HDAC6 Regulates the Chaperone-Mediated Autophagy to Prevent Oxidative Damage in Injured Neurons after Experimental Spinal Cord Injury

Figure 5

Deficiency of HDAC6 disrupts the CMA activity in response to HI. The PC12 and PC12/siHDAC6 cells were treated with HI for 24 h. Ctrl express vehicle-treated group. (a) The expression of LAMP-2a and HSC70 was analyzed by Western blot. The level of LAMP-2a and HSC70 was further observed in HDAC6 knockdown cells by siRNA. Results of the densitometric quantification are represented as mean ± SEM (). , versus control group; indicates the significance among the groups indicated in the bar chart. (b) Immunoprecipitated RNase A (an identified CMA substrate) from the treated cells and then assay RNase A activity by ELISA. Group data represent mean ± SEM (); versus corresponding control group; indicates the significance among the groups indicated in the bar chart. (c) Double immunostained with antibody to LAMP-1 (green) and LAMP-2a (red) as indicated. Superimposed confocal images (merge) demonstrate the colocalization of LAMP-2a with LAMP-1 (a lysosome marker). Arrows indicate LAMP-2a mainly clustered in the perinuclear lysosomal membrane. Scale bar: 15 μm. The fluorescence intensity of LAMP-1 and LAMP-2a from experiments results shown. At least 60 cells were included for analysis from five images per group. Group data represent mean ± SEM (); , , and versus corresponding control group.