Fig 1: Deguelin promotes Mcl-1 ubiquitination in NSCLC cells.a HCC827 cells were transfected with various constructs as indicated and treated with deguelin for 24 h, then incubated with MG132 for another 6 h. WCE were harvested and subjected to in vivo ubiquitination assay. b HCC827 cells were transfected with various His-Ub mutant constructs as indicated. MG132 was added to the cell culture medium for 6 h before harvest. WCE was subjected to in vivo ubiquitination assay. c HCC827 (left) and H1975 (right) cells were treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was collected and subjected to endogenous Mcl-1 ubiquitination analysis. d HCC827 cells were treated with deguelin or/and SB216763 for 24 h, followed by incubation with MG132 for another 6 h. WCE was collected and subjected to endogenous Mcl-1 ubiquitination analysis. e HCC827 sh-GFP and sh-GSK3β stable cells were treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was harvested and subjected to endogenous ubiquitination analysis. f HCC827 cells were transfected with GSK3β WT or GSK3β S9A construct, treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was harvested and subjected to endogenous ubiquitination analysis.
Fig 2: Deguelin attenuates Mcl-1 expression in NSCLC cells.a Human NSCLC cells, including HCC827, H3255, H1975, and A549 cells, were treated with deguelin for 24 h, WCE were collected and subjected to IB analysis. b HCC827 cells were transfected with siCtrl or siMcl-1, followed by treatment with deguelin for 24 h, WCE were collected and subjected to IB analysis. c HCC827 cells were transfected with Mcl-1 construct, followed by treatment with deguelin for 24 h, WCE were collected and subjected to IB analysis. d HCC827 cells were treated with DMSO, PD98059, deguelin, or LY294002 for 24 h, WCE were collected and subjected to IB analysis. e HCC827 cells were transfected with Myr-Akt1 construct, followed by treatment with deguelin for 24 h, WCE were collected and subjected to IB analysis. f HCC827 cells were treated with deguelin or SB216763 for 24 h, WCE were collected and subjected to IB analysis as indicated. g HCC827 cells stable expression sh-GFP or sh-GSK3β were treated with deguelin or DMSO for 24 h, WCE were collected and subjected to IB analysis as indicated. h HCC827 cells were transfected with GSK3β WT or GSK3β S9A construct, followed by treatment with deguelin for 24 h, WCE was collected and subjected to IB analysis.
Fig 3: FBW7 is required for deguelin-induced Mcl-1 down-regulation.a HCC827 cells were treated with deguelin or DMSO for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to co-immunoprecipitation (co-IP) analysis. b HCC827 cells were transfected with various constructs and treated with deguelin or DMSO for 24 h, followed by incubation with MG132 for another 6 h. WCE was subjected to in vivo ubiquitination assay. c HCC827 sh-GFP and sh-FBW7 stable cells were treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was harvested and subjected to endogenous ubiquitination analysis. d HCC827 cells were transfected with various constructs and treated with deguelin for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to co-IP analysis. e HCC827 cells were treated with deguelin or/and SB216763 for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to co-IP analysis. f HCC827 cells were transfected with various constructs and treated with deguelin for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to endogenous ubiquitination analysis. g HCC827 cells were transfected with various constructs and treated with deguelin for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to in vivo ubiquitination analysis. h HCC827 cells were transfected with various constructs and treated with cycloheximide (CHX). WCE was harvested at different time points and subjected to IB analysis. i, j Cell viability i, and colony formation j of HCC827 cells transfected with Mcl-1 WT or Mcl-1 5KR and treated with deguelin. *p < 0.05, **p < 0.01.
Fig 4: Effect of AAT on Mcl-1 phosphorylation, the activity of MAP kinases and caspases.Neutrophils from healthy volunteers (2.5 × 106/ml) were cultured in medium supplemented with patient serum (3 mg protein/ml; 1%) and those containing low levels of AAT (AAT-reduce serum; 1%) in the presence of STS (0.2 μM). After 3 h, the expression of pMcl-1 (A, n = 7), pAkt (B, n = 8) and pERK1/2 (C, n = 10) were analyzed by western blot. Expression levels of the phosphorylated proteins were normalized to that of the unphosphorylated forms. GAPDH was used as loading control. One representative blot is displayed. *p<0.05; **p<0.01; n.s. = not significant. D. After 4 h of incubation the activities of caspase-9 and caspase-3/-7 were quantified. Results are presented as means ± SEM of eight independent experiments. No significant differences were found (one-way ANOVA with Newman keuls post-hoc test).
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