Figure 4. Effects of Kaletra on muscle IGF1-related molecular changes in skeletal muscle and cultured myotubes. (A) Messenger RNA expression of IGF-1Ea and related muscle atrogenes in tibialis anterior (TA), quadriceps (Quad) and gastrocnemius (Gastro) muscle groups. Expression of each gene was first normalized to house-keeping gene HPRT and presented as a fraction of the control. (B, left panel) Western analysis of phosphor-Foxo3aser318/321, phosphor-Foxo1ser256, and total Foxo3a in quadriceps muscle, with GAPDH as loading control. (B, right panel) Western analysis for phosphor-mTORser2448, phosphor-eiF4Gser1108, phosphor-eiF4Bser422, and phosphor-S6ser235/236 in quadriceps, with GAPDH as loading control. Results are representative of N = 6 for each group with three of each group for display. (C) Expression of IGF1, myogenin (MyoG), MAFbx and MuRF1 in cultured C2C12 myotubes exposed to different concentration of lopinavir (lopinavir:ritonavir = 4:1). The experiment was repeated twice in duplicates. Results were normalized to house-keeping gene HPRT and shown as means+/- se, a > b > c, p < 0.05, analyzed by One-way ANOVA with post hoc Tukey’s test. (D) Western analysis of C2C12 myotubes treated with low and high dose of IGF1 for the phosphorylation of relevant downstream targets. Myotubes were pre-incubated with lopinavir/ritonavir (4:1) at 3 ug/ml of lopinavir in 2% horse serum for 24 h. Cells were then washed with serumfree medium and incubated with serum-free medium added the same concentration of lopinavir/ritonavir for two hours. IGF1 was then added and cells were harvested after 30 mins. Results are representative of three independent experiments.
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