Fig 1: P53 regulates miR-199a-3p expression and then regulates SERPINE2 expression. (A) When miR-199a-3p expression is downregulated (lane 4), the P53 downregulating effects on SERPINE2 expression and the subsequent pathways are offset, compared with NC. (B) When miR-199a-3p expression is upregulated (lane 4), the siP53 upregulating effects on SERPINE2 expression and the subsequent pathways are offset compared with NC. (C) Dual-luciferase assay of the putative miR-199a-3p promoter in CRL-4025 cells transfected with P53 and empty vector controls. (D) Chromatin immunoprecipitation analysis demonstrated that P53 occupies the putative miR-199a-3p promoter in CRL-4025 cells. All data are presented as the mean ± standard deviation. *P<0.05, as indicated. P53, tumor protein p53; miR, microRNA; SERPINE2, serine protease inhibitor E2; NC, negative control.
Fig 2: PN-1 protein expression in human IVD tissue.Representative images of PN-1 protein from paraffin-embedded IVD tissue sections (n = 12, 3 for each grade of IVD) that was detected by immunohistochemistry staining. Grade 2 represents a non-degenerated IVD, whereas grades 3, 4, and 5 signify mild, moderate, and severe degeneration, respectively. (Alcian blue staining 100×; IHC 100×, 400×).
Fig 3: Comparison of immunoblotting for quantitative analysis of relative protein change in NP cells.Cells were treated with IL-1ß (10 ng/mL) alone or combined with PN-1 at different concentrations (0, 10, 50, 100, or 200 ng/mL). (A) Equal amounts of protein extract were resolved by SDS-PAGE, and MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and COL2 proteins were detected by western blot analysis. (B–H) Densitometric analysis shows the suppression of MMP3 (B), ADAMTS-4 (C), MMP-13 (D), MMP-9 (E), and ADAMTS-5 (F) Protein levels in NP cells treated by IL-1ß and PN-1. (G,H) Densitometric analysis shows the promotion effect of Aggrecan (G) and COL2 (H) Protein levels in NP cells treated by IL-1ß and PN-1. Data are representative of three independent experiments, and p-values are shown: *p < 0.05; **p < 0.01 compared to IL-1ß stimulation alone.
Fig 4: SERPINE2 is a direct target of miR-199a-3p. (A) Sequence alignment of miR-199a-3p with the SERPINE2 3'-UTR. The seed-recognizing sites (underlined) in the SERPINE2 sequence matched with the seed regions of miR-199a-3p. (B) Following transfection of CRL-4025 cells with a miR-199a mimic, the expression of miR-199a was upregulated, whereas transfection with an inhibitor effectively suppressed the expression of miR-199a-3p. **P<0.01 vs. NC. (C) Luciferase assay in CRL-4025 cells demonstrated that miR-199a-3p mimics significantly suppressed luciferase activity in wild type reporter constructs, compared with NC. (D) miR-199a-3p inhibitor significantly promoted luciferase activity in wild type constructs, compared with NC. *P<0.05 vs. NC. (E) Reverse transcription-quantitative polymerase chain reaction demonstrated that the level of SERPINE2 mRNA in CRL-4025 cells was not affected by transfection with miR-199a-3p mimics or miR-199a-3p inhibitor. (F) Immunohistochemistry revealed that SERPINE2 was downregulated in DN microvasculature compared with volunteers. (G) Transfection of CRL-4025 cells with miR-199a-3p mimics or miR-199a-3p inhibitor affects SERPINE2 and tPA protein expression levels. Protein expression of SERPINE2 was negatively associated with the expression of miR-199a-3p. All data are presented as the mean ± standard deviation. SERPINE2, serine protease inhibitor E2; miR, microRNA; UTR, untranslated region; NC, negative control; DN, diabetic neuropathy; tPa, tissue plasminogen activator.
Fig 5: Down-regulation of IL-1ß-induced MMP expression in NP cells by PN-1.Cells were treated with IL-1ß (10 ng/mL) alone or combined with PN-1, ERK inhibitor, or NF-kB inhibitor. (A) Equal amounts of protein extract were resolved by SDS-PAGE, and MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and COL2 proteins were detected by western blot analysis. (B–F) Densitometric analysis shows the suppression of MMP3 (B), ADAMTS-4 (C), MMP-13 (D), MMP-9 (E), and ADAMTS-5 (F) protein levels in NP cells treated with IL-1ß, IL-1ß + PN-1, IL-1ß + ERK inhibitor, and IL-1ß + NF-kB inhibitor. Densitometric analysis shows the promotive effect on Aggrecan (G) and COL2 (H) protein levels in NP cells treated with IL-1ß, IL-1ß + PN-1, IL-1ß + ERK inhibitor, and IL-1ß + NF-kB inhibitor. Data are representative of three independent experiments, and p-values are shown: *p < 0.05; **p < 0.01 compared to IL-1ß stimulation alone.
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