Journal: iScience
Article Title: Regulation of the endoplasmic reticulum stress sensor ATF6α through multiple oxidoreductases in the ER
doi: 10.1016/j.isci.2026.116290
Figure Lengend Snippet: C618 dimers suppress C467 dimerization and its transport to the Golgi apparatus (A) Evaluation of ATF6α glycan levels. ATF6α-KO cells were transfected with p3×FLAG-tagged WT ATF6α and pretreated with 2 mM BADAP and 50 μM PF429242 (S1P inhibitor) for 1 h prior to the induction of ER stress with 5 μM Tg, and the results were analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. The cell lysates were left untreated or treated with Endo H. g and u indicate glycosylated and unglycosylated proteins, respectively. Original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study are contained in . (B) Transport and C467 dimerization of the ATF6α mutant during ER stress. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or C618A mutant ATF6α, pretreated with 50 μM PF429242 for 1 h prior to the induction of ER stress with 5 μM Tg for 0, 15, and 30 min, and analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (C) The intensity of the C467 dimer bands, as shown in (B). The bands were quantified using the ImageJ software and normalized to the total intensity (monomer, C618 dimer, and C467 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (D) The intensity of the C618 dimer bands, as shown in (B). The bands were quantified using ImageJ, normalized to the total intensity (monomer, C618 dimer, C467 dimer, and Golgi-C467 dimer), and plotted against time. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (E) The intensity of the Golgi-C467 dimers, as shown in (B). The bands were quantified using ImageJ, normalized to the total intensity (monomer, C618 dimer, C467 dimer, and Golgi-C467 dimer), and plotted against the time. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (F) Interaction between ATF6α and BiP. ATF6α-KO cells were transfected with p3×FLAG-tagged ATF6α and Myc-tagged BiP, and cell lysates were prepared for immunoprecipitation with an anti-Myc antibody. The immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies under nonreducing and reducing conditions. Representative immunoblots from three independent experiments are shown ( n = 3). (G) The intensity of each ATF6α band, as shown in (F). The bands were quantified using ImageJ software and normalized to the BiP (Input/IP) intensity. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (H) Transport of ATF6α mutant during ER stress. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or mutant ATF6α, pretreated with 50 μM PF429242 for 1 h prior to the induction of ER stress with 5 μM Tg for 1 h, and analyzed by immunoblotting with anti-FLAG antibodies under reducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (I) The intensity of the Golgi-ATF6α bands, as shown in (H). The bands were quantified using the ImageJ software and normalized to the total intensity (ATF6α-F and Golgi-ATF6α). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (J and K) Immunofluorescence of HeLa cells exogenously expressing GFP-tagged WT or mutant ATF6α. HeLa cells were transfected with N-terminal GFP-fused ATF6α (WT and various mutants). For ER localization analysis, cells were fixed and stained with calnexin (CNX), an ER-resident protein, without Tg treatment (J). For Golgi localization analysis, cells were pretreated with 50 μM PF429242 for 1 h and subsequently treated with 500 nM Tg for 30 min before staining with GM130, a Golgi marker (K). All cells were counterstained with Hoechst 33342 and immunostained with the indicated antibodies. Scale bars, 5 μm. (L) The percentage of cells showing colocalization between GFP-fused ATF6α and GM130, as shown in (K). The means ± standard error of the mean (SEM) of at three independent experiments, each performed with 10 cells. Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05). (M) ATF6α-KO cells were transfected with p3×FLAG-tagged WT or mutant ATF6α, pretreated with 5 μM Tg for 0, 30, and 60 min, and analyzed by immunoblotting with anti-FLAG antibodies under reducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (N) The intensity of ATF6α-N bands at 30 min after Tg treatment, as shown in (M). The bands were quantified using ImageJ, normalized to the total intensity (ATF6α-F and ATF6α-N). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05). (O) BiP mRNA levels in ATF6α-KO cells overexpressing WT or mutants under Tg treatment conditions. After treatment with 500 nM Tg for 8 h (+Tg), BiP mRNA levels were quantified by qPCR. The expression levels were normalized to GAPDH and compared to those in untreated cells (−Tg). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01).
Article Snippet: The DOI for the Mendeley Data is listed in the . contains original uncropped western blot images used for quantification (n = 3 independent biological replicates), and contains original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study.
Techniques: Glycoproteomics, Transfection, Western Blot, Mutagenesis, Control, Software, One-tailed Test, Immunoprecipitation, SDS Page, Immunofluorescence, Expressing, Staining, Marker
