Figure 6

Inhibition of NF-κB subunit by siRNA downregulates Mcl-1 expression and suppresses the viability of TE-1 cells. (A, B) Time course experiments performed with p50 or p65 siRNA utilized. Representative Western blots of endogenous p50, p65 or Mcl-1expression in TE-1 cells at various time points following transfection with 100 nM p50 (A) or p65 (B) siRNA. β-actin was used as a loading control. Data shown are representative of two independent experiments. Statistical significance: * p < 0.05, compared with the siRNA-transfected TE-1 cells at 0 h. (C) Growing TE-1 cells were transiently transfected with 100 nM of control, p50 or p65 siRNA and cultured in the medium for 72 h. Knockdown of endogenous p50 or p65 and expression of Mcl-1 were analyzed by Western blotting. β-actin was used as a loading control. Data represented the mean ± S.D. of two separate experiments. Statistical significance: * p < 0.05 compared with the si-Ctrl-transfected TE-1 cells. (D, E) Reintroduction of Mcl-1 to Mcl-1-downregulated TE-1 cells caused by NF-κB subunit siRNA restored cell viability. TE-1 cells were sequentially transfected with NF-κB subunit siRNA and Mcl-1 expression plasmid as described in Methods. Cells were analyzed the Mcl-1 levels at 120 h after siRNA transfection by Western blotting (D) and measured cell viability by WST-1 assay at 24 h-intervals up to 120 h after siRNA transfection (E). Data represented the mean ± S.D. of two separate experiments. Statistical significance: * p < 0.05, compared with the si-Ctrl and pCMV6-A-Puro empty vector co-transfected TE-1 cells. # p < 0.05, compared the si-p50 and pCMV6-A-Puro empty vector co-transfected with the si-p50 and pCMV6-A-Puro-Mcl co-transfected TE-1 cells. ** p < 0.05, compared the si-p65 and pCMV6-A-Puro empty vector co-transfected with the si-p65 and pCMV6-A-Puro-Mcl co-transfected TE-1 cells.