GAS5 suppresses c-Myc expression at protein level.
(A) GAS5 was knocked down by siRNA in HEK-293T cells, and global protein translation was measured by [3H]-leucine incorporation assay. Bars represent mean ±SD from 3 replicates for Q-PCR and 6 replicates for [3H]-leucine incorporation assay. The experiments were performed two times. (B) pRF and pRF-SL plasmid were transfected into HEK-293T cells and cap-dependent protein translation efficiency was evaluated by luciferase assay. Bars represent mean ±SD from 4 replicates. The data were repeated three times. (C) The protein level of c-Myc (P = 0.007), Mcl1 (P = 0.88), survivin (P = 0.47) and Bcl2 (P = 0.47) was assessed by western blot (n = 3) after the cells were treated with GAS5 or control siRNA. (D) The mRNA level of c-Myc (P = 0.10), Mcl1 (P = 0.75), survivin (P = 0.31) and Bcl2 (P = 0.19) was quantified by Q-PCR after the cells were treated with GAS5 or control siRNA. Bars represent mean ±SD from 3 replicates. The experiment was repeated three times. (E) The protein level of c-Myc (P = 0.005) was assessed by western blot (n = 3) after the cells were transfected with in vitro transcribed GAS5 RNA. (F-G) The mRNA level of c-Myc (P = 0.37) (F) and GAS5 (G) was quantified by Q-PCR after the cells were transfected with in vitro transcribed GAS5 RNA. Bars represent mean ±SD from 3 replicates. The experiment was performed three times.
