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. 2024 Jan 1;14(2):861–878. doi: 10.7150/thno.87317

Figure 5.

Figure 5

SGK3/NF-κB signaling pathway regulated the nuclear transcription of Pit-1 in mouse VSMCs. Mouse VSMCs treated with 20 µmol/L NF-κB inhibitor BAY11-7085. A. The mRNA levels of Pit-1 measured by qPCR. B. The protein levels of Pit-1 and NF-κB were measured by western blot. C. Mouse VSMCs transiently transfected with SGK3-S486D plasmid and then treated with or without 20 µmol/L BAY11-7085. The mRNA levels of Pit-1 measured by qPCR. D-E. Mouse VSMCs transiently transfected with SGK3-S486D plasmid (D) for 48 h, in presence or absence of BAY11-7085 (E). The protein levels of NF-κB, Pit-1 and SGK3 were measured by western blot. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. S486D group. F-G. Mouse VSMCs transiently transfected with SGK3-S486D plasmid (F), or treated with SGK3-PROTAC1 (G), in presence or absence of high Pi for 24 h. Representative immunofluorescence microscopy images showed NF-κB protein expression and localization in mouse VSMCs. Green labeling, NF-κB expression; blue labeling, nuclei. Scale bars: 20 μm. H. Mouse VSMCs transiently transfected with SGK3-S486D plasmid, in presence or absence of Pi for 48 h. The nuclear protein levels of NF-κB was measured by western blot. *P < 0.05, ***P < 0.001 vs. control group; #P < 0.05 vs. S486D group. I. Mouse VSMCs treated with 3 mM Pi and without or with additional treatment with 20 µmol/L NF-κB inhibitor BAY11-7085 for 7 days. Representative original images showing Alizarin Red S staining. J. Mouse VSMCs were treated with BAY11-7085 for 24 h, in presence or absence of high Pi. The protein levels of NF-κB, Pit-1 and RUNX2 were measured by western blot. *P < 0.05, **P < 0.01 vs. control group; △△△P < 0.001 vs. Pi group.