Journal: bioRxiv

Article Title: The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in HCT116 colon cancer cells

doi: 10.1101/2023.03.17.533075

Figure Lengend Snippet: A . Immunofluorescence experiments performed on HCT116 cells treated, or not, with oxaliplatin (0.5 μM) monitoring g-H2AX (yellow). DAPI (blue) was used to stain DNA and the nuclei. Quantification of the staining is shown on the right side and is represented as the corrected nuclear fluorescence. Data are represented as the mean ± SEM. (n=3). Unpaired two-tailed Student’s t-test; ** p<0.01. B . Immunofluorescence experiments performed on HCT116 cells treated, or not, with oxaliplatin (0.5 μM) monitoring 53BP1 (grey). DAPI (blue) was used to stain DNA and the nuclei. Quantification of the staining is shown on the right side and is represented as the corrected nuclear fluorescence. Data are represented as the mean ± SEM. (n=3). Unpaired two-tailed Student’s t-test; *** p<0.001. C . Western blot analysis showing protein expression of TEAD4 and p53 family of proteins in HCT116 treated (Oxa), or not (NT) with oxaliplatin (0.5 μM). GAPDH was used as a loading control (n=3). D. Heat map corresponding to the genes differentially expressed in HCT116 cells after 24h of oxaliplatin treatment at IC50 (see Supplemental Table 1). The three replicates for the non-treated (NT) and treated (Oxa) are shown.

Article Snippet: Antibodies used were rabbit anti-53BP1 (1/100; CST #4937), mouse anti-phospho-Histone H2AX clone JBW301 (1/200; Millipore #05-636), anti-TAZ (1/100; CST #4883), mouse anti-TEAD4 (1/50; Santa Cruz #sc101184), and rabbit anti-YAP (1/100; CST #14074).

Techniques: Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Western Blot, Expressing

Journal: Cancers

Article Title: Inhibition of Checkpoint Kinase 1 (CHK1) Upregulates Interferon Regulatory Factor 1 (IRF1) to Promote Apoptosis and Activate Anti-Tumor Immunity via MICA in Hepatocellular Carcinoma (HCC)

doi: 10.3390/cancers15030850

Figure Lengend Snippet: CHK1 inhibition induces DNA damage to cause apoptosis in HCC cells. ( a ) Western blot and quantitative analysis for γ-H2AX level is shown in Huh-7 cells induced by cisplatin (5 µM) for 24 h. The γ-H2AX expression is measured by western blot in HepG2 ( b ) and Huh-7 cells ( c ) induced by prexasertib with a dose of 5 µM for 24 h. ( d ) Representative image of FACS analysis of apoptotic Huh-7 cell rate treated with DMSO or prexasertib with a dose of 5 µM for 24 h are shown. ( e ) The statistical summary of apoptotic Huh-7 cell rate is shown with t test ( n = 3). Data represent mean ± SD, * p < 0.05. Western blot image shown are representative of 3 experiments. FACS assay shown are representative of 3 independent experiments. The uncropped blots are shown in .

Article Snippet: Antibodies used for western blot were antibodies against CHK1 (25887-1-AP), MICA (12619-1-AP), and β-actin (20536-1-AP) (Proteintech, Wuhan, China); Anti-IRF1 (ab243895, abcam, Burlingame, CA, USA); anti-γ-H2AX (#80312) and GAPDH (CST, Danfoss, MA, USA).

