Journal: The Journal of cell biology

Article Title: PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

doi: 10.1083/jcb.202407166

Figure Lengend Snippet: Figure 3. PITPβ promotes contact between the ER and COPI buds on the Golgi. Quantitative data are shown as mean ± SD, with the number of in- dependent experiments indicated. Statistics was performed using the two-tailed Student’s t test: ****P < 0.0001, ***P < 0.001, **P < 0.01, ns (non-significant) P > 0.05. (A) Colocalization of PITPβ with ER marker (Sec61β) and Golgi marker (giantin) as assessed by confocal microscopy using Airyscan, PITPβ (blue), Sec61β (green), giantin (magenta), n = 6. Representative images with scale bars are shown with inset highlighting PITPβ colocalizing with both Sec61β and Giantin (indicated by arrowheads). A reconstruction of this region is also shown, with a line scan along the dotted line providing quantitative information, as well as single-channel images. (B) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a Golgi marker (Giantin). Quantitation of a representative experiment is shown, n = 3. (C) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a TGN marker (TGN46). Quantitation of a representative experiment is shown, n = 3. (D) EM tomography showing COPI buds at the Golgi in close proximity to the ER membrane. A representative tomographic image slice is shown in the upper left panel with arrowheads pointing to COPI buds, bar = 200 nm. 3D reconstruction of the Golgi, COPI buds, and ER elements is shown in the lower left panel. Quantitation of a representative experiment is shown on the right, comparing the distance between COPI buds and ER membranes versus the distance between Golgi cisternal margins and ER membranes, n = 3. (E) Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and giantin. Quantitation of a representative experiment is shown on right, n = 3. Representative confocal images are shown on left, PLA signal (red),

Article Snippet: VAP-A (104447) and VAP-B (104448) in pEGFP-C1 and mEmerald-tagged Sec61β in pEGFP-C1 (90992) were obtained from Addgene.

Techniques: Two Tailed Test, Marker, Confocal Microscopy, Quantitation Assay, Tomography, Membrane, Proximity Ligation Assay

Journal: Cellular oncology (Dordrecht)

Article Title: Single-cell RNA profiling identifies diverse cellular responses to EWSR1/FLI1 downregulation in Ewing sarcoma cells

doi: 10.1007/s13402-021-00640-x

Figure Lengend Snippet: A, Subpopulation studies reveal down-regulation of FAM134B and up-regulation of LC-3-like modifiers (MAPLC3B, GABARAPL2) and ER stress response proteins in cluster C2 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA, error bars indicate S.D.); B, Fluorescence images of EW-8 cells transfected with si-Control or si-EWSR1/FLI1. EW-8 cells were fixed in D1, immunostained and subsequently imaged via fluorescent microscopy to study the induction of the autophagy marker MAPLC3B and the lysosomal marker CD63; C, Quantification of FAM134B co-localization with LCB3, presented as Pearson’s correlation coefficient (r); **** p < 0.0001, t-test, n = 15 fields (~150 cells) in the dormant cells shown in A using the colocalization tool in imageJ; D, EW-8 cells were treated with si-EWSR1/FLI1 or si-Control 48 h before being stained for the ER resident protein SEC61B. The expansion of ER is shown following EWSR1/FLI1 downregulation (boxed areas). E, Quantification of cells with expanded ER after masking nuclei. **** p < 0.0001, t-test, error bars indicate S.D., n = 150 cells.

Article Snippet: As secondary antibody Alexa Fluor 488 (ab150077; Abcam) against FAM134B, SEC61B (sc-393633; Santa Cruz) was used.

Techniques: Fluorescence, Transfection, Control, Microscopy, Marker, Staining

Journal: bioRxiv

Article Title: Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture

doi: 10.1101/2023.12.21.572811

Figure Lengend Snippet: BAF controls rupture diffusion in a size-dependent manner. A) Measurements of the width of the NE rupture gap in BJ5ta cells expressing GFP-Sec61β. Number of cells analyzed: siControl, n=15; siBAF, n=14. Error bars indicate ± SEM from triplicate experiments. B) Representative images of BJ5ta cells expressing either Hsp90-GFP or α-Tubulin-GFP after laser-induced NE rupture. Scale bar; 10 µm. C) Quantification of the nuclear-to-cytoplasmic ratio of cells expressing either Hsp90-GFP or α-tubulin-GFP treated with either siControl (n = 29 and 14, respectively) or siBAF (n = 21 and 16, respectively) from triplicate experiments. Error bars indicate ± SEM. D) Initial rate of increase into the nucleus following NE rupture for cells in B. E) Representative images of BJ5ta cells expressing Hsp90-GFP or α-tubulin-GFP during FLIP. Green circle indicates area of photobleaching in the cytoplasm. Red circle indicates area of measurement. F) FLIP measurements showing relative mobility of Hsp90-GFP (n = 16 cells) or α-tubulin-GFP (n = 16 cells). The slope of each line x was used to factor relative mobility between Hsp90-GFP and α-tubulin-GFP. G) Mobility of Hsp90-GFP and α-tubulin-GFP (measured as the best line-of-fit slope for each individual cell). H) Initial rate of increase into the nucleus following NE rupture for cells in D normalized by mobility. Error bars indicate ± SEM. Statistical significance: *, P<0.05; **<0.005; ***, P<0.0005 using an unpaired student t-test.

