Validation of RAD51C subcellular localization and population redistribution upon treatment with IPZ and inhibition of DDR signaling, related to Fig. 3. (A) Representative images showing the specificity of the RAD51C antibody ab72063 by siRNA-mediated depletion. HeLa-K cells were transfected with control or RAD51C siRNA and stained with antibodies against RAD51C (ab72063) and GM130; DNA was stained with Hoechst 33342. Scale bar, 10 μm. (B) Quantification of RAD51C sum intensity after RAD51C depletion from A. Data represent the mean ± SEM. (n = 3 biologically independent experiments with a total of 831 cells analyzed). (C) Western blot analysis showing the RAD51C protein level, in control and RAD51C-depleted HeLa-K cells, detected with RAD51C antibody ab95069 (n = 3 biologically independent experiments). Statistical significance was determined using a two-tailed unpaired Student’s t test. (D) Western blot analysis showing the subcellular distribution of RAD51C in membrane (M), nuclear (N), and cytoplasmic (C) fractions from HeLa-K cells treated with DMSO or DOX (40 μM; 3 h). Fraction purity was confirmed using GM130 (membrane), Lamin B1 (nuclear), and α-tubulin (cytoplasmic) as markers. The protein levels were normalized against the respective fraction control (RAD51C^mem/GM130; RAD51C^nuc/Lamin B1; RAD51C^cyt/tubulin). Data represent the mean ± SEM (n = 2 biologically independent experiments). (E) Quantification of RAD51C membrane-nuclear distribution ratio after treatment with DOX, calculated from isolated fractions. Data represent mean ± SEM. (n = 2 biologically independent experiments.) (F) Representative images of the RAD51C protein redistribution upon treatment with IPZ and DOX. HeLa-K cells were stained with antibodies against RAD51C and GM130. Cells were treated with DMSO or IPZ prior to a 3-h treatment with DOX. Yellow arrowheads denote the Golgi membrane; white denotes nuclear foci. Scale bar, 10 μm. The DMSO control image shown in this panel is the same control image shown in Fig. 3 K, as these panels derive from the same experiment and were split between the main and supplemental figures for comparison with the corresponding IPZ and DOX treatment conditions. (G) Representative images of the RAD51C protein redistribution upon treatment with DDR signaling inhibitors and DOX. HeLa-K cells were treated with DMSO (control) alone or with ATM inhibitor (KU55933), ATR inhibitor (VE-821), or DNA-PK inhibitor (NU7441) prior to a 3-h treatment with DOX. Yellow arrowheads denote the Golgi membrane; white denotes nuclear foci. Scale bars, 10 μm. The DMSO control image shown in this panel is the same control image shown in Fig. 3 N, as these panels derive from the same experiment and were split between the main and supplemental figures for comparison with the corresponding inhibitor and DOX treatment conditions. Source data are available for this figure: SourceData FS6.