Heat shock protein 60 manipulates Foot-and-Mouth disease virus replication by regulating mitophagy

Figures

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  Fig. 1. Parkin-dependent mitophagy is induced during FMDV infection and further suppresses FMDV replication. (A) PK-15 cells were infected with FMDV type O at an MOI of 1. The samples were collected at different time points, and the indicated protein levels were determined via Western blotting. (B) GFP-LC3 colocalizes with mitochondria in PK-15 cells. Transfection of the GFP-LC3 plasmid was performed. After 20 h, the cells were infected with FMDV O for 4 h and 6 h. The cells were stained with MitoTracker and examined by confocal microscopy. Scale bars, 5 μm. (C) FMDV infection induced the mitochondrial translocation of Parkin. After infecting with FMDV O at an MOI of 1 for 6 h, the PK-15 cells were subjected to a mitochondria isolation assay. The mitochondrial or cytoplasmic levels of the indicated proteins were detected by Western blotting. (D) Parkin-dependent mitophagy suppressed FMDV replication. PK-15 cells were transfected with siRNA targeting Parkin (siParkin) or negative control (NC). At 36 h posttransfection, the PK-15 cells were infected with FMDV O at an MOI of 1. The samples were collected at 6 h postinfection, and the indicated protein levels were determined via Western…

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  Fig. 2. Knocking down HSP60 activates Parkin-dependent mitophagy. (A-B) HSP60 depletion caused mitophagy. PK-15 cells were transfected with siRNA targeting HSP60 (siHSP60) or negative control (NC). After 48 h posttransfection, the samples were collected to assess the indicated protein levels via Western blotting. PK-15 cells were treated with 3-MA at 3 mM for 4 h. (C) HSP60 knockdown promoted Parkin translocation to mitochondria. PK-15 cells were transfected with an NC or siHSP60 for 48 h. Next, a mitochondria isolation assay was performed. The mitochondrial or cytoplasmic levels of the indicated proteins were detected via WB. Data are the means of results of three independent experiments, and error bars indicate standard deviations (SD). *, P < 0.05

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  Fig. 3. HSP60 deficiency enhances the phosphorylation of Drp1 at Ser616 and promotes Drp1 translocation to mitochondria. (A) Mdivi-1 treatment abrogates the mitochondrial accumulation of Parkin in HSP60-depleted cells. PK-15 cells were transfected with NC or HSP60. Moreover, PK-15 cells were treated with Mdivi-1 or DMSO. Then, a mitochondria isolation assay was performed. The indicated protein levels were detected by WB. (B) The aggregation of Parkin at the mitochondrial membrane induced by HSP60 depletion was inhibited after Drp1 knockdown. PK-15 cells were transfected with NC, siHSP60, or siDrp1 for 48 h, mitochondria isolation assay was performed. The mitochondrial or cytoplasmic levels of the indicated proteins were detected. (C) Drp1 is essential for mitophagy induced by HSP60 depletion. PK-15 cells were transfected with NC, HSP60, or siRNA targeting Drp1 (siDrp1) for 48 h. The indicated protein levels were detected via WB. Data are the means of results of three independent experiments, and error bars indicate standard deviations (SD). *, P < 0.05

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  Fig. 4. HSP60 depletion induces Drp1 phosphorylation at Ser616 in a CaMKII-dependent manner. (A) The active CaMKII protein level is increased in HSP60-depleted PK-15 cells. (B) HSP60 depletion led to the phosphorylation of Drp1 at Ser616 in a CaMKII-dependent manner. PK-15 cells were transfected with NC, HSP60, or siRNA targeting CaMKII (siCaMKII) for 48 h. WB was used to detect the indicated protein levels. (C) HSP60 depletion induced the accumulation of Drp1 and p-Drp1-Ser616 in mitochondria in a CaMKII-dependent manner. PK-15 cells were transfected with NC, siHSP60, or siCaMKII for 48 h. A mitochondria isolation assay was performed. The mitochondrial or cytoplasmic levels of the indicated proteins were detected. Data are the means of results of three independent experiments, and error bars indicate standard deviations (SD). *, P < 0.05