Research Paper Volume 13, Issue 11 pp 14892—14909

Inhibition of CDK9 attenuates atherosclerosis by inhibiting inflammation and phenotypic switching of vascular smooth muscle cells

CDK9 inhibitor reduces inflammatory responses and phenotype switch of VSMCs exposed to ox-LDL by suppressing NF-κB pathway. (A–B) VSMCs were treated with the Bay (10μM) for 1 h, and then stimulated with ox-LDL (50μg/mL) for 24 h. The expressions of α-SMA, Vimentin, and PCNA were detected by western blot ((n = 3; ##P *P **P C–D) transfected with siRNA against CDK9 and then incubated with ox-LDL (50 μg/mL) for 1 h. The IκB-α protein level in cell lysate was detected by western blot; and the nuclear fraction was isolated and the nuclear level of NF-κB p65 was measured by western blot (n = 3; ##P *P **P E) VSMCs were pretreated with LDC000067 (2.5 or 5 μM) for 1 h, and then stimulated with ox-LDL (50 μg/mL) for 1 h. Nuclear translocation of NF-κB p65 was measured by immunofluorescence staining (scale bar = 10 μm). (F–G) NF-κB-RE-EGFP reporter VSMCs were transfected with siRNA against CDK9 and then stimulated with ox-LDL (50 μg/mL) for 6 h. NF-κB activity is shown as mean fluorescence intensity (MFI) in flow cytometry histogram (n = 3; ##P **P

Figure 5. CDK9 inhibitor reduces inflammatory responses and phenotype switch of VSMCs exposed to ox-LDL by suppressing NF-κB pathway. (AB) VSMCs were treated with the Bay (10μM) for 1 h, and then stimulated with ox-LDL (50μg/mL) for 24 h. The expressions of α-SMA, Vimentin, and PCNA were detected by western blot ((n = 3; ##P < 0.01, compared to control; *P < 0.05, **P < 0.01, compared to ox-LDL). (CD) transfected with siRNA against CDK9 and then incubated with ox-LDL (50 μg/mL) for 1 h. The IκB-α protein level in cell lysate was detected by western blot; and the nuclear fraction was isolated and the nuclear level of NF-κB p65 was measured by western blot (n = 3; ##P < 0.01, compared to control; *P < 0.05, **P < 0.01 compared to ox-LDL). (E) VSMCs were pretreated with LDC000067 (2.5 or 5 μM) for 1 h, and then stimulated with ox-LDL (50 μg/mL) for 1 h. Nuclear translocation of NF-κB p65 was measured by immunofluorescence staining (scale bar = 10 μm). (FG) NF-κB-RE-EGFP reporter VSMCs were transfected with siRNA against CDK9 and then stimulated with ox-LDL (50 μg/mL) for 6 h. NF-κB activity is shown as mean fluorescence intensity (MFI) in flow cytometry histogram (n = 3; ##P < 0.01, compared to control; **P < 0.01, compared to ox-LDL).