Fig. 6: R-Ras is a direct transcriptional target of FOXF1.

a Violin plots show decreased R-RAS mRNA in EC of IPF (n = 4 lungs) compared to donor (n = 8 lungs), GSE 122960 datasets. R-RAS expression was log normalized. b R-RAS mRNA was decreased in FACS-sorted ECs from IPF lungs (n = 6) compared to donor lungs (n = 3) as shown by qRT-PCR. Data presented as mean ± SD, *p = 0.238, Mann–Whitney Two-tailed test. c Immunostaining for R-RAS, FOXF1 and CD31 shows co-localization of FOXF1 and R-RAS in ECs of donor lungs (n = 5). Neither R-RAS nor FOXF1 are detected in ECs within IPF fibrotic lesions (n = 5). Nuclei are counterstained with DAPI. Bar = 20 μm. d qRT-PCR shows that shRNA-mediated knockdown of FOXF1 (shFOXF1) in HUVECs decreased R-RAS mRNA compared to control (Scr). ACTB mRNA was used for normalization (n = 3), **p < 0.01 and ***p < 0.001 by Student’s T test (two-tailed). e qRT-PCR shows that siRNA-mediated knockdown of Foxf1 in mouse MFLM-91U ECs decreased Rras mRNA. Actb mRNA was used for normalization (n = 3). f ChIP-seq shows direct binding of FOXF1 protein to Rras promoter region in MFLM-91U cells. Binding of FOXF1 to Rras promoter is associated with H3K4me3 marks but not H3K27me3 marks. g Schematic drawing of the pGL2-Rras-Luc construct with the −762/+13 bp Rras promoter region containing the FOXF1-binding site (top panel). In co-transfection experiments, CMV-FOXF1 expression vector increased transcriptional activity of the −762/+13 Rras promoter region compared to CMV-empty vector (bottom panel), n = 3, ***p < 0.001. h Overexpression of R-Ras decreased Ccl2 and TNFα mRNAs in mock-transfected cells and prevented upregulation of Ccl2 and TNFα in cells transfected with Foxf1-specific siRNA. MFLM-91U cells were transfected with non-targeting siRNA (siControl) or siFoxf1, and EEV-empty vector or EEV-Rras. Actb mRNA was used for normalization (siControl+EEV-empty Foxf1, n = 6; siControl+EEV-empty Rras, n = 6; siControl+EEV-empty Ccl2, n = 3; siControl+EEV-empty Tnf, n = 6; siControl+EEV-Rras Foxf1, n = 6; siControl+EEV-Rras Rras, n = 6; siControl+EEV-Rras Ccl2, n = 6; siControl+EEV-Rras Tnf, n = 3; siFoxf1+EEV-empty Foxf1, n = 6; siFoxf1+EEV-empty Rras, n = 3; siFoxf1+EEV-empty Ccl2, n = 3; siFoxf1+EEV-empty Tnf, n = 3; siFoxf1+EEV-Rras Foxf1, n = 6; siFoxf1l+EEV-Rras Rras, n = 3; siFoxf1l+EEV-Rras Ccl2, n = 6; siFoxf1+EEV-Rras Tnf, n = 3), *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test (two tailed). Source data are provided as a Source Data file.