Figure 2. The Q75E PIK3CA mutation exhibits an activating phenotype in an HNSCC platform.

(A) Generation of isogenic models in PCI-52-SD1 cells. Immunoblot analysis of the indicated proteins using extracts from cells expressing LUC, WT PIK3CA, canonical E545K mutant, or noncanonical Q75E mutant, following growth in the presence or absence of doxycycline (Dox, 1 μg/mL) for 24 hours. GAPDH, loading control. The fold-changes of the ratio of pAKT/(total AKT) were quantified by densitometry 3 times independently and normalized to no Dox treatment (n = 3). Data are shown as mean ± SD. (B) Serum-dependence assays. Cells were cultured for 72 hours in medium containing Dox with either normal FBS (10%) or low FBS (1%–2%) followed by crystal violet staining. The cell growth rate in low FBS was normalized to the individual control of normal FBS (n = 6). Data are shown as the mean ± SD. The experiment was repeated 3 times with similar results. (C) Colony formation assays. Cells were cultured in the absence or presence of Dox for 3 weeks followed by crystal violet staining. Colonies were quantified using ImageJ and the relative colony counts were normalized to no Dox treatment (n = 3). Data are shown as the mean ± SD. (D) Cell migration assays. Cells were applied to Boyden chambers and incubated for 48 hours in the absence or presence of Dox followed by crystal violet staining. Images were taken from the bottom side of transmembrane chambers. Scale bar: 100 μm. The migrated cells were quantified using ImageJ and the relative cell migration level was normalized to no Dox treatment (n = 3). Data are shown as the mean ± SD. The experiments in C and D were repeated twice with similar results. In all bar graphs, *P < 0.05, **P < 0.01, ***P < 0.001, NS ≥ 0.05 for 1-tailed Student’s pairwise t test.