Techniques: Inhibition, Western Blot, Expressing

Journal: Oncoscience

Article Title: A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells

doi:

Figure Lengend Snippet: Intra-assay Imprecision

Article Snippet: The following reagents were used for electrophoresis: glycine, Tween 20, Tris, 10x Tris/Glycine/SDS electrophoresis running buffer, precision plus protein standards (10 – 250 kDa), Tris-HCL and 4-15% TGX Criterion ™ precast (18-well) gels from Bio-Rad Laboratories (Hercules, CA). anti-human ATM serine-1981 phosphorylation antibody, clone EP1890Y, Abcam (Cambridge, MA); purified mouse monoclonal anti-human/mouse pan-ATM antibody, clone 2C1, GeneTex (Irvine, CA); biotinylated rabbit monoclonal anti-human/mouse/rat/monkey histone H2AX serine-139 phosphorylation, clone 20E3, Cell Signaling Technology (Danvers, MA); mouse monoclonal anti-human/mouse/rat histone H2AX antibody, clone 322105, R&D Systems (Minneapolis, MN); rabbit monoclonal anti-human/rabbit/mouse ATR, clone E1S3S, Cell Signaling Technology (Danvers, MA); SMG-1, (Q25), Cell Signaling Technology (Danvers, MA); IRDye 800CW conjugated goat anti-rabbit, and IRDye 680RD conjugated goat anti-mouse LI-COR Biotechnology-US (Lincoln, NE).

Techniques: Intra Assay

Journal: Oncoscience

Article Title: A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells

doi:

Figure Lengend Snippet: Cellular lysates, prepared from lllltreated (A,D) and 2 Gy irradiated PBMCs (B,E,) from subject B were serially diluted and processed by ATM (A,B) and H2AX (C,D) immunoblot analysis. Fold activation ofATM (C) and H2AX (F) were analyzed as a function ofPBMCs per gel lane.

Article Snippet: The following reagents were used for electrophoresis: glycine, Tween 20, Tris, 10x Tris/Glycine/SDS electrophoresis running buffer, precision plus protein standards (10 – 250 kDa), Tris-HCL and 4-15% TGX Criterion ™ precast (18-well) gels from Bio-Rad Laboratories (Hercules, CA). anti-human ATM serine-1981 phosphorylation antibody, clone EP1890Y, Abcam (Cambridge, MA); purified mouse monoclonal anti-human/mouse pan-ATM antibody, clone 2C1, GeneTex (Irvine, CA); biotinylated rabbit monoclonal anti-human/mouse/rat/monkey histone H2AX serine-139 phosphorylation, clone 20E3, Cell Signaling Technology (Danvers, MA); mouse monoclonal anti-human/mouse/rat histone H2AX antibody, clone 322105, R&D Systems (Minneapolis, MN); rabbit monoclonal anti-human/rabbit/mouse ATR, clone E1S3S, Cell Signaling Technology (Danvers, MA); SMG-1, (Q25), Cell Signaling Technology (Danvers, MA); IRDye 800CW conjugated goat anti-rabbit, and IRDye 680RD conjugated goat anti-mouse LI-COR Biotechnology-US (Lincoln, NE).

Techniques: Irradiation, Western Blot, Activation Assay

Journal: Oncoscience

Article Title: A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells

doi:

Figure Lengend Snippet: Blood tubes from three subjects were 2 Gy irradiated and PBMCs were isolated and stored at −70°C. Immediately after irradiation and over 46 weeks, PBMC pellets were analyzed for ATM ( A ) and H2AX activation ( B ).

Article Snippet: The following reagents were used for electrophoresis: glycine, Tween 20, Tris, 10x Tris/Glycine/SDS electrophoresis running buffer, precision plus protein standards (10 – 250 kDa), Tris-HCL and 4-15% TGX Criterion ™ precast (18-well) gels from Bio-Rad Laboratories (Hercules, CA). anti-human ATM serine-1981 phosphorylation antibody, clone EP1890Y, Abcam (Cambridge, MA); purified mouse monoclonal anti-human/mouse pan-ATM antibody, clone 2C1, GeneTex (Irvine, CA); biotinylated rabbit monoclonal anti-human/mouse/rat/monkey histone H2AX serine-139 phosphorylation, clone 20E3, Cell Signaling Technology (Danvers, MA); mouse monoclonal anti-human/mouse/rat histone H2AX antibody, clone 322105, R&D Systems (Minneapolis, MN); rabbit monoclonal anti-human/rabbit/mouse ATR, clone E1S3S, Cell Signaling Technology (Danvers, MA); SMG-1, (Q25), Cell Signaling Technology (Danvers, MA); IRDye 800CW conjugated goat anti-rabbit, and IRDye 680RD conjugated goat anti-mouse LI-COR Biotechnology-US (Lincoln, NE).

