Fig. 2

I3A increases B7-H3 mRNA, total protein, and cell-surface protein expression in LM7 cells. a B7-H3 expression per cell after treatment with 22 doses of I3A for 6, 24, or 48 h. Total B7-H3 signal per cell (left panel) was normalized by averages of DMSO-treated wells. Filled circles represent detection by B7-H3-specific antibody and unfilled circles isotype IgG control. Black arrows represent treatment with 0.11 µM I3A, and green arrows represent treatment with 30 µM I3A (N = 4). Representative LM7 fluorescent images (right panel) after treatment with indicated concentrations of I3A at 6, 24, or 48 h are shown (scale bar = 100 µm). b Flow cytometry evaluation of B7-H3 surface expression on LM7-KO or LM7 cells treated with DMSO or 0.1 µM I3A. Representative flow plots (left panel) and summary of replicates (right panel; N = 3) are shown. c B7-H3 mRNA expression in LM7 cells post-treatment with DMSO or 0.5 µM I3A for 12 or 48 h (N = 3). B7-H3 mRNA expression was measured by RT-qPCR and normalized by the GAPDH mRNA expression and normalized again by averages of DMSO-treated samples. d. B7-H3 protein in LM7 cells after treatment with DMSO or 0.5 µM I3A for 48 h (N = 3). Relative B7-H3 protein expression to GAPDH protein expression was normalized by DMSO-treated LM7 samples. Summary data (left panel) and representative western blot (right panel) are shown. Data represents mean ± SD. **p < 0.01, ***p < 0.001, ns, non-significant by two-way ANOVA (b), unpaired t-test (c), or one-way ANOVA (d)