Journal: Cell communication and signaling : CCS
Article Title: Nesfatin-1 enhances vascular smooth muscle calcification through facilitating BMP-2 osteogenic signaling.
doi: 10.1186/s12964-024-01873-7
Figure Lengend Snippet: Fig. 8 STAT3 occupies nesfatin-1 promoters and contributes to its upregulation in VC. (A) Schematic of procedures for mass spectrometry (MS) screening in VSMCs. (B) Screening identified STAT3 bound to the nesfatin-1 promoters. m/z, mass/charge ratio. (C) The motif of STAT3 in Jaspar database. (D) Western blot analysis of STAT3, nesfatin-1, RUNX2, and BMP-2 after overexpression of STAT3. (E) Western blot analysis of STAT3, nesfatin-1, RUNX2, and BMP-2 after downregulation of STAT3. (F) Effects of STAT3 overexpression or downregulation on the luciferase reporter gene activity of nesfatin-1. (G) Fold enrichment of STAT3 within the promoters of nesfatin-1 in high Pi-incubated VSMCs or calcifying aortas. (H) Effects of STAT3 downregulation on calcium deposition in VSMCs incubated with high Pi. (I) Effects of STAT3 downregulation on ALP activity in VSMCs incubated with high Pi. (J) Predicted STAT3 and nesfatin-1 promoter binding regions. The P-value was calculated by unpaired two-tailed Student’s t-test (D-G). Differences between groups were assessed with ANOVA followed by Bonferroni post-hoc test (H-I). *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group. n = 6
Article Snippet: Upon 50–60% confluence, VSMCs were cultured in serum-free DMEM and transfected with lentivirus-mediated control shRNA (forward, 5′- T T T C T C T T C A C T C A A T A A A-3′; reverse, 5′- T T T A T T G A G T G A A G A G A A A-3′), SYTL4 shRNA1 (forward, 5′- G C G T G T A A G T T G T C T A C A A-3′; reverse, 5′- T T G T A G A C A A C T T A C A C G C-3′), SYTL4 shRNA2 (forward, 5′- G G A A G T A G A T G A T C T A G T A; reverse, 5′- T A C T A G A T C A T C T A C T T C C-3′), SYTL4 shRNA3 (forward, 5′- G C T A T A A G T T A G A A G G T T A-3′; reverse, 5′- T A A C C T T C T A A C T T A T A G C-3′), SYTL4 overexpression plasmid (TL712429, Origene), STAT3 overexpression plasmid (RR206813L3, Origene), K278A mutation plasmid (Genepharm), K287A mutation plasmid (Genepharm), K290 mutation plasmid (Genepharm), con siRNA (sc-108080, Santa Cruz), STAT3 siRNA (sc-270027-V, Santa Cruz) (10 μL/mL, multiplicity of infection: 50) according to the manufacturer’s instructions.
Techniques: Mass Spectrometry, Western Blot, Over Expression, Luciferase, Activity Assay, Incubation, Binding Assay, Two Tailed Test
