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SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification <t>of</t> <t>XBP-1s</t> and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.
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SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification <t>of</t> <t>XBP-1s</t> and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.
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SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification <t>of</t> <t>XBP-1s</t> and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.
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SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification <t>of</t> <t>XBP-1s</t> and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.
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SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification <t>of</t> <t>XBP-1s</t> and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.
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SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification of XBP-1s and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.

Journal: Journal of Lipid Research

Article Title: S-nitrosylation contributes to ER stress and aggresome formation in secosterol-B-mediated endothelial dysfunction

doi: 10.1016/j.jlr.2026.101017

Figure Lengend Snippet: SEC-B induces UPR activation, leading to ER stress. A: Confocal microscopy of ER-Tracker Green-stained HUVECs after 4 and 24 h of treatment with 10 μM SEC-B. The scale bar represents 25 μm. B: TEM images also showed the disorganization and enlargement of ER membranes as indicated by the red arrow (the scale bar represents 0.5 μm). C and D: Western immunoblotting analysis and quantification of XBP-1s and ATF6 expression levels in HUVECs treated with 10 μM SEC-B. Values are the mean ± SD of the values obtained from five independent experiments. ∗∗∗ P < 0.001 versus untreated cells. E: Confocal images of GRP78 immunostaining in HUVECs after 24 h treatment with 10 μM SEC-B. The scale bar represents 20 μm. F: Aggresome formation detected by the ProteoStat® Aggresome Detection Kit after 24 h of 10 μM SEC-B treatment. The scale bar represents 25 μm. Quantitative analysis of positive fluorescent area per cell, in HUVECs treated with 10 μM SEC-B for 24 h. Data are expressed as mean ± SD (n = 3). ∗∗ P < 0.01 versus untreated cells.

Article Snippet: Blots were probed with the following antibodies: anti-spliced X-box binding protein 1 (XBP-1s) (E9V3E, #40435), ATF6 (D4Z8V, #65880), BiP/GRP78 (C50B12, #3177), and protein disulfide isomerase (PDI) (#2446) from Cell Signaling Technology; anti-endothelial NOS (eNOS) (A1548) from ABclonal; and anti-NOS2 (N-20, sc-651) from Santa Cruz Biotechnology.

Techniques: Activation Assay, Confocal Microscopy, Staining, Western Blot, Expressing, Immunostaining