Cancer cachexia is regulated by selective targeting of skeletal muscle gene products
J. Clin. Invest. Swarnali Acharyya, et al. 114:370 doi:10.1172/JCI20174 [Go to this article.]

Figure 3
TNF/IFN inhibits MyHC transcription through the concomitant reduction of MyoD. (A) C2C12 cells were transfected in triplicate with a MyHC IIb–luciferase reporter plasmid as described in Methods. The next day, cells were differentiated and at the indicated times were harvested for luciferase assays. (B) Myoblasts were transfected with MyHC IIb–luciferase and subsequently differentiated for 48 hours, at which time cells were left untreated or treated with TNF/IFN. At the indicated times, cells were harvested and luciferase levels determined. (C) Myotubes were either untreated or treated with TNF (5 ng/ml), IFN (50 U/ml), or TNF plus IFN for 48 hours. Northern blots probing for MyoD were performed (25), and GAPDH was used as a loading control. (D) Myoblasts were transfected under conditions similar to those described in B with a MyHC IIb–luciferase reporter along with empty vector or MyoD wild-type or mutant (mut) expression plasmids (50 ng each). Cells were differentiated for 48 hours and subsequently left untreated or treated with TNF and IFN for an additional 24 hours, at which time cell extracts were prepared and luciferase activities determined. (E) Myotubes were either untreated or treated with TNF and IFN in methionine/cysteine–free DM, then pulsed for 1 hour with [³⁵S]-methionine/cysteine prior to collection of cells. Labeled MyHC was detected by immunoprecipitation. (F) Myotubes were pulsed with DM containing [³⁵S]-methionine/cysteine, then chased with fresh DM with or without TNF/IFN. For histograms, the data are representative of experiments performed a minimum of three times, plotted as mean ± SD.