Fig. 1

PACT sustains proliferation in prostate cancer cells. (A) LNCaP or (B) C4-2B PCa cells were treated with negative control siRNA (si-NC) or two different PACT siRNAs (si-PACT#4 and si-PACT#6) for one day and then harvested for RNA extraction or for seeding into appropriate plates for and various times for (i) western blot for PACT expression, β-actin loading control, 72 h, see Supplementary Fig. S6A-B for original blots; (ii) RT-qPCR validation of si-PACT knockdown at 1 d post-transfection. Expression of PACT mRNA is normalised to GAPDH housekeeping gene expression, calculated using the 2^-ΔΔCt method, and relative to si-NC; (iii) cell titre proliferation assay (72 h); and (iv) xCELLigence assay (72 h). (C) Comparison of LNCaP parental and CRISPR PACT knockout (PACT KO) cells at various times post-plating of equal number of cells (i) western blot for PACT expression, tubulin loading control, 72 h, see Supplementary Fig. S6C for original blot; (ii) cell titre proliferation assay (72 h); and (iii) colony forming assays (~ 2 weeks). (D) Comparison of LNCaP parental and PACT KO cells stably overexpressing empty vector (EV) or PACT cDNA (PACT OE) at various time points post-plating of equal number of cells (i) western blot for PACT expression, β-actin loading control, 72 h, red line indicates non-adjacent lanes from western blot used for figure (see Supplementary Figure S6D); (ii) cell titre proliferation assay (72 h); and (iii) colony forming assays (~ 2 weeks). Error bars = SD (or SE for RT-qPCR) and are representative of three independent experiments. Data analysis was with an unpaired two-tailed student’s t-test; with significance denoted as: *p < 0.05, **p < 0.005 relative to si-NC or parental cells; or ^##p < 0.005 PACT KO PACT OE relative to PACT KO EV.