Supplementary Figure 1: A20 does not affect the canonical TGF-β/Smad signaling, but stabilizes the Snail1 protein.

(a) A20-knockdown and shGFP-expressing NMuMG cells were transfected with a Smad- specific CAGA-Luc reporter. Cells were treated with TGF-β1 (5 ng ml⁻¹) for 24 h, and luciferase activities were measured and normalized. n.s., not significant. (b) NMuMG cells were reverse-transfected with 20 nM of control siRNA (siCON) or four different siRNAs targeting mouse A20 mRNA. Knockdown efficiency was confirmed by immunoblot analysis with anti-A20 antibody. (c–e) Quantitative real-time RT-PCR analysis of indicated target genes, induced by the TGF-β/Smad-dependent signaling pathway, in A20-knockdown NMuMG cells treated with TGF-β1 for 24 h. The data in a,c,d, and e were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. (f) Stability of the Snail1 protein was measured in A20-knockdown and shGFP-expressing control NMuMG cells in the presence of TGF-β1, followed by treatment of protein translation inhibitor, cycloheximide (CHX, 50 μg ml⁻¹) for the indicated times. Cell lysates were immunoblotted by the indicated antibodies (upper). Data were quantified using ImageJ software (lower). For normalization, expression of β-actin was used as a control. (g) A20-knockdown NMuMG cells were treated with TGF-β1 for 24 h, followed by exposure to proteasomal inhibitor MG132 (10 μM) for 6 h. Cell lysates were immunoblotted with the indicated antibodies. (h) A plasmid encoding Flag-Snail1 was co-transfected with increasing amounts of HA-A20 plasmid into HEK293 cells. Cell lysates were immunoblotted. (i) Panc-1 cells were reverse-transfected with 20 nM of control siRNA or two independent A20 siRNAs (siA20 #1 and siA20 #3) and treated with TGF-β1 for the indicated times. Cell lysates were immunoblotted. (j) A20-knockdown and shGFP-expressing control NMuMG cells were transfected with Flag-Snail1 and then treated with TGF-β1 for 24 h. Cell lysates were immunoblotted. Expression of β-actin was used as a loading control for all immunoblot analysis shown in this figure. Immunoblot images are representative of n = 3 independent experiments. Statistics source data for a and c–e are available in Supplementary Table 3. Unprocessed original scans of blots in b and f–j are in Supplementary Fig. 9.