Fig. 2

Effects of bortezomib on the spheroid growth, colony formation, and stemness of glioma cells. a Left part, representative images of U251 and U87 cell spheroids treated with 0 (Control), 10, and 20 nmol/L bortezomib. Right part, the growth speed is represented by the fold changes of the spheroid area compared with its own area on day 1. Images of spheroids were taken every 2 days (scale bar, 200 μm). Average fold change of the spheroid area was compared between every two groups detected on the same day. ^ɑP < 0.01, ^βP < 0.05, compared with control group; ^#P < 0.01, ^πP < 0.05, compared with 10 nmol/L bortezomib group. b Bortezomib reduced glioma cell colony formation. Left part, representative images of U251 and U87 colonies on the 10th day (with phase-contrast mode, scale bar, 200 μm). Cells were cultured in medium with 0 (Control, DMSO), 10, or 20 nmol/L bortezomib for the first 3 days and then in normal medium for another 7 days. Right part, the average number of cell colonies observed under 5 random microscopic fields was calculated. c Upper part, representative images of stem-like cells/spheroids derived from U251 and U87 cells in stem cell culture medium (scale bar, 200 μm). Cells were treated with 0 (Control, DMSO), 10, or 20 nmol/L bortezomib for 24 h before seeding. Bottom part, the average number of stem-like cells/spheroids (more than 20 cells) observed under 5 random microscopic fields on day 6 was calculated. d, e RT-qPCR and Western blotting detected the mRNA and protein levels of cells/spheroids enriched after stem cell medium culture. *P < 0.05; **P < 0.01. All experiments were repeated at least three times. DMSO dimethyl sulfoxide, RT-qPCR reverse transcription-quantitative polymerase chain reaction, GAPDH glyceraldehyde-3-phosphate dehydrogenase