Figure 2.

VCaP^ER are characterized by increased mitochondrial activity and decreased translation. (A) Mass spectrometry analysis showing differentially expressed proteins in VCaP^ER cells compared to VCaP^CRPC and VCaP (left). Volcano plot showing fold change between VCaP^ER cells and VCaP^CRPC (right). Significantly up- and downregulated proteins are marked by red and blue dots respectively. (B) Network analysis showing clusters formed by proteins highly expressed in VCaP^ER compared to VCaP^CRPC. Main clusters are identified and highlighted in pink. (C) OCR of VCaP^ER and VCaP^CRPC cells during mitochondrial stress test (left) and basal and maximal OCR in VCaP^CRPC and VCaP^ER (right). Results are shown as the average and standard error of the mean (SEM) of 3 independent experiments performed in triplicates. (D) Mitochondrial DNA and nuclear DNA ratio in VCaP^CRPC and VCaP^ER cells. Results are shown as the logged average and SEM of 3 independent experiments performed in triplicates, normalized to VCaP^CRPC. (E) Network analysis shows clusters formed by proteins lowly expressed in VCaP^ER compared to VCaP^CRPC and VCaP. Main clusters are identified and highlighted in blue. (F) Schematic of the effect of eIF2α and 4E-BP1 phosphorylation on translation. (G) Western-blot showing phosphorylation of 4E-BP1 and (H) eIF2α in VCaP^CRPC and VCaP^ER cell lines (left). Quantification of signal intensities (right), relative to VCaP^CRPC. 4E-BP1 was normalized to total protein and p-eIF2α, to eIF2α. n = 3 biological replicates. (I) Polysomal profiles of VCaP^CRPC (blue) and VCaP^ER (red) (left) and quantification of area under curve of polysomal area (top right), normalized to sub-polysomal area and relative to VCaP^CRPC. n = 4 biological replicates. (J) Puromycylation assay on VCaP^CRPC and VCaP^ER cell lines (left) and quantification (right) relative to VCaP^CRPC and normalized to total protein. n = 3 biological replicates. *P-value < 0.05