Journal: Developmental cell

Article Title: Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition

doi: 10.1016/j.devcel.2019.07.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies are listed in the . table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies NRF2 Abcam Cat# ab62352; RRID: AB_944418 20-s Proteasome UBPBio Y2010 P62 Novus Cat# H00008878-M01; RRID: AB_548364 LC3 Novus Cat# NB100-2220; RRID: AB_10003146 ATG5 Cell Signaling Technology Cat# 9980S; RRID: AB_10829153 ATG7 Cell Signaling Technology Cat# 8558; RRID: AB_10831194 ATG12 Cell Signaling Technology Cat # 2010s; RRID: AB_2059086 STX17 Sigma-Aldrich Cat# HPA001204; RRID: AB_1080118 FIP200 Novus Cat# NBP1-31583; RRID: AB_2300812 PTEN Santa Cruz Biotechnology Cat# sc-7974; RRID: AB_628187 FOXO3a Cell Signaling Cat# 3938; RRID: AB_2106669 β-Actin Sigma-Aldrich Cat# A5441; RRID: AB_476744 β-Tubulin Sigma-Aldrich Cat# T5168; RRID: AB_477579 Mouse-IgG Cell Signaling Technology Cat# 7076; RRID: AB_330924 Rabbit-IgG Cell Signaling Technology Cat# 7074; RRID: AB_2099233 Chemicals, Peptides, and Recombinant Proteins Bafilomycin A1 Sigma-Aldrich B1792; CAS RN: 88899-55-2 Chloroquine MP-Biomedicals 93919; CAS RN: 50-63-5 Protease Inhibitor Cocktail Roche 11836153001 Bortezomib Selleck Chemicals S1013 Critical Commercial Assays MEGAscript T7 Transcription Kit Thermo Fisher AM1354 MEGclear Transcription Clean Up Kit Thermo Fisher AM1908 Wizard SV Gel PCR clean up kit Promega A9282 Lipofectamine CRISPR Max Cas9 transfection reagent Thermo Fisher CMAX00003 RNeasy RNA isolation kit Qiagen 74104 QuantiTect Reverse Transcription Kit Qiagen 205311 SYBR Green CFX for QPCR Applied Biosystems Applied Biosystems 4472942 Seahorse XF Mitochondrial Stress Test Agilent 103015–100 Proteasome Activity Fluorometric Assay Kit II UBPBio J4120 CellROX green flow cytometry assay kit Thermo Fisher {"type":"entrez-nucleotide","attrs":{"text":"C10492","term_id":"1535563","term_text":"C10492"}} C10492 Experimental Models: Cell Lines HCT116 ATCC NCI-DTP Cat# HCT-116; RRID: CVCL_0291 MCF7 ATCC NCI-DTP Cat# MCF7; RRID: CVCL_0031 HT1080 ATCC CLS Cat# 300216/p517_HT-1080; RRID: CVCL_0317 NCIH292 Kindly provided from Dr. Joaquin Espinosa ECACC Cat# 91091815; RRID: CVCL_0455 MAF-794 Kindly provided from Dr. Jean Mulcahy-Levy N/A BT549 ATCC NCI-DTP Cat# BT-549; RRID: CVCL_1092 SJSA-1 Kindly provided from Dr. Joaquin Espinosa ATCC Cat# CRL-2098; RRID: CVCL_1697 NCIH-1650 ATCC ATCC Cat# CRL-5883; RRID: CVCL_1483 Experimental Models: Organisms/Strains Athymic Nude mice (nu/nu) Jackson Laboratory Stock number: 007850 Oligonucleotides Oligonucleotides for qRT-PCR primers: Table S1 this study; Fitzwalter et al., 2018 ; Oslowski and Urano, 2011 ; Radhakrishnan et al., 2010 ; Vangala et al., 2014 .

