Supplementary Figure 7: A20 expression is induced by the Smad-independent noncanonical pathway upon TGF-β1 treatment.

(a) Gating strategy of CD44(+)/CD24(-) cancer cell populations in A20-depleted and control M4 (MCF10CA1a) cells. M4 cells were initially gated by FSC-A versus FSC-H for single cells and these separated cells were further gated by FSC-A versus SSC-A for the exclusion of debris. Live cells were finally gated by using fixable dye, APC-Cy7. (b) After NMuMG cells were pre-treated with the TGF-β type I receptor inhibitor SB431542 (10 μM) for 1 h, they were treated with TGF-β1 (5 ng ml⁻¹) for the indicated times. A20 expression and Smad2 phosphorylation were monitored by immunoblot analysis. (c,d) NMuMG cells expressing Smad4-specific shRNAs or GFP-specific control shRNA were treated with TGF-β1 for the indicated times. A20 expression was analyzed by immunoblot (c) and quantitative real-time RT-PCR (d) analysis. In qRT-PCR analysis, expression of Gapdh mRNA was used for normalization. The data in d were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. All data of immunoblot analysis shown in this figure are representative of n = 3 independent experiments. Expression of β-actin was used as a loading control for the immunoblot analysis. Statistics source data for d are available in Supplementary Table 3. Unprocessed original scans of blots in b and c are in Supplementary Fig. 9.