Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Rapamycin reduces DNA damage of in vitro matured oocytes by promoting Rad51 -mediated homologous recombination

doi: 10.1186/s12958-025-01428-6

Figure Lengend Snippet: The impact of rapamycin on the DNA damage response and autophagy level in IVM oocytes. ( A ) The fluorescence images of γH2AX in IVM oocytes from the control and rapamycin groups. Scale bar: 10 μm. ( B ) The comparison of γH2AX fluorescence intensity between the two groups, *** P < 0.001. ( C-E ) The mRNA levels of the HR-associated genes Brca1 and Rad51 and the NHEJ-associated gene Dnapk in the two groups. ** P < 0.01. ( F-G ) The fluorescence images of LC3B in IVM oocytes and comparison of LC3B puncta among the control, 10 nM rapamycin, and 10µM rapamycin groups. Scale bar: 25 μm, *** P < 0.001

Article Snippet: IBR2 (MedChemExpress, New Jersey, USA) was used to inhibit RAD51, and LTURM34 (MedChemExpress, New Jersey, USA) was used to inhibit DNAPK.

Techniques: Fluorescence, Control, Comparison

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Rapamycin reduces DNA damage of in vitro matured oocytes by promoting Rad51 -mediated homologous recombination

doi: 10.1186/s12958-025-01428-6

Figure Lengend Snippet: The impact of RAD51 inhibitor and DNAPK inhibitor on IVM oocytes. ( A ) The images of IVM oocytes treated with RAD51 inhibitor (IBR2) and DNAPK inhibitor (LTURM34). Scale bar: 50 μm. ( B ) The fluorescent images of γH2AX in IVM oocytes from the three groups. Scale bar: 10 μm. ( C ) The comparison of maturation rates of IVM oocytes treated with RAD51 inhibitor (IBR2) and DNAPK inhibitor (LTURM34), ** P < 0.01. ( D ) The γH2AX fluorescence levels of IVM oocytes in control group and treated with IBR2 or LTURM34. ** P < 0.01

Article Snippet: IBR2 (MedChemExpress, New Jersey, USA) was used to inhibit RAD51, and LTURM34 (MedChemExpress, New Jersey, USA) was used to inhibit DNAPK.

Techniques: Comparison, Fluorescence, Control

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Rapamycin reduces DNA damage of in vitro matured oocytes by promoting Rad51 -mediated homologous recombination

doi: 10.1186/s12958-025-01428-6

Figure Lengend Snippet: The impact of RAD51 inhibition on the improving effects of rapamycin in IVM oocytes. ( A ) Images of oocytes IVM from the control group, rapamycin group, IBR2 group, and R + I group. Scale bar: 50 μm. ( B ) γH2AX fluorescence staining of IVM oocytes from the four groups. ( C ) Comparison of the maturation rates of the four groups. ( D ) Comparison of the γH2AX fluorescence levels of the four groups. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: IBR2 (MedChemExpress, New Jersey, USA) was used to inhibit RAD51, and LTURM34 (MedChemExpress, New Jersey, USA) was used to inhibit DNAPK.

Techniques: Inhibition, Control, Fluorescence, Staining, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Venetoclax enhances DNA damage induced by XPO1 inhibitors: A novel mechanism underlying the synergistic antileukaemic effect in acute myeloid leukaemia

doi: 10.1111/jcmm.17274

Figure Lengend Snippet: Effect of short‐term inhibition of XPO1 on the protein and mRNA levels of c‐Myc and DDR pathway components in AML cells. (A) MV4‐11 cells were treated with KPT‐330 or KPT‐8602, for 4 or 8 h, and then subjected to Annexin V‐FITC/PI staining and flow cytometry analysis. Mean per cent Annexin V+ cells ± SEM are shown. ** indicates p < 0.01 and *** indicates p < 0.001 compared with vehicle control. (B–E) MV4‐11 and THP‐1 cells were treated with KPT‐330 or KPT‐8602, for 4 or 8 h. Whole‐cell lysates were subjected to Western blot analysis and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β‐actin and then compared with vehicle control, are graphed below the corresponding blot. (F) MV4‐11 and THP‐1 cells were treated with KPT‐330 or KPT‐8602, for 8 h. Total RNA was extracted and c ‐ Myc , CHK1 , WEE1 , RAD51 , RRM2 and GAPDH transcripts were determined by real‐time RT‐PCR. The relative changes in transcripts, normalized to GAPDH, in comparison with control samples were quantified. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 compared with vehicle control. (G) MV4‐11 and THP‐1 cells were treated with KPT‐330, KPT‐8602 and MG‐132, alone or in combination, for 8 h, and then subjected to Western blot analysis and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β‐actin and then compared with vehicle control, are graphed below the corresponding blot

