Fig. 4 |. Ectopic NONO^C145S attenuates the activity of (R)-SKBG-1 in cancer cells coexpressing endogenous NONO^WT.

a, Western blot showing coexpression of endogenous NONO (lower-molecular-weight band) and exogenous FLAG-tagged NONO^WT or NONO^C145S (higher-molecular-weight band). b, Concentration-dependent effects of (R)-SKBG-1 on AR-FL (left) and AR-V7 (right) mRNA content of 22Rv1 cells coexpressing exogenous NONO^WT or NONO^C145S. Data for sgControl, sgNONO and empty vector control cells are shown for comparison. AR mRNAs were quantified by QuantiGene assays at 6 h after compound treatment, and expression was normalized relative to DMSO treatment for each experimental group. Data are shown as mean values for two replicates of a single experiment representative of two independent experiments. c, Number of genes that were substantially and significantly decreased by (R)-SKBG-1/(S)-SKBG-1 (log₂ (fold change) ≤ −0.75, P_adj ≤ 0.01) in 22Rv1 cells coexpressing exogenous NONO^WT or NONO^C145S compared to empty vector control cells. d, Effects of (R)-SKBG-1 and (S)-SKBG-1 on the growth of 22Rv1 cells coexpressing exogenous NONO^WT or NONO^C145S compared to empty vector control cells. Cells were treated with the indicated concentrations of compounds or DMSO, and, after 6 d, cell growth was determined by CellTiterGlo. Data are shown as mean values ± s.e.m. normalized to DMSO-treated control cells for two independent experiments with four replicates per experiment. EC₅₀ values are reported with 95% confidence intervals shown in parentheses. e, PCA plot of RNA-seq data for the indicated cell models, revealing that (R)-SKBG-1 effects in 22Rv1 cells coexpressing exogenous NONO^C145S shift to resemble the effects of the inactive enantiomer (S)-SKBG-1. f, Pie chart showing the effects of coexpression of exogenous NONO^C145S and endogenous NONO^WT in 22Rv1 cells on genes stereoselectively regulated by (R)-SKBG-1 in empty vector 22Rv1 cells (genes with (R)-SKBG-1/(S)-SKBG-1 log₂ (fold change) ≤ −0.75, P_adj ≤ 0.01). Rescue refers to genes for which NONO^C145S coexpression substantially nullified the effects of (R)-SKBG-1. g, Bar graphs for representative (R)-SKBG-1-regulated genes that were unaffected by coexpression of NONO^C145S. Data are shown as mean values ± s.e.m. for three independent replicates; TPM, transcript per million.