Article Snippet: GFP-Sec61β in pBabe puro (a generous gift from Indra Chandrasekar) was created by PCR amplifying Sec61β (a gift from Gia Voeltz, Addgene plasmid #49154) and recombining into XhoI-SalI cut GFP pBabe puro.

Techniques: Diffusion-based Assay, Expressing

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Misaligned chromosomes outside the exclusion zone are ensheathed in endomembranes. (A) Confocal image of a mitotic RPE-1 cell stably coexpressing GFP-Sec61β (green) Histone H3.2-mCherry (DNA, red) and stained with SiR-Tubulin (gray). Scale bar, 10 µm. (B) SBF-SEM imaging of mitotic cells and subsequent segmentation reveals the endomembranes (ER, blue) and mitochondria (Mito, orange) beyond the exclusion zone boundary (EZ, pink), with the chromosomes (DNA, gray) within. Angle of rotation about y axis is shown. Scale bar, 2 µm. (C) Confocal image of an untreated HeLa cell coexpressing GFP-Sec61β (green) and Histone H2B-mCherry (magenta) with a spontaneously occurring ensheathed chromosome. (D) SBF-SEM imaging of an untreated HeLa cell with a spontaneously occurring ensheathed chromosome. Model shows the position of two ensheathed chromosomes (red) away from the metaphase plate; height of slice 232 is indicated. Scale bar, 2 µm. Segmentation shows endomembranes (green and lilac surrounding the chromosome marked with a star), rendered in 3D (reconstruction). Scale bars, 1 µm. See and .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Induction of misaligned chromosomes in stably diploid RPE1 cells by pretreatment with a CENP-E inhibitor. (A) Polar, misaligned chromosomes can be induced by treatment with CENP-E inhibitor GSK923295 (150 nM, 3 h) and subsequent washout (1 h). (B) Confocal micrographs to show that these misaligned chromosomes (SiR-DNA, red) are either outside the exclusion zone delineated by GFP-Sec61β (green), termed ensheathed, or at the boundary and inside the exclusion zone, termed free. Scale bars, 10 µm; 1 µm (inset). (C) Spatially averaged 3D view of all CENP-C–positive kinetochores in the dataset; see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (free, blue). Spindle poles are shown in black. (D) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Chromosome misalignment was induced by pretreatment with GSK923295 (150 nM). Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log2 scale. Dots represent kinetochore ratios from 31 RPE-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Inset: Schematic diagram to show how the position of kinetochores relative to the exclusion zone boundary was calculated. C is the centroid of aligned kinetochores, P is a kinetochore, and Q is the point along the 3D path ( CP ) that intersects the exclusion zone boundary. The ratio of CP to CQ is taken for each kinetochore (aligned kinetochores, gray; free, blue; and ensheathed, orange). (E) Single SBF-SEM image showing an ensheathed chromosome. Boxed region is shown expanded and modeled (zoom). Single slice and a 3D model (bottom right) of slices 87–126 are shown. Scale bar, 2 µm (black) and 500 nm (white). (F) Modeled substacks from SBF-SEM images showing a chromosome outside the exclusion zone, ensheathed in ER. Slices shown and angles and axes of rotation are indicated (see ). Scale bar, 2 µm.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Ensheathed chromosomes in DLD-1 cells after targeted missegregation of Y-chromosome. (A) Schematic diagram after , showing how reexpression of a CENP-A mutant (C-H3) in DLD-1 cells where CENP-A is degraded causes selective misalignment of the Y-chromosome. WT and C-H3 lines were further modified to express mCherry-Sec61β. (B) Western blot of lysates from WT or C-H3 DLD-1 cells treated with doxycycline (Dox) and/or indole-3-acetic acid (IAA) as indicated. Upper blot shows anti-CENP-A detection of endogenous CENP-A fused to EYFP-AID tag (66 kD) and expression of untagged CENP-A (either WT or C-H3). Lower blot shows GAPDH loading control. (C) Typical FISH images locating the Y-chromosome in the main nucleus in control cells and in a micronucleus in cells expressing C-H3 CENP-A. Scale bar, 10 µm. (D) Western blot of lysates from stable cell lines expressing mCherry-Sec61β derived from WT (G2) or C-H3 (G11). Detection of Sec61β or mCherry is shown as indicated with actin loading controls. Migration of Sec61β and mCherry-Sec61β is indicated by black and red arrowheads, respectively. Note that the expression of mCherry-Sec61β downregulates endogenous Sec61β. (E) Deconvolved wide-field microscopy images showing an ensheathed chromosome in G11 cells but not in G2 cells treated with Dox/IAA. Scale bars, 10 µm; 2 µm (insets). (F) Spatially averaged view of all kinetochores in the G11 DLD-1 Dox/IAA dataset (see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (blue). Spindle poles are shown in black. (G) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log 2 scale. Dots represent kinetochore ratios from 50 DLD-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Source data are available for this figure: .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Mutagenesis, Modification, Western Blot, Expressing, Stable Transfection, Derivative Assay, Migration, Microscopy

