Journal: Oncogene

Article Title: NCOA3 coactivator is a transcriptional target of XBP1 and regulates PERK–eIF2α–ATF4 signalling in breast cancer

doi: 10.1038/onc.2016.121

Figure Lengend Snippet: Upregulation of NCOA3 during UPR is mediated by the IRE1–XBP1 pathway. ( a ) MCF7 cells were either untreated (CTRL) or treated with (1.0 μ m ) TG in the absence and presence of IRE1 inhibitors, (10 μ m ) 4μ8C (4 U) and (100 μ m ) STF083010 (STF) for 24 h. Cells were harvested and expression of XBP1 (unspliced and spliced) was analysed by RT–PCR followed by gel electrophoresis. ( b ) MCF7 cells were treated with TG (1.0 μ m ) in the absence and presence of (25 n m ) GSK-PERK inhibitor (PI) for indicated time points. Whole-cell lysates were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting using PERK antibody. ( c ) MCF7 cells were either untreated (CTRL) or treated with TG (1.0 μ m ) and (1.0 μg/ml) TM in the absence and presence of IRE1 inhibitors as in ( a ), and the expression level of NCOA3 was quantified by real-time RT–PCR, normalizing against RPLP0. Error bars represent mean±s.d. from three independent experiments performed in triplicate. ( d ) MCF7 cells were either untreated (CTRL) or treated with (1.0 μ m ) TG in the absence and presence of (25 n m ) GSK-PERK inhibitor (PI) for 24 h. The expression level of NCOA3 was quantified by real-time RT–PCR, normalizing against RPLP0. Error bars represent mean±s.d. from three independent experiments performed in triplicate. ( e ) MCF7 cells were transfected with (CTRL) pcDNA3-FLAG or plasmids expressing indicated UPR transcription factors (ATF4, ATF6, CHOP, NRF2 and XBP1-S). Cells were harvested 24 h post transfection normalizing against RPLP0. Error bars represent mean±s.d. from three independent experiments performed in triplicate. Equivalent amounts of cell lysates were resolved by SDS–PAGE and immunoblotting was performed using antibodies against FLAG, ATF6, spliced XBP1 and β-actin. ATF4, CHOP and NRF2 are FLAG-tagged. β-Actin served as a loading control. ( f ) MCF7-control (PKLO) and MCF7-XBP1 knockdown (XKD) cells were treated with (1.0 μ m ) TG for indicated time points. The expression levels of GRP78, XBP1-S and NCOA3 was quantified by real-time RT–PCR, normalizing against RPLP0. Error bars represent mean±s.d. from three independent experiments performed in triplicate. * P <0.05, two-tailed unpaired t -test compared with untreated cells; ‡ P <0.05 for one-way ANOVA; ** P <0.05, two-tailed unpaired t -test comparing respective time points; NS, not significant at P <0.05.

Article Snippet: To inhibit IRE1 endoribonuclease activity, cells were treated with IRE1 inhibitor (4μ8C) (Cat # 412512; Merck Millipore Ltd, Cork, Ireland) and STF083010 (Cat# 412510; Merck Millipore Ltd).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Quantitative RT-PCR, Transfection, Control, Knockdown, Two Tailed Test

Journal: Oncogene

Article Title: NCOA3 coactivator is a transcriptional target of XBP1 and regulates PERK–eIF2α–ATF4 signalling in breast cancer

doi: 10.1038/onc.2016.121

Figure Lengend Snippet: IRE1–XBP1 signalling is required for E2-mediated growth and expression of NCOA3. ( a ) MCF7 cells were treated with (10 n m ) oestrogen in the absence and presence of IRE1 inhibitors, (10 μ m ) 4μ8C (4 U) and (100 μ m ) STF083010 (STF) for indicated time points. Line graphs show the absorbance in cells at the indicated time points after the treatment with IRE1 inhibitors. ( b ) MCF7 cells were treated as in ( a ), for 24 h. Expression of XBP1-S, GREB1 and NCOA3 was quantified by real-time RT–PCR, normalizing against RPLP0. Error bars represent mean±s.d. from three independent experiments performed in triplicate. ( c ) MCF7-control (PKLO) and MCF7-XBP1 knockdown (XKD) cells were synchronized as described in the Materials and methods section. After synchronization, cells were treated with (10 n m ) E2 in 1% dextran-coated charcoal-stripped fetal bovine serum (DCC-FBS) supplemented medium. Line graphs show the absorbance in cells at the indicated time points after the E2 treatment. Error bars represent mean±s.d. from three independent experiments performed in triplicate. ( d ) MCF7-control (PKLO) and MCF7-XBP1 knockdown (XKD) cells were treated as in ( c ), for indicated time points. Expression of spliced XBP1 (XBP1-S), total XBP1 (XBP1-F), GREB1 and NCOA3 was quantified by real-time RT–PCR, normalizing against RPLP0. Error bars represent mean±s.d. from three independent experiments performed in triplicate. * P <0.05, two-tailed unpaired t -test compared with untreated cells; ‡ P <0.05 for one-way ANOVA; ** P <0.05, two-tailed unpaired t -test comparing respective time points.

Article Snippet: To inhibit IRE1 endoribonuclease activity, cells were treated with IRE1 inhibitor (4μ8C) (Cat # 412512; Merck Millipore Ltd, Cork, Ireland) and STF083010 (Cat# 412510; Merck Millipore Ltd).

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Two Tailed Test