Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Steady‐state levels of gastric cancer cell lines. Using Western blot assay, steady protein levels of BRCA1, BRCA2 and c‐MET are analysed in primary gastric cancer cell lines HS746T and AGS. Protein levels were normalized against actin

Article Snippet: For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Low levels of c‐MET partially sensitize GC cell lines in PARP inhibition. A, HS746T/AGS cells, control‐siRNA‐Hs746T/AGS cells and si‐c‐MET Hs746T/AGS cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of c‐MET expression (by Western blot analysis) revealed down‐regulation of the c‐MET receptor in both cell lines (HS746T and AGS); (B) HS746T cells, control‐siRNA‐Hs746T cells and si‐BRCA1/2 Hs746T cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of BRCA1 and BRCA2 expression (by Western blot analysis) revealed down‐regulation of the BRCA1/2 in HS746T cell line; (C) HS746T cells, control‐siRNA‐Hs746T, siBRCA1/2‐Hs746T and siMET/BRCA1/2‐Hs746T cells were cultured with the indicated concentrations of NU1025 (5, 10 and 20 μmol/L) for 48 h for determination of cell viability (MTT metabolic activity assay). Error bars represent SD

Article Snippet: For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).

Techniques: Inhibition, Control, Metabolic Assay, Expressing, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Co‐inhibition of c‐MET (SU11274) and PARP (NU1025) sensitizes GC cells after knockdown BRCA1/2. Knocking down BRCA1 or BRCA2 sensitizes cells to PARP and c‐MET inhibition in HS746T cells expressing low levels of c‐MET (AGS cells, c‐MET knockdown Hs746T cells) to PARP inhibition. A, HS746T cells, control‐siRNA‐Hs746T cells and siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cells were exposed to 5 µmol/L of NU1025 and/or 5 µmol/L of SU11274 for 48 h for determination of cell viability (MTT metabolic activity assay). Results are expressed as percentages. Average values of three experiments ± SD are shown; (B) Western blot analysis of PARP and cl.caspase‐3 in Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cell lines. Cells were cultured with the indicated drugs (5 μmol/L NU1025, 5 μmol/L SU11274 alone or in combination for 24 h of treatment). Protein levels were normalized against actin

Article Snippet: For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).

Techniques: Inhibition, Knockdown, Expressing, Control, Metabolic Assay, Western Blot, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: GSK3B directs DNA repair choice and determines tumor response to PARP1 inhibition independent of BRCA1

doi: 10.1172/JCI189956

Figure Lengend Snippet: ( A ) Representative images of 53BP1 –/– U2OS cells reconstituted with HA-tagged WT 53BP1 and irradiated with 6 Gy. Cells were costained for BRCA1 and p-T334 phospho-specific antibodies. ( B ) Number of BRCA1 foci per cell stratified by pT334 status at different time points following radiation and ( C ) relative intensity of BRCA1 foci at various time points. ( D ) Representative images of 53BP1 –/– U2OS cells reconstituted with HA-tagged WT 53BP1 and irradiated with 6 Gy. Cells were costained for RAD51 and T334 phospho-specific antibodies. ( E ) Number of RAD51 foci per cell stratified by pT334 status at different time points following radiation and ( F ) relative intensity of RAD51 foci at various time points. Quantification of foci and intensity were calculated for each cell by the Image J software. n = 3. ( G ) Schematic of I-SceI HR reporter assay. Assessment of chromosomal HR repair of DSB as measured by percentage GFP-positive 53BP1 –/– U2OS cells reconstituted with various 53BP1 alleles with or without BRCA1 knockdown. DSBs were induced by adenoviral delivery of I-SceI, and the ability of cells to resolve DSBs by HR was assessed 48 hours later. n = 3. Data were analyzed by 2-way ANOVA followed by Tukey’s multiple comparison. Values are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Magnification ×65 ( A and D ).

Article Snippet: For BRCA1 knock down, Signal Silence BRCA1 siRNA was purchased from Cell signaling, # 12519.

