Fig. 4

PKD1 was not required for the proliferation and survival of HNSCC cells. a and b Knockdown of endogenous PKD1 did not affect proliferation of UMSCC-1 cells. Cells were transiently transfected with non-targeting siRNA (si-nt) and three PKD1 siRNAs (si-D1–1, si-D1–2 and si-D1–3). Two days later, transfected cells were replated in triplicates in 24-well plates. Cell growth was determined by counting cell numbers for 7 consecutive days. The experiment was repeated thrice and representative data from one experiment are shown. ns, not significant (p ≥ 0.05) by Student’s t test. Knockdown of PKD1 was confirmed by Western blotting. Cell lysates were subjected to immunoblotting for endogenous PKD1–3 and GAPDH (a). c PKD1 induction by Dox in UMSCC-1 stable PKD1 expression clone (D1-c1). Cells were treated with increasing concentrations of Dox for 48 h before harvesting for Western blotting analysis. d Induced PKD1 expression did not alter the proliferation of UMSCC-1 cells. Control (Con-c9), PKD1 (PKD1-c1 and PKD1-c45), and constitutive-active PKD1 (PKD1-CA-c37) expression clones were pre-treated with 500 ng/ml Dox for 2 days. Cells were replated for proliferation assay, as described above. e The induction of PKD1 expression by Dox was confirmed by Western blotting. f Induction of PKD1 correlated to enhanced basal phosphorylation. Stable inducible clones were treated with Dox at 500 ng/ml for 5 days. The cells were lysed and subjected to Western blotting using p-S738/742-PKD1 and p-S916-PKD1 antibodies. g and h Induced PKD1 expression did not affect the proliferation of 686LN cells. Control (Con-c1) and PKD1 expression clones (PKD1-c14 and PKD1-c16) were pre-treated with 50 ng/ml Dox for 2 days. Cells were replated for analysis of proliferation by counting cell numbers. The induction of PKD1 expression by Dox was confirmed by Western blotting (g). Representative data from one of three independent experiments are shown. ns, not significant (p ≥ 0.05) between Dox-treated and -untreated clones. i and j Induced PKD1 expression did not affect the sensitivity of UMSCC-1 cells to cisplatin and erlotinib. UMSCC-1 cells were seeded in triplicate into 96-well plates and treated with varying concentrations of cisplatin (i) and erlotinib (j). Media containing fresh cisplatin and erlotinib were replenished every 48 h. The number of viable cells was measured after 72 h using MTT assay. One of two independent experiments is shown. ***P < 0.001