Fig 1.
Excessβ-glycerophosphate promotes VSMCs osteogenic differentiation and VC in VSMCs.
A and B. Representative alizarin red S staining images and quantified calcification area demonstrated calcium salt deposition in VSMCs for different duration. C. ALP activity incubated with β-glycerophosphate for 0, 3, 7, 10 days. D. SENRC mRNA expression with β-glycerophosphate-induced calcification was remarkably increased in VSMCs (n = 3). One-way analysis of variance and Tukey’s post hoc test were used to compare multiple groups. *P < 0.05, **P < 0.01 vs. Control, NS no significance.
Fig 2.
Phenotypic switching and endoplasmic reticulum stress in VSMCs.
A, B, C, D. mRNA levels of SM22-α, Msx-2, Runx2, and ColI in VSMCs (n = 3). E. Representative bands of Western blotting of Runx2, α-SMA, Msx-2 and SM22α in VSMCs (n = 3). F. Representative bands of Western blotting of p-PERK, PERK, GRP78 and ATF4 in VSMCs (n = 3). One-way analysis of variance and Tukey’s post hoc test were used to compare multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control.
Fig 3.
4.PBA attenuatedβ-glycerophosphate-induced phenotypic transition and ERS in VSMCs.
A and B. Protein expression levels of p-PERK, PERK, GRP78, and ATF4 in the 4-PBA-treated group (n = 3). C and D. Representative bands from western blotting for Runx2, α-SMA, Msx-2, and SM22-α in groups (n = 3). One-way analysis of variance and Tukey’s post hoc test were used to compare multiple groups. *P < 0.05, **P < 0.01 vs. Control.
Fig 4.
si-SENRC reversed β-glycerophosphate-mediated calcification in VSMCs by inhibiting ERS in vitro.
A. Transfection efficiency of SENRC small interfering RNA lentivirus in VSMCs. Alizarin red S staining (B) and cell scratch repair assays (C) were measured to quantify the calcified areas and cell migration ability of VSMCs transfected with sh-SENRC relative to sh-NC group. D. mRNA expression levels of SM22-α, Msx-2, and Runx2 in VSMCs transfected with sh-SENRC (n = 3). E. Representative bands of Western blotting of Runx2, α-SMA, Msx-2, and SM22-α (n = 3). F. Representative bands of Western blotting of p-PERK, PERK, GRP78, and ATF4 in sh-SENRC group (n = 3). One-way analysis of variance and Tukey’s post hoc test were used to compare multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5.
SENRC promoted the ERS-mediated calcification by suppressing miR-4731-5p expression in VSMCs.
A. Effect of SENRC knockdown or overexpression on miR-4731-5p expression (n = 3); B. miR-4731-5p expression level by RT-qPCR after treatment with miR-4731-5p inhibitor or mimic (n = 3). C and D. Representative bands of Western blotting of cellular ERS-related and phenotypic transformation-related proteins in VSMCs after treatment with miR-4731-5p inhibitor or mimic (n = 3). One-way analysis of variance and Tukey’s post hoc test were used to compare multiple groups. *P < 0.05, **P < 0.01, NS: no significance.