Fig. 3: PD-L1 sustains GBM capacity to form spheroids.

a Immunoblot of p-Akt and p-S6K1 in TM-GBM and SVZ-GBM cells. Levels of phospho-enzymes were higher in SVZ-GBM cells than in TM-GBM cells. b Expression of PD-L1 and CD133 in TM-GBM and SVZ-GBM cells. Levels of both proteins were higher in SVZ-GBM cells than in TM-GBM cells. c Flow cytometry measurements of PD-L1 expression in TM-GBM and SVZ-GBM cells. Graph columns represent mean fluorescence intensities with related p-values (N = 3). A representative histogram is shown in overlay. d RT-qPCR results for mRNA levels of PD-L1 in TM-GBM and SVZ-GBM cells. Graph columns represent normalized quantification relative to TM-GBM cells (N = 3). e Representative images of formed spheroids taken using an optical microscope and spheroid counts in TM-GBM and SVZ-GBM cultures (scale bar represents 100 μm). Graph columns represent spheroid numbers relative to TM-GBM cells (N = 3). A western blot of Sox-2 levels shows increased levels of the stemness marker in the spheroids (+), in comparison with adherent cells (−). f Quantification using flow cytometry of PD-L1 levels, in TM-GBM-formed spheroids and SVZ-GBM-formed spheroids. Graph columns represent MFI (N = 3). g Spheroid formation assay with TM-GBM and SVZ-GBM cells silenced or not silenced for PD-L1. Graph columns represent the relative spheroid numbers after a 4-day culture. NSRNA-treated cells were used as the reference sample. Each experiment was performed at least three times and in triplicate