Techniques: Irradiation, Software, Reporter Assay, Knockdown, Comparison

Journal: The Journal of Clinical Investigation

Article Title: GSK3B directs DNA repair choice and determines tumor response to PARP1 inhibition independent of BRCA1

doi: 10.1172/JCI189956

Figure Lengend Snippet: ( A ) 53BP1 –/– U2OS cells reconstituted with WT or T334A 53BP1 with or without BRCA1 knockdown and in the presence or absence of GSK3B inhibitor following PARPi were assessed by colony formation assay with survival fractions calculated. n = 3. ( B ) Number of RAD51 foci per cell and ( C ) relative intensity of RAD51 foci in BRCA1 -deficient MDA-MB-436 cells with or without BRCA1 reconstituted and with or without GSK3B inhibitor incubated with 15 μM of olaparib. n = 3. ( D ) BRCA1 -deficient MDA-MB-436 cells with or without BRCA1 reconstituted and with or without GSK3B inhibitor were incubated with the indicated concentrations of olaparib, and survival fractions were assessed with colony formation assay. n = 3. ( E ) Mouse 4T1 in vivo tumor study scheme. ( F ) Tumor volumes and ( G ) weights in C57BL6 mice injected with 4T1 cells with or without BRCA1 knock down in the fat pad. Mice were treated with Lithium (30 mg/kg) and/or Olaparib (50 mg/kg). Values are mean ± SEM. Statistical significance was assessed using 1-way ANOVA followed by Tukey’s multiple comparison test and 2-way ANOVA with Bonferroni’s correction. ### P < 0.001; ** P < 0.01; *** P < 0.001; **** P < 0.0001. * indicates comparing same biological group under treatment. # indicates comparing 2 different biological groups under treatment.

Article Snippet: For BRCA1 knock down, Signal Silence BRCA1 siRNA was purchased from Cell signaling, # 12519.

Techniques: Knockdown, Colony Assay, Incubation, In Vivo, Injection, Comparison

Journal: Nature Communications

Article Title: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

doi: 10.1038/s41467-018-03951-0

Figure Lengend Snippet: BARD1/BRCA1 is upregulated in tamoxifen-resistant breast cancer cells. a The growth curve of MCF7-Pa and MCF7-Re cells under tamoxifen treatment. b mRNA expression profile analysis shows upregulated proliferation-related genes in MCF7-Re cells. c Kaplan–Meier survival curves of breast cancer patients with low (black) and high (red) BARD1 or BRCA1 expression based on Curtis data set. d The mRNA and protein expression of BARD1 and BRCA1 in MCF7-Pa and MCF7-Re breast cancer cells. In a , d , data show means ± s.d. ( n = 3) ** p < 0.01; *** p < 0.001 compared with MCF7-Pa by Student’s t test

Article Snippet: Cells were then transfected with 50 nmol l −1 negative control small interfering RNA (siRNA), or 50 nmol l −1 BARD1 siRNA or BRCA1 siRNA (RiboBio, China) using Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies, USA).

Techniques: Expressing

Journal: Nature Communications

Article Title: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

doi: 10.1038/s41467-018-03951-0

Figure Lengend Snippet: Tamoxifen-resistant cells are resistant to DNA-damaging chemotherapy. a Cell viability assay showing the effect of tamoxifen on MCF7-Re cells when transfected with negative control siRNA (nc), BARD1, and/or BRCA1 siRNAs. b MCF7-Pa and MCF7-Re cells were treated with different concentrations of cisplatin (cis), adriamycin (ADM), or paclitaxel, and the proportions of viable cells were examined by cell viability assay. c Apoptosis assay by AnnexinV-FITC/PI staining showing the percentages of apoptotic cells when MCF7-Pa and MCF7-Re cells were treated with cisplatin. d Comet assay showing the DNA damage when MCF7-Pa and MCF7-Re cells were treated with cisplatin. Scale bar indicated 100 μm. e Western blot showing the expression of γH2AX when MCF7-Pa and MCF7-Re cells were treated with cisplatin. f Representative immunofluorescence staining of γH2AX, Rad51, and BRCA1 in MCF7-Pa and MCF7-Re cells treated with cisplatin at 0 h, 2 h, 8 h. Scale bar indicated 25 μm. g Percentages of positive nuclei (10 foci or more per nucleus being considered positive) from three fields per sample are indicated. In a – d , g , data show means ± s.d. ( n = 3) * p < 0.05; ** p < 0.01; compared with control or indicated lines by Student’s t test

Article Snippet: Cells were then transfected with 50 nmol l −1 negative control small interfering RNA (siRNA), or 50 nmol l −1 BARD1 siRNA or BRCA1 siRNA (RiboBio, China) using Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies, USA).

