Fig. 4

2IgB7-H3 increases in GBM recurrence and confers resistance to temozolomide. a Representative image of a western blot (WB) analysis of 4IgB7-H3 and 2IgB7-H3 in newly diagnosed GBM (ND, N = 11), in patient-matched recurrence (R), and in non-tumoral patients (CTRL, N = 3, 12 to 14). Vertical dashed lines delimit each gel. b–d WB quantification of 2IgB7-H3 or 4IgB7-H3 in ND vs R. Dots represent the values obtained for each of the variables in specific condition (ND vs R) and are annotated with respective patient number (b and d). ND and R tissues were also compared with CTRL tissues with graphs representing mean ± SD (c). NS = not significant and *p < 0.05 (t-test for b and d and ANOVA1 for c). e Representative images of a WB analysis of 4IgB7-H3 and 2IgB7-H3 in U87MG or GB138 cells treated with 250 µM TMZ, 20 µM etoposide (Eto) or vehicle (DMSO) during 3, 6, 24 or 48 h (N = 3). f, g WB quantification of 4IgB7-H3 expression respectively in U87MG or GB138 cells treated as described in (e). Normalized quantification is relative to DMSO condition. h, i Graphs quantifying cell death using AnnexinV/DAPI staining protocol followed by FACS in GB138 cells transduced with short hairpin RNA (shRNA) against B7-H3 (shB7-H3) or non-target (shNT) and vector over-expressing (OE) 2IgB7-H3 (OE2Ig) or control (OEctrl) after a 48-h exposure to TMZ or Eto, respectively. The percentage of cell death was relative to shNT OEctrl condition and expressed as a percentage. Actin was used as internal control for WB analysis. Graphs represent mean ± SD and are representative of three independent experiments (N = 3). ***p < 0.001 (ANOVA1)