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. 2018 May 1;9(33):23029–23046. doi: 10.18632/oncotarget.25196

Figure 5. c-MYC is upregulated in cSCC cell lines and promotes altered splicing following interference with the spliceosome.

Figure 5

(A) c-MYC protein expression in normal skin cells (NHF, NHK and RDEBK) and a panel of cSCC cell lines was analysed 72 hours after plating. NHF1 and 2 and NHK1 and 2 were from different donors. c-MYC protein expression was higher in cSCC cells compared to normal cells with the exception of SCCIC1Met, SCCIC12 and SCCIC18 cell lines. (B) Cell death at 10 nM pladienolide B (taken from Figure 2). Normal cells and two of the three cSCC cell lines with low c-MYC expression were relatively resistant to pladienolide B. (C) SCCRDEBMet cells were transfected with the indicated combinations of siRNAs. RNA was extracted 48 hours after transfection. One set of cells were treated with cycloheximide (20 μg/ml) 6 hours before harvesting to inhibit NMD (right panel). PCR was carried out using primers complementary to the indicated exons (E). (D) SCCRDEBMet cells were transfected with non-targeting siRNA (Control) or siRNA c-MYC (B) 24 hours before the addition of 10 nM pladienolide B (PD). One set of cells was treated with cycloheximide 6 hours before the end of the incubation (right panel). Samples were harvested for RNA extraction 24 hours after the initiation of pladienolide B treatment. c-MYC knockdown partially reversed alterations in mRNA splicing caused by interference with the spliceosome.