Journal: British Journal of Cancer

Article Title: Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

doi: 10.1038/bjc.2014.239

Figure Lengend Snippet: EDPs influence on Hsp90, pro-MMP-2 and uPA secretion. ( A ) Gelatin zymography, gelatin plasminogen zymography and western blotting analysis of conditioned media and actin content in total cell extracts from HT-1080 cells cultured for 6, 24 and 48 h in the absence (−) or presence of 50 μ g ml −1 Kel (+) as described in Materials and Methods section. ( B ) Quantitative evaluation of pro-MMP-2 secretion by HT-1080 cells on gelatin zymography. Results (mean±s.e.m.) were expressed as the percentage of control 6 h (EDP-untreated cells). * P <0.005, ** P <0.001, significantly different from control. Lighter bars, untreated cells; black bars, EDP-treated cells. ( C ) Quantitative evaluation of uPA secretion by HT-1080 cells on gelatin plasminogen zymography. Results (mean±s.e.m.) were expressed as the percentage of control for 6 h (EDP-untreated cells). * P <0.005, ** P <0.001, significantly different from control. Lighter bars, untreated cells; black bars, EDP-treated cells. ( D ) Quantitative evaluation of Hsp90 secretion by HT-1080 cells by western blotting. Results (mean±s.e.m.) were expressed as the percentage of control for 6 h (EDP-untreated cells). ** P <0.001, significantly different from control. Lighter bars, untreated cells; black bars, EDP-treated cells. ( E ) Dose-effect study of EDP treatment (Kel 0–100 μ g ml −1 ) on pro-MMP-2, uPA and Hsp90 secretion. Gelatin zymography, gelatin plasminogen zymography and western blotting analysis of conditioned media and actin content in cell extracts from HT-1080 cells cultured for 24 h in the absence (Ctl) or in the presence of 1–100 μ g ml −1 Kel. ( F ) Gelatin zymography, gelatin plasminogen zymography and western blotting analysis of conditioned media from HT-1080 cells cultured for 24 h in the absence (Ctl) or in the presence of 50 μ g ml −1 Kel (Kel) or synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK). All synthetic peptides were used at 200 μ g ml −1 . Quantitative evaluation of protein secretions by HT-1080 cells on gelatin zymography, gelatin plasminogen zymography and by western blotting. Results (mean±s.e.m.) were expressed as the percentage of control (EDP-untreated cells).

Article Snippet: Human fibrosarcoma cells (HT-1080) and breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Zymography, Western Blot, Cell Culture, Control

Journal: British Journal of Cancer

Article Title: Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

doi: 10.1038/bjc.2014.239

Figure Lengend Snippet: Evaluation of the intracellular Hsp90, MMP-2 and uPA expression in HT-1080 cells. ( A ) Western blotting analysis of intracellular Hsp90 using a rabbit anti-Hsp90 monoclonal antibody and probing with anti-actin antibody were carried out as described in Materials and Methods section. ( B ) Western blotting analysis of intracellular Hsp90 at 24 h using a rabbit anti-Hsp90 monoclonal antibody and probing with anti-actin. ( C – F ) Localisation of Hsp90, pro-MMP-2 and uPA in HT-1080 cells in the presence ( D , F ) (Kel 50 μ g ml −1 ) or absence ( C , E ) (control) of EDPs. Cells were cultured on glass slides, fixed with paraformaldehyde and labelled with an anti-Hsp90 (green), anti-pro-MMP-2 form (red) ( C , D ), anti-uPA (red) ( E , F ). Yellow staining corresponds to areas where Hsp90, pro-MMP-2 or uPA colocalised. Scale bar: 10 μ m.

Article Snippet: Human fibrosarcoma cells (HT-1080) and breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Control, Cell Culture, Staining