Techniques: Viability Assay, Transfection, Negative Control, Apoptosis Assay, Staining, Single Cell Gel Electrophoresis, Western Blot, Expressing, Immunofluorescence, Control

Journal: Nature Communications

Article Title: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

doi: 10.1038/s41467-018-03951-0

Figure Lengend Snippet: Reduced BARD1 or BRCA1 expression sensitizes tamoxifen-resistant cells to cisplatin. MCF7-Re cells were transfected with negative control siRNA (nc), BARD1, and/or BRCA1 siRNAs. a , b Cell viability assay showing the sensitivity to cisplatin ( a ) and ADM ( b ). c Apoptosis assay by AnnexinV-FITC/PI staining showing the effect of cisplatin on the percentages of apoptotic cells. d The expression of γH2AX induced by cisplatin. e Comet assay showing the DNA damage induced by cisplatin. Scale bar indicated 100 μm. In a – c , e , data show means ± s.d. ( n = 3) ** p < 0.01 compared with control by Student’s t test

Article Snippet: Cells were then transfected with 50 nmol l −1 negative control small interfering RNA (siRNA), or 50 nmol l −1 BARD1 siRNA or BRCA1 siRNA (RiboBio, China) using Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies, USA).

Techniques: Expressing, Transfection, Negative Control, Viability Assay, Apoptosis Assay, Staining, Single Cell Gel Electrophoresis, Control

Journal: Nature Communications

Article Title: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

doi: 10.1038/s41467-018-03951-0

Figure Lengend Snippet: PI3K inhibition decreases BRCA1/BARD1 expression and sensitizes tamoxifen-resistant cells to cisplatin. a , b The protein ( a ) and mRNA ( b ) expression of BARD1/BRCA1 in MCF7-Re cells treated with BKM120 or BYL719. c – f MCF7-Re cells were treated with control, BKM120, or BYL719 and then treated with cisplatin. The sensitivity to increasing concentrations of cisplatin ( c ), apoptotic cells ( d ), γH2AX expression ( e ), and DNA damage shown by comet assay ( f ) were measured. Scale bar indicated 100 μm. g – i MCF7-Re cells were treated with Dinaciclib and cisplatin. The sensitivity to increasing concentrations of cisplain ( g ), apoptotic cells ( h ), and γH2AX expression ( i ) were measured. In b – d , f – h , data show means ± s.d. ( n = 3) ** p < 0.01 compared with control by Student’s t test

Article Snippet: Cells were then transfected with 50 nmol l −1 negative control small interfering RNA (siRNA), or 50 nmol l −1 BARD1 siRNA or BRCA1 siRNA (RiboBio, China) using Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies, USA).

Techniques: Inhibition, Expressing, Control, Single Cell Gel Electrophoresis

Journal: Nature Communications

Article Title: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

doi: 10.1038/s41467-018-03951-0

Figure Lengend Snippet: PI3K inhibitor restores the sensitivity to cisplatin in xenografts of tamoxifen-resistant tumor in vivo. MCF7-Pa and MCF7-Re cells were inoculated into the mammary fat pads of Balb/c nude mice. When tumors became palpable, tumors were treated with control, cisplatin, or cisplatin with BKM120. The picture ( a ), tumor volume ( b ), and immunohistochemical staining of BARD1/BRCA1 and Tunel staining ( c ) of xenografts derived from MCF7-Pa and MCF7-Re cells with indicated treatment. d IHC scores of BRCA1 and BARD1 and Tunel-positive cell number counts in indicated xenografts. In b , d , data show means ± s.d. ( n = 6) ** p < 0.01 by Student’s t test. Scale bar indicated 400 µm

Article Snippet: Cells were then transfected with 50 nmol l −1 negative control small interfering RNA (siRNA), or 50 nmol l −1 BARD1 siRNA or BRCA1 siRNA (RiboBio, China) using Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies, USA).

Techniques: In Vivo, Control, Immunohistochemical staining, Staining, TUNEL Assay, Derivative Assay

Journal: Nature Communications

Article Title: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

doi: 10.1038/s41467-018-03951-0

Figure Lengend Snippet: The expression of BARD1 and BRCA1 are increased in metastatic breast cancer and associated with poor prognosis in ER+ breast cancer patients received radiotherapy. a , b Representative pictures ( a ) and quantification ( b ) of IHC staining of BARD1, BRCA1, and p-AKT in paired primary and metastatic breast cancer tissues. c , d Correlation analysis of p-AKT expression with BARD1 ( c ) or BRCA1 ( d ) expression in primary and metastatic breast cancer samples. e Overall survival of breast cancer patients in Curtis data set with or without radiotherapy whose tumors had high or low BARD1/BRCA1 expression. In b , data show means ± s.d. ( n = 27) * p < 0.05; ** p < 0.01; *** p < 0.001 compared with that in primary breast cancer by Student’s t test. Scale bar indicated 400 µm

Article Snippet: Cells were then transfected with 50 nmol l −1 negative control small interfering RNA (siRNA), or 50 nmol l −1 BARD1 siRNA or BRCA1 siRNA (RiboBio, China) using Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies, USA).

Techniques: Expressing, Immunohistochemistry