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. 2019 Mar 7;20(5):1164. doi: 10.3390/ijms20051164

Figure 5.

Figure 5

Inhibitory effect of Tie-2 inhibitor and γ-T3 on autophagic flux. Protein expression of p62 in PC-3 (A) and C42B (B) cells after treating with different combinations of Tie-2 inhibitor and γ-T3 was examined by Western blotting (representative Western blots are shown). (C) Measurement of autophagic flux using tandem fluorescent-tagged LC3: PC-3 cells transfected with fluorescent-tagged LC3 (mRFP-EGFP-LC3) before being treated with Tie-2 inhibitor/γ-T3 or both compounds for another 72 h (40× objective). Representative fluorescent images are shown. Red fluorescence signal represents autolysosomes (mRFP), while the green fluorescence signal represents autophagosomes (EGFP). Yellow fluorescence signal represented the merger of both mRFP and EGFP. (D) Cells with more than 5 puncta were quantified and at least 50 cells were counted in each group. Each experiment was repeated at least three times, and the results are presented as the mean ± SD. (p values: *** < 0.0005). Scale bars, 50 μM.