Supplementary Figure 2: DEC2 and p53 expression in HNSCC and breast cancer cell lines and in HNSCC patient samples.

(a) Lysates from 4T1, 4T07 and F3II cell lines were probed by immunoblot (IB) for the indicated antigens. Numbers on top of blots = [ERK/p38] ratio quantification(b-c) QPCR for DEC2 (b) and p53 (c) mRNAs in the indicated cell lines after 24 h in culture, n = 6 RNA samples per condition were assessed over 3 independent experiments, error bars denote s.e.m., *p<0.05, **p<0.01, ***p<0.001 by Mann Whitney test. (d) Left panel: p38α immunoblot in scrambled (scr) or p38α-siRNA transfected BM-D1 cells. Right and middle panels: qPCR for DEC2 and p53 mRNA in BM-D1 cells 24 h after transfection with the indicated siRNAs. n = 2 RNA samples (middle panel) 3 RNA samples (right panel) per condition were assessed over 2 independent experiments, *p<0.05 by Mann Whitney test. See Supplementary Table S4 for the statistic source data. (e) IB anti-HA (left panel) and anti-V5 (right panel) in Lu-HEp3 cells transfected with GFP, p38α constitutively active construct (p38α-CA-HA) or a DEC2-V5 construct. (f) P-H3 (left column) and Cleaved Caspase-3 (C-C3) (right column) immunohistochemistry in Lu-HEp3 cells transfected with vector (upper panels) or a p38α constitutively active construct (p38α-CA-HA) (lower panels). Scale bar: 40μm. Quantification of marker-positive cells is shown in the graphs on the right panels. Y axis shows the number of positive cells (for either Cleaved Caspase-3 (C-C3) or P-H3) per field of view (FOV). Graph: mean±s.e.m. (n = 100 cells assessed/section. 15 sections assessed from 3 different tumour/condition) **p<0.01 by Mann Whitney test. (g) Representative DEC2 staining in primary tumours (upper row) and lymph node metastasis (lower row) from 4 HNSCC patients. (S = stroma, T = primary tumour, M = Metastasis). Scale bar: 120μm.