Techniques: Recombinant, Protease Inhibitor, CRISPR, Transfection, Isolation, SYBR Green Assay, Activity Assay, Flow Cytometry, shRNA, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Quantitative EM analysis of ER–mitochondria contacts in HT-1080 and COS-7 cells. (A) Representative EM images of HT-1080 and COS-7 cells. Insets show rough ER–mitochondria contacts (RER-mito) in HT-1080 cells (green arrowheads) and smooth ER–mitochondria contacts (SER-mito) in HT-1080 and COS-7 cells (red arrowheads). (B) Quantification of contact width, contact length, contact length relative to mitochondria perimeter, and number of contacts per mitochondria profile are shown for SER-mito and RER-mito contacts in HT1080 and COS-7 cells. (C) The relative ratio of SER-mito and RER-mito contacts in HT-1080 and COS-7 cells based on the number of contacts per mitochondria or length of contacts. (D) The number of ribosomes per RER-mito contact is plotted versus the length of the contact in nm for HT-1080 and COS-7 cells. RER-mito contacts with five or less ribosomes are shown in red; those with more than five ribosomes are specific to HT-1080 cells and are shown in blue and defined as riboMERCs. n = 27 images from two independent biological replicates; ±SEM; ***P < 0.001; ****P < 0.0001; B: one-way ANOVA; C: Chi2 test. Bar = 500 nm; inset: 200 nm.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: MCS-DETECT analysis of sub-precision contacts. (A) 3D STED images of HT-1080 and COS-7 showing overlap between mitochondria (magenta) and ER (green). Insets show STED sections at 0.5 μm Z spacing. Bars = 10 μm. (B) Two objects (red and green discs) are shown at corresponding sub-precision distances. Intensity profiles (top row), second derivatives (Laplacian), and Spearman correlations of the negative part of the Laplacian (bottom row) are shown. Note how the Spearman response overlaps and changes consistently with the sub-precision distance. (C) The detection algorithm (orange) with additional stages that each address a specific confounding factor introduced by the acquisition (bleed through) or sample (vesicle removal).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Parameter study of the proposed method. (A) We test two representative HT-1080 and COS-7 cells, with known distinct contact types. We vary the analysis window, a 3D cube of 5k × 5k × k over the mitochondria and contact channel. (a) The surface coverage ratio stays stable for a range of k. (b) The user can set a significance and statistical power threshold. This increases the minimum significance at which voxels can be detected, as well as the minimum correlation considered to be observable. As expected, when this threshold increases, expected differences between two representative cells decrease, as does the overall number of correlation voxels. At the limit of 100% confidence, no information would be left. To avoid false responses by bleed-through of signal in nearby Z-planes, as well as high background intensity in low SNR conditions, we apply an adaptive threshold in z-space. (c) We observe that at low values, the inclusion of false responses masks any differences (z = 1.5). At high values (3.5) the ER channel was visually degraded, we see that after z = 3 the difference between the two cells is maximal and converged. (B) The full reference algorithm pseudocode listing of each stage of the proposed method, enabling reproduction in any implementation. The actual Julia implementation used adds non-algorithm stages to deal with parallelization, optimization, error handling, and recording intermediate stages, which are out of scope for the purposes of this listing. The source code is available under AGPL v3 license at https://github.com/bencardoen/SubPrecisionContactDetection.jl .

