Fig. 7: LXRα and HIVEP2 are functionally essential targets of YTHDF2 in cell proliferation, invasion, cholesterol dysregulation, and tumorigenesis of GBM cells.

a Western blotting of YTHDF2, LXRα, and HIVEP2 in GSC11 cells stably expressing shCtrl, shYTHDF2, or shYTHDF2 plus shLXRA or/and shHIVEP2. Representative blot of three independent experiments is shown. b Proliferation of the cells in (a) was measured. Data are mean ± S.E.M., n = 6 biologically independent experiments. c In vitro invasion assay for GSC11 cells expressing shCtrl, shYTHDF2, or shYTHDF2 plus shLXRA or/and shHIVEP2. Data are mean ± S.E.M., n = 4 wells examined over three independent experiments. d, e mRNA levels of LXRA and its downstream targets in shCtrl and shYTHDF2 GSC11 cells (d) or in LN229 cells expressing vector or YTHDF2 plasmid (e). Data are mean ± S.E.M., n = 3 biologically independent experiments (unpaired two-sided t test). f, g Relative cellular cholesterol of GSC11 cells stably expressing shCtrl, shYTHDF2, shLXRA, or shYTHDF2 plus shLXRA (f) or LN229 cells expressing vector or YTHDF2 plasmid (g). Data are mean ± S.E.M., n = 6 (f) or n = 3 (g) biologically independent experiments. h, i Quantification of LDL uptake in GSC11 cells expressing shCtrl, shYTHDF2, or shYTHDF2 plus shLXRA (h) or in LN229 cells expressing wild-type (WT) or m⁶A recognition defective (MUT) YTHDF2 (i). Data are mean ± S.E.M., n = 3 biologically independent experiments. j In vitro invasion assay for GSC11 cells treated with conditioned culture medium from GSC11 cells transfected with siCtrl, siYTHDF2, siAPOE, or siYTHDF2 plus siAPOE. Data are mean ± S.E.M., n = 3 biologically independent experiments. k, l mRNA levels of HIVEP2 and its downstream target SSTR2 in shCtrl and shYTHDF2 GSC11 cells (k) or in LN229 cells expressing vector or YTHDF2 plasmid (l). Data are mean ± S.E.M., n = 3 (k) or n = 4 (l) biologically independent experiments (unpaired two-sided t test). m Nude mice intracranial tumor assay using shCtrl, shYTHDF2, and shYTHDF2 plus shLXRA and shHIVEP2 GSC11 cells. Brain sections stained with H&E show representative tumor xenografts. Tumor volumes were calculated. Data are mean ± S.D. n In vivo invasion assay for shCtrl, shYTHDF2, and shYTHDF2 plus shLXRA and shHIVEP2 GSC11 cells. Representative H&E staining showing edges of the mice brain tumors (left), and the relative invasive fingers per tumor were counted (right). Scale bar = 200 μm. Data are mean ± S.D. o Overall survival of mice injected with shCtrl, shYTHDF2, and shYTHDF2 plus shLXRA and shHIVEP2 GSC11 cells (two-sided log-rank test). For b, c, f, g, h, i, m, and n, data were analyzed by one-way ANOVA Tukey’s post hoc test. For m–o, n = 10 mice per group examined over two independent experiments. Source data are provided as a Source data file.