Figure 3.

Effects of knockdown of identified hits on Mcl1 mRNA and protein. (a and b) IMR5 and NLF cells were transfected with siRNAs to the indicated genes for 72 h, and protein and mRNA were harvested. PSMD14 knockdown leads to increased Mcl1 protein (relative to β-tubulin loading control) and mRNA, while knockdown of the spliceosome components PRPF8, UBL5 or SART1 leads to (a) reduced Mcl1 protein but with (b) increased MCL1-L mRNA for most, rather than reduced mRNA. (c and d) Complementary QPCR and RT-PCT methods concordantly demonstrate increased splicing toward MCL1-S following knockdown of spliceosome complex members PRPF8, UBL5 or SART1 leading to an increased MCL1-S/MCL1-L ratio. siSham, non-targeting (control) siRNA; error bars, S.E.M. ND, not determined as siMcl1 led to loss of all viable cells. *, P-values ≤0.05 versus siSham. Data are representative of at least two independent experiments