Fig. 2. macroH2A2 antagonizes self-renewal in GBM and is suppressed in stem-like cells.

a Volcano plot highlighting differentially expressed genes after 7 days of MACROH2A2/macroH2A2 knockdown in G523 cells. b RNA-seq was used to determine transcriptional levels of the top genes of the state metamodules from ref. ⁸ at 7 days following MACROH2A2/macroH2A2 knockdown. Two biological replicates were used per condition. c–j Confocal microscopy images of macroH2A2 and SOX2 in two primary patient tumors (SM4491 (c–f) and SM4691 (g–j)). Scale bars: 20 µ. The experiment was performed once on three independent clinical samples. k–m Limiting dilution assay results after 14 days of doxycycline induction in G523 glioblastoma cells (k), GSC2 (l), and GSC3 (m). The center point represents a calculated estimate of sphere formation. P value was determined by the Chi-square test with the tool ELDA (see Methods). Error bars: 95% confidence interval. Statistics from six technical replicates; the experiment was repeated three times. n Schematic of in vivo limiting dilution assay. o Overview of in vivo limiting dilution assay results. Mice were transplanted orthotopically with either shScr or shMH2A2a-transduced GSCs. P value and chi-square value obtained by Chi-square test. p Orthotopic xenograft experiments to assess the effects of MACROH2A2 knockdown on survival of transplanted mice. Patient-derived GSCs carrying either scrambled control shRNA constructs (shScr; n = 10; one mouse censored) or independent shRNAs targeting MACROH2A2 (shMH2A2a/b; n = 10 mice per group) were transplanted orthotopically in immunocompromised mice. P values were calculated with the log-rank test.