Skip to main content
. 2016 Apr 25;35(45):5860–5871. doi: 10.1038/onc.2016.121

Figure 3.

Figure 3

NCOA3 is a transcriptional target of XBP1. (a) Schematic representation of wild type (pGL3-NCOA3-WT) and XBP1-binding site mutant (pGL3-NCOA3-MT) human NCOA3 promoter reporter constructs. The nucleotide sequence of human NCOA3 promoter from position −119 to −98 relative to the transcription start site is shown. (b) 293T cells were transfected with pGL3-NCOA3-WT along with control (FLAG) or expression plasmid for indicated UPR transcription factors. Luciferase activity was measured 24 h after transfection and normalized luciferase activity (Firefly/Renilla) relative to control is shown. Error bars represent mean±s.d. from three independent experiments performed in duplicate. (c) 293T cells were transfected with pGL3-NCOA3-WT along pcDNA3 (control) and pIRE1-ΔC (IRE1-DN). After 24 h of transfection, cells were either untreated (UN) or treated with (1.0 μm) TG for indicated time points. Normalized luciferase activity (Firefly/Renilla) relative to untreated control is shown. Error bars represent mean±s.d. from three independent experiments performed in duplicate. (d) 293T cells were transfected with pGL3-NCOA3-WT or mutant pGL3-NCOA3-MT (MT5 and MT7) along with control (PCDNA3) or spliced XBP1 (XBP1) expression plasmid. Luciferase activity was measured 24 h post transfection and normalized luciferase activity (Firefly/Renilla) relative to control is shown. Error bars represent mean±s.d. from three independent experiments performed in duplicate. (e) 293T cells were transfected with pGL3-NCOA3-WT or mutant pGL3-NCOA3-MT (MT5 and MT7). Twenty-four hour post transfection, cells were either untreated (UN) or treated with (1.0 μm) TG for indicated time points. Normalized luciferase activity (Firefly/Renilla) relative to untreated control is shown. Error bars represent mean±s.d. from three independent experiments performed in duplicate. *P<0.05, two-tailed unpaired t-test compared with untreated cells.