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Impact of ensheathed chromosomes on cell division. (A) Mitotic timing of RPE-1 cells. Cumulative frequencies for NEB to metaphase (NEB-Meta) and metaphase to anaphase (Meta-Ana) are shown. RPE-1 stably expressing GFP-Sec61β were treated with 150 nM GSK923295 for 3 h before washout. Three classes of metaphase were seen: all chromosomes aligned (Aligned, n = 29), cells with one or more free chromosomes (Free, n = 11), and cells with one or more ensheathed chromosome (Ensheathed, n = 107). Timing of untreated parental (Parental, n = 69) and stable RPE-1 (Control, n = 52) cells is also shown. Inset in Meta-Ana shows same data on an expanded time scale. Comparison of NEB-Meta and Meta-Ana timing distributions for ensheathed vs. control, P = 1.9 × 10 −57 and 7.8 × 10 −23 , Kolmogorov–Smirnov test. (B) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, red) at kinetochores (CENP-C, blue) in cells stably expressing GFP-Sec61β (green); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (C) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the GFP-Sec61β signal. Dots represent kinetochores, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: n A = 132, n F = 30, n E = 37; (Mad2: n A = 103, n F = 20, n E = 31). (D) Stills from live-cell imaging experiments to track Mad2 levels at kinetochores of ensheathed chromosomes. A GSK923295-pretreated RPE-1 cell is shown, stably coexpressing GFP-Mad2 (green) and mCherry-Sec61β (red); DNA is stained using SiR-DNA (blue). Time relative to anaphase is shown in minutes. Insets show 2× zoom of the indicated ROI. Scale bars, 10 µm; 2 µm (insets). (E) Quantification of live Mad2 imaging experiments. Kaplan–Meier plot to show congression times of the last misaligned chromosome to align. Measurement of mCherry-Sec61β (mean ± SD) and GFP-Mad2 is shown for the misaligned that congressed and those that were missegregated (misseg). A linear regression fit with 95% confidence intervals is shown for GFP-Mad2. All plots are shown in time (minutes) relative to anaphase onset. Total cells with misaligned chromosomes, n = 72; cells where all chromosomes congressed, n = 56; and where there was missegregation, n = 16.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Expressing, Immunofluorescence, Staining, Live Cell Imaging, Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Spindle assembly checkpoint and micronucleus formation in DLD-1 cells. (A) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, green) at kinetochores (CENP-C, blue) in cells stably expressing mCherry-Sec61β (red); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (B) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the mCherry-Sec61β signal. Dots show kinetochore measurements, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: n A = 52, n F = 49, n E = 52; (Mad2: n A = 55, n F = 57, n E = 55). (C) Stills from a video showing an example of ensheathed chromosomes in G11 DLD-1 cells forming micronuclei following Dox/IAA treatment. Scale, 10 µm.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Ensheathed chromosomes do not have stable microtubule-kinetochore attachment. (A) Micrographs of RPE-1 cells stably expressing GFP-Sec61β (gray) pretreated with GSK923295 immunostained for tubulin (red) and CENP-C (green); DNA stained with DAPI. Examples show end-on attachments at aligned kinetochores and potential lateral kinetochore-MT contacts for ensheathed chromosomes. (B) Micrographs of RPE-1 cells stably expressing GFP-Sec61β (gray) pretreated with GSK923295 immunostained for kinastrin (red) and CENP-C (green); DNA stained by DAPI. Scale bars, 10 µm; 2 µm (insets). (C) Frequency distributions of the proximity of the nearest kinastrin punctum to each kinetochore (CENP-C punctum). Kinetochores (n, % with kinastrin <600 nm): aligned (3,124, 26.8%); free (74, 4.1%); ensheathed (227, 6.2%). ( D and E ) Still images from live-cell imaging experiments of RPE-1 cells stably expressing GFP-Sec61β (green) and Histone H3.2-mCherry (gray), pretreated with 150 nM GSK923295 and stained with SiR-Tubulin (red). Similar results were recorded in 25 cells with free chromosomes and 16 cells with ensheathed chromosomes. Scale bars, 10 µm; 2 µm (insets).

