Research Paper Volume 10, Issue 12 pp 4224—4240

ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma

Figure 3. Depletion of p63 reduces the responsiveness of HNSCC cells to ligand stimulation. (A) Fadu cells were transfected with siScr or different p63 (sip63#1, sip63#2, siΔNp63) siRNAs. Forty-eight h after transfection, cells were serum starved for 4 h, and then stimulated with 5 nM IGF1 (upper panel) or 500 ng/ml insulin (lower panel) for 10 min. Protein amounts of p63, IRS1 and p-IRS1 were detected by western blot analysis. β-actin served as loading control. Blots are representative of three individual experiments. (B) Fadu cells were transfected with siScr, sip63#1 and sip63#2, serum starved for 4 h and then stimulated with 5 nM IGF1 for 10 min. Cellular extracts were analysed with the following antibodies: anti-IRS1, anti-p-IRS1, anti-p-AKT, anti-AKT, anti-p-S6 Ribosomal Protein, anti-S6, anti-p44/42 MAPK (p-ERK1/2), anti-ERK1/2, p63 and β-actin as loading control. Blots are representative of three individual experiments. (C) Fadu cells were transfected with siScr or siIRS1 (upper panel) and with sip63#1, ΔNp63, or siScr (lower panel). Forty-eight h after transfection, cells were seeded in 6-cm plates at 500,000/plate and growth was followed until day 6.