Journal: International Journal of Molecular Sciences

Article Title: DRG2 Depletion Promotes Endothelial Cell Senescence and Vascular Endothelial Dysfunction

doi: 10.3390/ijms23052877

Figure Lengend Snippet: Effects of DRG2 deficiency on the proliferation, apoptosis, and senescence of endothelial cells. Lung endothelial cells (mLECs) derived from wild-type and DRG2 −/− mice were cultivated under normal conditions as described in the without any treatment. The mLECs were analyzed by ( A ) MTS assay (n = 3 independent experiments in each group) and ( B ) Ki67 staining for cell proliferation (n = 40 cells per 3 independent experiments in each group), ( C ) Annexin-V staining and FACS analysis for apoptosis (n = 3 independent experiments in each group), ( D ) senescence-associated β-gal (SA-β-gal) staining for cellular senescence (n = 120 cells per 3 independent experiments in each group), and ( E ) FACS analysis for cell cycle arrest (n = 10 independent experiments). ( F ) Detection of SA-β-gal activity in uteruses from wild-type and DRG2 −/− mice. Images in ( B , D ) are representative (Original magnification 600× and 200×, respectively) of Ki67-stained cells and SA-β-gal-stained cells, respectively. The data are expressed as the mean ± SD. * p < 0.05; *** p < 0.001. ns, not significant.

Article Snippet: They were then incubated with Alexa Fluor 488-conjugated anti-Ki67 antibody (#11882, Cell Signaling, Danvers, MA, USA) for 1 h. Confocal images were obtained using an Olympus 1000/1200 laser-scanning confocal system (Olympus, Shinjuku, Japan).

Techniques: Derivative Assay, MTS Assay, Staining, Activity Assay

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: (A) Alleles of Kras G12D (K), Trp53 R172H (P), Pdx1-Cre (C), and Gα13fl (G). (B) Gα13 expression levels in the pancreas of mice of indicated genotypes were determined by western blotting. Heat shock protein (Hsp90) was used as an endogenous control. The expression of Gna13 was determined by qRT-PCR. GAPDH was used as an endogenous control. One-way ANOVA (n = 6 for KPCG +/+ , n = 6 for KPCG fl/fl , and n = 5 for KPCG fl/+ genotype). ***p ≤ 0.001, mean ± SD. (C) The whole profile of pancreas tissue from KPCG +/+ , KPCG fl/fl , and KPCG fl/+ mice at 3 months was scanned, and the relative lesion burden was quantified (n = 9, 9, and 9, respectively). One-way ANOVA, * p ≤ 0.05, mean ± SD. Scale bar = 1 mm (H&E), 100 μm (IHC). Ki67 (n = 5, 5, and 5, respectively) stains of the pancreatic tissue from KPCG +/+ , KPCG fl/fl , and KPCG fl/+ mice at 3 months. (D), H&E, trichrome, Ki67 (n = 8, 6, 7), CK19 (n = 6, 6, 6), and E-cadherin (n = 6, 6, 6) stains of the pancreatic tissue from KPCG +/+ , KPCG fl/fl, and KPCG fl/+ mice at the survival endpoints. One-way ANOVA, * p ≤ 0.05, ** p ≤ 0.01, mean ± SD. Scale bar = 100 μm. Immunofluorescence co-staining of CK19 and Ki67 of pancreatic tissue from KPCG +/+ (n = 4), KPCG fl/fl (n = 4), and KPCG fl/+ (n = 4) mice at the survival endpoint, and the double-positive (CK19 + /Ki67 + ) cells were quantified. Scale bar = 50 μm. One-way ANOVA, * p ≤ 0.05, mean ± SD. (E) Kaplan-Meier survival analysis of KPCG +/+ (n = 24), KPCG fl/+ (n = 28), and KPCG fl/fl (n = 16) mice using log-rank test.

