Fig. 7: USP42 modulates alternative splicing of cancer-related genes through regulating PLRG1.

A Examples of alternative exon and alternative use of 3′ ss affected by USP42. Numbers of exon-junction reads and PSI were indicated. B Quantification of different AS events affected by USP42. C Gene-ontology of USP42-regulated AS targets. Fisher exact P values were plotted. D Gene ontology of AS targets regulated by both USP42 and PLRG1. Fisher exact P values were plotted. E Functional association network of USP42- and PLRG1-regulated AS targets. F Validation of candidate AS events by semi-quantitative RT-PCR using parental and USP42-Cas9 H1299 cells. The PSI means from three independent experiments were plotted (P values obtained from paired t-test). Error bars represent standard error of the mean. G The splicing change of SS18 was measured with parental and USP42-Cas9 H1299 cells. The protein levels of USP42 were examined by immunoblotting. GAPDH was also probed as loading control. H The proliferation of H1299 cells stably transfected with SS18-S-expressing or control vector was determined by colony formation assays. All experiments were performed with three biological repeats, with quantified relative colony numbers plotted. P value was calculated by Student’s t test. Error bars represent standard error of the mean. I H1299 cells stably expressing SS18-S or control were grown for 6 days, with cell numbers measured every day. The changes of cell numbers were compared to day 0. The means from three replicates were plotted and P value was calculated by Student’s t test. Error bars represent standard error of the mean. J USP42 levels from six pairs of NSCLC (T) and adjacent normal (N) tissues were analyzed by immunoblotting. β-actin blot shows equal loading. K Total RNAs were isolated from paired NSCLC tumours and normal tissues. Splicing of SS18 was assayed by RT-PCR, with below column chart showing PSI values. L The mRNA levels of USP42 and PLRG1 in lung adenocarcinoma patients as reported from TCGA were analyzed and plotted. P values were calculated with unpaired t-test. M linear regression analysis of the relative levels of USP42 and PLRG1 in GSE11969 and GSE31210 datasets was shown. R = 0.37, P < 8.25 × 10⁻⁸. N The groups of lung cancer patients with distinct USP42 or PLRG1 levels were obtained from GSE11969 and GSE31210 datasets to analyze the overall survival (P values were calculated by log-rank test).