Figure 7: Dub3 is critical for IL-6 induced Snail1 stabilization.

(a) MDA-MB157 and MDA-MB231 cells were serum starved for 24 h, then treated with IL-6 for different time intervals. Expression of endogenous Snail1 and Dub3 were assessed by western blot. (b) MDA-MB231 and MDA-MB157 cells with Dub3-knockdown by two individual shRNAs or vector control were serum starved for 24 h followed by IL-6 treatment for 4 h. Expression of endogenous Snail1 and Dub3 were assessed by western blot. (c) A predicted model structure of Dub3 and WP1130 complex. The Dub3-UCH domain structure (yellow) is shown as ribbons while WP1130 (magenta) and the catalytic triad residues (sky blue: Cys89, His334, and Asp350) are shown as sticks. The inhibitor molecule binds to the opening surface of the active site where the ubiquitin cleavage site is presented. A zoom-up view of the interaction network is shown in the inset where the interacting residues are represented by sticks. These interactions include hydrogen bonds with backbone atoms (dotted lines) and potential halogen (Br) bonding interactions with the neighbouring electronegative atoms. Normalized melting curves (Right panel) depicting dose-dependent shifted thermal stability of Dub3 by WP1130 (coloured lines) from that of the apo protein (grey line), but not by a furan compound (dotted line; negative control). Tm for apo protein and ΔTm values for compound-bound proteins are indicated. (d) MDA-MB231 and MDA-MB157 cells stably expressing control vector or Dub3 shRNA were treated with 0.5 μM WP1130 for 24 h and analysed for cell migration using a wound healing assay. Graphic representation is the percentage of migration (mean±s.e.m. in three separate experiments). (e) MDA-MB231 and MDA-MB157 cells stably expressing control vector or Dub3 shRNA were treated with 0.5 μM WP1130 for 4 h and analysed for cell invasion. Graphic representation is the percentage of invasive cells (mean±s.e.m. in three separate experiments in duplicates). (f) MDA-MB231 and MDA-MB157 cells stably expressing control vector or Dub3 shRNA were treated with 0.5 μM WP1130 and analysed for tumorsphere-formation. Graphic representation is the number of tumorspheres (mean±s.e.m. in three separate experiments). (g,i) MDA-MB231-luc cells were injected into the mammary fat pad of SCID mice. When tumours reached 100 mm³, mice were divided into two groups and treated with WP1130 (50 mg kg⁻¹) or solvent, respectively. Tumour size was recorded by bioluminescence imaging before or after 2-week of treatment (g). Tumour growth (h) and weights (i) were measured. Presented data are the mean±s.d. from six mice. *P≤0.05, **P≤0.01; Student's t-test.