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Expressing, Staining, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Ensheathed chromosomes promote formation of micronuclei. (A) Stills from live-cell imaging experiments to track the fate of ensheathed chromosomes. A control or GSK923295-pretreated GFP-Sec61β RPE-1 cell is shown; DNA is stained using SiR-DNA (red). Scale bars, 10 µm; 2 µm (insets). Shown in and . (B) Sankey diagram to show the fate (right) of cells in each of the three metaphase classes (left). Fates include normal division, micronucleus formation, death, and other defects (lagging chromosome, cytokinesis failure). Note that the fate of cells (and not chromosomes) is tracked. A cell with three misaligned chromosomes, only one of which is ensheathed, is classified as ensheathed. Parental RPE-1 cells (Parental, n = 92) and untreated RPE-1 stably expressing GFP-Sec61β (Control, n = 69) are from two and three independent overnight experiments, respectively. Fates of GSK923295-pretreated GFP-Sec61β cells ( n = 186) were compiled from seven experiments. Fates of individual chromosomes are shown in .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Live Cell Imaging, Staining, Stable Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Missegregation of an ensheathed chromosome results in a micronucleus with a disrupted NE. (A) Confocal images showing examples of an intact or a disrupted micronucleus as indicated. Images show mCherry-BAF or LBR-mCherry (red) stably coexpressed with GFP-Sec61β (green) in RPE-1 cells; H3K27ac was detected by immunofluorescence (blue), and DNA was stained with DAPI. XY view is through the center of the micronucleus; YZ (right) and XZ (below) are orthogonal views at the positions indicated. Scale bar, 10 µm. (B) Scatter plots to show the fluorescence intensity of H3K27ac (blue) and either mCherry-BAF or LBR-mCherry (red) vs. GFP-Sec61β intensity. Data are plotted as the log 2 ratio of intensity at the micronucleus vs. main nucleus. For RPE1 GFP-Sec61β mCherry-BAF, n = 71 cells, and LBR-mCherry, n = 73 cells, from three independent experiments in each cell type.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Inducible relocalization of ER in mitotic cells. (A) Schematic diagram of the ER clearance procedure. Rapamycin induces the heterodimerization of the ER-resident FKBP-GFP-Sec61β and the plasma-membrane localized Stargazin-mCherry-FRB. (B) Cumulative histogram showing the time to detection of ER clearance. An automated segmentation procedure was used to monitor ER localization in mitotic cells. The time at which the largest decrease in ER localization occurred was taken ( n = 35−37, see Materials and methods). Random occurrence is shown for comparison. The median (IQR) ER clearance time in rapamycin-treated cells was 15 (12–24) min; rapamycin is applied after the first frame ( T = 0). (C) Induced relocalization of FKBP-GFP-Sec61β to the plasma membrane causes ER clearance. Typical immunofluorescence micrographs of mitotic HCT116 cells pretreated with GSK923295, expressing FKBP-GFP-Sec61β (green) and Stargazin-mCherry-FRB (blue), treated or not with rapamycin (200 nM). Cells were stained for ER markers KDEL or Calnexin as indicated (red), DNA was stained with DAPI (gray). Insets are 2× expansions of the ROI shown. Scale bars, 10 µm; 1 µm (insets). (D) SBF-SEM imaging of control or ER-cleared (rapamycin) mitotic HCT116 cells. A single slice is shown with segmentation of ER (green), plasma membrane (yellow), mitochondria (blue), and chromosomes (red). Scale bars, 5 µm; 1 µm (insets). Insets are 2× expansions of the indicated ROI shown without segmentation; green arrowheads indicate ER attachment to the plasma membrane.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Immunofluorescence, Expressing, Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Rescue of ensheathed chromosomes by the induced relocalization of ER. (A) Stills from live-cell imaging of ER clearance experiments. FKBP-GFP-Sec61β (green), Stargazin-mCherry-FRB (red), and SiR-DNA (gray) are shown. Insets are 2× expansions of the ROI shown. Scale bars, 10 µm; 1 µm (insets). See . (B) Semiautomated 4D tracking of misaligned chromosome location is used to monitor congression. Two tracks from the cells in A are shown. The shortest Euclidean distance from the centroid of the misaligned chromosome to the edge of the main chromosome plate is plotted as a function of time. (C) Fate of misaligned chromosomes in control or rapamycin-treated cells. Rescue of misaligned chromosomes was detected in 26 of 30 rapamycin-treated cells. Coloring in B and C is with the color scale shown. Tracks terminate at 90 min or when the chromosome merges with the plate. Median termination time was 93 min (control, n = 36) and 45 min (rapamycin, n = 30); P = 7.1 × 10 −9 , Wilcoxon rank test. Rapamycin is applied after the first frame ( T = 0).

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Stable transgene expression in RPE1 cells and fate of misaligned chromosomes in RPE1 cells stably expressing GFP-Sec61β. (A–D) Western blots to examine expression of proteins in parental RPE1 cells or clonal cells stably expressing GFP-Sec61β alone or with Histone3.2-mCherry, LBR-mCherry, or mCherry-BAF, as indicated. Membranes were probed for GFP, Sec61β, mCherry, LBR, BAF. Actin or tubulin is shown as a loading control. Green or red arrowheads indicate the expected position of GFP- or mCherry-tagged protein; black arrowheads indicate the untagged protein. (E) Mitotic timing of RPE1 cells stably expressing transgenes. Cumulative frequencies for NEB to metaphase, metaphase to anaphase, and NEB to anaphase are shown. Parental, n = 69; GFP-Sec61β alone, n = 52; GFP-Sec61β and LBR-mCherry, n = 66; GFP-Sec61β and mCherry-BAF, n = 51. (F) Sankey diagram to show the fate (right) of RPE1 cells in each of the three metaphase classes (left). Fates include normal division, micronuclei formation, death, and other defects (lagging chromosome, cytokinesis failure). Note that the fate of cells (and not chromosomes) is tracked. LBR-mCherry/GFP-Sec61β, n = 51; mCherry-BAF/GFP-Sec61β, n = 67; pooled from three experiments. (G) Sankey diagram to show the fate (right) of chromosomes in each of the three metaphase classes (left) after GSK923295 pretreatment. Fates include rescue, micronuclei formation, death, and other defects (lagging chromosome, cytokinesis failure). Number of chromosomes: free, 146; ensheathed, 207; lagging, 9. The same dataset was analyzed for the outcome of cells (classified by the final misaligned chromosome) in . Note that ensheathed chromosomes at metaphase that were rescued all became “free” chromosomes before rescue. Source data are available for this figure: .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Expressing, Stable Transfection, Western Blot