Article Snippet: CHK1 (Hs00967506_m1), RAD51 (Hs00153418_m1) and WEE1 (Hs01119384_g1) transcripts were quantitated using TaqMan probes (Thermo Fisher Scientific).

Techniques: Inhibition, Staining, Flow Cytometry, Control, Western Blot, Quantitative RT-PCR, Comparison

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 1D, levels of Rad51 and BRCA1 were maintained in E7 and E6/E7-expressing cells 351

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 4C, while knockdown of Rad51 had no detectable effect on episome maintenance in 416

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques: Knockdown

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 1. High-risk HPV positive keratinocytes exhibit higher levels of Rad51 and 830

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques:

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 2. Rad51 and BRCA1 promoter activity and transcript levels are increased in 852

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques: Activity Assay

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 3. The half-life of Rad51 and BRCA1 protein is extended in HPV31 positive cells. 870

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques:

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 5. Cell cycle distribution is not affected by knockdown of Rad51 or BRCA1. 905

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques: Knockdown

Journal: Journal of Virology

Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

doi: 10.1128/jvi.02495-15

Figure Lengend Snippet: Figure 6. Inhibition of Rad51 DNA binding activity leads to a defect in productive 915

Article Snippet: Proteins were 225 visualized using primary antibodies as follows: Rad51, Involucrin, GAPDH (Santa Cruz); 226 BRCA1 (Calbiochem); cleaved caspase-7 D198, caspase-7 (Cell Signaling Technology); 227 phospho-ATM S1981 (Abcam); ATM (Bethyl Laboratories).

Techniques: Inhibition, Binding Assay, Activity Assay

Journal: Science Advances

Article Title: A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair

doi: 10.1126/sciadv.aau7566

Figure Lengend Snippet: ( A ) Asynchronous cells stably expressing Flag-Plk1 were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour. Plk1 were immunoprecipitated using an α-Flag antibody, and samples were probed with the indicated antibodies. ( B ) Inhibition of ATR pathway by using the ATR inhibitor VE-821 (10 μM) resulted in premature mitotic entry. Synchronized G 2 cells were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour and an addition with or without VE-821 treatment for 2 hours. The changes of K209me1 and pT210 levels were examined by Western blot. ( C , F , and H ) The indicated cells were treated with VP-16 (40 μM for 1 hour) and allowed to release into normal medium for the indicated times and analyzed using immunofluorescent staining with an α-γH2A.X, α-RPA2, or α-RAD51 antibody, respectively. ( D ) Quantification of γH2A.X-positive cells in (C) using ImageJ. The data represent means ± SD ( n > 100 each) from three independent experiments. ( E ) The indicated cells that were treated as described in (C) were analyzed using Western blotting. ( G and I ) Quantification of RPA2 or RAD51 foci numbers in individual cells described in (F) or (H) using ImageJ. The boxes designate cells with more than 10 foci, whose percentage is indicated above each box. *** P < 0.001. ( J ) The indicated cells were treated as described in (C), the chromatin fractions were collected, and chromatin-bound RPA2 and RAD51 levels were examined using Western blotting.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: The α-Plk1 (F-8) (sc-17783), α-HA (Y-11) (sc-805), and α-Wee1 (B-11) (sc-5285) antibodies were purchased from Santa Cruz Biotechnology; α-Flag (F7425-2MG) and α-Aurora A (A1231) antibodies were purchased from Sigma-Aldrich; α-G9a/EHMT2(C6H3) (3306S) and α-pCdc2 (Y15) (4539T) antibodies were purchased from Cell Signaling Technology; α-cyclin B1 (1495-1), α-phospho histone H3 pS10 (1173-1), and α-phospho Plk1 pT210 (3646-1) antibodies were purchased from Epitomics; α-BubR1 (ab172581), α-RAD51 (ab133534), and α-H3K9me2 (ab1220) antibodies were purchased from Abcam; α-eGFP (50430-2-AP), α-RPA2 (10412-1-AP), α-Myc (60003-2-Ig), and α-Actinβ (60008-1-Ig) antibodies were purchased from Proteintech; α-H3K9me2 (A2359) and mouse α-GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; α-H3 (39163) antibody was purchased from Active Motif; pan-mono/dimethyl (TM602) antibody was purchased from PTM Biolabs; α-HEC1(9G3.23) (GTX70268) antibody was purchased from GeneTex; α-Plk1 K209me1 antibody was generated by ABclonal by immunizing rabbits with a K209-monomethylated peptide (DGERKK(me1)TLC) conjugated with keyhole limpet hemacyanin; secondary horseradish peroxidase–conjugated α-mouse or α-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; the α-FLAG M2 affinity gel (A2220), thymidine, and nocodazole were purchased from Sigma-Aldrich; the α–hemagglutinin (HA) affinity gel (B23301) was purchased from BioTool; BIX-01294 was purchased from Selleck; VP-16 and VE-821 were purchased from TargetMol; and CPT was purchased from Aladin.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Inhibition, Western Blot, Staining