Techniques: Irradiation, Isolation, Activation Assay

Journal: Oncoscience

Article Title: A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells

doi:

Figure Lengend Snippet: PBMCs were obtained from three patients treated with doxorubicin/ifosfamide. Samples were processed for immunoblot analysis for detection of ATM ( A ) and H2AX activation ( B ). ( C ) Image of H2AX immunoblots in PBMCs from patient#2.

Article Snippet: The following reagents were used for electrophoresis: glycine, Tween 20, Tris, 10x Tris/Glycine/SDS electrophoresis running buffer, precision plus protein standards (10 – 250 kDa), Tris-HCL and 4-15% TGX Criterion ™ precast (18-well) gels from Bio-Rad Laboratories (Hercules, CA). anti-human ATM serine-1981 phosphorylation antibody, clone EP1890Y, Abcam (Cambridge, MA); purified mouse monoclonal anti-human/mouse pan-ATM antibody, clone 2C1, GeneTex (Irvine, CA); biotinylated rabbit monoclonal anti-human/mouse/rat/monkey histone H2AX serine-139 phosphorylation, clone 20E3, Cell Signaling Technology (Danvers, MA); mouse monoclonal anti-human/mouse/rat histone H2AX antibody, clone 322105, R&D Systems (Minneapolis, MN); rabbit monoclonal anti-human/rabbit/mouse ATR, clone E1S3S, Cell Signaling Technology (Danvers, MA); SMG-1, (Q25), Cell Signaling Technology (Danvers, MA); IRDye 800CW conjugated goat anti-rabbit, and IRDye 680RD conjugated goat anti-mouse LI-COR Biotechnology-US (Lincoln, NE).

Techniques: Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

doi: 10.1371/journal.pone.0005245

Figure Lengend Snippet: A) Dot plots representing γH2AX (H2AX phosphorylated in Ser 139) and DNA content in live NFAT5 +/+ and NFAT5 −/− lymphocytes after 24 hours in isotonic or hypertonic conditions. Bars on the right represent the percentage of γH2AX + cells after 24 hours in isotonic or hypertonic medium (values are the mean±SEM of seven independent experiments; * = p<0.05). B) T cells grown in isotonic or hypertonic conditions during 24 hours were stained with the DNA dye Hoechst 33342 and annexin-V-Fluos, and analyzed by flow cytometry. The experiment shown is representative of three independently performed. C) Single-cell alkaline gel electrophoresis assay (comet assay) done on the population of live cells isolated from cultures of NFAT5 −/− and wild-type cells after 6 or 24 hours in isotonic or hypertonic conditions. Etoposide-treated wild-type cells are included as a positive control. The experiment shown is representative of three independently performed. D) Cell cycle distribution of viable, γH2AX-negative cells after 24 hours in hypertonic medium (representative of seven independent experiments).

Article Snippet: Rabbit polyclonal anti-phospho-p53 (Ser15) (Cat. 9284), mouse monoclonal anti-p53 (Cat. 2524) and mouse monoclonal anti-cyclin D3 (Cat. 2936) were from Cell Signaling Technology (Danvers, MA, USA); mouse monoclonal anti-phospho-histone H2AX (Ser139, γH2AX) (Cat. 05-636) was purchased from Upstate Technologies (Lake Placid, NY, USA); mouse monoclonal anti-BrdU antibody (Cat. 555627) was purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Staining, Flow Cytometry, Nucleic Acid Electrophoresis, Single Cell Gel Electrophoresis, Isolation, Positive Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

