Fig. 2: Knockdown of SMURF1 enhances UPR signaling mediated-cell death. | Cell Death & Disease

Fig. 2: Knockdown of SMURF1 enhances UPR signaling mediated-cell death.

From: SMURF1 attenuates endoplasmic reticulum stress by promoting the degradation of KEAP1 to activate NRF2 antioxidant pathway

Fig. 2: Knockdown of SMURF1 enhances UPR signaling mediated-cell death.

A The LN229 and U343 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h or transfected with Flag-SMURF1 or Flag vector plasmid for 24 h, and treated with or without TG (1 μM) for 12 h. The whole cell extracts were analyzed by western blotting with antibodies against phospho-IRE1/IRE1, phospho-JNK/JNK, phospho-eIF2α/eIF2α, SMURF1, Flag and Actin. B, C Quantification of relative intensity of phospho-IRE1/IRE1, phospho-JNK/JNK and phospho-eIF2α/eIF2α in (A). D The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h or transfected with Flag-SMURF1 or Flag vector plasmid for 24 h, and treated with or without TG (1 μM) for 12 h. The whole cell extracts were analyzed by western blotting with antibodies against XBP1, ATF4, CHOP, SMURF1, Flag and Actin. E, F Quantification of relative intensity of sXBP1, ATF4 and CHOP in (D). G, H The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h (G) or transfected with Flag-SMURF1 or Flag vector plasmid for 24 h (H), and treated with or without TG (1 μM) for 12 h. The relative mRNA levels of sXBP1, ATF4, CHOP and SMURF1 were conducted by qRT-PCR analysis. I The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h and treated with or without TG (1 μM) for 16 h. The whole cell extracts were analyzed by western blotting with antibodies against Caspase3, BCL-2, SMURF1 and Actin. J Quantification of relative intensity of Cleaved Caspase3 and BCL-2 in (I). K The LN229 cells were transfected with Flag-SMURF1 or Flag vector plasmid for 24 h and treated with or without TG (1 μM) for 16 h. The whole cell extracts were analyzed by western blotting with antibodies against Caspase3, BCL-2, Flag and Actin. L Quantification of relative intensity of Cleaved Caspase3 and BCL-2 in (K). M The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h or transfected with Flag-SMURF1 or Flag vector plasmid for 24 h, and treated with or without TG (1 μM) for 16 h, then stained with Annexin-V/ propidium iodide (PI). The apoptotic cells were analyzed by flow cytometry. N Quantification of apoptosis in (M). O The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h and treated with or without 4-phenylbutyric acid (4-PBA) (10 μM) for 24 h. The whole cell extracts were analyzed by western blotting with antibodies against phospho-eIF2α/eIF2α, phospho-JNK/JNK, SMURF1 and Actin. P Quantification of relative intensity of phospho-eIF2α/eIF2α and phospho-JNK/JNK in (O). Q The LN229 cells were transfected with SMURF1 or scramble siRNA oligos for 72 h and treated with or without ISRIB (trans-isomer) (200 nM) for 12 h. The whole cell extracts were analyzed by western blotting with antibodies against ATF4, CHOP, SMURF1 and Actin. R Quantification of relative intensity of ATF4 and CHOP in (Q). Data are presented as mean ± SD, (*p < 0.05, **p < 0.01 and ***p < 0.001).

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