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. 2021 Aug 17;10:e64846. doi: 10.7554/eLife.64846

Figure 5. Fosl1 knock-out (KO) impairs cell growth and stemness in vitro and increases survival in a orthotopic glioma model.

(A) Cell viability of control and Fosl1 KO p53-null KrasG12V neural stem cells (NSCs) measured by MTT assay; absorbance values were normalized to day 1. Data from a representative of three independent experiments are presented as mean ± SD (n = 10, technical replicates). Two-way ANOVA, relative to sgCtrl for both sgFosl1_1 and sgFosl1_3: ***p≤0.001. (B) Quantification of cell cycle populations of control and Fosl1 KO p53-null KrasG12V NSCs by flow cytometry analysis of PI staining. Data from a representative of two independent experiments are presented as mean ± SD (n = 3, technical replicates). Student’s t test, relative to sgCtrl: *p≤0.05; **p≤0.01; ***p≤0.001. (C) Representative limiting dilution experiment on p53-null KrasG12V sgCtrl and sgFosl1_1 NSCs, calculated with extreme limiting dilution assay (ELDA) analysis; bar plot inlet shows the estimated stem cell frequency with the confidence interval; chi-square p<0.0001. (D) Heatmap of expression of stem cell (yellow) and lineage-specific (neuronal – purple, astrocytic – green, and oligodendrocytic – orange) genes, comparing sgCtrl and sgFosl1_1 p53-null KrasG12V NSCs. Two biological replicates are shown. (E) Quantification of pixel area (fold-change relative to sgCtrl) of CD44, GFAP, and OLIG2 relative to DAPI pixel area per field of view in control and Fosl1 KO p53-null KrasG12V NSCs. Data from a representative of two independent experiments; Student’s t test, relative to sgCtrl: ***p≤0.001. (F) Kaplan–Meier survival curves of nu/nu mice injected with p53-null KrasG12V sgCtrl (n = 9) and sgFosl1_1 (n = 6) NSCs. Log-rank p=0.0263. (G) Western blot analysis using the indicated antibodies of four sgCtrl and four sgFosl1_1 tumors (showing low or no detectable expression of FRA-1); vinculin was used as loading control. (H) Representative images of IHCs using the indicated antibodies. Scale bars represent 100 µm. (I) mRNA expression of MES genes in the samples sgCtrl–T4 (higher FRA-1 expression) and sgFosl1_1–T3 and –T4 (no detectable FRA-1 expression). (J) mRNA expression of PN genes in samples as in (H). Data from a representative of two experiments are presented as mean ± SD (n = 3, technical replicates), normalized to Gapdh expression. Student’s t test for sgFosl1_1 tumors, relative to sgCtrl–T4: ns = not significant, *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 5—source data 1. Source data of Figure 5G.
Figure 5—source data 2. Source data of Figure 5A–C, E, F, I, J.

Figure 5.

Figure 5—figure supplement 1. Fosl1 loss is associated with the reduction of proliferative genes and increase in differentiation genes.

Figure 5—figure supplement 1.

(A) Heatmap showing a reduction in expression of cell cycle regulators in sgFosl1_1 as compared to sgCtrl p53-null KrasG12V neural stem cells (NSCs). (B) Representative images of immunofluorescence staining of the indicated markers in sgCtrl and sgFosl1_1 p53-null KrasG12V NSCs plated on laminin-coated coverslips. Scale bars represent 50 µm.