Fig. 5: Androgens are sufficient for the growth of PFA-EPN in vitro.
From: Androgen activity in the male embryonic hindbrain drives lethal PFA ependymoma
a,b, Western blots showing AR expression in PFA9 (a) and PFA4 (b) cells using whole-cell lysate. T, testosterone; Veh, vehicle. c,d, Western blots showing AR expression in PFA9 (c) and PFA4 (d) cells using subcellular protein fractionation extract. Cyt, cytoplasmic fraction; LMNB1, lamin B1; Nuc, nuclear fraction; WC, whole cell. a–d, Western blots shown are representative of n = 4 biological replicates with similar results. e, PFA-EPN cells were treated with 50 nM testosterone, oestradiol or progesterone for in vitro LDA to assess the frequency of colony-forming stem cells. n = 8 technical replicates per condition. f, PFA-EPN cell lines were cultured with testosterone for seven days. g, PFA-EPN cells were treated with 5 µM enzalutamide or MTX-23 for LDA. n = 6 technical replicates per condition. h, PFA-EPN cells were treated with 5 µM enzalutamide or MTX-23 for LDA. Cells were pre-treated with 50 nM testosterone before drug exposure. n = 8 technical replicates (control); n = 6 technical replicates (drug). e,g,h, Statistical comparisons were performed using a two-sided extreme limiting dilution analysis (ELDA) Chi-squared test. Solid lines represent maximum-likelihood estimates of clonogenic frequency from ELDA; dashed lines indicate 95% confidence intervals. i,j, PFA-EPN cell lines were pre-treated with 50 nM testosterone for seven days, followed by treatment with enzalutamide (i) or MTX-23 (j) for seven days. f,i,j, Cell counts are normalized to the vehicle control for each line. n = 2 biological replicates, each with 3 technical replicates. Data are mean ± s.d. Statistical comparisons were performed using a one-way ANOVA on replicates followed by a Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant. Exact P values are provided in the Source Data.