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Subprecision contact detection identifies distinct contact profiles in HT-1080 and COS-7 cells. (A) Volume-rendered MCS-DETECT views of cells expressing ERmoxGFP (green) and labeled for TOM20 (magenta) with contact sites overlaid (white) are shown for COS-7, HT-1080, and OMM–ER linker transfected COS-7 cell ROIs from the whole view image are shown volume rendered in adjacent panels. COS-7 mitochondria display numerous small contact zones while mitochondria in HT-1080 and OMM–ER linker transfected COS-7 cells present more extended contact zones (bar = 10 µm whole cell; 1 µm insets). (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in COS-7, HT-1080, and OMM–ER linker transfected COS-7 cells (averaged over cell, two-sided non-parametric Mann Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; ***P < 0.001). (C) 2D KDE plots of mean contact size over mean anisotropy and mean Spearman response, with a linear regression overlayed, are shown for COS-7 (red) versus HT-1080 (blue) cells or COS-7 (red) versus OMM–ER linker transfected COS-7 (blue) cells.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Expressing, Labeling, Transfection, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Identifying and filtering mitochondria. (A) For each contact, size and mean intensity of the adjacent mitochondria is plotted for two replicates, to indicate consistency across replicates. Note that this is by default larger than a segmentation method would compute. The size and mean intensity of mitochondria in HT-1080 and COS-7 cells present a clearly separable group of small, low-intensity mitochondria structures. To report results on what are clearly and unambiguously mitochondria, corresponding to the mitochondria observed by EM, mitochondrial structures smaller than thresholds 9 (ln size) and 0.2 (mean intensity) were eliminated. (B) 3D STED images show labeling of ER (green), mitochondria (magenta), and contact sites (white) of representative COS-7 and HT-1080 cells before and after mitochondrial filtering. Bar = 10 μM (insets: 1 μM).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 regulation of riboMERCs. (A) Volume-rendered MCS-DETECT views of cells expressing ERmoxGFP and labeled for TOM20 (magenta) with contact sites overlaid (white) are shown for HT-1080 and Gp78 KO HT-1080 cells and for untransfected COS-7 cells and COS-7 cells overexpressing WT Gp78 or Gp78 RM. Bar = 10 μm whole cell; 1 μm insets. (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in HT-1080 and Gp78 KO HT-1080 cells and for untransfected COS-7 cells and COS-7 cells overexpressing Gp78 WT or Gp78 RM. (C) 2D KDE plots of mean contact size over mean anisotropy and mean Spearman response, with a linear regression overlayed, are shown for HT-1080 (blue) versus Gp78 KO HT-1080 (green) cells or COS-7 (red) versus COS-7 overexpressing either Gp78 WT (green) or Gp78 RM (blue). Averaged over cell, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001, two-sided non-parametric Mann–Whitney test. (D) COS-7 cells were transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Integrated density of MitoView 633 per cell was quantified. n = 3 independent biological replicates; >35 cells/condition per experiment; *P < 0.05; ****P < 0.0001; Tukey post hoc test.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Expressing, Labeling, MANN-WHITNEY, Transfection, Control

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 overexpression western blot and representative Mito V iew images. (A) Western blots of COS-7 cells transfected with pcDNA3, Flag-Gp78 WT, or Flag-Gp78 RM were probed with antibodies to Flag tag to reveal Gp78 and to β-actin. (B) Representative images of COS-7 cells transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Corresponding GFP or RFP images (FP) are shown and cell boundaries outlined . (C) Representative images of HeLa cells transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Corresponding GFP or RFP images (FP) are shown and cell boundaries outlined . (D) Representative images of HT-1080 cells transfected with either siControl or siRRBP1 and labeled with MitoView633 are shown and cell boundaries outlined . Bars (B, C, D) = 20 μm. Source data are available for this figure: .

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Over Expression, Western Blot, Transfection, FLAG-tag, Control, Labeling

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 expression in COS-7 and HeLa cells, siRRBP1 western blot, and shape features for Q95 Gp78 RM overexpressing cells. (A) COS-7 and HeLa cells were transfected in parallel with Flag-Gp78 WT, Flag-Gp78 RM, or the RFP-tagged OMM–ER linker and fixed after 24 h. The Flag-Gp78 transfected cells were labeled for anti-Flag and the OMM–ER linker transfected cells left unlabeled. Cells were imaged and anti-Flag or RFP labeling density was quantified. n = 3; >36 cells per sample; ****P < 0.0001; Student t test. (B) Western blots of RRBP1 and β-actin show a reduction of RRBP1 in HT-1080 and Gp78 KO HT-1080 cells following transfection of siRRBP1 relative to siCTL. Graph shows densitometric quantification of band intensity. n = 3; *P < 0.05; **P < 0.01. (C) Shape features, height, sphericity, and planarity for the Q95V contacts of a representative cell closest to the mean Q95V for control HeLa cells or HeLa cells overexpressing Gp78 WT or Gp78 RM were analyzed. The comparison shows that the large contacts induced by Gp78 RM in HeLa cells have a shape signature comparable to control cells and not to the riboMERCs induced by Gp78 overexpression. Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3; ***P < 0.001. Source data are available for this figure: .