Journal: ACS pharmacology & translational science

Article Title: Inhibition of Protein Disulfide Isomerase (PDIA1) Leads to Proteasome-Mediated Degradation of Ubiquitin-like PHD and RING Finger Domain-Containing Protein 1 (UHRF1) and Increased Sensitivity of Glioblastoma Cells to Topoisomerase II Inhibitors.

doi: 10.1021/acsptsci.2c00186

Figure Lengend Snippet: Figure 2. Chemical or genetic inhibition of PDIA1 leads to the downregulation of UHRF1 in glioma cells. (A) GSEA plots showing that treatment of U87 cells with PACMA31 or knockdown of P4HB (PDIA1) in U87 cells negatively correlates with enrichment of DNA repair gene set. NES: normalized enrichment score. (B) Treatment with BAP2 or PACMA31 or knockdown of P4HB using siRNA (in U87) or doxycycline-inducible shRNA (in U87 or D54) negatively correlates with enrichment of GO_DNA_REPAIR including 44 common downregulated core enrichment genes among which is UHRF1. The Bru-seq datasets used in this analysis are listed in Table S1. (C) Box plot representing UHRF1 expression in GBM samples (red) versus matched normal data (black) (TCGA + GTEx) using GEPIA online tool. Asterisk (*) indicates p < 0.05. TPM:

Article Snippet: Primary antibodies: UHRF1 (Rabbit pAb, ABclonal), PDIA1 (Rabbit mAb, Cell signaling), RAD51 (Rabbit mAb, Cell signaling), GAPDH (Rabbit mAb, Cell signaling), β-tubulin (Rabbit mAb, Cell signaling), cleaved caspase-3 (Rabbit mAb, Cell signaling), cleaved PARP (Rabbit mAB, Cell signaling), cleaved caspase-9 (Rabbit mAB, Cell signaling), SLC7A11 (Rabbit mAb, Cell signaling), ATF4 (Rabbit mAb, Cell signaling), GPX4 (Rabbit mAb, Abcam), γH2AX (Rabbit mAb, Cell signaling).

Techniques: Inhibition, Knockdown, shRNA, Expressing

Journal: ACS pharmacology & translational science

Article Title: Inhibition of Protein Disulfide Isomerase (PDIA1) Leads to Proteasome-Mediated Degradation of Ubiquitin-like PHD and RING Finger Domain-Containing Protein 1 (UHRF1) and Increased Sensitivity of Glioblastoma Cells to Topoisomerase II Inhibitors.

