Fig 4

Tp blocks the autophagic flux.

A tandem fluorescent-labelled plasmid pmCherry-EGFP-LC3B construct was transfected into HMC3 cells to quantify the numbers of different LC3 puncta and to assess the impact of Tp on the autophagic flux in HMC3 cells. In the absence of autophagy, the cells emitted diffuse yellow fluorescence. GFP (green fluorescence) is sensitive to the acidic environment of the lysosome and mCherry (red fluorescent) exhibits significant stability. After the initiation of autophagy, yellow puncta (RFP+ GFP+) indicated early autophagosomes, whereas red puncta (RFP+ GFP-) indicated late autophagosomes, showing that LC3 had been delivered to the lysosomes and that autophagosomes and lysosomes had fused to form autolysosomes. (A) The expression of LC3-I and LC3-II after HMC3 cells were treated with different concentrations of Tp for 24 h was measured by Western blotting. (B) Expression of the autophagy-related proteins Beclin1, LC3-I, LC3II and P62 after HMC3 cells were treated with Tp (MOI of 50:1) for different times was measured by Western blotting. (C) Expression of Lamp2 after HMC3 cells were treated with different concentrations of Tp for 24 h was measured by Western blotting. (D) HMC3 cells were transfected with the pmCherry-EGFP-LC3B plasmid, cocultured with or without Tp (MOI of 50:1) for 24 h, and analysed by confocal fluorescence microscopy. HMC3 cells were treated with BAF (bafilomycin, 80 nM) for 24 h as a positive control. Scale bar: 20 μm. The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.