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID; 37 proteins identified as RNA binding in terms of molecular function are listed in Dataset . ( B ) Interactions of Derlins with Sec61β. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C ) Endogenous interaction of Derlin-1 with Sec61β. Immunoprecipitation (IP) with anti-Sec61β antibody or control (Ctrl) IgG using Protein G Sepharose and immunoblotting (IB) with indicated antibodies in HepG2 cells treated with or without 200 nM Tg and/or 500 nM MG132 for 16 h. ( D – F ) IB of ERpQC substrates in HEK293 cells transfected with indicated siRNAs and plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Black arrowhead, signal peptide-uncleaved NHK QQQ ( S NHK QQQ ); white arrowhead, signal peptide-cleaved NHK QQQ ( C NHK QQQ ). ( G ) IB of ERpQC substrate in wild-type (WT) or Derlin-1, -2 , and -3 triple knockout (TKO) HEK293 cells transfected with indicated siRNAs and plasmid for NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Expression levels of S NHK QQQ were calculated and shown as the percentage of S NHK QQQ out of the total amount of NHK QQQ ( S NHK QQQ and C NHK QQQ ). Black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot, Triple Knockout, Plasmid Preparation, Expressing

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) and MG132 treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID. eIF4A1 and Sec61β are included among 43 proteins identified as RNA binding in terms of molecular function. Related to Fig. . ( B ) Interactions of exogenous Derlins with endogenous eIF4A1 and eIF4E. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C , D ) Endogenous interactions of Derlin-1 ( C ) or Derlin-2 ( D ) with eIF4A1 ( C , D ) and eIF4E ( D ). HepG2 cells treated with or without 200 nM Tg and 500 nM MG132 for 16 h were IPed with an anti-Derlin-1 or an anti-Derlin-2 antibody and immunoblotted with indicated antibodies. ( E ) Interaction of endogenous Derlin-1 with exogenous eIF4E. HEK293 cells transfected with Flag-eIF4E and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were IPed with an anti-Flag antibody and immunoblotted with indicated antibodies.

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Volcano plots of the quantitative proteomic analysis of ER stress-induced Sec61β interactome. Plots indicate the fold change in the abundance of identified proteins in Tg and MG132-treated samples compared with that in DMSO-treated samples (log2 fold change [Tg_MG132/DMSO], x axis) against its significance (−log10 P value, y axis) ( n = 4). Orange, significant fold change (Tg_MG132/DMSO) ≥ 2, P value ≤ 0.05; red, ARIH1. See also Dataset . ( B ) Domain structures of human ARIH1 and truncated forms with or without mutation. UBA-L, ubiquitin-associated domain-like; RING1, RING domain 1; IBR, in-between RING domain; RING2, RING domain 2; C357S (CS), catalytically inactive serine mutant of Cys357, the active site of Ub ligase activity; ΔAri, mutant lacking inhibitory Ariadne domain. ( C ) Endogenous interaction of Sec61β with ARIH1 during ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in WT or 3× Flag-tagged Sec61β knock-in HEK293 cells and treated with or without 50 nM Tg and/or 200 nM MG132 for 16 h. ( D ) Interaction of endogenous Sec61β and Derlin-1 with exogenous ARIH1 during ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with Flag-ARIH1 and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-ARIH1 were normalized by the amount of input for each protein and shown as fold increases relative to the control lane. ( E ) Endogenous interaction of Sec61β and ARIH1 with Derlin-1 during ER stress. Wild-type C57BL/6 J mice (12 to 14-week-old), matched for sex, were given a single 2 μg/gram body weight intraperitoneal injection of a 0.1 mg/ml suspension of tunicamycin (Tun) in PBS or vehicle (PBS) alone. After 20 h, mice were deeply anesthetized and transcardially perfused with PBS. Whole cell lysates were prepared by homogenizing livers for 60 s × five times in lysis buffer. Cell lysates were IPed with anti-Derlin-1 antibody or control IgG using Protein G Sepharose. All samples were immunoblotted with indicated antibodies. ( F , G ) Interaction of endogenous ARIH1 ( F ), Sec61β ( F , G ), or Derlin-1 ( F , G ) with exogenous 4EHP in HEK293 cells ( F ) and ARIH1-deficient HEK293 cells ( G ). IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated siRNAs ( G ) and Flag-4EHP ( F , G ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-4EHP were normalized by the amount of input for each protein and shown as fold changes relative to the control lane. ( H , I ) Interaction of endogenous Derlin-1 with exogenous eIF4E in ARIH1- ( H ) or Sec61β- ( I ) deficient cells. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated siRNAs and Flag-eIF4E and treated with 50 nM Tg and 200 nM MG132 for 16 h. The amount of co-IPed Derlin-1 with Flag-eIF4E was normalized by the amount of input Derlin-1 and shown as fold increase relative to the control lane. .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Mutagenesis, Ubiquitin Proteomics, Activity Assay, Knock-In, Transfection, Control, Injection, Suspension, Lysis

