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. 2020 Jan 9;6(1):e03115. doi: 10.1016/j.heliyon.2019.e03115

Figure 4.

Figure 4

TRIM34 enhanced the mitochondrial depolarization and release of cytochrome c. (A) TRIM34-induced loss of MMP was measured by Rhodamine 123 method. HEK293T cells were transfected with 5′FLAG-pcDNA3.1- TRIM34 or 5′FLAG-pcDNA3.1 vectors for 24 h. Thereafter, the cells were stained with Rhodamine-123 and analyzed by flow cytometry. Representative histograms were shown and the percentage of cells in depolarized zone (M1) was illustrated. (B) Quantification of the cells with low MMP in Figure 4A. Results were presented as mean ± SD in triplicates. *P < 0.05, compared to the control group. (C) TRIM34-induced mitochondrial depolarization was measured by JC-1 method. HEK293T cells were transfected with 5′FLAG-pcDNA3.1-TRIM34 or 5′FLAG-pcDNA3.1 for 24 h. Then the cells were stained with JC-1 and subjected to flow cytometry analysis. Flow cytometry analysis showed the distribution of JC-1 aggregates (healthy cells, red fluorescence) and JC-1 monomers (apoptotic cells, green fluorescence). (D) Histograms showed the percentage of JC-1 monomer-positive cells. Results were presented as mean ± SD (n = 3). *P < 0.05, difference versus control vectors. (E) TRIM34-induced mitochondrial depolarization was measured by MitoTracker Deep Red method. HEK293T cells were transfected with pEGFP-N3-TRIM34 or pEGFP-N3 vectors (control) for 24 h. Thereafter, the cells were stained with MitoTracker Deep Red and analyzed by flow cytometry. FSC/SSC plot was used to gate (R1) the viable HEK293T cells. Next, the R1 gated events were analysed for the EGFP positive cells (R2). The MMP was measured by the MFI of MitoTracker Deep Red, gating of events using R2. Overlays of the TRIM34-EGFP group (blue) and the pEGFP-N3 control group (red) were shown. (F) Histogram showed the MFI of MitoTracker Deep Red between the TRIM34-EGFP group (blue) and the pEGFP-N3 control group, gating on the EGFP-positive cells. Results were presented as mean ± SD (n = 3). *P < 0.05, difference versus control vectors. (G) HEK293T cells were transfected with 5′FLAG-pcDNA3.1-TRIM34 (TRIM34), 5′FLAG-pcDNA3.1 (vector) or subjected to mock transfection for 24 h. The mitochondria-free cytoplasmic fraction was prepared and the cytochrome c level was examined by western blot analysis. Representative results were presented and two additional experiments yielded similar results.