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: The Journal of Cell Biology

Article Title: Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation

doi: 10.1083/jcb.200507057

Figure Lengend Snippet: Characterization of Derlin-2 and -3. (A) HeLa cells were trans-fected with plasmid to express each Derlin tagged with the c-myc epitope at the respective NH 2 terminus. Transfected cells were fixed and stained with anti-myc and anti-Sec61β antibodies. (B) HEK293 cells were trans-fected with plasmid to express each Derlin tagged with the c-myc epitope at the respective COOH terminus. Postnuclear supernatant of transfected cells was incubated with increasing amounts of trypsin (0, 4, 8, and 16 μg for lane 1, 2, 3, and 4, respectively) for 15 min at 4°C. Immunoblotting analysis of the samples was performed using anti-myc, anti–NH 2 terminus of calnexin (CNX[N]), anti–COOH terminus of calnexin (CNX[C]), and anti-calreticulin (CRT) antibodies. (C) HEK293 cells were transfected with plasmid to express Derlin-2 (a) or Derlin-3 tv1 or tv2 (b) tagged with the c-myc epitope at either the NH 2 (N) or COOH (C) terminus. 36 h later, transfected cells were pulse labeled with 35 S-methionine and cysteine for 15 min and then chased for the indicated periods. Cells were lysed with buffer containing 1% NP-40 and subjected to immunoprecipitation analysis using anti-myc antibody.

Article Snippet: 36 h later, cells were fixed with 4% formaldehyde at room temperature for 10 min, permeabilized with 0.2% Triton-X 100 in PBS, reacted with mouse anti-myc antibody (Santa Cruz Biotechnology, Inc.) and rabbit anti-Sec61β antibody (Upstate Biotechnology) for 1 h, and incubated with FITC-conjugated anti–rabbit IgG antibody (MP Biomedicals) or rhodamine-conjugated anti–mouse IgG antibody (Cappel).

Techniques: Plasmid Preparation, Transfection, Staining, Incubation, Western Blot, Labeling, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Formation of stacked ER cisternae by low affinity protein interactions

doi: 10.1083/jcb.200306020

Figure Lengend Snippet: A point mutation that converts GFP to mGFP inhibits formation of OSER structures. (a) Immunoblot analysis of cells expressing unmutated or mGFP-Sec61γ lysates of equal amounts of cells untransfected (no DNA) or expressing either construct were loaded on the same gel and probed with anti-GFP or an antibody against native Sec61β as a control for equal loading. (b–d) Representative confocal micrographs of transiently transfected COS-7 cells expressing (b) mYFP-Sec61β, (c) mGFP-Sec61γ, and (d) C1(1-29)P450-mGFP reveal the absence of whorls, short karmellae, and loop structures. Other amorphous, often perinuclear, structures that are generally of much lower fluorescence intensity than cells expressing the unmutated GFP counterparts are visible. Bar, 5 μm.

Article Snippet: The polyclonal rabbit antibody against Sec61β was prepared (Lampire Biological Laboratories) against the synthetic peptide PGPTPSGTNC (residues 2–10 plus a cysteine) of canine Sec61β conjugated to keyhole limpet hemocyanin using standard protocols.

Techniques: Mutagenesis, Western Blot, Expressing, Construct, Control, Transfection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis

doi: 10.1074/jbc.RA119.007996

Figure Lengend Snippet: BioID candidate ribosome-interacting chimeras label small and large ribosomal subunit proteins of translationally active ribosomes. A, ribosomal protein biotin-labeling patterns for the LRRC59-BioID and Sec61β-BioID chimera were determined by sucrose density gradient fractionation of reporter cell extracts prepared following 16-h doxycycline induction and a 3-h biotin-labeling period. Ribosomal subunits were recovered by ultracentrifugation of pooled gradient fractions and biotinylated proteins determined by SDS-PAGE separation, transfer to nitrocellulose, and streptavidin detection. The 80S ribosomal fractions depict the biotin-labeling patterns of ammonium chloride–washed, puromycin-treated 80S ribosomes. The data are representative of two independent biological replicates. The vertical line in the gradient fraction gels is to indicate that the 80S fraction is derived from a separate gel. RNA detection was by SYBR Green staining. B, the distribution of biotinylated ribosomes in the polyribosome fractions was examined by sucrose density gradient ultracentrifugation. rRNA and biotin-labeled protein distributions were analyzed as above. The data are representative of two independent biological replicates. C, MS/MS-identified biotinylated ribosomal proteins were mapped onto a PDB structure of the ribosome bound to the translocon (PDB code 3J7R). Several ribosomal features are labeled for orientation. MS experiments were performed in duplicate. D, summary table of high-confidence MS-identified, biotinylated ribosomal proteins depicted in C.