doi: 10.1021/acsptsci.2c00186

Figure Lengend Snippet: Figure 3. PDIA1 inhibition leads to proteasome-mediated downregulation of UHRF1 and RAD51 levels. (A) Bar graph (top) and representative Western blot (bottom) indicating downregulation of UHRF1 by BAP2 and bepristat-2a (bep2a) which is rescued by MG132 treatment. RAD51 was also blotted but not displayed in the bar graph. GB-1, DBTRG, and U87 cells were treated with BAP2 (10 μM), bepristat-2a (30 μM), and MG132 (5 μM) for 24 h. The band intensity of each sample was normalized to β-tubulin, which is used as a loading control, and each bar represents the logFC of UHRF1 relative to the control sample. The error bars represent standard deviation of three biological replicates. The asterisk (*) indicates p < 0.05. Uncut Western blots for three biological replicates are shown in the Supporting Information. (B) Bar graph showing the log2 fold change of UHRF1 in cells treated with the indicated ER-stress-inducing agents in comparison to control (p-value < 0.05 for all). The cell line corresponding to each dataset is shown on top of the bar. The six datasets used for this analysis are listed in Table S3. (C) Western blot analysis of GB-1 and DBTRG cells treated with tunicamycin (5 μg/mL) with or without MG132 (5 μM) for 24 h. UHRF1 and RAD51 levels are repressed by tunicamycin and rescued by MG132 treatment. The band intensity of each sample was normalized to β-tubulin, and the numbers above each band represent the fold change of UHRF1 and RAD51 relative to the control sample.

Article Snippet: Primary antibodies: UHRF1 (Rabbit pAb, ABclonal), PDIA1 (Rabbit mAb, Cell signaling), RAD51 (Rabbit mAb, Cell signaling), GAPDH (Rabbit mAb, Cell signaling), β-tubulin (Rabbit mAb, Cell signaling), cleaved caspase-3 (Rabbit mAb, Cell signaling), cleaved PARP (Rabbit mAB, Cell signaling), cleaved caspase-9 (Rabbit mAB, Cell signaling), SLC7A11 (Rabbit mAb, Cell signaling), ATF4 (Rabbit mAb, Cell signaling), GPX4 (Rabbit mAb, Abcam), γH2AX (Rabbit mAb, Cell signaling).

Techniques: Inhibition, Western Blot, Control, Standard Deviation, Comparison

Journal: ACS pharmacology & translational science

Article Title: Inhibition of Protein Disulfide Isomerase (PDIA1) Leads to Proteasome-Mediated Degradation of Ubiquitin-like PHD and RING Finger Domain-Containing Protein 1 (UHRF1) and Increased Sensitivity of Glioblastoma Cells to Topoisomerase II Inhibitors.

doi: 10.1021/acsptsci.2c00186

Figure Lengend Snippet: Figure 6. PDIA1 inhibition results in increased sensitivity of tumor cells to Top-II inhibitors through degradation of UHRF1. Treatment of tumor cells with PDIA1 inhibitors results in ER stress leading to transcriptional repression of UHRF1 gene and proteasome-mediated degradation of UHRF1 protein. PDIA1 inhibition results in resistance to ferroptosis, and downregulation of UHRF1 impacts DNA repair pathways resulting in the accumulation of DNA damage induced by Top-II inhibitors ultimately leading to apoptosis.

Article Snippet: Primary antibodies: UHRF1 (Rabbit pAb, ABclonal), PDIA1 (Rabbit mAb, Cell signaling), RAD51 (Rabbit mAb, Cell signaling), GAPDH (Rabbit mAb, Cell signaling), β-tubulin (Rabbit mAb, Cell signaling), cleaved caspase-3 (Rabbit mAb, Cell signaling), cleaved PARP (Rabbit mAB, Cell signaling), cleaved caspase-9 (Rabbit mAB, Cell signaling), SLC7A11 (Rabbit mAb, Cell signaling), ATF4 (Rabbit mAb, Cell signaling), GPX4 (Rabbit mAb, Abcam), γH2AX (Rabbit mAb, Cell signaling).