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Expressing, Western Blot, Transfection, Labeling, Control, Comparison, Over Expression, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: RRBP1 knockdown reduces riboMERCS independent of Gp78. (A) Representative EM images of HT-1080 and HT-1080 Gp78 KO cells treated with either siControl or siRRBP1. Images highlight the presence of riboMERCS in both HT-1080 WT and Gp78 KO cells, which are almost completely lost upon RRBP1 knockdown. (B) Quantification of the number of riboMERCs per mitochondria and the ratio of riboMERC length to mitochondrial perimeter for the conditions in A. (C) Quantification of the MERC width for both riboMERCs and smooth MERCs for the conditions in A. n = 31 images from two independent biological replicates; **P < 0.01; ***P < 0.001; ****P < 0.0001; unpaired t test. Bar = 200 nm.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Knockdown

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: MCS-DETECT captures MERC changes induced by RRBP1 knockdown. (A) Volume-rendered MCS-DETECT views of HT-1080 WT and Gp78 KO cells treated with either siControl or siRRBP1. Mitochondria are labeled with TOMM20 (red) and MERCS are visualized in white. Bar = 10 µm whole cell; 1 µm insets. (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in HT-1080 WT and Gp78 KO cells treated with either siControl or siRRBP1. Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001. (C) HT-1080 cells transfected with either siControl or siRRBP1 were labeled with MitoView633. Integrated density of MitoView633 per cell was quantified. n = 3 independent biological replicates; >50 cells/condition per experiment; ***P < 0.0001; ****P < 0.0001; Tukey post hoc test.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Knockdown, Labeling, MANN-WHITNEY, Transfection

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Large MERCs induced by Gp78 and the OMM–ER linker present distinct shape signatures. (A) The 95th quantile of MERC volume per cell (Q95V; largest 5% of MERCs per cell) and number of MERCs per cell larger than the average 500-voxel size of HT-1080 Q95V MERCs are shown for HT-1080 and COS-7 cells, COS-7, and COS-7 cells overexpressing either Gp78 WT, Gp78 RM, or the OMM–ER linker, HeLa and HeLa cells overexpressing either Gp78 WT, Gp78 RM, or the OMM–ER linker, HT-1080, and Gp78 KO HT-1080 cells, HT-1080 cells transfected with siCTL and siRRBP1, and Gp78 KO HT-1080 cells transfected with siCTL and siRRBP1. (B) Representative cells whose Q95V is closest to the mean Q95V for HT-1080 cells, for COS-7 or HeLa cells overexpressing Gp78 WT, and for COS-7 cells overexpressing the OMM–ER linker were selected for analysis. For the Q95V contacts of each cell, we compute shape features: height, sphericity, and planarity. The comparison shows that the COS-7 OMM–ER linker–induced contacts have a markedly different shape signature compared to those present in HT-1080 and Gp78 overexpressing COS-7 or HeLa cells (i.e., riboMERCS). Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Comparison, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 induces convoluted, tubular riboMERCs. Representative whole-cell views of Q95V MERCs (color-coded for increasing size from 500 to 5,613 voxels) from HT-1080 cells, COS-7, or HeLa cells overexpressing Gp78 WT and COS-7 cells overexpressing the OMM–ER linker as well as representative individual Q95V MERCs alone or adjacent to transparent (pink) or solid mitochondria (magenta) to highlight intercalation of riboMERCs with mitochondria. Rotating videos of these MERCs are included as Supplemental , , , , , , , , , , , and . Bar = 1 μm.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of MERCs larger than the 500 voxels in complete HT-1080 cell, transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . Video was rendered in Imaris 10.0 software using the contacts channel outputted from MCS-DETECT (30 frames/s). MERCs are color-coded for increasing size from 500 (blue) to 5,613 voxels (red).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in an HT-1080 cell transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . MERCs display a high degree of complexity, with multiple branch points and extending over several Z slices. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in an HT-1080 cell transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . MERCs display a high degree of complexity, with multiple branch points and extending over several Z slices. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a COS-7 cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a COS-7 cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a HeLa cells overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (10 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (magenta), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a HeLa cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (10 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (magenta), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software