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A – D ) Representative fluorescence images of interaction between Sec61β and ARIH1 ( A , B ) or 4EHP ( C , D ) detected by a proximity ligation assay (PLA). HepG2 cells transfected with GFP-KDEL, Flag-Sec61β and HA-ARIH1 ( A , B ) or HA-4EHP ( C , D ) were treated with 50 nM Tg and 200 nM MG132 for 16 h. PLA was performed using anti-Flag and anti-HA antibodies. GFP-KDEL (green, ER marker), PLA signal (red, Flag/HA) and DAPI (blue, nuclei) are shown. Scale bars, 3–10 μm. ( E – G ) RNA immunoprecipitation (RIP) of Flag-4EHP with DUSP6 mRNA ( E ) and ERpQC substrate TTR mRNA ( F ). RIP of Flag-4EHP with TTR mRNA in Sec61β-deficient cells ( G ). HepG2 cells were transfected with Flag-4EHP alone ( E , F ) or together with siCtrl or siSec61β ( G ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h ( E , F ) or with 50 nM Tg and 200 nM MG132 for 16 h ( G ). Flag-4EHP was IPed using an anti-Flag antibody, and the levels of the indicated mRNAs (normalized to input) in Flag-4EHP-bound mRNA were analyzed by RT-qPCR ( E , F ; n = 3, G ; n = 4). ( H , I ) Interaction of exogenous ( H ) or endogenous ( I ) 4EHP and endogenous Sec61β with ARIH1 mutants upon ER stress. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated plasmids and treated with 50 nM Tg and 200 nM MG132 for 16 h. The amounts of co-IPed proteins with Flag-ARIH1 were normalized by the amount of IPed Flag-ARIH1 and the input amount of each protein and shown as fold changes relative to the intensity observed in IP with Flag-ARIH1(CC) retaining its E3 ligase activity. Details regarding the ARIH1 mutants are described in Fig. . ΔAri, mutant lacking the inhibitory Ariadne domain; CS, catalytically inactive serine mutant of Cys357; S427D, phospho-mimetic mutant of Ser427 to Asp on the Ariadne domain. ( J ) IB of ERpQC substrate in HEK293 cells transfected with indicated siRNAs and plasmids and then treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. 4KR, non-ISGylatable 4EHP mutant (Lys121/130/134/222 to Arg). Data are means ± SEM. * P < 0.05, ** P < 0.01; n.s., not significant. Two-tailed unpaired t test for DMSO vs Tg+MG ( E , F ) and for siCtrl vs siSec61β ( G ).

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Fluorescence, Proximity Ligation Assay, Transfection, Marker, RNA Immunoprecipitation, Quantitative RT-PCR, Activity Assay, Mutagenesis, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A , B ) IB of ERpQC substrate in HEK293 cells transfected with siRNAs against ARIH1 ( A ), 4EHP ( B ), or Sec61β ( A , B ) and Flag-NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of the expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). ( C ) IB of ERpQC substrates in HEK293 cells transfected with siRNA against 4EHP and cDNAs for Flag-NHK QQQ and HA-4EHP and treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). ( D , E ) RIP of Flag-4EHP with ERpQC substrate mRNA, α1AT . HepG2 cells were transfected with Flag-4EHP alone ( D ) or together with siCtrl or siSec61β ( E ) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h ( D ) or with 50 nM Tg and 200 nM MG132 for 16 h ( E ). Flag-4EHP was IPed using an anti-Flag antibody, and the levels of the indicated mRNAs (normalized to input) in Flag-4EHP-bound mRNA were analyzed by RT-qPCR ( D ; n = 3, E ; n = 4). ( F ) IB of ERpQC substrates in HEK293 cells transfected with siRNA against ARIH1 UTR and cDNAs for Flag-NHK QQQ and HA-ARIH1ΔAri(CC) or (CS) and treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siARIH1 ( A ), siCtrl vs si4EHP ( B ) or siCtrl vs siSec61β ( A , B ); one-way ANOVA with Tukey’s multiple comparisons test ( C ). Two-tailed unpaired t test for DMSO vs Tg+MG ( D ) or siCtrl vs siSec61β ( E ); one-way ANOVA with Tukey’s multiple comparisons test ( F ). For ( A – C , F ), black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A – D ) IB of ERpQC substrate in HEK293 cells transfected with indicated siRNAs and plasmids and then treated with or without 50 nM Tg and 200 nM MG132 for 16 h; samples were immunoblotted with indicated antibodies. Bar graph: Ratio of the expression level of S NHK QQQ to the total amount of NHK QQQ ( n = 3 for A , C , D and n = 4 for B ). ( E ) Domain structure of human Sec61β and its truncated form. IDR intrinsically disordered region, TM transmembrane domain. ( F ) Interaction of endogenous ARIH1 and 4EHP with exogenous Sec61β. IP with an anti-Flag antibody and IB with indicated antibodies in HEK293 cells transfected with indicated plasmids and treated with 50 nM Tg and 200 nM MG132 for 16 h. Two endogenous 4EHP bands were detected in the input, and both interacted with Sec61βWT (arrows). These bands may reflect post-translational modified 4EHP, including previously reported ubiquitination or ISGylation, in addition to the possibility of splicing products. Arrows, 4EHP; asterisk, a non-specific band unrelated to 4EHP that appears due to overexpression of 3× Flag-Sec61β. ( G ) IB of ERpQC substrate in HEK293 cells transfected with Sec61β UTR siRNA and indicated plasmids treated with or without 50 nM Tg and 200 nM MG132 for 16 h. For exogenous Flag-Sec61β, lysates were IPed with an anti-Flag antibody and immunoblotted with an anti-Flag antibody; input samples were immunoblotted with indicated antibodies. Bar graph: ratio of the expression level of S NHK QQQ to the total amount of NHK QQQ was calculated and shown ( n = 3). Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. One-way ANOVA with Tukey’s multiple comparisons test. For ( A – D , G ), black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Transfection, Expressing, Modification, Ubiquitin Proteomics, Over Expression

