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. 2018 Sep 12;293(44):17008–17020. doi: 10.1074/jbc.RA118.004706

Figure 2.

Figure 2.

CDK9 regulates CHOP expression. A, immunoblot analysis of CHOP protein to screen for inhibition of SFA-induced CHOP induction with a kinase inhibitor library containing >140 compounds. VSMCs were pretreated with 10 μm of each inhibitor for 2 h prior to co-treatment with 500 μm C18:0 and 10 μm of each inhibitor for 6 h. CHOP expression is expressed as a fold value relative to that of vehicle-treated (DMSO-treated, fold value is 1) cells with GAPDH correction by band densitometry. The experiments were repeated three times. B, immunoblot analysis of CHOP protein to screen for inhibition of SFA-induced CHOP induction. VSMCs were pretreated with 10 μm of each inhibitor for 2 h prior to co-treatment with 500 μm C18:0 and 10 μm of each inhibitor for 6 h. Total cell lysates of VSMCs were then prepared. BSA (Veh) with DMSO (lane 1), C18:0 with DMSO (lane 2), C18:0 with triacin C (acyl-CoA–specific synthetase inhibitor, lane 3), C18:0 with H89 (PKA inhibitor, lane 4), C18:0 with Gö6983 (PKC inhibitor, lane 5), C18:0 with Akti-1/2 (Akt inhibitor, lane 6), C18:0 with U0126 (MEK inhibitor, lane 7), C18:0 with rapamycin (mTOR inhibitor, lane 8), C18:0 with SB2003580 (p38 MAPK inhibitor, lane 9), C18:0 with SP600125 (JNK inhibitor, lane 10), C18:0 with AT7519 (pan-CDK inhibitor, lane 11), C18:0 with SL0101–1 (RSK inhibitor, lane 12), and C18:0 with CHIR99021 (GSK-3 inhibitor, lane 13) are as indicated. C, immunoblot analysis of CHOP protein to screen for CDK-specific inhibition of SFA-induced CHOP induction. VSMCs were pretreated with 10 μm of each inhibitor for 2 h prior to co-treatment with 500 μm C18:0 and 10 μm of each inhibitor for 6 h. Total cell lysates of VSMCs were then prepared. BSA (Veh) with DMSO (lane 1), C18:0 with DMSO (lane 2), AT7519 (lane 3), flavopiridol (lane 4), dinaciclib (lane 5), RO-3306 (lane 6), AZD5438 (lane 7), roscovitine (lane 8), palbociclib (lane 9), CAY10574 (lane 10), and LDC000067 (lane 11) in the presence of 500 μm C18:0 are as shown. D, immunoblot analysis of ATF4, CHOP, and GAPDH proteins in VSMCs co-treated with SFA and CDK9 inhibitor. VSMCs were pretreated with DMSO (−) or 30 μm CAY10574 (CDK9 inhibitor) for 2 h prior to co-treatment with 250 μm C18:0 and DMSO or 30 μm CAY10574 for 6 h. Total cell lysates of VSMCs were then prepared. qRT-PCR analyses of CHOP (E), sXBP-1 (F), and BiP (G) in VSMCs co-treated with SFA and CDK9 inhibitor are as shown. VSMCs were pretreated with DMSO or 30 μm CAY10574 (CDK9 inhibitor) for 2 h prior to co-treatment with 250 μm C18:0 and 30 μm CAY10574 for 6 h. Total RNA of VSMCs was then prepared (n = 4). One-way ANOVA with a Student-Newman post hoc test was used for statistical analysis. *, p < 0.05.