Fig 1: Blocking of PARP16 mitigates intimal hyperplasia after balloon injury. (A)–(E) Knocking down of Parp16 mitigated intimal hyperplasia after rat carotid artery balloon injury. Representative H&E-stained sections in carotid arteries from rat that underwent sham, balloon injury, and Parp16 siRNA (siPARP16) treatment for 14 days after balloon injury, scale bars: 200 µm. (A); treatment with siPARP16 led to a reduction of neointima/media ratio (I/M), &P < 0.05, n = 10/group (B); Western blot analysis of the PARP16, p-PERK, p-eIF2a, p-IRE1a, and spliced XBP-1 protein levels in arteries from rat that underwent sham, balloon injury, and siPARP16 treatment for 14 days after balloon injury (C); Western blot analysis of the cyclin D1, PCNA, MMP9, cyclin E and VCAM-1 protein levels in arteries from rat carotid arteries (D); immunofluorescent staining with anti-cyclin D1 in rat carotid arteries, the arrowheads indicate the positive cells staining in intimal hyperplasia, scale bars: 100 µm (E); above all Western blot data are represented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham; &&P < 0.01, &&&P < 0.001 vs. balloon injury + siCTL, n = 8/group. (F)–(J) The inhibiting PARP16 abrogated intimal hyperplasia in carotid artery ligation mice. Carotid artery ligation mice were administered 5 and 10 mg/kg/day EGCG by oral gavage 28 days until the end of the experiment. Representative hematoxylin and eosin (H&E) staining of partial left carotid arteries from mouse with sham, ligation and EGCG treatment, scale bars: 100 µm (F); EGCG treatment decreased neointima/media ratio (I/M), &&P < 0.01, n = 10/group (G); representative EVG staining and immunohistochemical staining of collagen III of partial left carotid arteries from mouse with sham, ligation or EGCG treatment, scale bars: 100 µm (H); immunofluorescent staining with PARP16 (green) and cyclin E (red) of partial left carotid arteries from different-treated mice, scale bars: 100 µm (I).
Fig 2: Inhibition of PARP16 alleviates PDGF-BB-induced rVSMC proliferation and migration through ER stress. Pretreated with EGCG (30 µmol/L) for 4 h, rVSMCs were treated with 20 ng/mL PDGF-BB for 24 h, cell extracts were collected for determining the protein levels of PCNA, cyclin E, cyclin D1 and MMP9 by Western blot (A); the protein levels of two branches PERK, IRE1a/XBP-1 of UPR signaling were determined by Western blot (B); the EdU positive cells showed green color and cell nuclei stained with DAPI showed blue color, scale bars: 200 µm (C); the migratory ability of rVSMCs was determined by Transwell assay, scale bars: 200 µm (D) and wound healing assay, scale bars: 400 µm. (E). All data are represented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. PDGF, each acquired from three individual experiments.
Fig 3: PARP16 and ER stress are involved in SMC proliferation and migration as well as neointima formation. (A) rVSMCs were treated with PDGF-BB (20 ng/mL) for 24 and 48 h, cell extracts were collected for determining the protein levels of PARP16 and activation of three signaling branches of UPR: PERK, IRE1a/XBP-1 and ATF6 by Western blot. (B) mRNA levels of UPR target genes in rVSMCs after PDGF-BB treatment for 2 h. All data are represented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 h, each acquired from three individual experiments. (C) The protein levels of PARP16 and activation of three signaling UPR branches PERK, IRE1a/XBP-1 and ATF6 were detected in hVSMCs after 20 ng/mL PDGF-BB stimulation for indicated time using Western blot; (D) Western blot analysis of MMP9, cyclin D1, cyclin E, PCNA protein levels in hVSMCs after 20 ng/mL PDGF-BB treatment. (E)–(G) PARP16 and ER stress were involved in intimal hyperplasia after rat carotid artery balloon injury. Western blot analysis of the PARP16, cyclin D1, p-PERK, p-IRE1a, spliced-XBP-1 protein levels in arteries from rat with sham operation or balloon injury for 14 days (E); mRNA levels of UPR target genes in arteries from rat with sham operation or balloon injury for 14 days (F); all data are represented as means ± SEM; *P < 0.05, ***P < 0.001 vs. Sham group, n = 7 in each group. (G) Immunofluorescent staining with PARP16 (green) and MMP9 (red) in the rat carotid artery from the sham operation or 14-day post-balloon injury. All sections were counter-stained by DAPI to visualize nuclei (blue), scale bars: 100 µm.
Fig 4: Deficiency of Parp16 suppresses PDGF-BB-induced rVSMC proliferation and migration through ER stress. Following transfection with control siRNA (siCTL) or Parp16 siRNA (siPARP16) for 72 h, rVSMCs were treated with 20 ng/mL PDGF for 24 h, cell extracts were collected for determining the protein levels of PARP16, PCNA, MMP9, cyclin D1, and cyclin E by Western blot (A); cell lysates were immunoblotted with p-PERK, p-eIF2a, BIP, p-IRE1a, spliced XBP-1 and calnexin antibodies (B). rVSMCs were transfected with Parp16 siRNA (siPARP16) or control siRNA (siCTL) for 72 h, and then stimulated with 20 ng/mL PDGF-BB for 24 or 48 h, the EdU positive cells were showed green color and cell nuclei stained with DAPI showed blue color, scale bars: 200 µm (C); the migratory ability of rVSMCs was determined by Transwell assay, scale bars: 200 µm (D) and wound healing assay, scale bars: 400 µm (E). All data are represented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; &&P < 0.01, &&&P < 0.001 vs. PDGF + siCTL, each acquired from three individual experiments.
Fig 5: Overexpression of PARP16 results in VSMC proliferation and migration accompany with ER stress. rVSMCs were transfected with lentivirus-mediated Parp16 cDNA (PARP16 OE) or vector (Control) for 72 h, cell lysates were immunoblotted with antibodies against PARP16, p-PERK, p-eIF2a, p-IRE1a, spliced XBP-1, and BIP (A); PCNA, MMP9, cyclin E and cyclin D1 (B); rVSMCs migration ability was tested by wound healing assay, scale bars: 400 µm. (C) and Transwell assay, scale bars: 200 µm (D); rVSMCs proliferation ability was detected by EdU assay, scale bars: 200 µm (E); All data are represented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control, each acquired from three individual experiments. (F)–(H) PARP16 selectively ADP-ribosylated PERK and IRE1a during the UPR. hVSMCs with overexpression FLAG-PARP16 were treated PDGF-BB for 36 h, the PARP16 localization was demonstrated by co-immunostaining with calnexin, scale bars: 50 µm. (F); Coimmunoprecipitation using antibody against FLAG in lysates of hVSMCs induced by PDGF (G); Coimmunoprecipitation using anti-ADP-ribose binding reagents in lysates of PDGF-BB-induced hVSMCs (H).
Supplier Page from Abcam for Anti-PARP16 antibody