Fig 1: P4HA3 is a target gene of miR-205 in rat atrial fibroblasts. After transfection with miR-205 mimic or miR-205 inhibitor, the mRNA expression of miR-205 was detected by qRT-PCR (A), and the mRNA and protein levels of P4HA3 were measured by qRT-PCR (B) and Western blot (C). RIP experiments verified that Ago2 antibody significantly enriched the mRNA of miR-205 and P4HA3 (D,E). The binding site of miR-205 and P4HA3 was predicted by TargetScan, and sequences were designed according to the binding site (F). Dual-luciferase reporter gene assay was applied to verify the binding site of miR-205 and P4HA3 (G); *P < 0.05, **P < 0.01, ***P < 0.001; RIP, RNA immunoprecipitation.
Fig 2: Rat atrial fibrosis model had elevated expression of miR-205 and decreased expression of P4HA3. The blood pressure of rats was measured (A) (n = 6). The pathology of rat atrial tissues was observed by H&E staining (B) (n = 6), and the collagen fiber deposition of rats was detected by Masson staining (C) (n = 6). qRT-PCR was utilized to inspect the mRNA level of miR-205 (D) (n = 6). The mRNA and protein levels of collagen I, a-SMA, and P4HA3 were analyzed by qRT-PCR (E) and Western blot (F) (n = 6); *P < 0.05, **P < 0.01.
Fig 3: Microcystin-LR antagonizes activation of the TGF-ß signaling pathway and expression of fibrotic markers in rats with bleomycin-induced pulmonary fibrosis.Rats were treated as explained in Fig. 1. a Expression of TGF-ß signaling pathway molecules, including TGF-ß1, p-Smad2, Smad2, p-Smad3, Smad3 and prolyl 4-hydroxylase subunit aIII (P4HA3) in rat pulmonary tissues was measured by western blot (one-way ANOVA; TGF-ß1: F4,10 = 3.726, P = 0.042; p-Smad2: F4,10 = 5.790, P = 0.011; p-Smad3: F4,10 = 5.720, P = 0.012; P4HA3: F4,10 = 3.861, P = 0.038; n = 3). b Protein levels of the fibrotic markers, including fibronectin, collagen 1a1 and a-smooth muscle actin (aSMA) in rat pulmonary tissues was measured by western blot (one-way ANOVA; fibronectin: F4,10 = 3.742, P = 0.041; collagen 1a1: F4,10 = 5.808, P = 0.011; aSMA: F4,10 = 4.722, P = 0.021; n = 3). c–e Expression of fibronectin, collagen 1a1 and aSMA at the mRNA level in rat pulmonary tissues was examined by quantitative RT-PCR (qRT-PCR). One-way ANOVA, fibronectin: F4,28 = 7.024, P < 0.000; collagen 1a1: F4,28 = 3.317, P = 0.024; aSMA: F4,28 = 2.841, P = 0.043. n = 5 (bleomycin) or n = 7 (all the other treatments). *P < 0.05, **P < 0.01 determined by one-way ANOVA with S-N-K multiple-comparison test.
Fig 4: P4HA3 is epigenetically activated by Slug in gastric cancer. A, Regression analysis of the expression between P4HA3 and SNAI2 in Cancer Genome Atlas-Stomach Cancer (TCGA-STAD). B-C, Quantitative polymerase chain reaction (qPCR) analysis of P4HA3 messenger RNA (mRNA) expression in MKN-45 (B) and AGS (C) cells 36 hours after infection of lentiviral Slug expression particles (B) or lentiviral Slug short hairpin RNA (shRNA) (C). D-E, Western blot analysis of Slug and P4HA3 expression in MKN-45 (D) and AGS (E) cells 48 hours after infection of lentiviral Slug expression particles (D) or lentiviral Slug shRNA (E). F, Predicted high score Slug binding sites in the P4HA3 promoter region. G-H, Fold-enrichment of Slug binding at the P4HA3 promoter relative to the background in MKN-45 (G) and AGS (H) cells was measured by ChIP-qPCR. The primer set for chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) is indicated in figure F. Upon normalization to GAPDH, results were expressed as n-fold compared to IgG. I, The luciferase reporter constructs carrying intact or truncated P4HA3 promoter sequences were introduced into HEK-293 cells preinfected with lentiviral Slug expression particles. Luciferase activity was measured 24 hours posttransfection. PWM indicates position weight matrix. **, P < .01.
Fig 5: P4HA3 upregulation is associated with gastric cancer metastasis. A, Heatmap of P4HA3 exon expression in Cancer Genome Atlas-Stomach Cancer (TCGA-STAD). B, P4HA3 exon expression in patients with metastasis (M1, N = 27) and without metastasis (M0, N = 367). C, Western blot analysis of P4HA3 expression in MKN-45 and AGS cells 48 hours after infection of lentiviral P4HA3 expression particles or lentiviral shRNA. D-G, Quantitative results of wound healing assay (D and F) and transwell assay (E and G) that were conducted 48 hours after indicating infection in MKN-45 and AGS cells. The relative wound areas at 24 hours compared to 0 hours after scratch were calculated to reflect the speed of wound healing. The relative proportion of invaded cells in P4HA3 overexpression/shRNA groups compared to Vector/shNC groups was calculated to reflect the capability of cell invasion. **, P < .01.
Supplier Page from Abcam for Anti-P4HA3 antibody