Fig. 6
From: Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90
Tumor cell-released Hsp70/90-expressing EVs induce muscle catabolism in myotubes. a CD9-positive EVs induce catabolic response in C2C12 myotubes. EVs isolated from LCM were subjected to immunoprecipitation (IP) against CD9 with pre-immune IgG as control. Resulted pellet and supernatant (Sup) were used to treat C2C12 myotubes and compared with PBS and input EVs for catabolic activity using western blotting. Data (n = 3) were analyzed by analysis of variance (ANOVA) and * denotes a difference from PBS treatment (P < 0.05). b Hsp70/90 release by cachexia-inducing tumor cells is dependent on EV release. Rab27a and Rab27b were knocked down by transfecting LLC and C26 cells with control or specific siRNAs. EVs and Hsp70/90 released into conditioned media were quantified after Brij98 treatment. EVs were quantified by AchE. Data (n = 3) were analyzed by Student t-test. c Tumor cell-induced catabolic response in myotubes is dependent on EV release. C2C12 myotubes were treated with conditioned media of Rab27-deficient LLC or C26 cells and analyzed for p38 MAPK activity (at 1 h), atrogin1 (at 8 h) and MHC levels (72 h). Conditioned medium of non-tumorigenic NL20 cells (NCM) was used as control. Data (n = 3) were analyzed by ANOVA. * denotes a difference (P < 0.05) from NCM-treated control. # denotes a difference between bracketed columns
