Fig 2: CDC40 knockdown results in up-regulation of splicing and translation related genes and down-regulation of DNA damage response and proliferation related genes. (a) RNA-seq workflow using H460 cells. (b) Gene expression level changes after knockdown compared to shCTR. Left: Number of genes up-regulated after induction of shCDC40-1 and -5 for 24 h and 36 h in comparison to shCTR. Right: Number of genes down-regulated after induction of shCDC40-1 and -5 for 24 and 36 h in comparison to shCTR. (d) Pathway enrichment via GSEA using genes whose expression levels changed with both shCDC40-1 and -5 after 36-h induction and (c) visualization using a Cytoscape Enrichment Map. (e) RT-qPCR validation of RNA-Seq up-regulated gene (SMNDC1) and down-regulated genes (PIGQ and CSPG4) in response to shCDC40-1, shCDC40-5 and shCTR induction after 8, 24 and 36 h. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; n = 2, ± SD, Student’s T-test).

Fig 3: CDC40 knockdown leads to cell cycle distribution changes in multiple lung cancer cell lines. (a) The effect of Flag-CDC40 over-expression on cell cycle distribution of H460 cells after 48 h of shCDC40-1 and -5 induction in comparison to shCTR and shPLK1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; n = 3, ± SD, Student’s T-test) (b) Western blot analysis showing p21 expression levels after 4 days of CDC40 knockdown using shCDC40-5 (representative) in H460 cells. (see Supplementary Fig. 14 for source blot images).

Fig 4: Knockdown of CDC40 results in increased intron retention events. (a) Up-regulation (yellow) and down-regulation (blue) of different splicing events in H460 cells after 36 h induction of shCDC40-1 and shCDC40-5. (b) RNA-seq results of CDCA5 transcripts (grey) mapped to the CDCA5 human locus (in blue, genome assembly GRCh38, hg38) showing mapping to exonic (rectangle) and intronic (line) regions visualized using the Integrative Genomics Viewer (IGV). (c) Left: DNA gels of RT-PCR products amplified using CDCA5 primers. Right: Intron retention ratio of CDCA5 intron 1 after 36 h of shCDC40-1 and shCDC40-5 induction in comparison to shCTR. (d) Western blot analysis shows the effect of CDC40 knockdown using shCDC40-5 on CDCA5 and p21 expression after 48 h of DOX induction in H460 cells. (see Supplementary Fig. 15 for source blot images) (e) Jess analysis shows the effect of CDC40 knockdown using shCDC40-5 on CDC40, CDCA5 and p21 expression after 48 h of DOX induction in various cell lines. (see Supplementary Fig. 15 for source blot images).

Fig 5: Knockdown of CDC40 reduces proliferation of H460 lung cancer cells. (a) Assessing the effect of CDC40 knockdown on H460 cell proliferation after 4 days of shCDC40 induction. (n = 3, ± SD, Student’s T-test) (b) Western blot analysis showing the effect of Flag-CDC40 overexpression on the expression of Flag and CDC40 after 4 days of DOX induction (see Supplementary Fig. 12 for source blot images). Results are representative of at least two independent experiments.