Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: A A schematic illustration showing the retention of Str‐Ii‐SCOTIN‐SBP‐EGFP in the ER membrane and its release to the membrane of another organelle after biotin treatment. B–D Representative immunofluorescence images of SCOTIN ‐KO cells expressing Str‐Ii‐SCOTIN‐SBP‐EGFP or Str‐Ii‐VSVG‐SBP‐EGFP (green) with 40 μM D‐biotin treatment for the indicated times. (B, C) Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. (D) The localization of SCOTIN‐SBP‐EGFP or VSVG‐SBP‐EGFP (green) relative to giantin (red) is shown in cells treated with biotin for the indicated times. The arrow indicates the EGFP signal on the Golgi. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm. E Representative images of SCOTIN‐SBP‐EGFP trafficking from the ER (Sec61β, magenta) to late endosomes (CD63, red) after biotin treatment for the indicated times. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. Scale bar: 10 μm. Scale bars in the magnified images: 5 μm. F The colocalization of EGFP and Sec61 or CD63 was calculated as the Pearson correlation coefficient from the experimental data in (E). The error bars indicate the SEMs. n : number of cells analyzed. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, ** P < 0.01 and **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Sec61β , Atlas antibodies , Cat# HPA049407, RRID: AB_2680750.

Techniques: Membrane, Immunofluorescence, Expressing, Clinical Proteomics, Two Tailed Test, Labeling

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: Schematic illustration of the proteinase K protection assay. Representative images of Rab5 Q79L‐EGFP‐ and SCOTIN‐DsRed‐expressing cells with early endosomes (EEA1, white) and late endosomes (CD63, magenta). The arrowhead indicates the area of crowded small vesicles attached to enlarged endosomes. Scale bar: 30 μm. Scale bars in the magnified images: 1.5 μm. Schematic illustration of knock‐in FP 11 to endogenous SCOTIN loci. Left: Representative high‐resolution microscopy images of SCOTIN‐FP 11 (green) and Sec61β, EEA1, CD63, TfR, and LAMP1 (red) in SCOTIN‐FP 11 knock‐in cells. Right: Plot analysis of SCOTIN‐FP 11 relative to the organelle marker signal shown. The graph indicates the fluorescence intensity of each channel on the lines. Scale bars: 20 μm. Scale bars in the magnified images: 5 μm. Colocalization of SCOTIN‐FP 11 (green) and EEA1, CD63, TfR, or LAMP1 was calculated as Pearson correlation coefficients. Data information: n : number of cells analyzed. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Sec61β , Atlas antibodies , Cat# HPA049407, RRID: AB_2680750.

Techniques: Expressing, Knock-In, Microscopy, Marker, Fluorescence, Two Tailed Test, Labeling

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: Left, representative images of Str‐Ii‐SCOTIN‐SBP‐EGFP along with the ER and late endosomes in SCOTIN ‐KO cells. Cells were stained with the ER tubular protein RTN4 (white) and the late endosomal marker CD63 (red). Untreated (−Biotin) or biotin‐treated (+Biotin 4 h). Arrows indicate EGFP signal with RTN4 and arrowheads indicate EGFP signal with CD63. Right, Schematic illustration of the relative localization of SCOTIN in Str‐Ii‐SCOTIN‐SBP‐EGFP‐expressing cells with or without biotin treatment. Scale bars: 20 μm. Representative images of VAPA‐Rab7 PLA signals taken from Str‐Ii‐SCOTIN‐SBP‐EGFP transfected SCOTIN ‐KO cells after untreated (−Biotin) or biotin treatment (+Biotin 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Representative images of Sec61β‐Emerald‐ or Sec61β‐PRD‐Emerald‐transfected SCOTIN ‐KO cells. Cells were stained with RTN4 (red). Arrowheads indicate the accumulated ER membrane. All scale bars: 20 μm. Representative images of Rab5 Q79L‐EGFP‐ and CD63‐ or CD63‐PRD‐Myc (red)‐transfected SCOTIN ‐KO cells. Arrowheads indicate the CD63‐ or CD63‐PRD‐Myc localization on the endosome membrane. All scale bars: 20 μm. Representative images of Sec61 or Sec61‐PRD‐Emerald with CD63 or CD63‐PRD‐Myc (red) cotransfected SCOTIN ‐KO cells. A colocalization image was obtained using the colocalization finder ImageJ Plugin. The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n : number of cells analyzed. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Sec61β , Atlas antibodies , Cat# HPA049407, RRID: AB_2680750.

Techniques: Staining, Marker, Expressing, Transfection, Particle Size Analysis, Membrane, Two Tailed Test, Labeling

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet:

Article Snippet: Sec61β , Atlas antibodies , Cat# HPA049407, RRID: AB_2680750.

Techniques: Recombinant, Fluorescence, Electron Microscopy, Knock-Out, Knock-In, Plasmid Preparation, Software, Microscopy

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: PLoS ONE

Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

doi: 10.1371/journal.pone.0151806

Figure Lengend Snippet: P1 WT mice retinas were co-transfected with GFP-fused (green) plasmids and a mCherry tagged ER reporter (mCh-Sec61b: red). Retinas were harvested and sectioned at P30 (A) Confocal images of retinas show co-localization (merge: yellow) of GFP-fused mutant D94Y-TULP1 and F491L-TULP1 with mCh-Sec61b, the ER resident protein, in the inner segments. Scale bar = 10μM. (B-C) Activation of the ER-UPR stress markers in transgenic mice expressing mutant TULP1 protein. qRT-PCR indicates a 1.3–5 fold increase of multiple ER-UPR stress markers in mutant D94Y-TULP1 (B) or mutant F491L-TULP1 (C) retinas vs WT-TULP1 injected retinas. qRT-PCR analysis was normalized to GAPDH (as a mRNA quantity control) and GFP (as a transfection efficiency control). The ΔΔCt method was employed to calculate fold changes. Samples were assayed in triplicates for each ER-UPR marker and qRT-PCR experiments were repeated twice. Statistical significance was assessed by using the two-tailed Student’s t -test. * = p<0.001; n.s. not statistically significant.

Article Snippet: For co-localization studies, WT or mutant TULP1 plasmids were co-electroporated with the mCherry-tagged ER-marker Sec61b plasmid DNA (mCherry-Sec61b, a gift from Dr. Gia Voeltz (Addgene #49155)).

Techniques: Transfection, Mutagenesis, Activation Assay, Transgenic Assay, Expressing, Quantitative RT-PCR, Injection, Marker, Two Tailed Test