Techniques: Inhibition

Journal: International journal of radiation oncology, biology, physics

Article Title: Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway.

doi: 10.1016/j.ijrobp.2022.12.029

Figure Lengend Snippet: Fig. 1. Cancer-associated fibroblasts (CAFs) promote breast cancer radioresistance. (A) Morphologic features of primary CAFs isolated from fresh breast cancer tissues. The CAF1 were isolated from luminal B breast cancer and the CAF2 from lumi- nal A breast cancer. Scale bar = 50 mm. (B) Collection of CAF-derived conditioned media (CM), which was used to maintain breast cancer cells. The cells were incubated in CAF-derived conditioned media for 24 hours. (C) The expression of E-cadherin, N-cadherin, vimentin in breast cancer cells were detected by western blotting. The cells were cultured in CAF-derived condi- tioned media. (D) Colony numbers and (E) survival fractions of breast cancer cells cultured in CAF-derived conditioned media. Values represent means § SDs (n = 3). *P < .05 and **P < .01, Student t test. (F) Establishment of a xenograft mouse model. The MDA-MB-231 cancer cells were used in the xenograft mouse model. Tumors in the radiation (RT) group were locally treated with 2-Gy radiation daily for 5 treatments. (G) Tumor growth curve. Data show the mean tumor volume of

Article Snippet: The primary antibodies against E-cadherin (#60335-1), vimentin (#60330-1), N-cadherin (#66219-1), and RAD51 (#14961-1) were purchased from ProteinTech Group (Rosemont, IL).

Techniques: Isolation, Derivative Assay, Incubation, Expressing, Western Blot, Cell Culture

Journal: International journal of radiation oncology, biology, physics

Article Title: Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway.

doi: 10.1016/j.ijrobp.2022.12.029

Figure Lengend Snippet: Fig. 3. Inhibition of the hepatocyte growth factor (HGF)/c-Met signaling axis abrogates cancer-associated fibroblast (CAF) −induced radioresistance. (A) p-c-Met, c-Met, p-AKT, AKT, E-cadherin, and vimentin expression in MDA-MB-231 and SKBR3 cells was detected by western blotting. Cells were treated with CAF1-derived conditioned media and/or HGF neutraliz- ing antibody for 24 hours. The CAF1 were isolated from luminal B breast cancer. (B, C) Colony numbers and (D) survival frac- tion of MDA-MB-231 and SKBR3 cells. Cells were treated as in (A). (E) p-ATM, ATM, RAD51, and gH2AX expression in MDA-MB-231 and SKBR3 cells were detected by western blotting. Cells were treated with CAF1-derived conditioned media and/or HGF neutralizing antibody and then exposed to 6-Gy radiation for 24 hours. (F) p-c-Met, c-Met, p-AKT, AKT, E-cad- herin, and vimentin expression in MDA-MB-231 and SKBR3 cells were detected by western blotting. Cells were treated with CAF1-derived conditioned media and/or c-Met inhibitor JNJ-38877605. (G-H) Colony numbers and (I) survival fraction of MDA-MB-231 and SKBR3 cells. Cells were treated as in (F). (J) p-ATM, ATM, RAD51, and gH2AX expression in MDA-MB-

Article Snippet: The primary antibodies against E-cadherin (#60335-1), vimentin (#60330-1), N-cadherin (#66219-1), and RAD51 (#14961-1) were purchased from ProteinTech Group (Rosemont, IL).

Techniques: Inhibition, Expressing, Western Blot, Derivative Assay, Isolation

Journal: Journal of Biomedical Science

Article Title: O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma

doi: 10.1186/s12929-020-00699-y

Figure Lengend Snippet: MGMT inhibition suppressed CDDP-induced RAD51 expression in NPC cells. a A heat map showing the expression changes of DNA repair genes with statistical significance, by using RT 2 Profiler PCR Array, in HONE-1 cells with or without O6BG treatment, respectively. After HONE-1 cells were treated with O6BG (120 μM) for 8 h, the extracted mRNA was subjected to gene expression analyses. b HONE-1 and c TW01 cells were treated with O6BG at indicated concentrations for 8 h. d HONE-1 and e TW01 cells were treated with O6BG (60 μM), CDDP (10 µM), or a combination treatment for 8 h. The IC 50 concentration of O6BG in both HONE-1 and TW01 cells was 120 µM. After the indicated treatment, cell lysates were analyzed using Western blotting. Fold changes in protein levels listed under each blot were normalized to the levels of the actin control. Following single-drug or combination treatment as indicated in d and e , the mRNA levels of f HONE-1 and g TW01cells were quantified using qPCR. Representative results of at least three independent experiments are presented. Bar values are presented as mean ± SD of at least three independent experiments. * P < 0.05

