Fig. 3

PKC inhibition abrogates I3A induced B7-H3 protein expression. a LM7 cells were treated with 22 doses of PDBu and evaluated with the immunofluorescence screen. Total B7-H3 signal per cell (left panel; N = 4) was normalized by averages of DMSO-treated wells. Filled circles represent detection by B7-H3-specific antibody and unfilled circles isotype IgG control. Representative LM7 fluorescent images (right panel) after treatment with indicated concentrations of PDBu are shown. b, c Immunofluorescence analysis for the combinational treatment of 8 doses of I3A and 12 doses of sotrastaurin was performed. b B7-H3 signal heatmap and c synergistic score heatmap (ZIP model) are shown. The total B7-H3 signal per cell was normalized by DMSO-treated samples (0 µM Sotrastaurin, 0 µM I3A; 0%) and 0.37 µM I3A with 0 µM Sotrastaurin (100%). Data are shown as means of quadruplicates. Negative synergistic scores indicate antagonistic effects. d Western blot analysis for the combinational treatment of I3A and sotrastaurin. B7-H3 protein expression relative to β-actin normalized by 0 h treated samples (left panel) and representative western blots (middle and right panels) are shown. Data represents mean ± SD (a and d). *p < 0.05, ****p < 0.0001, ns, non-significant by two-way ANOVA (d)