Nucb2

Human melanoma cell lines Me1007, Mel-RM, A375, Mel-CV, and SK-Mel-28 were obtained as previously described [21 (link)]. Studies on a panel of melanoma cell lines revealed that ER stress did not induced significantly apoptosis in Mel-RM, A375, Mel-CV, SK-Mel-28 but Me1007. Me1007 was reported to be a ER stress non-resistant melanoma cell line [22 (link)]. These cells were cultured in DMEM containing 10% FBS (ExCell Bio, Lot: FSP500) and 0.1% Savelt™ (Hanbio Co. LTD., 1:1000). The following antibodies were used in this study: antibodies against GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778, 1:1000); PARP (Santa Cruz Biotechnology, SC-8007, 1:1000); KLF4 (Santa Cruz Biotechnology, SC-20691, 1:10 for ChIP); KLF4 (Cell Signaling Technology, #12173S, 1:500); NUCB2 (Abcam, ab229683, 1:300); KLF8 (Abcam, ab168527, 1:300); KLF5 (Proteintech, 21,017–1-AP 1:500); KLF6 (Proteintech, 14,716–1-AP 1:500); GRP78 (Santa Cruz Biotechnology, SC-13968,1:1000). Tunicamycin (TM, Lot:T7765) and thapsigargin (TG, Lot:T9033) were purchased from Sigma Chemical Co. They was dissolved in DMSO and made up in stock solutions of 3 mmol/L for Tunicamycin and 1 mmol/L for thapsigargin. The cells were treated with 3 μmol/L TM or 1 μmol/L TG as the indicated times.

The human melanoma cell lines Mel-RM, Mel-CV, ME4405, A375 and Me1007 were cultured in Dulbecco’s modified Eagle’s medium (Gibco Invitrogen) containing 10% foetal bovine serum (ExCell Bio). Sk-Mel-28 and H1299 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco Invitrogen) containing 10% foetal bovine serum (ExCell Bio). The following antibodies were used in this study: GAPDH (Santa Cruz Biotechnology, SC-25778, 1:1000), ITIH5 (Sigma, SAB1303414, 1:50), actin (Santa Cruz Biotechnology, sc-1616, 1:1000), KLF4 (Cell Signalling Technology, 12173 S, 1:500), NUCB2 (Abcam, ab229683, 1:300), Flag (Sigma, F3165, 1:1000) and GFP antibodies (Proteintech, 66002-1-Ig, 1:1000).