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. 2018 Sep 29;144(5):1037–1048. doi: 10.1002/ijc.31781

Figure 3.

Figure 3

Inhibition of BRCA1 exacerbates double‐strand DNA breaks and suppresses HR but does not affect gene amplification in HSR‐containing MTX‐resistant cells. a. Western blot analysis of BRCA1 protein level and densitometry values in HSR‐containing cells: control and two BRCA1‐depleted clones (n = 3, *p<0.025). b. IF assay for γH2AX protein in HSR‐containing control and two BRCA1‐depleted clones (n ≥ 100, **p<0.005). c. Western blot for γH2AX protein level and densitometry values in HSR‐containing control and two BRCA1‐depleted clones (n = 3, *p<0.025). d. Western blot for MRE11 protein level and densitometry values in HSR‐containing control and two BRCA1‐depleted clones (n = 3, *p<0.025). e. IF assay for RAD51 in HSR‐containing control and two BRCA1‐depleted clones (n ≥ 100, **p<0.005). f. Western blot for RAD51 nuclear protein level and densitometry values in HSR‐containing control and two BRCA1‐depleted clones (n = 3, **p<0.005). g. Real‐time PCR analysis of DHFR amplification in HSR‐containing control and two BRCA1‐depleted clones (n = 3, p>0.025). h. Western blot analysis of DHFR protein level and densitometry values in HSR‐containing control and two BRCA1‐depleted clones (n = 3, p>0.025). i. Real‐time PCR analysis of other genes that co‐localized with DHFR in chromosome 5, including RAD1, PLK2, MSH3, ZFYVE16, CCNH, GLRX and CAST, in HSR‐containing control and two BRCA1‐depleted clones (n = 3, p>0.025). j. Western blot analysis of BRCA1 protein level in 2 × 10−6 M MTX‐resistant control, BRCA1‐depleted clone and sh‐BRCA1 clone adding 4 × 10−6 M MTX cells. k. Real‐time PCR analysis of DHFR amplification in 2 × 10−6 M MTX‐resistant control, BRCA1‐depleted clone and sh‐BRCA1 clone adding 4 × 10−6 M MTX cells (n = 3, *p<0.025). l. FISH analysis of HSR formation in 2 × 10−6 M MTX‐resistant control, BRCA1‐depleted clone and sh‐BRCA1 clone adding 4 × 10−6 M MTX cells. BAC‐containing DHFR was used as a probe and is marked in red; nuclei were stained with DAPI and are marked in blue. Yellow arrow points HSR.