Article Snippet: Tissue sections were blocked with a mixture of goat serum and bovine serum albumin for 1 h. Tissue sections were incubated with a mixture of antibodies for CK19 (DSHB TROMA-III, RRID: AB_2133570,1:100) and Ki67 (Cell Signaling #12202, RRID: AB_2620142, 1:500) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: (A) Western blot for Gα13 in pancreatic cancer cell lines established from tumors developing in KPCG +/+ (2138 and 3213) and KPCG fl/fl (3458 and 3566) mice. Equal numbers of 2138, 3213, 3458, and 3566 cells were grown in 3D type I collagen, and pictures were taken after 5 days. The cells were extracted out of collagen and counted. This experiment was repeated at least 3 times. (B) The cells were implanted subcutaneously in the flank of B6 mice (6–8 weeks old). The tumor sizes were measured using a caliper, harvested day ~24, and photographed, and wet weight was measured (2138 n = 3, 3213 n = 5, 3458 n = 3, and 3566 n = 5 mice; 2 tumors per mouse). One-way ANOVA for tumor growth, mean ± SD, ***p ≤ 0.001, ****p ≤ 0.0001. t test, mean ± SD for tumor weights. **p ≤ 0.01, ****p ≤ 0.0001. (C) H&E and immunostains and quantification of Ki67 in the KPCG +/+ and KPCG fl/fl tumors. t test, mean ± SD. Scale bar = 100 μm. *p ≤ 0.05, **p ≤ 0.01 (D and E) Trichrome, CK-19, and E-cadherin stains of tumors developing in the KPCG +/+ and KPCG fl/fl mice. t test, mean ± SD. Scale bar = 100 μm. *p ≤ 0.05, **p ≤ 0.01. The tumors were also analyzed for E-cadherin expression by western blotting.

Article Snippet: Tissue sections were blocked with a mixture of goat serum and bovine serum albumin for 1 h. Tissue sections were incubated with a mixture of antibodies for CK19 (DSHB TROMA-III, RRID: AB_2133570,1:100) and Ki67 (Cell Signaling #12202, RRID: AB_2620142, 1:500) for 2 h at room temperature.

Techniques: Western Blot, Expressing

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet: (A) Syngeneic KPCG +/+ and KPCG fl/fl tumors were analyzed for p-Pdk1, Pdk1, p-mTOR, mTOR, p-Rps6, and Rps6 by western blotting using Hsp90 as a loading control. (B and C) The KPCG +/+ and KPCG fl/fl cells were implanted subcutaneously in the flank of B6 mice (6–8 weeks old) and treated with vehicle control or rapamycin (60 mg/kg daily; arrow indicates the start of treatment), once the tumors reached ~100 mm 3 . The tumor sizes were measured using a caliper, harvested day 27, and photographed, and wet weight was measured. (n = 5 mice per group; 2 tumors per mouse). Representative whole profile of H&E stains of vehicle control and rapamycin-treated tumors. One-way ANOVA, mean ± SD. **p ≤ 0.01, ****p ≤ 0.0001. (D) Tumors treated with vehicle control or rapamycin were analyzed for p-Pdk1, Pdk1, p-mTOR, mTOR, p-Rps6, and Rps6 by western blotting using Hsp90/GAPDH as loading controls. (E) The effect of rapamycin on proliferation was analyzed by immunostains and quantification of Ki67 in the KPCG +/+ and KPCG fl/fl tumors. One-way ANOVA (n = vehicle control KPCG +/+ and KPCG fl/fl [4 and 5], rapamycin KPCG +/+ and KPCG fl/fl [5 and 5]), t test, mean ± SD. Scale bar = 100 μm. *p ≤ 0.05, **p ≤ 0.01. (F) Whole profile of cleaved-caspase 3 (c-C3) stains of vehicle control and rapamycin-treated tumors. Higher magnification and quantification of c-C3 stains in the treated tumors. One-way ANOVA (n = 5, 5, 5, and 5, per treatment group). ****p < 0.0001.

Article Snippet: Tissue sections were blocked with a mixture of goat serum and bovine serum albumin for 1 h. Tissue sections were incubated with a mixture of antibodies for CK19 (DSHB TROMA-III, RRID: AB_2133570,1:100) and Ki67 (Cell Signaling #12202, RRID: AB_2620142, 1:500) for 2 h at room temperature.

Techniques: Western Blot, Control

Journal: Cell reports

Article Title: Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin

doi: 10.1016/j.celrep.2022.110441

Figure Lengend Snippet:

Article Snippet: Tissue sections were blocked with a mixture of goat serum and bovine serum albumin for 1 h. Tissue sections were incubated with a mixture of antibodies for CK19 (DSHB TROMA-III, RRID: AB_2133570,1:100) and Ki67 (Cell Signaling #12202, RRID: AB_2620142, 1:500) for 2 h at room temperature.