doi: 10.12659/MSM.905370

Figure Lengend Snippet: Effect of XIST knockdown on cell proliferation and radiosensitivity of NPC cells. ( A ) Transfection efficiency of si-XIST-1 and si-XIST-2 in CNE1 and CNE2 cells was detected by qRT-PCR. ( B ) CCK-8 assay was performed to determine cell proliferation at 24 h, 48 h, and 72 h in si-XIST- or si-con-transfected CNE1 and CNE2 cells. ( C ) Colony formation assay was used to measure colony survival rate 2 weeks after CNE1 and CNE2 cells transfected with si-XIST or si-con were exposed to the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy). ( D ) CNE1 and CNE2 cells transfected with si-XIST or si-con were irradiated with 4-Gy X-rays and then subjected to immunofluorescent staining for γ-H2AX expression. ( E ) Western blot analysis of γ-H2AX expression level in CNE1 and CNE2 cells transfected with si-XIST or si-con under 4 Gy irradiation. * P <0.05.

Article Snippet: After being blocked with 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies, including mouse monoclonal anti-γ-H2AX (No. 2577; 1: 1000 dilution, Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-GAPDH (No. 97166; 1: 2000 dilution, Cell Signaling Technology) at 4°C overnight, and then by probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (sc-2379; 1: 1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Reactive protein expression was visualized and detected using a chemiluminescence kit (Millipore).

Techniques: Transfection, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Irradiation, Staining, Expressing, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

doi: 10.12659/MSM.905370

Figure Lengend Snippet: Effect of miR-29c overexpression on cell proliferation and radiosensitivity of NPC cells. ( A ) CCK-8 assay was carried out to evaluate cell proliferation at 24 h, 48 h, and 72 h in CNE1 and CNE2 cells transfected with miR-29c or miR-con. ( B ) The clonogenic survival curves were compared in CNE1 and CNE2 cells transfected with miR-29c or miR-con with the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy) treatment. ( C ) Immunofluorescent staining for γ-H2AX expression in CNE1 and CNE2 cells transfected with miR-29c or miR-con under 4-Gy irradiation. ( D ) Western blot analysis of γ-H2AX level in CNE1 and CNE2 cells transfected with miR-29c or miR-con with 4-Gy irradiation. * P <0.05.

Article Snippet: After being blocked with 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies, including mouse monoclonal anti-γ-H2AX (No. 2577; 1: 1000 dilution, Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-GAPDH (No. 97166; 1: 2000 dilution, Cell Signaling Technology) at 4°C overnight, and then by probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (sc-2379; 1: 1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Reactive protein expression was visualized and detected using a chemiluminescence kit (Millipore).

Techniques: Over Expression, CCK-8 Assay, Transfection, Irradiation, Staining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: DNA damage to bone marrow stromal cells by antileukemia drugs induces chemoresistance in acute myeloid leukemia via paracrine FGF10–FGFR2 signaling

doi: 10.1016/j.jbc.2022.102787

Figure Lengend Snippet: Antileukemia drugs induce DNA damage in bone marrow stromal cells in vitro . HS-5 and HUVECs cells were exposed to 200 μg/l ADR, 20 μg/l IDA, 400 μg/l Ara-C or NS as a vehicle control for 24 h. A , cells were probed with antibodies recognizing γ-H2AX foci ( red and pink signals ) by immune fluorescence, and nuclei were counterstained with DAPI ( blue ). The scale bar represents 20 μm. Quantitative analysis of γ-H2AX-TRITC fluorescence intensity was performed, and data were shown normalized to cells treated with NS as a control. Western blotting analysis of γ-H2AX protein levels in HS-5 cells ( B ) and HUVECs ( C ) after drug exposures. β-actin was the loading control. D , DNA damage marker PARP1 was analyzed in HS-5 cell lysates after drug exposures by Western blotting. Western blots were quantified using β-actin or histone H3 as a loading control, and data were normalized to 1.0 using samples treated with NS as a negative control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. ADR, adriamycin; Ara-C, cytarabine; NS, normal saline; DAPI, 4′,6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; IDA, idarubicin; PARP1, poly(ADP-ribose) polymerase 1; TRITC, tetramethylrhodamine-isothiocyanate.