Article Snippet: TaqMan gene expression assay kits for RAD51 (Hs00947967_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Inhibition, Expressing, Gene Expression, Concentration Assay, Western Blot, Control

Journal: Journal of Biomedical Science

Article Title: O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma

doi: 10.1186/s12929-020-00699-y

Figure Lengend Snippet: MGMT modulated CDDP-induced RAD51 expression in NPC cells. a HONE-1 and b TW01 cells transfected with scrambled or two independent MGMT -targeted siRNAs were treated with CDDP for 8 h. Fold changes in protein levels listed under each blot were normalized to the levels of the actin control. Following treatment with CDDP as indicated in a , b , the mRNA levels of c HONE-1 and d TW01cells transfected with scrambled or MGMT -targeted siRNA were quantified using qPCR. Representative results of at least three independent experiments are presented. Bar values are presented as mean ± SD of at least three independent experiments. * P < 0.05. e The correlation between MGMT and RAD51 expression levels in patients with NPC (GSE102349) was analyzed using Pearson correlation analyses. The correlation coefficient and P are indicated

Article Snippet: TaqMan gene expression assay kits for RAD51 (Hs00947967_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Control

Journal: Journal of Biomedical Science

Article Title: O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma

doi: 10.1186/s12929-020-00699-y

Figure Lengend Snippet: MGMT inhibition suppressed HR activity in NPC cells. The representative images of immunofluorescence foci of γ-H2AX and RAD51 in a HONE-1 and b TW01 cells are shown. Following treatment with O6BG (60 μM), CDDP (10 µM), or a combination of both for 24 h, the immunofluorescence foci of γ-H2AX and RAD51 were captured at ×60 on a confocal immunomicroscope. DAPI was used to detect the nuclei. Representative images of at least three independent experiments are shown. c The percentages of γ-H2AX-positive NPC cells in a and b , indicating the levels of DSB formation, were quantified in response to drug treatment (left panel). Only cells with more than five foci of γ-H2AX were considered positive. The percentages of γ-H2AX-positive cells with the recruitment of RAD51 foci in a , b were quantified in terms of DNA repair activity (right panel). Following CDDP or combination treatment, cells with more than five colocalized foci of γ-H2AX and RAD51 were considered positive. At least 100 cells per sample were counted in each experiment. d The representative images of immunofluorescence foci of γ-H2AX and RAD51 in MGMT -proficient or -deficient HONE-1 cells are shown. After cells were transfected with scrambled or MGMT -targeted siRNA for 24 h, these cells were treated with CDDP for another 24 h. The immunofluorescence foci of γ-H2AX and RAD51 were detected using confocal immunomicroscope as indicated in a . Scale bars indicate 10 µm. e The levels of DNA damage (upper panel) and DNA repair activity (low panel) in d were quantified by measuring the immunofluorescence foci of γ-H2AX and RAD51as indicated in c . Following treatment with the f indicated concentration of O6BG or g transfection of MGMT-targeted siRNA for 24 h, HR activities in the tested cells were determined using a plasmid-based recombination reporter assay. The value of the control cells was set as 100% after internalization with a backbone plasmid. Bar values are represented as mean ± SD of at least three independent experiments. * P < 0.05

Article Snippet: TaqMan gene expression assay kits for RAD51 (Hs00947967_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Inhibition, Activity Assay, Immunofluorescence, Transfection, Concentration Assay, Plasmid Preparation, Reporter Assay, Control

Journal: Journal of Biomedical Science

Article Title: O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma

doi: 10.1186/s12929-020-00699-y

Figure Lengend Snippet: MGMT inhibition with CDDP treatment retarded NPC tumor growth. a Tumor volume and b body weight in mice bearing tumors were measured twice weekly. Tumor mass was measured with a caliper and calculated as π/6 × length (mm) × width (mm) 2 . c After 21 days of treatment, the mice were sacrificed, and tumor sizes were measured. The average tumor sizes in each treatment group are presented as a bar histogram (right panel). Bar values are presented as mean ± SD. * P < 0.05. d The expression of MGMT, RAD51, and p-BRCA1 was analyzed in paraffin-embedded tumor sections obtained from mice in each treatment group through immnunohistochemical staining. The slides were then examined under a fluorescence microscope, and photomicrographs were taken at a magnification of ×400