Techniques: Recombinant, Membrane, Reverse Transcription, Plasmid Preparation, Software

Journal: Science Advances

Article Title: PTPN2 elicits cell autonomous and non–cell autonomous effects on antitumor immunity in triple-negative breast cancer

doi: 10.1126/sciadv.abk3338

Figure Lengend Snippet: ( A ) CD45 − CD24 + CD29 hi MECs were isolated from the mammary glands of 12-week-old Ptpn2 fl/fl and MMTV-Cre; Ptpn2 fl/fl females and transplanted contralaterally into the cleared mammary fat pads of 3-week-old FVB/n female mice. The transplanted fat pads were collected after 12 weeks and fixed in formalin, and ( B ) whole mounts stained with carmine alum to assess epithelial repopulation or processed for either ( C ) immunohistochemistry and stained for PTPN2, STAT-3 Y705 phosphorylation (p-STAT-3), and Ki67 (counterstained with hematoxylin) or ( D and E ) immunofluorescence microscopy and stained for CK8, CK14, and DNA [4′,6-diamidino-2-phenylindole (DAPI)] to identify nuclei. In (C), p-STAT-3– or Ki67-positive MECs were quantified; three mice per genotype were scored, with three randomly selected fields of view scored per section. (E) Quantification of perturbed ductal structures in Ptpn2 fl/fl and MMTV-Cre; Ptpn2 fl/fl MEC outgrowths; four repopulated fat pads per mouse were scored per genotype with at least five randomly selected fields of view and 50 structures scored per gland. Results in (C) and (D) are means ± SEM; significance was determined using two-tailed Student’s t tests; * P ≤ 0.05; *** P ≤ 0.001.

Article Snippet: Rabbit p-STAT-1 (Tyr 701 ) (clone 58D6) [Research Resource Identifier (RRID): AB_2198287), rabbit p-STAT-3 (Tyr 705 ) (clone D3A7) (RRID: AB_2255568], mouse STAT-3 (clone 124H6) (RRID: AB_331757), rabbit STAT-1 (#9172) (RRID: AB_2198300), rabbit Ki67 (clone D3B5) (RRID: AB-2687446), mouse Ki67 (clone #8D5) (RRID:AB_2797703), and rabbit Vinculin (clone E1E9V) (RRID: AB_2728768) antibodies were from Cell Signaling Technology (Danvers, MA); mouse actin (clone ACTN05) (RRID: AB_1954910) antibody was from Thermo Fisher Scientific (Waltham, MA); rabbit CK8 (clone EP1628Y) (RRID: AB_869901), mouse CK14 (clone LL002) (RRID: AB_306091), and rabbit CD3ε (clone SP7) antibodies were from Abcam (Cambridge, UK) (RRID: AB_446487); rabbit FLEX CD3ε Ready-to-Use was from Agilent Dako (RRID: AB_2732001) (Glostrup, Denmark); and mouse PTPN2 6F3 antibody was from MediMabs (Quebec, Canada).

Techniques: Isolation, Staining, Immunohistochemistry, Phospho-proteomics, Immunofluorescence, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: ( a ) Schematic representation of primers for genotyping and targeting strategy. ( b ) miR-31 genotyping using P1/P2, 1,064 bp band for miR-31 fl/fl and 235 bp band for miR-31 fl/fl /K5-Cre (cKO). DNA samples were prepared from total skin. ( c ) Cre-mediated tissue-specific deletion of miR-31 in epidermis. DNA samples were prepared from either epidermis or dermis. ( d ) miR-31 expression in epidermis derived from miR-31 fl/fl and cKO mice. ( e ) Phenotypic presentation of mouse back skin for miR-31 fl/fl or cKO mice treated with IMQ or vehicle for 7 days. ( f ) Splenomegaly and lymphadenopathy in miR-31 fl/fl or cKO mice treated with IMQ or vehicle for 7 days. Data are representative of more than five mice. ( g ) Skin thickness was measured on the days indicated. Symbols represent mean skin thickness±s.e.m. for five to six mice per group. ( h ) H&E staining of the back skin of miR-31 fl/fl or cKO mice treated with IMQ or vehicle. Dotted line indicates the border between the epidermis and the dermis. Scale bar, 100 μm. ( i ) Acanthosis of miR-31 fl/fl or cKO mice treated with IMQ or vehicle. ( j ) Dermal cellular infiltrates of miR-31 fl/fl or cKO mice treated with IMQ. ( k ) Immunostaining of Ki67 in lesional skin derived from miR-31 fl/fl (left panel) or cKO (right panel) mice treated with IMQ. Scale bar, 100 μm. ( l ) Quantitation of Ki67 + cells in epidermis. For all measurements, numbers of specifically stained Ki67 + cells in epidermis counted in three high-power fields for each section were used. ( m ) qPCR analysis of epidermal differentiation markers (Keratin 10, Loricrin and Filaggrin) in miR-31 fl/fl or cKO mice treated with IMQ or vehicle. Results ( d , m ) are presented as the ratio of miRNA to the small nuclear RNA U6 or of mRNA to the β-actin, relative to that in miR-31 fl/fl mice. * P <0.05, ** P <0.01, *** P <0.001, two-tailed Student's t -test for d , i , j , l , m , one-way ANOVA for g . Data ( g , i , j , l , m ) are representative of two independent experiments with four to six mice per group in each (mean and s.e.m.).

