Fig. 2. p63-dependent regulation of HA/CD44 pathway favors EGF-R activation.
a The indicated basal-type breast tumor cells transfected with scrambled (SCR) or p63 siRNA oligos (sip63) were analyzed for the expression of CD44 by qRT-PCR. We utilized the following CD44 oligos. CD44 total: forward 5′-CAACTCCATCTGTGCAGCAAA-3′; rev 5′-GTAACCTCCTGAAGTGCTGCTC-3′. CD44v6 forward 5′-AGTACAACGGAAGAAACAGCTA-3′; rev 5′-TGTCCCTGTTGTCGAATGG-3′. Bars represent the mean of four independent experiments (n = 4 biological replicates) ± SD. No data were excluded from the analysis. *p-value < 0.05. b The indicated basal-type breast tumor cells treated as in a were analyzed by immunoblotting using the antibodies for the indicated proteins. Mouse monoclonal antibody anti-CD44 (8E2) (Cell Signaling Technology) or mouse monoclonal anti-HCAM (CD44) (DF1485) (Santa Cruz Biotechnology) were utilized. c HCC1937 and HCC1954 cells were transfected as in a and total protein lysates were immunoblotted utilizing antibodies for the indicated proteins. Rabbit polyclonal anti-EGF Receptor (Cell Signaling Technology) and rabbit monoclonal anti-Phospho-EGF Receptor (Tyr1068) (D7A5) (Cell Signaling Technology) were utilized; d HCC1954 cells were transfected with scrambled (SCR), or p63 siRNA (sip63) oligos for 48 h and then transfected cells were plated for the sphere-forming assay. Briefly, breast cancer cells were plated in low-attachment 24-well culture plates at a density of 1000 cells per milliliter, in DMEM supplemented with 5 μg/mL insulin, 0.5 μg/mL hydrocortisone, 2% (vol/vol) B27 (Invitrogen), 20 ng/mL EGF and bFGF (BD Bio-sciences), and 4 μg/mL heparin (Sigma). The medium was made semisolid by the addition of 1% (vol/vol) methylcellulose to prevent cell aggregation. Total protein lysates were immunoblotted, utilizing antibodies for the indicated proteins. Rabbit polyclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology) and rabbit monoclonal p44/42 MAPK (Erk1/2) (Cell Signaling Technology, clone 137F5) were utilized. e Representative image of clonogenic assay (left panel) performed by treating HCC1954 cells (500 cell per well) with the indicated concentration of 4-methylumbelliferone (4-MU, Sigma-Aldrich) for 1 week. Growth curve (middle panel) was performed by treating 1 × 105 cells with the indicated concentration of 4-MU for the indicated days. Cells were counted at the indicated time points in quadruplicates (n = 4 technical replicates). The mean ± SD at days 2 and 5 was calculated and plotted. The graph is representative of two independent experiments. *p-value < 0.05. In parallel, protein lysates were immunoblotted for the indicated proteins (right panel)