Effect of the −2055~+77 DNA fragment of human ENDOU-1 gene on reporter gene expression in stress conditions. (A). Histograms presenting the relative luc activity obtained from HEK-293T cells co-transfected with pE2.1p (100 ng/well) and phRG-TK (20 ng/well) and then treated with either DMSO (control) or Thapsigargin (TH; stress inducer) for 2 or 6 h. HEK-293T cells treated with DMSO served as the control group. The relative luc activity was represented by the fold increase of Flu/Rlu ratio over that obtained from the control group normalized as 1. Values representing DMSO- and TH-treated cells were calculated from four independent experiments and presented as mean ±SD (n = 4). Student’s t-test was used to determine significant differences between each group (**, p < 0.05; ***, p < 0.005). (B). Histograms presenting the relative luc activity obtained from zebrafish embryonic cells microinjected at the one-cell stage with pE2.1p (6.9 pg/embryo) combined with phRG-TK (2.3 pg/embryo), followed by heat shock at 72 hpf and luc quantification at 96 hpf. Zebrafish embryos treated with microinjection, but not treated with heat shock, served as the control group. The relative luc activity was represented by the fold increase in the Flu/Rlu ratio over that obtained from the control group normalized as 1. Each value was averaged from three independent experiments and presented as mean ± SD (n = 3). Student’s t-test was used to determine significant differences between each group (***, p < 0.005).

Promoter activity driven by the −2055~+77 DNA fragment of human ENDOU-1 could be induced by various stress-inducing drugs. (A) HEK-293T cells co-transfected with pE2.1p and phRG-TK plasmid for 24 h and then treated with DMSO, Thapsigargin (TH), Anisomycin (Am), CoCl₂ (Co), Poly I:C (IC), or CuSO₄ (Cu) for 6 h, followed by analysis of luc activity via a dual-luc assay. Cells treated with DMSO served as a control group. The relative luc activity was represented by the fold increase in the Flu/Rlu ratio over that obtained from the pCS2-transfected control group, normalized as 1. The values were averaged from three independent experiments and presented as mean ± S.D. (n = 3). The Student’s t-test was used to perform statistical analysis (***, p < 0.001; **, p < 0.005) (B) Western blot analysis of proteins, as indicated, in HEK-293T cells. The α-tubulin served as internal control.

Comparison of luc expression activity driven by the DNA fragments −2055~+77 and −1125~+77 of human ENDOU-1 gene during ER stress. (A) In vitro system. Histograms presenting the relative luc activity obtained from HEK-293T cells cotransfected with either pE2.1p (containing −2055~+77 fragment; 100 ng/well) combined with phRG-TK (20 ng/well) (above) or pE1.2p (containing −1125~+77 fragment; 100 ng/well) combined with phRG-TK (20 ng/well) (bottom) in the absence (empty) or presence (solid) of ER stress inducers. HEK-293T cells treated with DMSO served as the control group. (B) In vivo system. Histograms presenting the relative luc activity obtained from zebrafish embryos microinjected at the one-cell stage with either pE2.1p (containing −2055~+77 fragment; 6.9 pg/embryo) combined with phRG-TK (2.3 pg/embryo) (above) or pE1.2p (containing −1125~+77 fragment; 6.9 pg/embryo) combined with phRG-TK (2.3 pg/embryo) (bottom), followed by no treatment (untreated; empty) or heat shock (solid) at 72 hpf and detection of luc activity at 96 hpf. Microinjected zebrafish embryos not exposed to heat shock served as the untreated control group. Relative luc activity was represented by the fold change of Flu/Rlu ratio over that obtained from the control group normalized as 1. Values representing untreated control and heat-shock-treated embryos were calculated from three independent experiments and presented as mean ± SD (n = 3). Student’s t-test was used to determine significant differences between pE2.1p- and pE1.2p-injected groups (***, p < 0.001; *, p < 0.05).

Comparison of the promoter activity of ENDOU-1 driven by different internal deletion clones from −2.1 kb of the ENDOU-1 fragment. (A) In vitro system. Histograms presented the relative luc activity obtained from HEK-293T cells cotransfected either phRG-TK (20 ng/well) combined with pE2.1p or phRG-TK (20 ng/well) combined with different internal deletions (100 ng/well), as indicated on the left side. For example, plasmid pE2.1p-d1850/1750 contained a −2.1 kb fragment of ENDOU-1, but deletion of −1850~−1750 nucleotides. The luc activities obtained from control and five deletion clones, as indicated, were quantified both during normal (DMSO) (represented by empty box) and Thapsigargin (TH)-induced stress condition (represented by solid box). HEK-293T cells treated with DMSO served as control group. (B) In vivo system. Histograms presenting the relative luc activity obtained from zebrafish embryos microinjected at the one cell stage with either indicated plasmids (6.9 pg/embryo) and phRG-TK (2.3 pg/embryo) followed by heat-shock treatment at 72 hpf and luminescence detection at 96 hpf. Microinjected zebrafish embryos not exposed to heat shock served as the control group. The relative luc activity was represented by the fold change in the Flu/Rlu ratio over that obtained from the control group normalized as 1. Values representing control and heat-shock-treated embryos were averaged from three independent experiments. A one-way ANOVA, followed by Tukey’s multiple comparison test, was used to perform statistical analysis (***, p < 0.001; error bars indicate mean ± SD).