Journal: British Journal of Cancer

Article Title: Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

doi: 10.1038/bjc.2014.239

Figure Lengend Snippet: Hsp90 regulates migration and invasion of EDP-treated HT-1080 cells. ( A ) Geldanamycin and radicicol, two Hsp90 inhibitors, block the Matrigel invasion of EDP-stimulated HT-1080 cells. HT-1080 cells were incubated with 1.5 μ M geldanamycin or 10 μ M radicicol during 1 h before being stimulated or not for 6 h with 50 μ g ml −1 EDPs. At the end of the incubation period, cells were fixed with methanol and stained with crystal violet for 15 min. Cells remaining on the upper face of the membranes were scrapped and those on the lower face were counted. Data are expressed as mean±S.E.M. values from three independent experiments, each performed in triplicate. ** P <0.001, significantly different from Kel alone). Lighter bars, untreated cells; black bars, EDP-treated cells. ( B ) Heat-shock protein 90 inhibitors (geldanamycin and radicicol) inhibit the EDP-stimulated migration of HT-1080 cells. HT-1080 cells have been seeded in 12-well plates and a homogeneous wound was created in each well by scraping cells with a tip. Cells were incubated with 1.5 μ M geldanamycin or 10 μ M radicicol during 1 h before being stimulated or not for 24 h with 50 μ g ml −1 EDPs and cell migration was evaluated by microscopy. ( C ) Quantification of the wound closure. ** P <0.001, significantly different from Kel alone.

Article Snippet: Human fibrosarcoma cells (HT-1080) and breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Migration, Blocking Assay, Incubation, Staining, Microscopy

Journal: British Journal of Cancer

Article Title: Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

doi: 10.1038/bjc.2014.239

Figure Lengend Snippet: EDP increases extracellular Hsp90 and promotes pro-MMP-2 and uPA accumulation in HT-1080 culture media. ( A ) Gelatin zymography and gelatin plasminogen zymography analysis of Hsp90 inhibitors effect (1.5 μ M geldanamycin and 10 μ M radicicol) on the production of pro-MMP-2 and uPA in the presence (+: 50 μ g ml −1 ) or in the absence (−) of EDPs (24 h of treatment). ( B ) Quantitative evaluation of protein secretions by HT-1080 cells on gelatin zymography and gelatin plasminogen zymography. Results (mean±s.e.m.) were expressed as the percentage of control (EDP-untreated cells). * P <0.005, ** P <0.001, significantly different from Kel alone. ( C ) Gelatin zymography and gelatin plasminogen zymography analysis of monoclonal anti-Hsp90 antibody effect on the production of pro-MMP-2 and uPA in EDP-treated HT-1080 cells (50 μ g ml −1 ) (24 h of treatment). ( D ) Quantitative evaluation of protein secretions by HT-1080 cells on gelatin zymography and gelatin plasminogen zymography. Results (mean±s.e.m.) were expressed as the percentage of control (EDP-untreated cells). * P <0.005, ** P <0.001, significantly different from Kel alone. ( E ) Gelatin zymography and gelatin plasminogen zymography analysis of the effect of recombinant Hsp90 α protein on the production of pro-MMP-2 and uPA by HT-1080 cells (24 h of treatment). ( F ) Quantitative evaluation of protein secretions by HT-1080 cells on gelatin zymography and gelatin plasminogen zymography. Results (mean±s.e.m.) were expressed as the percentage of control (EDP-untreated cells). * P <0.005, ** P <0.001, significantly different from control.

Article Snippet: Human fibrosarcoma cells (HT-1080) and breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Zymography, Control, Recombinant

Journal: British Journal of Cancer

Article Title: Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

doi: 10.1038/bjc.2014.239

Figure Lengend Snippet: Hsp90 interacts with MMP-2 and uPA. Immunoprecipitation of extracellular Hsp90 from EDP-treated HT-1080 cells' conditioned media and bound MMP-2 and uPA analysis by western blotting. Output 100% represent the unbound protein after Hsp90 immunoprecipitation.

Article Snippet: Human fibrosarcoma cells (HT-1080) and breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: British Journal of Cancer

