Fig. 3.

Cytochrome c colocalizes and interacts with ANP32B in the cell nucleus upon DNA damage. (A) Colocalization assays between endogenous Cc and endogenous ANP32B in non-treated (control) and CPT-treated HeLa cells. Subcellular distribution of Cc and ANP32B was visualized with an anti-Cc antibody (green fluorescence) and an anti-ANP32B antibody (red fluorescence), respectively. Nuclei were stained with Hoechst (blue fluorescence). The merged images correspond to the overlaid images of Cc and ANP32B. Scale bars in panel A are 10 μm. (B) In situ Proximity Ligation Assay (PLA) for detection of Cc:ANP32B complexes in HeLa cells following CPT treatment for 6 h. Representative confocal maximal projections of untreated (top) and CPT-treated (bottom) cells, with PLA spots in red and nuclei in blue. As a negative control, goat ANP32B antibody was used together with an unspecific rabbit primary antibody and the two PLA probes (−/+). Scale bar represents 25 μM. (C) Scatter plot showing quantification of the PLA signal as the number of PLA spots per nucleus. Data were collected from three independent experiments as that shown in panel B. Black lines represent the mean of the data collected. ****p < 0.0001; n.s., non-significant. (one-way ANOVA followed by a Tukey's post hoc test). (D) Upper panel — Western-Blot against c-myc tag, encoded at the C-terminal of ANP32B, after performing the pull-down assays. Middle panel — Western-Blot against Cc, demonstrating that recombinant Cc was captured in the carboxymethyl cellulose in all cases. Lower panel — Western-Blot against c-myc tag of cell lysates as loading and transfection control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)