Fig. 1
From: MYC overexpression and SMARCA4 loss cooperate to drive medulloblastoma formation in mice
Loss of SMARCA4 or MYC overexpression does not increase proliferation of GCPs in vitro. (A) Tamoxifen-induced knockdown of SMARCA4 is evident in Western Blot of P7/8 Math1creERT2::Smarca4fl/fl GCPs compared to controls (Smarca4fl/fl) after tamoxifen injection at P3. Two SMARCA4 bands are detected as seen in previously published studies [19, 46]. (B) IF staining of knockdown GCPs at day 3 in culture shows loss of SMARCA4 protein and proliferation indicated by BrdU incorporation. White arrowheads mark SMARCA4-negative areas. (C) Evaluation of SMARCA4 knockdown in IF on day 3 in culture of 19 independent GCP cultures. (D) Proliferation as measured by BrdU incorporation in IF on day 1, 3, and 5 in culture, separately counted for SMARCA4-positive and -negative GCPs in knockdown cultures. Two-tailed paired t-tests were applied. (E) MYC expression is evident in Western Blot of wild-type P7/8 GCPs 72 h after transduction. (F) IF staining shows GFP signal 72 h after transduction of GCPs. (G) MYC transduction rates were evaluated in IF stainings of GCPs 72 h after transduction. The three groups include GCPs without tamoxifen (Tam) induction and GCPs of cre-negative (Smarca4fl/fl) and cre-positive (Math1-creERT2::Smarca4fl/fl) genotype after tamoxifen induction at P3. Tukey’s multiple comparisons test was applied. (H) Overall proliferation as measured by BrdU incorporation in IF of Math1creERT2::Smarca4fl/fl GCPs without tamoxifen induction 72 h after transduction with Mock or MYC constructs. Paired two-tailed t-test was applied. (I, J) IF staining of tamoxifen-induced Math1creERT2::Smarca4fl/fl GCPs 72 h after transduction with MYC virus. The subpopulation with SMARCA4 protein loss and GFP signal (white arrowheads) constitutes around 8.4% of the whole cell culture. (K) Overall proliferation of tamoxifen-induced Math1creERT2::Smarca4fl/fl GCPs 72 h after transduction with Mock or MYC constructs. Paired two-tailed t-test was applied. Scale bar in B corresponds to 20 μm, scale bars in F + I correspond to 50 μm