Fig 1.
Schematic representation of RBM10v1/v2 exons.
Exons are represented by grey boxes outlined in black. Box sizes are not representative of actual exon size. Black ovals represent approximate location of the epitopes recognized by the antibodies used in this study. Solid black lines represent the approximate location of shRNA RBM10 targets, which were not variant specific. Corresponding names of antibodies and shRNA are listed next to their approximate location. Arrow indicates position of the GTG codon coding for the valine residue that can be differentially spliced from RBM10 transcripts (last amino acid coded by exon 10).
Fig 2.
RBM10 RIP-Seq results and comparison to identified RBM5 targets.
(A) Successful immunoprecipitation of RBM10 from C4 cells, demonstrated by Western blot of input and output protein samples. Blots probed for RBM10, using the same RBM10 antibody used in the immunoprecipitation experiment (Sigma-Aldrich). Control immunoprecipitation performed, using a non-specific rabbit IgG antibody. (B) Overlap between RBM5 and RBM10 targets in C4 GLC20 subline. (C) GO Molecular Function gene sets enriched in common RBM5 and RBM10 RIP-Seq targets, as determined by FIDEA. Values indicated are Benjamini values.
Table 1.
Pathways and gene sets enriched in RBM10-only RIP-Seq targets.
Fig 3.
RBM10 expression and dehydrogenase activity in control and stable RBM10KD GLC20 sublines.
(A & B) RBM10 mRNA levels, determined by RNA-Seq, for all RBM10 variants combined (A) or for individual splice variants (B). ‘Control’ represents values from parental GLC20 cells and the control G300.3 GLC20 subline. RBM10KD sample used was the G29/30.4 subline, as it showed greatest decrease in RBM10 protein expression. (C & D) RBM10v1 protein levels were monitored in the G300.3, G29/30.1, G29/30.3 and G29/30.4 sublines using the Bethyl RBM10 antibody. (C) One representative Western blot result is presented for RBM10v1 and α-tubulin (loading control). (D) Densitometric results of the average RBM10v1 protein levels from three biological replicates performed in duplicate. Analysis was performed using the AlphaEaseFC, ‘1D-Multi’ analysis tool. Values of RBM10v1 were normalized to the α-tubulin of each biological replicate, and then made relative to the G300.3 control subline. Standard error is presented. Subline expression levels were compared using the Student’s unpaired t-test, between sublines. (E) G300.3, G29/30.1, G29/30.3 and G29/30.4 were grown for five days and dehydrogenase activity was monitored daily using an MTT assay. Absorbance was plotted relative to day 0. The average of three biological replicates performed in eight technical replicates with standard error is presented. A Two-way ANOVA was performed between the G300.3 and other sublines, with Bonferroni post-hoc analysis. * p < 0.05, ** p < 0.01, and **** p < 0.0001.
Fig 4.
Pathway analysis of genes differentially expressed upon RBM10KD in GLC20 cells.
(A) KEGG pathways significantly enriched upon RBM10KD in GLC20 cells. Analysis performed using subline RNA-Seq expression data and the FIDEA pathways analysis program. (B) MSigDB Hallmark gene sets enriched at a false discovery rate of below 5% upon RBM10KD in GLC20 cells. Analysis performed using subline RNA-Seq expression data and the GSAASeqSP pathway analysis program. (C) Relationship between select RBM10-altered pathways in GLC20 cells. Hypoxia is associated with decreased levels of mTORC1 signaling, and promotion of EMT and angiogenesis. In addition, hypoxia is associated with increased levels of glycolysis, and likewise, survival in hypoxic conditions is promoted by increased levels of glycolysis. The influence of RBM10KD on these pathways, as determined by RNA-Seq is indicated.
Fig 5.
Comparison of genes differentially expressed upon RBM10KD in GLC20 cells or RBM5 expression in GLC20 sublines.
Differentially expressed genes, based on RNA-Seq results, in GLC20 cells upon RBM10KD, and moderate (A) or high (B) expression levels of RBM5 expression.
Table 2.
Expression of select National Cancer Institute (NCI) ‘Genes of Interest in SCLC’ in Control and RBM10KD samples, as determined by RNA-Seq.
Fig 6.
RBM10 expression in the parental GLC20 cell line and stable RBM5-expressing sublines.
(A) Expression of all RBM10 mRNA variants, as determined by RNA-Seq. (B) mRNA expression of specific RBM10 splice variants, as determined by RNA-Seq. ‘Control’ in A and B refers to the GLC20 parental cell line and stable pcDNA3-transfected GLC20 subline. (C) Representative Western blot for RBM5 and RBM10 (Sigma antibody) protein expression levels. Alpha-tubulin was used as loading control. (D) Densitometric results of the average RBM10v1/v2 expression of three biological replicates each performed in technical duplicate. Analysis was performed using AlphaEaseFC, ‘1D-Multi’ analysis tool. Values of RBM10v1 and RBM10v2 were normalized to the α-tubulin of each biological technical duplicate, and then made relative to the pcDNA3 controls. Standard error is presented. One-way ANOVA was performed with Tukey post-hoc analysis, between sublines. * p < 0.05 and ** p < 0.01.
Fig 7.
RBM5 and RBM10 RIP-Seq results for RBM10 splice variants.
Graph representing the expression of the various RBM10 splice variants in RBM5 (A) and RBM10 (B) RIP-Seq experiments, which were carried out in C4 cells.