Article Title: Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

doi: 10.1038/bjc.2014.239

Figure Lengend Snippet: Effect of Hsp90 knockdown on the EDP-regulated pro-MMP-2 and uPA secretion by HT-1080 cells. ( A ) Real-time PCR analysis of Hsp90α transcript 2 mRNA relative expression 48 h after treatment with Hsp90 siRNA vs negative control siRNA-treated cells (−). Results (mean±s.e.m.; n =3) were expressed as the ratio of Hsp90α transcript 1 mRNA to EEF1a1 mRNA. ( B ) Western blotting analysis and quantification of intracellular Hsp90 using a rabbit anti-Hsp90 monoclonal antibody and probing with anti-actin 72 h after treatment with Hsp90 siRNA vs negative control siRNA-treated cells (−) as described in Materials and Methods section. Results (mean±s.e.m.) were expressed as the percentage of negative control siRNA-treated cells (EDP-untreated cells). ( C ) Immunolocalisation of Hsp90 72 h after treatment with siRNA to Hsp90 mRNA vs negative control siRNA-treated cells (−). Cells were cultured on glass slides, fixed with paraformaldehyde and labelled with an anti-Hsp90 antibody (green). Scale bar: 20 μ m. ( D ) Gelatin zymography, gelatin plasminogen zymography and western blotting analysis of conditioned media in the presence (+: 50 μ g ml −1 ) or in the absence (−) of EDPs 72 h after treatment with Hsp90 siRNA vs negative control siRNA-treated cells. Quantitative evaluation of protein secretions by HT-1080 cells on gelatin zymography, gelatin plasminogen zymography and by western blotting. Results (mean±s.e.m.) were expressed as the percentage of control (negative control siRNA/EDP-untreated cells).

Article Snippet: Human fibrosarcoma cells (HT-1080) and breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Western Blot, Cell Culture, Zymography, 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: 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

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting AAVS1 and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Irradiation, Control

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Wasserstein distance of cells with DDR gene-targeting guides (Perturb) or control guides (Controls) to control cells for RAD51 foci in irradiated cells. Hits of RAD51 foci identified in the pooled screening are marked. (B) same as A) for BRCA1 foci. (C) Volcano plots showing variants yielding significant changes in the proportion of cells with high p21 expression in untreated (left) or irradiated (right) conditions. Significance is defined by p.adj < 0.05, Beta-Binomial test. The proportion of control cells with high p21 is marked with the vertical line (UNT: 0.376; IR: 0.576). sgRNA category and ClinVar category for significant guides are displayed by colors and shapes, respectively. (D) Correlation of RAD51 foci log2-fold change (L2FC) in guides delivered in the pooled library (pooled) and transduced individually (individual). The Pearson correlation (r) is 0.90. (E) same as D) for BRCA1 foci. The Pearson correlation (r) is 0.95. (F) MCF7-BE3 cells were lentivirally transduced with the DDR364 library, selected in puromycin-containing medium for 2 days, and cultured 2 days without puromycin. Cells were either untreated (UNT) or treated with DNA damaging agents Camptothecin (CPT), Olaparib (OLAP), Cisplatin (CISP) or Etoposide (ETOP) for 24 hours, then fixed before optical phenotyping and barcode detection. (G) Correlation between L2FC in RAD51 foci in CISP-treated cells and the Rule Set 2 on-target score, showing a negative correlation. All guides targeting RAD51 regulators including RAD51D, RAD51C, XRCC3, BRCA1, and BRCA2 are shown. The Pearson correlation (r) equals −0.29. (H) same as G) for OLAP-treated cells. The Pearson correlation (r) equals −0.30. (I) Volcano plot showing variants yielding significant changes in RAD51 foci in CISP-treated cells. Statistical significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.5, all AAVS1-targeting and non-targeting control (NTC) guides are shown. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. Same figure legend as in C). (J) same as I) for OLAP-treated cells. (K) Gene enrichment analysis in variants that result in significant changes in RAD51 foci. Fisher exact test. Enrichment is defined as p.adj <0.05. (L) Same as I) for variants yielding significant changes in large γH2AX foci in CISP-treated cells. (M) same as L) for OLAP-treated cells. (N) same as K) for large γH2AX foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Control, Irradiation, Expressing, Transduction, Cell Culture

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Immunoblots on guides targeting BRCA1 showing the reduction of full-length BRCA1 protein in the splice variant BRCA1.234, compared to the missense variants BRCA1.416 and.BRCA1.476, and the AAVS1 variant AAVS1.86. Cells transduced with Firefly siRNA (siFirefly) and BRCA1 siRNA (siBRCA1) were included to show the specificity of BRCA1 detection. Cells were treated with or without irradiation and the induction of DNA damage is shown with the phospho-KAP1 (pKAP1) staining. Tubulin is used as the loading control. (B) As in A) for BRCA2 variants showing the reduction of full-length BRCA2 protein in the nonsense variant BRCA2.207, compared to the missense variant BRCA2.438 and the AAVS1 variant AAVS1.86.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Western Blot, Variant Assay, Transduction, Irradiation, Staining, Control