Figure 1.
FOSL1 maintains the MES phenotype in GSCs
(A) Immunoblot (IB) analysis of FOSL1, CD44, ALDH1A3, SOX2, OLIG2, and UBC9 in NHAs, two MES GSCs, and two PN GSCs. α-Tubulin was used as internal control. (B) qRT-PCR analysis of FOSL1 mRNA expression in ALDH1-positive and ALDH1-negative subpopulations of MES 21 glioma spheres. (C) IB analysis of FOSL1 protein expression in ALDH1-positive and ALDH1-negative subpopulations of MES 21 glioma spheres. α-Tubulin was used as internal control. (D) Representative immunofluorescence (IF) images of FOSL1 and CD44 expression in MES 21 GSCs. FOSL1 is in red and CD44 in green. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (E) Representative IF images of FOSL1 and OLIG2 expression in PN 35 GSCs. FOSL1 is in red and OLIG2 in green. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (F) IB analysis of FOSL1 in MES 21 and MES 505 GSCs after transduction with two independent lentiviral shRNA constructs targeting FOSL1 (FOSL1 shRNA1 and shRNA2; one targeting the open reading frame and one targeting the 3′ UTR). α-Tubulin was used as internal control. (G) Representative images of primary or secondary neurosphere formation of MES 21 GSCs with indicated modifications. Scale bar, 25 μm. (H) Limiting dilution neurosphere-forming assay in MES 21 and MES 505 GSCs transduced with shCtrl or shFOSL1 (targeting the 3′ UTR), reconstituted with vector control or wild-type (WT) FOSL1. Stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals, where 1 = stem cell and x = all cells. (I) IB analysis of FOSL1, CD44, C/EBPβ, TAZ, p-STAT3 (Tyr705), and STAT3 in MES 21 and 505 GSCs transduced with shCtrl or shFOSL1 (targeting the 3′ UTR), with or without re-expression of an shRNA-resistant FOSL1. (J) Representative bioluminescent (BLI) images of intracranial GBM xenografts derived from luciferase-expressing MES 21 and MES 505 GSCs with indicated modifications. Colored scale bars represent photons/s/cm2/steradian. (K) Representative H&E-stained brain sections from mice intracranially implanted with MES 21 and MES 505 GSCs with indicated modifications. Red arrows indicate tumors. Scale bar, 1 mm. (L) Kaplan-Meier survival curves of mice intracranially injected with MES 21 and MES 505 GSCs with indicated modifications (n = 8). Data are presented as means ± SD of three independent experiments. ∗∗∗p < 0.001, two-tailed Student’s t test.