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. 2019 Jul 4;16(4):683–697. doi: 10.1080/15548627.2019.1635380

Figure 5.

Figure 5.

MIR145-3p induces autophagy and cell death through the inhibition of HDAC4 in MM cell. (A) LP-1 cells were transfected with MIR145-3p mimic, MIR control (MIRctrl), siRNA-HDAC4 or control siRNA (sictrl) for 72 h, and then cell apoptosis was analyzed by ANXA5 and 7-AAD staining. The percentage of ANXA5-positive cells was presented as mean ± SD from 3 independent experiments (*P < 0.05; **P < 0.01, NS, not significant). (B) After transfection with MIR145-3p mimic, MIRctrl, siRNA-HDAC4 or sictrl for 72 h, LP-1 cells were lysed and extracted. Western blotting was performed to detect the expression levels of the active cleaved CASP3 and BCL2L11. GAPDH was used as loading control. The experiments were performed in triplicate. (C) LP-1 cells expressing GFP-MAP1LC3B were transfected with siRNA-HDAC4 or sictrl and were treated with or without lysosomal inhibitor BAF (20 nM) for 4 h. Then cells were visualized with a fluorescence microscope. Representative images are shown. (D) Quantitative analysis of the experiments in (C) (mean ± SD of independent experiments, n = 3, **P < 0.01.). (E) LP-1 cells were transfected with MIR145-3p mimic, MIRctrl, siRNA-HDAC4 or sictrl for 72 h, in the absence or presence of 20 nM BAF, and then the protein levels of HDAC4, MAP1LC3B-I, MAP1LC3B-II, SQSTM1, RPS6KB1 and p-RPS6KB1 (Thr389) were determined by western blot analysis. GAPDH was used as loading control. The experiments were performed in triplicate. (F) LP-1 cells expressing GFP-MAP1LC3B were co-transfected with pcDNA-control (pcDNActrl) or pcDNA-HDAC4 vector and MIR145-3p mimic or MIRctrl and were treated with or without BAF (20 nM) for 4 h. Then cells were visualized with a fluorescence microscope. Representative images are shown. (G) Quantitative analysis of the experiments in (F) (mean ± SD of independent experiments, n = 3, **P < 0.01.). (H) LP-1 cells were co-transfected with either pcDNA-control (pcDNActrl) or pcDNA-HDAC4 vector and MIR145-3p mimic or MIRctrl for 72 h, in the absence or presence of 20 nM BAF, and then the protein levels of HDAC4, MAP1LC3B-I, MAP1LC3B-II, SQSTM1, RPS6KB1 and p-RPS6KB1 (Thr389) were determined by western blot analysis. GAPDH was used as loading control. The experiments were performed triplicate. (I) LP-1 cells were co-transfected with pcDNActrl or pcDNA-HDAC4 vector and MIR145-3p mimic or MIRctrl for 72 h, and then cell apoptosis was analyzed by ANXA5-7-AAD staining. The percentage of ANXA5-positive cells was presented as mean ± SD from 3 independent experiments (*P < 0.05; **P < 0.01) .