Article Snippet: TaqMan gene expression assay kits for RAD51 (Hs00947967_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Inhibition, Expressing, Staining, Fluorescence, Microscopy

Journal: Journal of Biomedical Science

Article Title: O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma

doi: 10.1186/s12929-020-00699-y

Figure Lengend Snippet: Schematic illustrating the role of MGMT in HR signaling in NPC cells. After the formation of platinum–DNA adducts, MGMT is involved in RAD51 and p-BRCA1 (ser 988) expression in HR signaling pathway. Through the participation in HR activation, MGMT could modulate CDDP and PARP inhibitor sensitivity in NPC cells

Article Snippet: TaqMan gene expression assay kits for RAD51 (Hs00947967_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Inhibition of ERβ Induces Resistance to Cisplatin by Enhancing Rad51–Mediated DNA Repair in Human Medulloblastoma Cell Lines

doi: 10.1371/journal.pone.0033867

Figure Lengend Snippet: Neutral comet assay (single cell electrophoresis) of exponentially growing Daoy cells (FBS) in which cisplatin treatment (1 µg/ml for 6 hours) was applied in the absence (Cis) or in the presence of 10 µM ICI182,780 (Cis+ICI). The histogram represents average Olive tail moment (with standard deviation) calculated from three experiments in duplicate (n = 6). In each experiment at least 100 cells were selected for the calculation (Automated Comet Assay; Loats Associates. Inc.). * indicates value statistically different from the sample labeled Cis. ** indicates value statistically different form Cis+ICI (paired student t-test; P≤0.05). Inset: Western blot showing effectiveness of Rad51 siRNA (100 nM for 48 hrs) tested in exponentially growing Daoy cells.

Article Snippet: In some experiments, expression of Rad51 and ERβ was inhibited by utilizing ON-TARGETplus SMARTpool siRNA against human Rad51 - target sequences: CCAACGAUGUAAGAAUU ; GCAGUGAUGUCCUGGAUAA ; CUAAUCAGGUGGUAGCUCA ; UAUCAUCGCCCAUGCAUCA (100 nM, Thermo Scientific); and human ERβ - target sequences: GGAAAUGCGUAGAAGGAAU ; UUCAAUUUCGAGAGUUA ; GCACGGCUCCAUAUACAUA ; GAACCCACAGUCUCAGUGA (200 nM; Thermo Scientific) delivered to the cells by Oligofectamine transfection reagent (Invitrogen).

Techniques: Neutral Comet Assay, Electrophoresis, Standard Deviation, Single Cell Gel Electrophoresis, Labeling, Western Blot

Journal: PLoS ONE

Article Title: Inhibition of ERβ Induces Resistance to Cisplatin by Enhancing Rad51–Mediated DNA Repair in Human Medulloblastoma Cell Lines

doi: 10.1371/journal.pone.0033867

Figure Lengend Snippet: Panel A: Fluorescent images of Daoy cells immunolabeled with anti-histone γH2AX (upper panel) and anti-IRS-1 (lower panel) antibodies. The nuclei are visualized by DAPI staining (blue fluorescence). The histograms represent quantification of the co-localization between γH2AX and DAPI; IRS-1 and DAPI. The data represent average percentage of nuclear voxels (3-D pixels) of γH2AX (red fluorescence) and IRS-1 (green fluorescence) calculated from three independent experiments (n = 3) in which ten randomly selected cells have been evaluated by the Mask analysis included in SlideBook 5 deconvolution software. * indicates value statistically different from the sample labeled Cis (paired student t-test; P≤0.05). Panel B: Fluorescent images of the cells labeled with anti-Rad51 (green fluorescence) and with anti-BrdU (red fluorescence) antibodies. Exponentially growing cultures of Daoy cells (10%FBS) were exposed for one hour to bromodeoxyuridine (BrdU) during the 6 hours treatment with cisplatin (1 µg/ml) in the absence (Cis) or in the presence of 10 µM ICI182,780 (Cis+ICI). The histogram represents quantification of Rad51 positive cells in which Rad51 nuclear foci co-localize with BrdU-labeled DNA. Note, almost 40% increase in the number of cells utilizing Rad51 to repair cisplatin-induced DNA damage (during DNA replication) when the cisplatin treatment is accompanied by ICI182,780. * indicates value statistically different from the sample labeled Cis (paired student t-test; P≤0.05).

