Fig 2: CD248 promotes NOX4 expression through NF-?B activation. A and B, (A) mRNA and (B) protein levels of NOX4 were measured after treated with siCD248 or siCRTL for 24 h before treatment with PDGF-BB (20 ng/mL) for another 48 h, or hPASMCs were treated with PDGF-BB and with or without ontuxizumab (10 µg/mL) for 48 h. C, An upper, schematic overview of a luciferase construct containing Nox4 promoter (-718 to +241) or a mutated NF-?B binding site (-511 to 501) is shown; below, the luciferase reporter activity in hPASMCs under treatment with or without PDGF-BB, the luciferase activity in pGL3 vector was set as 1. D and E, (D) reporter activity and (E) mRNA expression of Nox4 in hPASMCs transfected with CD248-expressing plasmid and administration of NF-?B inhibitor, BAY 11–7082 (10 nM). F, Reporter activity of Nox4 in hPASMCs treated with ontuxizumab or Isotype IgG (10 µg/mL). The results are expressed as mean ± SD (n = 3 each group), * P < .05, ** P < .01; one-way ANOVA with Tukey's multiple comparisons test or two-way ANOVA analysis

Fig 3: CD248 contributes to PDGF-BB-induced proliferation and migration of hPASMCs. A and B, hPASMCs were treated with different doses of PDGF-BB (1, 10, and 20 ng/mL) and (A) cell proliferative ability or (B) protein expression of CD248 were determined by BrdU or western blot (WB) assays, respectively; C, hPASMCs were transfected with siRNA targeting CD248 for 24 h before treatment with PDGF-BB (20 ng/ml) for another 24 h, or hPASMCs were treated with PDGF-BB and with or without ontuxizumab (10 µg/mL) for 24 h, cell proliferative ability was determined by BrdU assay; D, expression of indicated proteins were determined by WB; E, TUNEL staining was used to detect the apoptosis of hPASMCs (n = 6 for each group). F and G, hPASMCs were treated as in (C), and cell migration was measured in the (F) Transwell Boyden chamber and (G) Scratch-wound assay. The results are expressed as mean ± SD (n = 6 for each group), * P < .05, ** P < .01; one-way ANOVA with Tukey's multiple comparisons test or two-way ANOVA analysis. Scale bar: 50µm

Fig 4: CD248 regulates PDGF-BB-induced signal pathways and promotes a PDGF induced phenotype switch of PASMCs. A, After hPASMCs were treatment with PDGF-BB (20 ng/ml) for 0 or 30 min, CD248 (green) and PDGFR-ß (red) expression was detected by confocal microscopy. The cell nucleus was stained with DAPI (blue). B, Co-immunoprecipitation using CD248 antibody can pull down PDGFR-ß in hPASMCs. C, Coimmunoprecipitation using PDGFR-ß antibody can pull down CD248 in hPASMCs. IP: immunoprecipitant; Input: whole cell lysate. D, hPASMCs were transfected with siCD248 or siCRTL for 24 h before treatment with PDGF-BB (20 ng/mL) for another 30 min; left, p-PDGFR-ß, p-ERK ½, and p-NF-?B p65 protein levels were measured; right, relative expression of p-PDGFR-ß, p-ERK 1/2 and p-NF-?B p65; E, Left, p-ERK 1/2 and p-NF-?B p65 protein levels were measured after PDGF-BB induction of hPASMCs treated with ontuxizumab or Isotype IgG (10 µg/mL); right, relative expression of p-ERK 1/2 and p-NF-?B p65; F, hPASMCs were transfected as indicated, cultured, and administered with PDGF-BB (20 ng/mL), and the mRNA levels of proliferative and migration-related markers were detected by real-time PCR; G, hPASMCs were administered with PDGF-BB and treated with ontuxizumab or Isotype IgG (10 µg/mL), and the mRNA levels of proliferative and migration-related markers were detected by real-time PCR; H and I, hPASMCs were transfected as in (F), (H) the mRNA levels of synthetic and (I) contractile related markers were detected by real-time PCR; J and K, hPASMCs were treated as in (G), (J) the mRNA levels of synthetic and (K) contractile-related markers were detected by real-time PCR. The results are expressed as mean ± SD (n = 3 for each group), * P < .05, ** P < .01; one-way ANOVA with Tukey's multiple comparisons test or two-way ANOVA analysis

Fig 5: CD248 is upregulated in MCT-induced primary pulmonary arterial smooth muscle cells. A, Cd248 mRNA level in MCT-induced rat lungs; B, CD248 protein expression in rat lungs and pulmonary arteries (PAs) with MCT-induced PAH; C, Pearson comparison analyses display the association between CD248 levels (normalized to GAPDH) and RVSP during the development of MCT-induced PAH; D, representative immunofluorescence of CD248 (red), a-SMA (green), and DAPI (blue) in pulmonary arterioles from MCT-induced rat lungs, quantitative analysis of the mean fluorescent intensity (MFI) of CD248 (n = 5 rats per group, 10 arterioles per rat), and quantitative analysis of the colocalization of CD248 and a-SMA; E, PASMCs isolated from a rat during the development of MCT-induced PAH identified by immunofluorescence of a-SMA (green) and DAPI (blue); F, FCM analyses of CD248 expression on isolated rat PASMCs. The results are expressed as mean ± SD (n = 3 for each group), *P < .05, **P < .01; one-way ANOVA with Tukey's multiple comparisons test analysis