Fig. 3: SMURF1 knockdown enhances ROS production and impairs ERAD activity.

A The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h and treated with or without TG (1 μM) for 12 h. Cells were then stained with 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) and the ROS level was detected by flow cytometry. B Quantification of relative increase in mean fluorescence intensity in (A). C The LN229 cells were transfected with Flag-SMURF1 or Flag vector plasmid for 24 h and treated with or without TG (1 μM) for 12 h, then stained with DCFH-DA (10 μM), and the ROS level was detected by flow cytometry. D Quantification of relative increase in mean fluorescence intensity in (C). E The LN229 cells were transfected with CD3-δ-YFP plasmid for 12 h, and then transfected with SMURF1 or scramble siRNA oligos for 72 h. The whole cell extracts were analyzed by western blotting with antibodies against GFP, SMURF1 and Actin. F Quantification of relative intensity of CD3-δ-YFP in (E). G The 293A cells were transfected with CD3-δ-YFP plasmid for 12 h, and then transfected with SMURF1 or scramble siRNA oligos for 72 h and processed for immunofluorescence analysis. The nucleus was stained by DAPI (blue). Scale bar: 10 μm. YFP Excitation max (nm) 514 and Emission max (nm) 527. H The LN229 cells were transfected with CD3-δ-YFP plasmid for 12 h, and then transfected with SMURF1 or scramble siRNA oligos for 60 h. The LN229 cells were per-treated with TG (1 μM, 6 h) and then treated with cycloheximide (CHX, 100 μg/mL) for the indicated time. The whole cell extracts were analyzed by western blotting with antibodies against GFP, SMURF1 and Actin. I Quantification of CD3-δ-YFP band intensities relative to Actin. J The LN229 cells were transfected with CD3-δ-YFP plasmid for 12 h, and then transfected with HA-SMURF1 or HA vector plasmid for 24 h, per-treated with TG (1 μM, 6 h), followed by treatment with CHX (100 μg/ml). Cells were collected at the indicated time and analyzed by western blotting with antibodies against GFP, HA and Actin. K Quantification of CD3-δ-YFP band intensities relative to Actin. L The LN229 cells were transfected with CD3-δ-YFP plasmid for 12 h, and then transfected with SMURF1 or scramble siRNA oligos for 60 h, and treated with DMSO, NAC (2 mM) or tBHQ (20 μM) for 8 h. The whole cell extracts were analyzed by western blotting with antibodies against GFP, SMURF1 and Actin. M Quantification of CD3-δ-YFP band intensities relative to Actin. Data are presented as mean ± SD, (^*p < 0.05, ^**p < 0.01 and ^***p < 0.001).