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

Journal: Cell reports

Article Title: Scavenging of Labile Heme by Hemopexin Is a Key Checkpoint in Cancer Growth and Metastases

doi: 10.1016/j.celrep.2020.108181

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-P(Ser139)-H2AX (γH2AX) , Cell Signaling , Cat#: 9178; RRID:AB_2072132.

Techniques: Recombinant, Fractionation, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Expressing, Software

Journal: Gynecologic oncology

Article Title: Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

doi: 10.1016/j.ygyno.2016.07.088

Figure Lengend Snippet: A) The percentage of OVCAR-3 and SKOV-3 cells showing co-expression of RAD51 and pH2AX (positive was >5 foci). Cells were pre-treated with 0.5µM cisplatin (6h), then effects of the panobinostat pre-treatment (25nM; 24h)/panobinostat (25nM; 24h) and olaparib (10µM) co-treatment combination regimen (24h) determined by IF. B) IF analysis of GFP expression in cells co-transfected with pDRGFP and I-Sce1 plasmids (both 1µg), then treated with panobinostat and/or olaparib as above. At least 100 cells were counted in 3 independent fields. Values in A) and B) are mean+SD for 3 independent experiments. * p<0.01 compared to vehicle; ap<0.01 relative to olaparib alone, bp<0.01 relative to panobinostat alone, Student’s t test. C) Representative images of GFP-positive SKOV-3 cells (green) and DAPI-stained nuclei (blue). Scale bars are 20µm.

Article Snippet: Immunofluorescence Following drug treatment and/or transient transfection with the HR reporter plasmid pDRGFP and endonuclease encoding pCBASce1 (I-Sce1) (both gifts from Maria Jasin; Addgene plasmids #26475 and #26477, respectively) [ 27 , 28 ] plasmids using Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), ovarian cancer cells were fixed, permeabilized and visualized for GFP expression, or stained with mouse monoclonal anti-phospho(p)-H2AX (Ser139) (pH2AX) (Millipore, Billerica, MA), rabbit polyclonal anti-RAD51 (Millipore), and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) as described [ 19 ].

Techniques: Expressing, Transfection, Staining

Journal: Gynecologic oncology

Article Title: Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

doi: 10.1016/j.ygyno.2016.07.088

Figure Lengend Snippet: A) IF analysis of the effects of panobinostat pre-treatment (25nM; 24h)/panobinostat (25nM; 24h) and olaparib (10µM) co-treatment combination regimen (24h) panobinostat (25nM)/olaparib (10µM) combination (24h) on pH2AX expression in OVCAR-3 cells. B) Representative images showing pH2AX staining (green) and DAPI-stained nuclei (blue). C) Western blot analysis pH2AX, cleaved PARP and cleaved caspase-3 expression in OVCAR-3 cells treated as above. Actin and total histone H3 were loading controls. D) IF analysis of cleaved caspase-3 expression in cells treated as above. Values for A) and D) are mean+SD for 3 independent experiments. At least 100 cells were counted (×40) for each drug treatment per experiment. *p<0.01 compared to vehicle; ap<0.01 relative to olaparib alone; bp<0.01 relative to panobinostat alone, all Student’s t test. E) Representative images of cleaved caspase-3 staining (green) and DAPI-stained nuclei (blue). Scale bars in B) are 20µm and in E) are 10µm.

Article Snippet: Immunofluorescence Following drug treatment and/or transient transfection with the HR reporter plasmid pDRGFP and endonuclease encoding pCBASce1 (I-Sce1) (both gifts from Maria Jasin; Addgene plasmids #26475 and #26477, respectively) [ 27 , 28 ] plasmids using Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), ovarian cancer cells were fixed, permeabilized and visualized for GFP expression, or stained with mouse monoclonal anti-phospho(p)-H2AX (Ser139) (pH2AX) (Millipore, Billerica, MA), rabbit polyclonal anti-RAD51 (Millipore), and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) as described [ 19 ].

Techniques: Expressing, Staining, Western Blot

Journal: Gynecologic oncology

Article Title: Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

doi: 10.1016/j.ygyno.2016.07.088

Figure Lengend Snippet: A) Schematic of drug treatment experiment in nude mice injected subcutaneously with SKOV-3 cells. Mice (10 per group) were pre-treated for one week with vehicle or panobinostat, and then subsequently with vehicle, panobinostat and/or olaparib for three weeks as shown (IP: intraperitoneal; PO: per os, oral gavage). B) Time course measurements of tumor volume at weekly intervals (single arrow: start of panobinostat pre-treatment; double arrow: start of full drug regimen). C) Tumor weights at sacrifice. Tumors are shown in D). E) Western blot analysis of cyclin E, BRCA1, cleaved PARP, PCNA and pH2AX protein expression in harvested tumors. Actin and histone H3 were loading controls. F) Densitometry analysis of expression relative to corresponding actin or histone H3 levels. Values are mean+ SD; *p<0.01 single drug effect relative to vehicle; ap<0.01 combination drug effect relative to olaparib; bp<0.01 combination drug effect relative to panobinostat, Student’s t test.

Article Snippet: Immunofluorescence Following drug treatment and/or transient transfection with the HR reporter plasmid pDRGFP and endonuclease encoding pCBASce1 (I-Sce1) (both gifts from Maria Jasin; Addgene plasmids #26475 and #26477, respectively) [ 27 , 28 ] plasmids using Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), ovarian cancer cells were fixed, permeabilized and visualized for GFP expression, or stained with mouse monoclonal anti-phospho(p)-H2AX (Ser139) (pH2AX) (Millipore, Billerica, MA), rabbit polyclonal anti-RAD51 (Millipore), and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) as described [ 19 ].

Techniques: Injection, Western Blot, Expressing