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Proteasome chymotrypsin-like peptidase activity of cell extracts from HepG2 cells transfected with siCtrl or siSec61β (#1, #2, or #3) and treated with 200 nM Tg for 16 h was measured using Suc-LLVY-AMC as a substrate. Fluorescence intensity was normalized to cell viability in each condition. Proteasome activity is shown as fold decrease relative to that of siCtrl-transfected cells ( n = 3). ( B ) Degradation of the cytoplasmic protein CL1 degron after a 15-min pulse of [ 35 S]-methionine/cysteine metabolic labeling, followed by the indicated chase periods. Lysates of HEK293 cells transfected with indicated siRNAs and Venus-CL1-Flag and stimulated with 50 nM Tg for 16 h were IPed with an anti-Flag antibody and resolved via SDS-PAGE. The relative radioactivity of Venus-CL1-Flag at different chase times was calculated and shown as fold decreases relative to the intensity observed at 0 h chase ( n = 3). ( C ) Representative fluorescence images of HepG2 cells transfected with siCtrl or siSec61β and stimulated with 50 nM Tg for 10 h, followed by staining with ProteoStat (green, protein aggregation) and DAPI (blue, nuclei). Scale bars, 25 μm. ( D , E ) Quantification of protein aggregation in Sec61β-deficient HepG2 cells using ProteoStat and flow cytometry. Mean ProteoStat fluorescence in HepG2 cells transfected with siCtrl or siSec61β and stimulated with 50 nM Tg for 4 h ( D ) and relative mean ProteoStat fluorescence in HepG2 cells transfected with siSec61β and indicated cDNAs and stimulated with 50 nM Tg for 4 h ( E ) were analyzed by flow cytometry and software. Boxes represent the 25th–75th percentiles with the median indicated; whiskers represent the minimum and maximum values ( n = 8 for D or n = 3 for E ). ( F ) Superimposed images of tail movements at 3 dpf and a scheme of the quantitative analysis of head-tail angle. Compared with larvae injected with water or control Sec61β-atg-5mis MO, Sec61β-deficient zebrafish exhibited impaired swimming behavior. ( G ) Histogram showing the maximum head-tail angles at 3 dpf. Sec61β-deficient zebrafish ( n = 17) showed reduced maximum head-tail angles compared to control animals ( n = 17 for water injection or n = 18 for Sec61β-atg-5mis MO injection). ( H ) Histogram showing the distribution of phenotype scores of zebrafish at 4 dpf. The degree of morphological and swimming abnormalities was scored from 0 to 3 as shown in Fig. . Sec61β-deficient zebrafish ( n = 49) showed greater abnormality scores than control animals ( n = 44 for water injection or n = 52 for Sec61β-atg-5mis MO injection). ( I ) Histogram showing the rescue of abnormal phenotype in Sec61β-deficient zebrafish by ARIH1 at 4 dpf. Exogenous expression of human ARIH1ΔAri(CC) ( n = 124), but not ARIH1ΔAri(CS) ( n = 113), reduced the abnormal phenotype score in Sec61β-deficient zebrafish ( n = 104). Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siSec61β ( A , B ); two-tailed paired t test for siCtrl vs siSec61β ( D ); two-tailed unpaired t test for Sec61β WT vs Sec61β ΔIDR ( E ); one-way ANOVA with Tukey’s multiple comparisons test ( G ); Fisher’s exact test followed by Bonferroni’s post hoc test ( H , I ). .

