Fig. 7: Knocking down of Ddit4 in C2C12 reversed abnormal mTOR signaling and attenuated autophagy.

A Western blot analysis of the expression of the indicated muscle-related genes between normal and cachectic mice. B C2C12 myotubes were treated with RPMI1640, CT26 CM, and C26 CM. Western blot analysis of the indicated genes were detected. C C2C12 myotubes were treated with CT26 CM, CT26 CM plus Ddit4 siRNA (two different siRNA of Ddit4 named #1 and #2) or scramble siRNA, C26 CM, C26 CM plus Ddit4 siRNA (two different siRNA of Ddit4 named #1 and #2) or scramble siRNA for 24 h. Western blot analysis of the indicated genes in C2C12 cells with Ddit4 knockdown and/or C26 CM challenge. D C2C12 myotubes were treated with control vector, Ddit4 overexpression plasmid, or C26 CM for 24 h. E Upper panel: the morphological changes in C2C12 myotubes were addressed by H&E staining (left panel). C2C12 cells were immunostained with the MHC (MF20) antibody. MHC, green, DAPI, blue (right panel). Scale bar: 50 μm. Lower panel: Western blot analysis of MHC in C2C12 myotubes. F C2C12 cells were pretreated with (+) or without (−) SB203580(0.5, 1, 2.5, and 5 µM) prior to C26 CM for 24 h. Cell lysates were subjected to Western blot analysis; G C2C12 cells were pre-treated with (+) or without (−) SB203580 (5 µM) and then transfected with Ddit4-overexpressing plasmid. Cell lysates were subjected to Western blot analysis. H Western blot detection of the indicated genes in the muscle of healthy people and the muscle of liver cancer patients with cachexia.