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. 2022 May 18;323(5):C1555–C1575. doi: 10.1152/ajpcell.00369.2021

Figure 6.

Figure 6.

Gain of chaperone-mediated autophagy (CMA) decreases cytotoxicity and cell death in cultured cardiomyocytes. Cardiomyocytes were treated with CoCl2 for 48 h and the effect of both the loss and gain of CMA function on cytotoxicity and cell death was determined. A: cardiomyocytes were transfected with either a Lamp2a or Ctrl siRNA and treated with vehicle (Veh) or without CoCl2. CoCl2-induced cytotoxicity was determined by lactate dehydrogenase (LDH) release assay in Lamp2a-silenced cardiomyocytes. B: cardiomyocytes transfected with a Lamp2a or a Ctrl siRNA were treated with or without CoCl2; and were dual stained using propidium iodide (PI), as a marker of necrotic cell death, and Hoechst, to determine total nuclei present. The bar graph shows the percent of PI-positive myonuclei over the total nuclei in each group. C: the percent of terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL)-positive nuclei (green) indicating apoptotic cell death was determined in the cardiomyocytes with Lamp2a-silenced and treated with or without CoCl2. D: bar graph shows the percent changes in TUNEL-positive nuclei over the total nuclei. Green represents TUNEL-positive nuclei, actin is stained in red and represents myocytes; nuclei were counterstained with DAPI (blue). E: CoCl2-induced cytotoxicity was determined by LDH release assay in Lamp2a- or control LacZ-overexpressing cardiomyocytes. F: bar graph shows the percent of PI-positive over the total nuclei in the Lamp2a- or LacZ-infected cells with or without CoCl2 treatment. G: percent of TUNEL-positive nuclei (green) was determined in the Lamp2a-overexpressing cardiomyocytes, with or without CoCl2 and (H) bar graph shows the percent changes in TUNEL-positive nuclei over the total nuclei. Results for all experiments are means ± SD from four independent groups (n = 4). *P < 0.05: significant difference between Veh and CoCl2 within similarly infected groups; #P < 0.05 and %P < 0.05: differences within Lamp2a knockdown or overexpressing cells treated with Veh or CoCl2, respectively.