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: Expressing, Derivative Assay, Staining, Immunostaining, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: ( a , b ) Western blotting of ppp6c expression in the epidermis of normal skin derived from four healthy individuals (Ctr-1 to 4) or in psoriatic lesions derived from four patients with psoriasis (Pso-1 to 4). ( c ) Immunohistochemical staining of ppp6c in skin sections derived from healthy or psoriatic skin. Scale bar, 100 μm. ( d ) Primary mouse keratinocytes were transfected with scramble siRNA (Ctr) or with ppp6c siRNA (siRNA). Cell lysates were immunoblotted with anti-ppp6c or anti-actin. ( e ) Cell cycle analysis of mouse primary keratinocytes transfected with scrambled siRNA or ppp6c siRNA. ( f ) Western blotting of ppp6c expression in epidermis derived from lentiviral shRNA-control (LV-Ctr) or lentiviral shRNA-ppp6c (LV-shRNA) treated mice. ( g ) H&E staining of the back skin injected with LV-Ctr or LV-shRNA in mice applied with IMQ. Dotted line indicates the border between the epidermis and dermis. Scale bar, 100 μm. ( h ) Acanthosis of the back skin injected with LV-Ctr or LV-shRNA in mice applied with IMQ. ( i ) Immunohistochemical staining of Ki67 in skin sections derived from mice injected with LV-Ctr or LV-shRNA prior to IMQ painting. Dotted line indicates the border between the epidermis and dermis. Scale bar, 50 μm. Values ( a , d , f ) were expressed as fold changes relative to Ctr-1 ( a ), to scramble siRNA ( d ), or to lentiviral shRNA-control ( f ), and normalized to β-actin. * P <0.05, ** P <0.01, two-tailed Student's t -test. Data ( c – i ) are representative of two independent experiments with three to five samples per group.

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: Western Blot, Expressing, Derivative Assay, Immunohistochemical staining, Staining, Transfection, Cell Cycle Assay, shRNA, Control, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: ( a ) Undifferentiated NHEK proliferation induced by various concentrations of IL-6 for 24 h, and analysed by BrdU incorporation assay. ( b ) NHEK were transfected with scramble siRNA (Ctr) or with p65 siRNA (p65 siRNA). Cell cycle analysis of NHEK or transfected NHEK treated without or with IL-6 for 24 h. ( c ) In vitro wound healing rate of NHEK treated without or with IL-6 for 16 h. ( d , e ) Three-dimensional organotypic culture of HaCaT keratinocytes treated without or with IL-6. Scale bar, 100 μm. ( f ) 1 μg recombinant mouse IL-6 (in 25 μl PBS) or PBS was injected i.d. in ears of C57BL/6J mice. Ear sections were prepared for Ki67 staining 3 days after IL-6 administration. Scale bar, 100 μm. ( g ) NHEK were transfected with scramble siRNA (Ctr) or with p65 siRNA (p65 siRNA). Western blotting of ppp6c expression in NHEK with or without IL-6 treatment for 24 h. ( h ) Cell cycle analysis of primary mouse keratinocytes derived from miR-31 fl/fl or cKO mice in absence or presence of IL-6. Values were expressed as fold changes relative to non-stimulated HaCaT keratinocytes ( e ) or to non-stimulated NHEK ( g ) and normalized to β-actin. IL-6 was used at the concentration of 50 ng ml −1 ( b – e , g , h ). ** P <0.01, *** P <0.001, two-tailed Student's t -test. Data are representative of at least two independent experiments.

