Fig. 5. Covalent binding between WIT and Cys239 accounts for WIT-induced tubulin degradation. (A) Selected amino acid sequences of β2-, β3-, β4-, β5 (β)-, and β6-tubulin isoforms. (B) HeLa cells were treated with increasing concentrations of WIT for 16 hours, and β2-, β3-, β4- and β6-tubulin protein levels were detected via Western blot. The right image depicts quantitative data on β2-, β3-, β4-, and β6-tubulin protein expression. Values are presented as mean ± 95% CI of three independent experiments. (C) HeLa cells were transiently transfected with MSCV-IRES-GFP vectors coexpressing GFP and WT-FLAG-β-Tubulin, C239S-FLAG-β-Tubulin, or C303S-FLAG-β-Tubulin for 24 hours. Cells were treated with or without 3 µM WIT for 16 hours, and GFP and FLAG-tubulin detected via Western blot. The right image depicts quantitative data on GFP and FLAG-tubulin. Values are presented as mean ± 95% CI of three independent experiments. (D) Chemical structure of dihydrowithaferin A (DWIT). (E) HeLa cells were treated with increasing concentrations of WIT or DWIT for 16 hours, and β-tubulin protein was detected via Western blot. The right image depicts quantitative data on β-tubulin protein. Values are presented as mean ± 95% CI of three independent experiments. (F) HeLa cells were treated with increasing concentrations of WIT or DWIT for 48 hours, and cell viability was assessed with the trypan blue exclusion assay.Values are presented as mean ± 95% CI of three independent experiments. β-Tub, β-tubulin; β2-Tub, β2-tubulin; β3-Tub, β-3tubulin; β4-Tub, β4-tubulin; β6-Tub, β6-tubulin.