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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: J Hepatol. 2015 Jul 26;63(6):1397–1404. doi: 10.1016/j.jhep.2015.07.020

Figure 2. Exposure to PM2.5 stimulates TGFβ signaling in macrophages and collagen production in HSC.

Figure 2

(A) Levels of secreted TGFβ1 in primary hepatocytes, RAW264.7 cells, and LX-2 cells upon the challenge of PM2.5 (5 µg/ml) for different time intervals as indicated. The levels of TGFβ1 in the culture medium were determined by ELISA and presented after normalization to cell numbers. Each time point represents the mean ± SEM (n=3 biological replicates). (B) qRT-PCR analysis of expression levels of the Tgfβ1, Tgfβ2, and Tgfβ3 mRNAs in RAW264.7 cells challenged with PM2.5 for different time intervals as indicated. Fold changes of mRNA levels were shown by comparing to the vehicle (PBS)-treated controls. Each bar represents the mean ± SEM (n=3 biological replicates). (C) Immunoblotting analysis of total and phosphorylated SMAD3 in LX-2 cells cultured in the conditioned medium from RAW264.7 cells exposed to PM2.5 for 12 or 24 hours. The graph beside the images showed fold changes of phosphorylated SMAD3 levels after normalization to total SMAD3 levels. Each bar denotes the mean ± SEM (n=3 biological replicates). (D) qRT-PCR analysis of expression levels of the Collagen 1α1 (Col1α1) and Collagen 4α4 (Col4α4) mRNA in LX-2 cells cultured in the conditioned medium from RAW264.7 cells exposed to PM2.5 for 12 or 24 hours. LX-2 cells were incubated with the conditioned medium for 36 hours before subjected to the real-time RT-PCR analysis. Fold changes of mRNA are shown by comparing to the vehicle control. Each bar denotes the mean ± SEM (n=3 biological replicates). (E) Immunoblotting analysis of Col1 protein levels in LX-2 cells cultured in the conditioned medium from RAW264.7 cells exposed to PM2.5 for 12 or 24 hours. The graph beside the images showed fold changes of Collagen protein levels after normalization to those of Tubulin. Each bar denotes the mean ± SEM (n=3 biological replicates). From A–E, * p<0.05; ** p<0.01. (F) Immunofluorescent analysis of Col1 production in the LX-2 cells cultured in the conditioned medium from RAW264.7 cells exposed to PM2.5 for 10 hours. LX-2 cells were incubated with the conditioned medium for 36 hours before subjected to the immunofluorecent analysis. LX-2 cells were cultured in the conditioned medium from RAW264.7 cells exposed to PBS (vehicle) as the control. The nucleus was stained with DAPI. The experiments were repeated 3 times, and representative images were shown.