Ab120663

A different batch of mice was divided into two groups: Basal (WT and KO mice) and Trained (WT and KO mice submitted to the acute exhaustive physical exercise protocol) with intraperitoneal injections of vehicle (0.9% saline) or colchicine (0.4 mg/kg/day: AB120663, ABCAM, Cambridge, UK) for 3 consecutive days. The final injection was performed 1h before the acute physical exercise protocol (20 (link)–23 (link)). After the exercise protocol and totalizing three hours from the last injection, the animals were anesthetized by an intraperitoneal administration of xylazine (10 mg/kg body weight) and ketamine (100 mg/kg body weight). As soon as the loss of pedal reflexes confirmed the effect of anesthesia, the gastrocnemius was removed, washed with saline, and used for the immunoblotting technique. The autophagic flux index was calculated as follows: Autophagy flux index = (LC3II expression levels with colchicine)/(LC3II expression levels without colchicine) (24 (link)). Each LC3II was normalized by its beta-actin expression level (24 (link)).

Mice were treated with intraperitoneal injections of colchicine (0.4 mg/kg/day; AB120663, ABCAM, Cambridge, UK) or vehicle (0.9% saline) for 3 consecutive days. The final injection was performed 4 h before the acute physical exercise protocols [44 (link),68 (link),69 (link),70 (link)]. The colchicine treatment inhibits autophagosome degradation, inducing an increase in the levels of LC3II and/or Sqstm1/p62 [71 (link)]. The mean colchicine values were subtracted from the vehicle values of corresponding protocols (i.e., mean CT with colchicine-mean CT with saline) for autophagic flux calculation. Immediately after the acute physical exercise protocols, the animals were anesthetized by an intraperitoneal administration of xylazine (10 mg/kg body weight) and ketamine (100 mg/kg body weight). As soon as the loss of pedal reflexes confirmed the effect of anesthesia, the gastrocnemius, heart (left ventricle isolation), and liver samples were removed, washed with saline, and used for the immunoblotting technique.

Forty-eight hours after the last exercise session of each training protocol, mice were treated with intraperitoneal injections of colchicine (0.4 mg/kg/day; AB120663, Abcam, Cambridge, UK) or vehicle (0.9% saline) for 3 consecutive days. One hour after the last injection of colchicine, the rodents were anesthetized as previously described, and the liver, heart (with subsequently left ventricle isolation), and EDL were harvested, washed with saline, and used for the measurement of the protein levels of LC3-II (the active/lipidated form of LC3-I) and p62 (autophagosome adaptor protein) by the immunoblotting technique. The schematic presentation of colchicine treatment is shown in Figure 1B. In addition, there were specific mice used in this analysis, and they did not perform physical tests. The colchicine values were subtracted from the vehicle mean values of corresponding protocols (for example, mean END with the colchicine–mean END with the saline) for autophagic flux calculation.