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. 2013 Oct 25;2(5):e000238. doi: 10.1161/JAHA.113.000238

Figure 4.

Figure 4.

ATF4 knockdown alters mineralization and osteoblastic differentiation of MOVAS‐1 cells induced by TNFα. A, Mineralization and (B) ALP activity of ATF4 knockdown MOVAS‐1 cells treated with TNFα (10 ng/mL) for 7 days. C, Phosphorylated eIF2α (p‐eIF2α), phosphorylated ATF4 (p‐ATF4), total ATF4 (ATF4), and CHOP protein expression in ATF4 knockdown MOVAS‐1 cells treated with TNFα (30 minutes for p‐eIF2α and p‐ATF4 expression, 3 hours for ATF4 expression, and 16 hours for CHOP expression). D, RT‐PCR analysis of XBP‐1 in ATF4 knockdown MOVAS‐1 cells treated with TNFα for 16 hours. The upper band is the expression of uXBP‐1. The lower band is the expression of sXBP‐1. E, ATF4 mRNA expression in MOVAS‐1 cells treated with adenoviruses containing wild‐type ATF4 (Ad‐ATF4), ATF4 mutant with S251A, and S254A (Ad‐ATF4 mutant) or empty (Ad‐empty) at a MOI of 40 for 6 hours. ***P<0.001 vs wild‐type ATF4. Four samples per group were analyzed. F, Mineralization of VSMCs overexpressing ATF4 mutant. MOVAS‐1 cells were treated with either Ad‐ATF4, Ad‐ATF4 mutant or Ad‐empty for 6 hours and then incubated for 7 days. **P<0.01 and ***P<0.001 vs wild‐type ATF4. Four samples per group were analyzed. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; eIF2α, eukaryotic initiation factor 2α; PCR, polymerase chain reaction; PERK, protein kinase RNA‐like endoplasmic reticulum kinase; RSK2, ribosomal S6 kinase 2; TNFα, tumor necrosis factor‐α; XBP, X‐box binding protein‐1.