Fig 1: FOXF1 is the target gene regulated by EnhAP1-OIS1. a UCSC screenshot of GRO-seq analysis of BJ-indRASG12V cells. BJ cells were treated with DMSO or 4-OHT for 14 days. Bi-directional transcription is represented by using positive and negative values for expression in the Crick and Watson strands, respectively. The genomic regions of EnhAP1-OIS1 and FOXF1 are enlarged. Note the enhancement in GRO-seq signal for both EnhAP1-OIS1 and FOXF1 in BJ + 4-OHT (brown track) compared to BJ + DMSO (blue track). b mRNA levels of FOXF1 are reduced in sgRNA-AP169 and sgRNA-AP171 targeted cells under 4-OHT treatment. Data shown represent mean (SD), n = 3. *p < 0.05. c BJ-indRASG12V cells transduced with the specified sgRNAs were treated with DMSO or 4-OHT for 14 days; FOXF1, p21, and HRas protein levels were measured by western blot. HSP90 was used as the loading control. The band of FOXF1 is marked with an arrow. ER-HRas indicates the induced version of HRas. d Targeting the FOXF1 and p53 genes caused OIS bypass as measured by ß-gal staining. Note the stronger effect of FOXF1ko compared to the effect elicited by targeting EnhAP1-OIS1 (Fig. 2d). Data shown represent mean (SD), n = 4. *p < 0.05. e Targeting FOXF1 and p53 gene resulted in enhanced proliferation as measured by BrdU staining. Data shown represent mean (SD), n = 4. *p < 0.05
Fig 2: Model of EnhAP1-OIS1 regulation of OIS. a In normal BJ fibroblast cells, hyper-activation of RAS induces MAPK signaling cascade, including AP-1 TFs. Activated AP-1 TFs control different cellular functions, including cell proliferation and apoptosis. AP1 is recruited, among other enhancers, to EnhAP1-OIS1 and stimulates its activity. This, in turn, promotes the expression of the target gene FOXF1, diverting oncogenic signals into the pre-senescent pathway. b Mutagenesis of the AP-1 binding site in EnhAP1-OIS1 abrogates its enhancer activity and thus leads to decreased expression of FOXF1. This results in compromised induction of OIS and thus cells continue uncontrolled cell proliferation [52]
Fig 3: EnhAP1-OIS1 and FOXF1 knockouts display expression profiles of senescence bypass. GSEA analysis of expression profiles measured in 4OHT-treated BJ-indRASG12V cells targeted by sgRNA-AP169, sgRNA-AP171, or sgRNA-FOXF1 compared to the profile of control 4OH-treated cells transduced with non-targeting sgRNA. A list of shared genes within each group is shown
Fig 4: Generation of Mural-like FOXF1-Derived MSCs(A) Flow cytometry analysis of MSCs and perivascular markers (red) of iPSCs differentiated into FD-MSCs (pass 6, day 30) with MesenCult-ACF. Isotype controls (blue).(B) qPCR results demonstrate increased smooth muscle marker (aSMA and SM22a) expression during differentiation of FOXF1+ mesoderm toward MSCs. Means ± SDs of three replicates shown.(C) Immunostaining of FD-MSCs with anti-smooth muscle marker antibodies. Counterstaining with DAPI (blue). Scale bar, 25 µm.(D) Alizarin red staining (calcium deposition) of FD-MSCs differentiated into osteoblasts. Scale bar, 200 µm.(E) Alcian blue staining (proteoglycan deposition) of FD-MSCs differentiated into chondrocytes. Scale bar, 50 µm.
Fig 5: Activation of FOXF1 suppresses the tumorigenesis of EJ cells in vivo. (A) The procedure of injection, weighting and measuring of xenograft tumor models. (B) EJ Lenti-dsControl and EJ Lenti-dsFOXF1-367 cells (6×106, 200 µl) were injected into the right flanks of the mice; the mice were examined for 30 days. (C) Subcutaneous tumors in each group were dissected and photographed. (D and E) Body weights and tumor volumes in each group. *P<0.05 and **P<0.01. The image in (A) was created using BioRender.com. FOXF1, forkhead box F1.
Supplier Page from Abcam for Anti-FOXF1 antibody [EPR7971]