Article Snippet: Plasmids were from the following sources: pCMV-Sport6-RPN1 (Transomic ID: pCS6- {"type":"entrez-nucleotide","attrs":{"text":"BC010839","term_id":"14789967","term_text":"BC010839"}} BC010839 , TransOMIC, Huntsville, AL), pCMV-Sport6-LRRC59 (Transomic ID: pCS6- {"type":"entrez-nucleotide","attrs":{"text":"BC017168","term_id":"16877877","term_text":"BC017168"}} BC017168 ), Sec61β (Transomic ID: pOTB7-BC {"type":"entrez-nucleotide","attrs":{"text":"BC001734","term_id":"12804622","term_text":"BC001734"}} BC001734 ), Neo-IRES-GFP-Sec62 (Richard Zimmerman, Saarland University, Homburg, Germany), pEYFP-N1-BirA* (Scott Soderling, Department of Cell Biology, Duke University Medical Center).

Techniques: Labeling, Fractionation, SDS Page, Derivative Assay, RNA Detection, SYBR Green Assay, Staining, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis

doi: 10.1074/jbc.RA119.007996

Figure Lengend Snippet: Ribosomes biotin-tagged by Sec61β and LRRC59 BioID reporters undergo translation-dependent and translation-independent ER–cytosol exchange. A, a biotinylation pulse-chase protocol was developed to examine ER–cytosol exchange of ribosomes biotin-tagged by the Sec61β and LRRC59 BioID reporters. A time course of biotin labeling was performed, where biotin was present in the culture medium either throughout the experiment (control; Full Biotin Label) or the medium was exchanged to medium with no added biotin (experimental; Biotin Pulse-Chase). At each time point, ribosomes were isolated by detergent extraction and ultracentrifugation, with biotin labeling determined as above. Relative labeling represents the streptavidin signal normalized to the 1-h biotin labeling time point. B, streptavidin blots of ribosomes obtained from the cytosol and membrane fractions of Sec61β and LRRC59 BioID reporter cell lines at 0, 0.5, and 1 h of chase. Labels represent small (S) or large (L) ribosomal proteins from the LRRC59 (L) or Sec61β (B) reporter lines. The arrowhead indicates the band that was quantified for the bar graph depictions of the relative fraction of labeled ribosomes in the cytosol (light bars) and membrane (dark bars) fractions (L-R2 or L-B1). C, to identify translation-independent cytosol–ER ribosome exchange, cycloheximide (elongation inhibitor) was added at the start of the chase period, and reporter cell lines were processed as in B. D, to assess translation-dependent ribosome exchange, reporter cell lines were treated with harringtonine (initiation inhibitor) and processed as described above. Error bars shown in all graphs mark the high and low measurements of streptavidin signal intensity from each experiment determined by densitometry analysis. Streptavidin blots depicted are representative of three independent biological replicates. Error bars, S.D.

Article Snippet: Plasmids were from the following sources: pCMV-Sport6-RPN1 (Transomic ID: pCS6- {"type":"entrez-nucleotide","attrs":{"text":"BC010839","term_id":"14789967","term_text":"BC010839"}} BC010839 , TransOMIC, Huntsville, AL), pCMV-Sport6-LRRC59 (Transomic ID: pCS6- {"type":"entrez-nucleotide","attrs":{"text":"BC017168","term_id":"16877877","term_text":"BC017168"}} BC017168 ), Sec61β (Transomic ID: pOTB7-BC {"type":"entrez-nucleotide","attrs":{"text":"BC001734","term_id":"12804622","term_text":"BC001734"}} BC001734 ), Neo-IRES-GFP-Sec62 (Richard Zimmerman, Saarland University, Homburg, Germany), pEYFP-N1-BirA* (Scott Soderling, Department of Cell Biology, Duke University Medical Center).

Techniques: Pulse Chase, Labeling, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis

doi: 10.1074/jbc.RA119.007996

Figure Lengend Snippet: RNA-Seq analysis of Sec61β and LRRC59 BioID reporter-labeled ribosomes reveals divergent transcriptomes and demonstrates that ER-bound ribosomes engage in the translation of cytosolic and secretory protein-encoding RNAs. A, biotin-labeled ribosomes from the membrane fractions of Sec61β and LRRC59 BioID reporter cell lines were affinity-isolated, total RNA was extracted, and RNA-Seq analyses of mRNA-enriched RNA were performed. Depicted is the relative fraction of trimmed read counts for each of the data sets aligning to a human reference genome, binned to coding, noncoding, and 7SL RNA sequences. B, subcellular distributions of proteins encoded by mRNAs represented as a percentage of the total. Stack plots of RNA-Seq TPM reveal an enrichment for cell membrane and organellar protein-encoding mRNAs (DeepLoc1.0) compared with the total mRNA distribution by TPM for membrane and total cell (LocTree3), using data sets from Reid and Nicchitta (15) and the Human Protein Atlas, respectively). The category “Organelle” encompasses mRNAs whose encoded proteins are localized to the ER, Golgi, lysosomes/vacuoles, plasma membrane, and mitochondria. C, table of the top 10 genes by log2 -fold change value for genes either enriched in a given ribosome fraction or present in both (shared), color-coded to indicate -fold enrichment over the control data sets. D, bubble plots depicting the −log(FDR) of reporter cell line–enriched GO molecular function enrichments for the transcriptome identified by each chimera. Red line, an FDR cutoff of 0.05. Human GO term IDs are listed in Table S1. Analysis is shown from three biological replicates performed as independent experiments, with individual bar-coded libraries combined for multiplexed deep sequencing.

Article Snippet: Plasmids were from the following sources: pCMV-Sport6-RPN1 (Transomic ID: pCS6- {"type":"entrez-nucleotide","attrs":{"text":"BC010839","term_id":"14789967","term_text":"BC010839"}} BC010839 , TransOMIC, Huntsville, AL), pCMV-Sport6-LRRC59 (Transomic ID: pCS6- {"type":"entrez-nucleotide","attrs":{"text":"BC017168","term_id":"16877877","term_text":"BC017168"}} BC017168 ), Sec61β (Transomic ID: pOTB7-BC {"type":"entrez-nucleotide","attrs":{"text":"BC001734","term_id":"12804622","term_text":"BC001734"}} BC001734 ), Neo-IRES-GFP-Sec62 (Richard Zimmerman, Saarland University, Homburg, Germany), pEYFP-N1-BirA* (Scott Soderling, Department of Cell Biology, Duke University Medical Center).

Techniques: RNA Sequencing Assay, Labeling, Isolation, Sequencing

Journal: bioRxiv

Article Title: Proteolytic processing of the Marburg virus glycoprotein depends on Sec61β and is required for cell entry

doi: 10.1101/2025.06.26.660697

Figure Lengend Snippet: ( A ) SEC61B -knockout (KO) single cell clones were generated from HEK293T cells using CRISPR/Cas9. Sec61β expression in lysates of the indicated SEC61B -KO single cell clones was analyzed by immunoblot using polyclonal anti-Sec61β antibody. Expression of β-actin was analyzed as loading control. Results were confirmed in two additional experiments. ( B ) The indicated SEC61B -KO cell lines were stained with CFSE and analyzed via flow cytometry at day 0, 1, 2 and 3. The results of a single experiment are shown and were confirmed in two separate experiments. ( C ) Phase contrast microscopy of Sec61β single cell clones. Similar results were obtained in one additional experiment.

Article Snippet: Here, we report that the Sec61 subunit Sec61β, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses.

Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Expressing, Western Blot, Control, Staining, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: Proteolytic processing of the Marburg virus glycoprotein depends on Sec61β and is required for cell entry

doi: 10.1101/2025.06.26.660697

Figure Lengend Snippet: ( A ) WT and SEC61B -KO cells were transfected with plasmids expressing C-terminal V5-tagged GPs of VSV, EBOV (Makona) and MARV (Musoke) or empty vector (EV) as control. Glycoprotein expression was detected by immunoblot using a mouse monoclonal anti-V5 antibody. Detection of β-actin served as a loading control. Results were confirmed by one to three additional experiments. ( B ) The experiment was conducted as for panel A but the effect of coexpression of Sec61β on MARV-GP expression and cleavage was examined. The results of a representative immunoblot are shown and were confirmed in two separate experiments. ( C-D ) The expression of MARV-GP and EBOV-GP with the indicated mutations in the furin cleavage sites was analyzed in cell lysates (C) and pseudotyped VSV particles (D). The expression of β-actin (cell lysates) and VSV-M (particles) served as loading control. Results were confirmed in two (EBOV-GP) or three (MARV-GP) additional experiments. ( E ) HEK293T cells were inoculated with VSV particles pseudotyped with the indicated glycoproteins. Luciferase activity in cell lysates were quantified at 18-20 h post inoculation. Luminescence signals were normalized to background (empty vector control). Shown is the mean ± SEM of four separate experiments each conducted with four technical replicates. Statistical significances were determined using unpaired, two-tailed t-test with Welch‘s correction (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **)

Article Snippet: Here, we report that the Sec61 subunit Sec61β, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay, Two Tailed Test