (1:20 dilution; SantaCruz), rabbit anti-Sox2 (1:200 dilution; Millipore, Bedford, MA), rabbit anti-cleaved caspase-3 (1:100 dilution; Cell Signaling Technology, Danvers, MA), rat anti-BrdU (1:200 dilution; Abcam), sheep anti-progranulin (1:20 dilution; R & D Systems, Minneapolis, MN), mouse anti-CD206 (1:50 dilution; Abcam), Alexa Fluor® 488 goat anti-mouse immunoglobulin G (IgG), Alexa Fluor® 546 goat anti-rat IgG, Alexa Fluor® 546 donkey anti-rabbit IgG, and Alexa Fluor® 647 donkey anti-sheep IgG (Invitrogen, Carlsbad, CA).

    Techniques: Injection, Immunostaining, Marker

    PLX3397 treatment decreased the number of microglia and BrdU-positive proliferative cells. A : PLX3397, a colony-stimulating factor 1 receptor (CSF1R) inhibitor, is treated to neonatal mice by intraperitoneal injection (twice daily) to deplete the microglia. B , C : The treatment decreases the number of Iba-1-positive microglia (red). D, E : PLX3397 decreases the number of 5-bromo-2′-deoxyuridine (BrdU)-positive proliferative cells (red). F , G : PLX3397 decreases the BrdU and retinal precursor cell markers, Chx10 or Pax6 (green) double-positive cells. The quantitative data show the decrease in the BrdU and Chx10 double-positive cells by PLX3397 treatment. H : PLX3397 does not alter the cleaved caspase-3-positive cells. Data are the mean ± standard error of the mean (SEM; n=4). # p

    Journal: Molecular Vision

    Article Title: Microglia increases the proliferation of retinal precursor cells during postnatal development

    doi:

    Figure Lengend Snippet: PLX3397 treatment decreased the number of microglia and BrdU-positive proliferative cells. A : PLX3397, a colony-stimulating factor 1 receptor (CSF1R) inhibitor, is treated to neonatal mice by intraperitoneal injection (twice daily) to deplete the microglia. B , C : The treatment decreases the number of Iba-1-positive microglia (red). D, E : PLX3397 decreases the number of 5-bromo-2′-deoxyuridine (BrdU)-positive proliferative cells (red). F , G : PLX3397 decreases the BrdU and retinal precursor cell markers, Chx10 or Pax6 (green) double-positive cells. The quantitative data show the decrease in the BrdU and Chx10 double-positive cells by PLX3397 treatment. H : PLX3397 does not alter the cleaved caspase-3-positive cells. Data are the mean ± standard error of the mean (SEM; n=4). # p

    Article Snippet: The following primary antibodies were used: mouse anti-Pax6 (1:300 dilution; Abcam, Cambridge, MA), mouse anti-Chx10 (1:200 dilution; SantaCruz, Dallas, TX), rabbit anti-Iba-1 (1:50 dilution; Wako Pure Chemical Industries, Ltd.), rabbit anti-CRX (1:20 dilution; SantaCruz), rabbit anti-Sox2 (1:200 dilution; Millipore, Bedford, MA), rabbit anti-cleaved caspase-3 (1:100 dilution; Cell Signaling Technology, Danvers, MA), rat anti-BrdU (1:200 dilution; Abcam), sheep anti-progranulin (1:20 dilution; R & D Systems, Minneapolis, MN), mouse anti-CD206 (1:50 dilution; Abcam), Alexa Fluor® 488 goat anti-mouse immunoglobulin G (IgG), Alexa Fluor® 546 goat anti-rat IgG, Alexa Fluor® 546 donkey anti-rabbit IgG, and Alexa Fluor® 647 donkey anti-sheep IgG (Invitrogen, Carlsbad, CA).

    Techniques: Mouse Assay, Injection