Skip to main content
. 2021 Aug 12;18(4):860–876. doi: 10.1080/15548627.2021.1961072

Figure 4.

Figure 4.

PA treatment stimulates SQSTM1 phosphorylation and the interaction of STING1 and SQSTM1. (A) Hep3B cells with TBK1-siRNA transfection for 48 h were treated with the indicated concentration of PA for 24 h, and lysates were immunoblotted for p-RPS6KB1 (T389), p-SQSTM1 (S403), SQSTM1, and ACTB. Cells treated with scrambled siRNA were used as control. (B) Coimmunoprecipitation and immunoassay of extracts of Hep3B cells transfected with MYC-STING1, Flag-SQSTM1, and HA-TBK1. (C) Coimmunoprecipitation and immunoassay of extracts of Hep3B cells transfected with HA-STING1, Flag-SQSTM1, and followed with PA treatment (0.5 mM, 24 h) with or without BX795 (10 μM). (D) Hep3B were treated with or without PA (0.5 mM, 24 h), or transfected with cGAMP (2 μg/ml, 6 h) in the presence or absence of TBK1-siRNA transfection for 48 h, or in the presence of absence of BX795 (10 μM, 8 h). Cells treated with scrambled siRNA were used as control (Ctrl-siRNA). STING1 was immunoprecipitated from whole-cell lysates, and levels of STING1, RPTOR and SQSTM1 in the precipitates were evaluated by immunoblotting. (E) Confocal microscopy of Hep3B cells treated with PA (0.5 mM, 24 h) or cGAMP (2 μg/ml, 6 h). Cells treated with scrambled siRNA were used as control. The cells were fixed 8 h later and stained with antibodies against TBK1 and RPTOR. Hoechst 33342 was used for nucleus staining. Representative images are shown. Scale bar: 10 μm. Quantitative analysis of colocalized puncta of TBK1 and RPTOR is shown on the right. The numbers of puncta per cell were quantified by blind counting. The data are presented as means of three views of counting. **p < 0.01, *p < 0.05, ns = not significant. (F) Coimmunoprecipitation and immunoassay of extracts of Hep3B cells transfected with various combinations of expression vector for MYC-STING1 and other indicated plasmids, and treated with PA (0.5 mM, 24 h). (G) Coimmunoprecipitation and immunoassay of extracts of Hep3B cells transfected with various combinations of expression vector for Flag-STING1, HA-RPTOR and other indicated plasmids.