Article Snippet: In some experiments, expression of Rad51 and ERβ was inhibited by utilizing ON-TARGETplus SMARTpool siRNA against human Rad51 - target sequences: CCAACGAUGUAAGAAUU ; GCAGUGAUGUCCUGGAUAA ; CUAAUCAGGUGGUAGCUCA ; UAUCAUCGCCCAUGCAUCA (100 nM, Thermo Scientific); and human ERβ - target sequences: GGAAAUGCGUAGAAGGAAU ; UUCAAUUUCGAGAGUUA ; GCACGGCUCCAUAUACAUA ; GAACCCACAGUCUCAGUGA (200 nM; Thermo Scientific) delivered to the cells by Oligofectamine transfection reagent (Invitrogen).

Techniques: Immunolabeling, Staining, Fluorescence, Software, Labeling

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: Serine miscoding is toxic in E. coli . Chimeric tRNA Ser variants were generated to decode at either the Ser (anticodon, GGA), Ala (UGC), or Thr (GGU) codon (top). Mutant tRNAs were expressed in wild-type E. coli , and growth was monitored (bottom). Both tRNA mutants caused growth defects in MG1655. All growth experiments were performed in triplicate, and error bars indicate the standard deviation of the replicates.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Generated, Mutagenesis, Standard Deviation

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: AlaRS fidelity is required for optimal growth in E. coli . (A) AaRS mutants and the AlaRS C666A complement strain (pLK- alaS ) were grown in LB, and their growth was monitored. In rich medium, AlaRS fidelity is required for optimal growth. (B) AlaRS C666A was grown in M9 minimal medium supplemented with exogenous amino acids to determine the toxicity of noncognate stress. (C) Serine mistranslation was observed in the AlaRS C666A mutant using a β-lactamase S68A mistranslation reporter. (D) The severity of the AlaRS C666A growth defect was enhanced when grown at 42°C. All growth experiments were performed in triplicate, and error bars indicate the standard deviation of the replicates. WT, wild type.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Mutagenesis, Standard Deviation

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: AlaRS-mediated mistranslation disrupts proteome homeostasis. Total proteome analysis was performed on wild-type and AlaRS C666A E. coli . Depicted is a volcano plot of the 833 significantly enriched or underrepresented proteins when AlaRS fidelity is impaired.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques:

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: Swimming motility is impaired in the absence of AlaRS fidelity. (A) Representative images (top) and quantification (bottom) of swimming motility data. In the absence of AlaRS proofreading, E. coli has a swimming defect, and this can be rescued by complementation. (B) The observed swimming defect is not exclusively due to small RNA inhibition, as an hfq mutant was unable to rescue the defect. All data were plotted relative to a wild-type control for each experiment. Bar graphs shown the averages of the data collected from three experiments, and error bars represent the standard deviation of those experiments.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Inhibition, Mutagenesis, Control, Standard Deviation

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: The E. coli membrane is affected when AlaRS fidelity is impaired. (A) The AlaRS C666A mutant (blue) is sensitive to an array of antibiotics, as observed using a disk diffusion assay compared to wild-type (black) and ThrRS C182A (red) E. coli . All experiments were performed in triplicate, with error bars indicating the standard deviation. The asterisks indicate statistical significance as determined using one-way analysis of variance (ANOVA) with Tukey post hoc comparison ( P < 0.05). (B) TEM analysis indicated altered nucleoid morphology in the absence of AlaRS fidelity.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Membrane, Mutagenesis, Diffusion-based Assay, Standard Deviation, Comparison