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Activity Assay, Transfection, Fluorescence, Labeling, SDS Page, Radioactivity, Staining, Flow Cytometry, Software, Injection, Control, Expressing, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Proteasome chymotrypsin-like peptidase activity of cell extracts from HepG2 cells transfected with siCtrl or siSec61β (#1, #2, or #3) and treated with 2 μg/ml tunicamycin (Tun) for 16 h was measured using Suc-LLVY-AMC as a substrate. Fluorescence intensity was normalized to cell viability in each condition. Proteasome activity is shown as fold decrease relative to that of siCtrl-transfected cells ( n = 7, 7, 6, and 7 from left to right, respectively). ( B ) Degradation of CL1 degron chased for the indicated periods after 15 min pulse of [ 35 S]-methionine/cysteine metabolic labeling shown in Fig. . Cell lysates of HEK293 cells transfected with indicated siRNAs and Venus-CL1-Flag and stimulated with 50 nM Tg for 16 h were IPed with an anti-Flag antibody, resolved by SDS-PAGE and analyzed by autoradiography. ( C ) Representative fluorescence images of HepG2 cells transfected with siSec61β and stimulated with 50 nM Tg for 6 h, followed by staining with ProteoStat (red, protein aggregation), calnexin (green, ER membranes) and DAPI (blue, nuclei). Scale bars, 25 μm. ( D ) Reduced expression of Sec61β in zebrafish by injection of antisense morpholino oligonucleotide (MO). Antisense MO was designed as described in Methods. MOs were injected into zebrafish embryos at 1- to 2-cell stages. The expression of Sec61β in zebrafish at 3 dpf was analyzed by IB using a polyclonal antibody against a peptide against zebrafish Sec61β (SAGTGGMWRFYTEDSPGLKV) raised in rabbits. Lysates were prepared by homogenizing 3 dpf fish for 60 s in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EGTA, and 1% Triton X-100) supplemented with 5 μg/mL leupeptin (Nacalai Tesque; 43449-62) on ice using a Micro Smash (TOMY; MS-100) (4500 rpm, 4 °C). Lysates were resolved by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBS-T (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.05% Tween-20), the membranes were probed with antibody against to zebrafish Sec61β (1/1000) or actin (1/5000) diluted in 5% BSA in TBS-T overnight at 4 °C. Secondary antibodies [IRDye 800CW Donkey anti-Rabbit IgG (H + L) (1/10,000) and IRDye 680RD Donkey anti-Mouse IgG (H + L) (1/10,000)] were diluted in 5% skim milk in TBS-T, and membranes were incubated 2 h at room temperature. Images were revealed and analyzed using Odyssey CLx (LICOR) and Empiria Studio software 3.0 (LICOR). ( E ) Representative images showing the typical morphology of zebrafish larvae injected with water, Sec61β-atg MO or Sec61β-5mis MO at 3 dpf. Arrowhead indicates the abnormal morphology observed in rare Sec61β-deficient zebrafish. ( F ) Histogram showing the average body length of zebrafish larvae relative to that of water-injected controls at 3 dpf. Sec61β-deficient zebrafish ( n = 13) showed a tendency toward reduced body length compared to control groups ( n = 10 for water injection or n = 24 for Sec61β-atg-5mis MO injection). ( G ) The behavior of zebrafish was observed at 4 dpf, and a phenotype scores were recorded manually. The scoring criteria were defined as follows: no obvious abnormality (0), abnormal swimming with head shaking and slightly smaller size (1), abnormal swimming and morphology (2), and no swimming and abnormal morphology (3). ( H ) Exogenous expression of ARIH1 mutants in zebrafish by injection of synthesized mRNAs. Synthesized mRNAs for human ARIHΔAri(CC) or (CS) were co-injected into zebrafish embryos at 1- to 2-cell stages. Lysates were prepared by homogenizing 3 dpf 10 fish for 60 s in lysis buffer due to weak expression of exogenous proteins. The expression of HA-ARIHΔAri was analyzed by IB using a rat monoclonal antibody against HA (clone 3F10) and secondary antibody (HRP-linked anti-rat IgG antibody). The membranes were detected by an ECL system, and images were revealed and analyzed using ChemiDoc Touch (BioRad). Data are means ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001; n.s., not significant. Two-tailed unpaired t test for siCtrl vs siSec61β ( A ); Kruskal–Wallis rank-sum test ( F ).

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Activity Assay, Transfection, Fluorescence, Labeling, SDS Page, Autoradiography, Staining, Expressing, Injection, Lysis, Blocking Assay, Incubation, Software, Control, Synthesized, Two Tailed Test

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: Under ER stress conditions, chaperones such as BiP and PDI continue to be synthesized by cotranslational translocation into the ER lumen (upper left). The signal sequence of nascent polypeptides emerging from the ribosome is recognized by SRP, and RNC–SRP complex docks onto SRα/β. When the SRP dissociates from the RNC, the nascent polypeptide is delivered into the ER, where it undergoes proper folding. By contrast, in ERpQC (upper right), some secretory proteins are degraded by the proteasome without translocation into the ER. ERpQC is triggered by recruitment of Derlins to the Sec61 translocon and SR in response to ER stress. Derlins interact with SRP54 on the RNC through their C-terminus, thereby rerouting the nascent polypeptide from the ER translocation pathway to the cytoplasmic degradation pathway (rerouting). Sec61β bound to Derlins recruits the E3 ligase ARIH1 via its IDR upon ER stress. ARIH1, guided in the vicinity of the ER membrane, enables 4EHP to associate with the 5ʹ cap structure of the ERpQC substrate mRNAs captured by Derlins, thereby causing translational repression of ERpQC substrates (translational repression). Rerouted ERpQC substrates that have already initiated translation are ubiquitinated by HRD1 and effectively transported to the proteasome by p97 and Bag6 (proteasomal degradation). Derlins–Sec61β–ARIH1 complex-mediated translational repression contributes to reduced proteasomal degradation load of cytosolic unfolded proteins, leading to inhibition of aggresome formation and maintenance of cytoplasmic proteostasis. Conversely, Sec61β deficiency allows eIF4F recruitment to the mRNA 5ʹ cap structure of the Derlins-bound RNC and polysome formation, resulting in the overproduction of ERpQC substrates and disruption of cytosolic proteostasis (lower).

Article Snippet: Wild-type and Derlin-1 , Derlin-2 , and Derlin-3 triple knockout HEK293 cells (Kadowaki et al, ) and 3× Flag-tagged Sec61β knock-in HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34).

Techniques: Synthesized, Translocation Assay, Sequencing, Membrane, Inhibition, Disruption