Research Paper Volume 12, Issue 21 pp 21423—21445

Figure 5. Smyd3 overexpression induces PARP16-mediating ER stress. (A) RAECs were transfected with lentivirus-mediated Smyd3 cDNA (Smyd3 OE) for 72 h, cell lysates were immunoblotted with antibody against Smyd3, PARP16, p21, VCAM-1, p-PERK, Spliced XBP-1. All data were shown as mean ± S.D of at least 4 independent experiments. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. control. (B) Knockdown of PARP16 blocked ER stress and the upregulation of p21 upon overexpression of Smyd3. Smyd3 overexpressed RAECs were transfected with control or Smyd3 siRNA, and then induced by Ang II, cell extracts were collected for determining the protein levels of PARP16, p-PERK, Spliced XBP-1, and p21 by Western blot. Data were shown as mean ± S.D of at least 4 independent experiments. ^#p < 0.05, ^###p < 0.001 vs. control; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Smyd3 OE cells. (C) Inhibition of Smyd3 or PARP16 decreased RAECs senescence markers. Pretreated with PARP16 inhibitor (EGCG) or Smyd3 inhibitor (EPZ031686) for 4 h, RAECs were treated with Ang II for 48 h, respectively, cell extracts were collected for determining the protein levels of p21, p53, and VCAM-1 by Western blot. All data were shown as mean ± S.D of at least 4 independent experiments. ^###p < 0.001 vs. control; ^*p < 0.05, ^***p < 0.001 vs. Ang II treated cells.