Fig 2: RBM10 transfection recovers the Bcl-xS to Bcl-xL ratio and the apoptotic response to EGFR TKI therapy.(A) qRT-PCR and (B) conventional RT-PCR analysis of the Bcl-xS to Bcl-xL ratio (mRNA levels) following genetic reconstitution of RBM10 or Bcl-xS in RBM10-deficient A014 cells. (C) A014 cells (RBM10-deficient) overexpressing Bcl-xS or reconstituted with RBM10 24 hours before treatment with osimertinib (500 nM) or the DMSO control for 48 hours. Cell lysates were harvested, and the indicated proteins were measured by Western blotting. (D) Caspase 3/-7 activity was measured with the Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the FC after normalization to the DMSO control. Data represent 3 independent experiments. *P < 0.05, by 1-way ANOVA.

Fig 3: Rbm5 and Rbm10 expression in Rbm5 shRNA stably transfected H9c2 clones. (A) RT-PCR results for Rbm5 and Rbm10 expression in the various RBM5 knockdown (KD) clones, individually from technical duplicates (i) and pooled (ii). Gapdh was used as reference gene. (B) Representative raw Western blot protein expression data for RBM5 and RBM10 using one anti-RBM10 antibody and various anti-RBM5 antibodies (antibody name or manufacturer indicated on the right of the blots). Precision Plus ladder (BioRad) was used, and ladder values refer to weight in kilodalton (kDa). (C) Densitometric analysis of protein expression in the various clones, individually (from technical duplicates for each clone except clone 87 and 104 in regards to RBM10 expression) (i) and pooled (ii). Results were normalized to alpha-tubulin. In the pooled figures (Aii, Cii), “No KD Clones and Controls” represents results from the scrambled control as well as clones which did not show at least 70% knockdown of Rbm5 at the RNA level (clones 12 and 104), whereas “5 KD Clones” represent results from those clones that did show mRNA knockdown of Rbm5 (clones 87, 9, and 100). Values represent mean ± standard error (SE). Asterisk placed directly above a bar indicates value is statistically different from control as determined by unpaired t test (**indicates p < 0.01).

Fig 4: Rbm5 and Rbm10 expression in Rbm5 shRNA/siRNA transiently transfected H9c2 cells. (A) RT-PCR results for Rbm5 and Rbm10 expression in the various transient RBM5 knockdown (KD) experiments, individually (from technical duplicates, except KD3 (one technical replicate)) (i) and pooled (ii). Gapdh was used as reference gene. (B) Representative raw Western Blot protein expression data for RBM5 and RBM10 using one anti-RBM10 antibody and various anti-RBM5 antibodies (antibody name or manufacturer indicated on the right of the blots). Precision Plus ladder (BioRad) was used, and ladder values refer to weight in kDa. (C) Densitometric analysis of protein expression in the various KD experiments, individually from one technical replicate (two for KD1 expression of RBM5) (i) and pooled (ii). Results were normalized to alpha-tubulin. Values represent mean ± SE. Asterisk placed directly above a bar indicates value is statistically different from control as determined by unpaired t test (** indicates p < 0.01).

Fig 5: Rbm5 and Rbm10 expression in Rbm5 transiently overexpressed H9c2 cells. (A) RT-PCR results for Rbm5 and Rbm10 expression in the various transient RBM5 overexpression (OE) experiments, individually from technical duplicates (i) and pooled (ii). Gapdh was used as reference gene. (B) Representative raw Western Blot protein expression data for RBM5 and RBM10 using one anti-RBM10 antibody and various anti-RBM5 antibodies (antibody name or manufacturer indicated on the right of the blots). Precision Plus ladder (BioRad) was used, and ladder values refer to weight in kDa. (C) Densitometric analysis of protein expression in the various OE experiments, individually from technical duplicates for RBM5 expression and one technical replicate for RBM10 expression (i) and pooled (ii). Results were normalized to alpha-tubulin. Values represent mean ± SE. Statistical significance was evaluated using an unpaired t test for each sample compared to their respective control.