Figure 6. Fosl1 overexpression upregulates the MES gene signature (MGS) and induces larger tumors in vivo.

(A) Western blot analysis of FRA-1 and Flag expression on Fosl1^tetON and Fosl1^WT neural stem cells (NSCs) derived from Kras^LSLG12V; Trp53^lox; ROSA26^{LSLrtTA-IRES-EGFP}; Col1a1^TetO-Fosl1 mice upon in vitro infection with Cre and induction of Fosl1 overexpression with 1 µg/mL doxycycline (Dox) for 72 hr; vinculin was used as loading control. (B) mRNA expression of Fosl1 and mesenchymal (MES) genes in Fosl1^tetON p53-null Kras^G12V cells upon 72 hr induction with 1 µg/mL Dox. (C) mRNA expression of PN genes in Fosl1^tetON p53-null Kras^G12V cells upon 72 hr induction with 1 µg/mL Dox. (D) Quantification of tumor area (µm²) of –Dox and +Dox tumors (n = 8/8). For each mouse, the brain section on the hematoxylin and eosin (H&E) slide with a larger tumor was considered and quantified using the ZEN software (Zeiss). (E) Western blot detection of FRA-1 expression in tumorspheres derived from a control (−Dox) tumor. Tumorspheres were isolated and kept without Dox until first passage, when 1 µg/mL Dox was added and kept for 19 days (+Dox in vitro). (F) mRNA expression of Fosl1 and MES genes in tumorspheres in the absence or presence of Dox for 19 days. (G) mRNA expression of PN genes in tumorspheres in the absence or presence of Dox for 19 days. (H) Western blot detection of FRA-1 expression in tumorspheres derived from a Fosl1 overexpressing (+Dox) tumor. Tumorspheres were isolated and kept with 1 µg/mL Dox until first passage, when Dox was removed for 19 days (−Dox in vitro). (I) mRNA expression of Fosl1 and MES genes in tumorspheres in the presence or absence of Dox for 19 days. (J) mRNA expression of PN genes in tumorspheres in the presence or absence of Dox for 19 days. qRT-PCR data from a representative of two experiments are presented as mean ± SD (n = 3, technical replicates), normalized to Gapdh expression. Student’s t test, relative to the respective control (−Dox in B, C, F, and G; +Dox in I and J): ns = not significant, *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 6—figure supplement 1. Characterization of Fosl1 overexpressing mouse tumors.

(A) Kaplan–Meier survival curves of C57BL/6J wildtype mice injected with p53-null Kras^G12V Fosl1^tetON neural stem cells (NSCs) subjected to doxycycline (Dox) diet (n = 8) or kept as controls (n = 8); log-rank p-value=0.814. (B) Hematoxylin and eosin (H&E) and immunohistochemical staining, using the indicated antibodies, of representative –Dox and +Dox tumors. Scale bars represent 100 µm.