Article Snippet: Primary antibodies to β-actin, phospho-Histone H2AX (Ser139) (clone JBW301), NF-κB P65, p-P65, IKBα, STAT3, p-STAT3 (Tyr705), PTEN, p-PTEN, ERK1/2, p-ERK1/2 (T202/Y204), P38, p-P38, AKT, p-AKT (S473), mTOR, p-mTOR, FGFR1, FGFR2, pFRS2α, CyclinD1, and CD44 were purchased from Cell Signaling Technology.

Techniques: In Vitro, Fluorescence, Western Blot, Marker, Negative Control

Journal: Antioxidants

Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

doi: 10.3390/antiox11102050

Figure Lengend Snippet: S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: PVDF membranes were incubated overnight with primary antibodies including Aqp5 (0.1 μg/mL, ab78486, Abcam, Waltham, MA, USA), Bax (1:1000, Abcam ab32503), Bcl2 (1:2000, Abcam ab182858), p-H2ax (1:1000, CST 80312, Danvers, MA, USA), cleaved Caspase3 (1:1000, CST 9664P), p53 (1:1000, 10442-1-AP, Proteintech, Rosemont, IL, USA), Nox4 (1:1000, Proteintech 14347), Nrf2 (1:1000, Proteintech 16396), Akt (1:1000, Proteintech 10176-2), p-Akt (1:2000, CST 4060T), Sod2 (1:1000, Abcam ab137037) eNOS (1:500, Abcam ab76198), p-eNOS (1:1000, CST 9570), and β-actin (1:50,000, Abclone AC026).

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

Journal: Antioxidants

Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

doi: 10.3390/antiox11102050

Figure Lengend Snippet: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: PVDF membranes were incubated overnight with primary antibodies including Aqp5 (0.1 μg/mL, ab78486, Abcam, Waltham, MA, USA), Bax (1:1000, Abcam ab32503), Bcl2 (1:2000, Abcam ab182858), p-H2ax (1:1000, CST 80312, Danvers, MA, USA), cleaved Caspase3 (1:1000, CST 9664P), p53 (1:1000, 10442-1-AP, Proteintech, Rosemont, IL, USA), Nox4 (1:1000, Proteintech 14347), Nrf2 (1:1000, Proteintech 16396), Akt (1:1000, Proteintech 10176-2), p-Akt (1:2000, CST 4060T), Sod2 (1:1000, Abcam ab137037) eNOS (1:500, Abcam ab76198), p-eNOS (1:1000, CST 9570), and β-actin (1:50,000, Abclone AC026).

Techniques: Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

doi: 10.3390/antiox11102050

Figure Lengend Snippet: S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: These samples were incubated overnight at 4 °C with following primary antibodies: anti-p-H2ax (1:200, 80312, CST, Danvers, MA, USA), anti-Nrf2 (1:50, 16396, Proteintech, Rosemont, IL, USA), anti-S1pr1 (1:500, PA11040, ThermoFisher Scientific, Waltham, MA, USA), anti-Cd31 (1:50, ab281583, Abcam, Waltham, MA, USA), and anti-F4/80 (1:100, bs7058R, Bioss, Beijing, China).

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

Journal: Antioxidants

Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

doi: 10.3390/antiox11102050

Figure Lengend Snippet: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: These samples were incubated overnight at 4 °C with following primary antibodies: anti-p-H2ax (1:200, 80312, CST, Danvers, MA, USA), anti-Nrf2 (1:50, 16396, Proteintech, Rosemont, IL, USA), anti-S1pr1 (1:500, PA11040, ThermoFisher Scientific, Waltham, MA, USA), anti-Cd31 (1:50, ab281583, Abcam, Waltham, MA, USA), and anti-F4/80 (1:100, bs7058R, Bioss, Beijing, China).

Techniques: Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR