Fig 1: PAXIP1‐AS1 positively regulated the expression of WIPF1 through assembling ETS1. (A) Protein levels of WIPF1 were determined in MCT‐induced rat lung tissues by Western blotting. (B) Western blotting analysis was performed to measure the protein levels of WIPF1 in hypoxia‐induced hPASMCs. (C) Pearson correlation analysis was conducted for correlation between PAXIP1‐AS1 and WIPF1. (D) Protein levels of WIPF1 were determined in hypoxia‐induced hPASMCs transfected with oe‐PAXIP1‐AS1 or sh‐PAXIP1‐AS1. (E) RIP assay was conducted for examining the interaction between PAXIP1‐AS1 and ETS1. (F) Prediction binding mode between ETS1 and WIPF1. (G) Dual‐luciferase reporter assay was conducted for interaction between ETS1 and WIPF1. (H) CHIP assay was subjected to the binding relationship between ETS1 and WIPF1 promoter. (I) CHIP was employed for the interaction between ETS1 and WIPF1 promoter when PAXIP1‐AS1 was knocked down. (J) Protein levels of WIPF1 and ETS1 were determined using Western blotting. Data are presented as the means ± SD of three independent experiments (n = 3). *P < .05, **P < .01 and ***P < .001
Fig 2: PAXIP1‐AS1 promoted cell viability and migration of hPASMCs through ETS1/WIPF1 signalling. (A) Cell viability was determined by CCK‐8 assay. (B‐E) BrdU assay and immunofluorescence of Ki67 were subjected to validate the cell proliferation in hPASMCs transfected with oe‐PAXIP1‐AS1, combined with sh‐ETS1 or sh‐WIPF1. (F and G) Cell migration was measured by wound healing and transwell assays in hPASMCs transfected with oe‐PAXIP1‐AS1, combined with sh‐ETS1 or sh‐WIPF1. Data are presented as the means ± SD of three independent experiments (n = 3). *P < .05, **P < .01 and ***P < .001
Fig 3: The interaction of WIPF1/RhoA. (A) Pearson analysis was conducted for correlation between WIPF1 and RhoA. (B) mRNA expression of RhoA was determined in cells transfected with sh‐WIPF1 by qRT‐PCR. (C and D) Protein levels of RhoA were determined in cells transfected with sh‐WIPF1 by Western blotting. (E) Protein levels of RhoA were determined in cells transfected with sh‐WIPF1 when MG132 was applied by Western blotting. (F) Co‐IP assay was conducted to confirm the interaction between WIPF1 and RhoA. Data are presented as the means ± SD of three independent experiments (n = 3). *P < .05, **P < .01 and ***P < .001
Fig 4: PAXIP1‐AS1 promoted the pulmonary vascular remodelling via ETS1/WIPF1/RhoA axis in vivo. (A) Right ventricle system pressure (RVSP) of healthy rats and PAH rats with/without fasudil or PAH administrated with plasmids expressing sh‐PAXIP1‐AS1. (B and C) H&E staining and Masson's trichrome staining, as well as IHC of RhoA and ɑ‐SMA, were conducted for MCT‐induced lung tissues. Scale bar, 200 μm. (D) Expression of PAXIP1‐AS1 in healthy rats, PAH rats with/without fasudil or PAH administrated with plasmids expressing sh‐PAXIP1‐AS1. (E) Levels of RhoA, ETS1 and WIPF1 in lung tissues from healthy rats and PAH rats with/without fasudil or PAH administrated with plasmids expressing sh‐PAXIP1‐AS1 were determined with Western blotting. Data are presented as the means ± SD of three independent experiments (n = 3). *P < .05, **P < .01 and ***P < .001
Fig 5: Inhibiting HCG18 suppresses the growth and MTS of tumor in vivo. (A) After the AGS cells with stable HCG18 knockdown were obtained, HCG18 expression (left) and miR‐141‐3p expression (right) were detected using qRT‐PCR. (B) The tumor size of the mice was detected every 7 days. (C) Weight of the tumors removed from the mice was measured. (D) The effect of HCG18 knockdown on tumor cells metastasizing to the lungs was evaluated. (E) The expressions of WIPF1, YAP, and TAZ in tumor tissues were detected using qRT‐PCR. (F) The protein expressions of WIPF1 and YAP/TAZ in tumor tissues were detected by Western blot. * P < .05, ** P < .01, *** P < .001
Supplier Page from Abcam for Recombinant Human WIPF1 protein (GST tag N-Terminus)