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. 2016 Aug 19;8(51):88386–88400. doi: 10.18632/oncotarget.11393

Figure 2. SH003 induces autophagy by suppressing STAT3 phosphorylation.

Figure 2

(A) MDA-MB-231 and HCC-38 cells were treated with different doses of SH003 for 15 minutes and then performed western blots with anti-p-STAT3 and STAT3. Actin was used for the internal control. (B) Cells were treated with SH003 for 24 hours and whole-cell lysates were immunoprecipitated with anti-Beclin1 antibody. The immunoprecipitants and input proteins were then blotted with the antibodies for STAT3, VPS34, BCl-2, Beclin1 and actin. (C) MDA-MB-231 and HCC-38 cells were treated with SH003 (0, 100, 250 and 500 μg/ml) for 24 hours and then stained with Cyto-ID fluorescence dye for 30 minutes at room temperature in the dark. Data analyzed using a FACSCalibur. Data were analyzed by ANOVA with P < 0.05. (D) MDA-MB-231 and HCC-38 cells were treated with 500 μg/ml of SH003 for 24 hours and then stained with anti-LC3B antibody (1 μg/ml) and anti-Alexa Fluor-488 (1:250) antibody. LC3 punctate in the cells were analyzed using Olympus FV10i Self Contained Confocal Laser System. The object was 20× and scale bar indicates 10 μm. *P < 0.05. (E) Analysis of autophagy-related molecules. Cells were treated with SH003 for 24 hours and whole-cell lysates were analyzed by western blots with anti-LC3A/B and p62/SQSTM1. Actin was used for the loading control. (F) Cells were transfected with STAT3-CA and treated with SH003 for 24 hours. Autophagosome formation was stained with Cyto-ID fluorescence. *P < 0.05. Experiments were performed in triplicate. Bars indicate means that standard deviations (SD).