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: BrdU Incorporation Assay, Transfection, Cell Cycle Assay, In Vitro, Recombinant, Injection, Staining, Western Blot, Expressing, Derivative Assay, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: Mice were injected subcutaneously with an irrelevant antagomir (NC) or an antagomir to miR-31 (anti-miR-31). The first injection was administered 3 days before the application of IMQ and thereafter was performed every other day until the end of the experiment. ( a ) H&E staining of the back skin derived from mice injected with NC (upper panel) or anti-miR-31 (lower panel). Scale bar, 100 μm. ( b , c ) Acanthosis and dermal cellular infiltrates were quantitated for mice treated with NC or anti-miR-31. ( d , e ) Ppp6c mRNA and protein levels in NC- or anti-miR-31-treated mice. ( f , g ) Immunohistochemical staining of ppp6c or Ki67 in skin sections derived from NC- or anti-miR-31-treated mice after induction of skin phenotype by IMQ ( n =8–9). Scale bar, 50 μm ( f ) or 100 μm ( g ). For all measurements ( c ), the median number of specifically stained dermal nucleated cells was counted in three high-power fields per section. Results ( d ) are presented as the ratio of mRNA to the β-actin, relative to that in NC-treated mice. * P <0.05, *** P <0.001, two-tailed Student's t -test. Data ( a – g ) are representative of at least two independent experiments with four to nine samples per group in each (mean and s.e.m.).

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: Injection, Staining, Derivative Assay, Immunohistochemical staining, Two Tailed Test

Journal: Oncology Letters

Article Title: Bufalin enhances the antitumor effect of gemcitabine in pancreatic cancer

doi: 10.3892/ol.2012.783

Figure Lengend Snippet: The combination treatment of gemcitabine with bufalin inhibited tumor growth in xenograft model. (A) The tumor volume in pancreatic cancer xenograft model after treatment. There was a statistically significant difference between the combination treatment group and the control group (P<0.05). (B) The expression of Ki-67 was decreased and ASK1 was increased in the combination group.

Article Snippet: The primary antibodies of anti-human Ki-67 and ASK1 were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Genome-wide analysis suggests a differential microRNA signature associated with normal and diabetic human corneal limbus

doi: 10.1038/s41598-017-03449-7

Figure Lengend Snippet: List of antibodies used in this study.

Article Snippet: Ki-67 , Rabbit mAb ab92742 , Abcam , 359 , 1:200.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Inhibition of Stat3‐mediated astrogliosis ameliorates pathology in an Alzheimer's disease model

doi: 10.15252/emmm.201809665

Figure Lengend Snippet: A, B The total density of astrocytes and microglia remained unchanged in APP/PS1‐Stat3KO compared to APP/PS1‐Stat3WT mice (Mann–Whitney test for both comparisons; APP/PS1‐Stat3WT, n = 8 (three females and five males) mice; APP/PS1‐Stat3KO, n = 8 (five females and three males) mice; age, 8–9 months). C, D Using an anti‐Ki67 antibody as a marker for cellular proliferation, we detected Ki67‐positive cells (arrows) in the hippocampal dentate gyrus as a positive control. However, no Ki67 signal was detected around plaques (marked by arrowheads) of either APP/PS1‐Stat3KO or APP/PS1‐Stat3WT mice, indicating little‐to‐no glial proliferation (scale bars, 50 μm; same mice as in A and B). Data information: Data are represented as mean ± SEM.

Article Snippet: All brain sections were blocked with 10% normal goat serum (Vector Labs) and 0.3% Triton X‐100 (Sigma) in PBS for 1 h. Mouse brain sections were incubated with rat anti‐GFAP (1:250; #130300; Invitrogen), rabbit anti‐GFAP (1:500; #Z0334, Dako), mouse anti‐Aβ (IC16; 1:250; provided by Dr. C. Pietrzik, Mainz University), rabbit anti‐pStat3 (1:500; #9145S, Cell Signaling), rabbit anti‐Iba1 (1:250; 019‐19741, Wako), rabbit anti‐Stat3 (1:500; #12640, Cell Signaling), rabbit anti‐RFP (1:300; #AB234, Evrogen), rat anti‐LAMP1 (1:750; #121602, BioLegend), mouse anti‐S100β (1:250; #S2532, Sigma), rat anti‐C3d (1:250; A0063, Dako), rabbit anti‐Ki67 (1:500; RM9106, Thermo Scientific), mouse anti‐NeuN (1:100; MAB377, Millipore), rabbit anti‐NG2 (1:250; AB5320, Millipore), and rabbit anti‐Olig2 (1:500; AB9610, Millipore) in 5% normal goat serum and 0.05% Triton X‐100 overnight at 4°C.

Techniques: MANN-WHITNEY, Marker, Positive Control