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NF-κB-Induced Loss of MyoD Messenger RNA: Possible Role in Muscle Decay and Cachexia
Denis C. Guttridge, Marty W. Mayo, Lee V. Madrid, Cun-Yu Wang, and Albert S. Baldwin Jr.

Supplementary Material

Supplemental Figure 1. TNF reduces the level of MyoD mRNA. C2C12 celler were switched to DM and treated with TNF. At indicated times. total RNA was extracted and probed for MyoD. To normalize for equal loading, the blot was stripped and reprobed for GAPDH mRNA.

Supplemental Figure 2. p65 inhibits MyoD transactivation. 10T1/2 fibroblasts were transfected with MyoD, NF-κB subunits, and IκαBSR expression plasmids along with a reporter plasmid containing MyoD-responsive promoter/enhancer elements: TnI-Luc, troponin-I enhancer element (upper panel); 4RTK-Luc, four E-box sites fused to a minimal thymidine kinase promoter (middle panel); and AchR-Luc, acetylcholine receptor enhancer element (bottom panel). Light units were normalizd to total cellular protein. Luciferase measurements are expressed as fold activation over that obtained from reporter plasmids in the absence of MyoD expression.

Supplemental Figure 3. Identifying the region inMyoD regulated by p65-dependent transcription. (A) Schematic representation of deletions generated in MyoD expression plasmids. MyoD cDNA was removed from pEMC11s containing an LTR element and ligated into pCDNA3 (Invitrogen), generating the plasmid pCMV-MyoD(1785). Convenient restriction sites in MyoD and pCDNA3 were utilized to generate 5´ and 3´ deletions in MyoD sequences. (B) Inhibition of MyoD requires TNF-dependent transcription. C2C12 cells were switched to DM containing actinomycin D (5 μg/ml) to inhibit transcription. After a 1-hour preincubation period, cells remained in actinomycin D and were treated with or without TNF. At indicated times, RNA was extracted and MyoD mRNA was probed. MyoD amounts wree quantitated by scanning densitometry and normalized to levels of GAPDH mRNA.

Supplemental Figure 4. Decline in MHC levels is not due to apoptosis. C2C12 myocytes were grown in 12-well culture plates and differentiated in DM for a 3-day period. Cells were replaced with DM alone or with DM containing cytokines (20 ng/ml TNF and 100 U/ml IFN-γ). Treatments were repeated at times 6, 12, and 24 hours. At 48 hours, cells were fixed in 4% paraformaldehyde and double immunostained to detect for MHC (red) and for apoptotic cells (TUNEL, green). TUNEL-positive cells were predominantly undifferentiated (non-MHC-positive) cells. Scale bar, 15 μm.