mef feeder layer  (ATCC)


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    ATCC mef feeder layer
    Mef Feeder Layer, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    mef feeder layer - by Bioz Stars, 2023-01
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    mef cell lines  (ATCC)


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    ATCC mef cell lines
    (a) (Top) Western blot detection <t>of</t> <t>Opa1</t> forms in indicated <t>MEF</t> cell lines using Opa1 antibody. (Bottom) Actin was used as loading control. (b) Genetic schematic and cartoon depictions of Opa1 forms present in MEF cell lines used in this study.
    Mef Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    mef cell lines - by Bioz Stars, 2023-01
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    1) Product Images from "In situ architecture of Opa1-dependent mitochondrial cristae remodeling"

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    Journal: bioRxiv

    doi: 10.1101/2023.01.16.524176

    (a) (Top) Western blot detection of Opa1 forms in indicated MEF cell lines using Opa1 antibody. (Bottom) Actin was used as loading control. (b) Genetic schematic and cartoon depictions of Opa1 forms present in MEF cell lines used in this study.
    Figure Legend Snippet: (a) (Top) Western blot detection of Opa1 forms in indicated MEF cell lines using Opa1 antibody. (Bottom) Actin was used as loading control. (b) Genetic schematic and cartoon depictions of Opa1 forms present in MEF cell lines used in this study.

    Techniques Used: Western Blot

    Mitochondria with distinguishable inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) visualized by cryo-ET. (a) (Right) Summed, projected central slices of cryo-electron tomograms visualizing representative mitochondria in indicated MEF lines. (Left) Three-dimensional (3D) rendering of segmented membranes with mitochondria shown across Z slices. Green and yellow surfaces indicate OMM and IMM, respectively. (Bottom left) Schematic of Opa1 forms present in respective cell lines. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Plot of mitochondria size (µm 2 ) observed in cryo-electron tomograms in MEF lines. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, **p<0.0, ****p< 0.0001. N: wild-type = 57, Opa1-OE = 17, l- Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.
    Figure Legend Snippet: Mitochondria with distinguishable inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) visualized by cryo-ET. (a) (Right) Summed, projected central slices of cryo-electron tomograms visualizing representative mitochondria in indicated MEF lines. (Left) Three-dimensional (3D) rendering of segmented membranes with mitochondria shown across Z slices. Green and yellow surfaces indicate OMM and IMM, respectively. (Bottom left) Schematic of Opa1 forms present in respective cell lines. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Plot of mitochondria size (µm 2 ) observed in cryo-electron tomograms in MEF lines. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, **p<0.0, ****p< 0.0001. N: wild-type = 57, Opa1-OE = 17, l- Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.

    Techniques Used: Tomography, MANN-WHITNEY

    Representative images of mitochondrial morphology in indicated MEF cell lines labeled with MitoTracker TM Deep Red FM. Insets show magnified view of regions indicated with dashed boxes. Scale bar = 10 µm. Inset scale bar = 5 µm.
    Figure Legend Snippet: Representative images of mitochondrial morphology in indicated MEF cell lines labeled with MitoTracker TM Deep Red FM. Insets show magnified view of regions indicated with dashed boxes. Scale bar = 10 µm. Inset scale bar = 5 µm.

    Techniques Used: Labeling

    (a) Gallery of representative mitochondrial morphology observed by TEM for indicated MEF cell lines. Matrix phenotype is indicated. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Graph bar representing the relative proportion of mitochondrial matrix density based on four categories: empty, uneven, dark and normal in indicated MEF cell lines. (d) Violin graphs plotting mitochondrial area, (e) number of mitochondria per cell and (f) mitochondrial area in µm 2 per cell in MEF cell lines included in this study. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001. For b: N wild-type, Opa1-OE and Opa1-KO = 301, l-Opa1* = 531, s-Opa1* = 359. For c: N wild-type and Opa1-OE = 20, l-Opa1* = 33, s-Opa1* = 32, Opa1-KO = 13. For d: N wild- type = 536, Opa1-OE = 337, l-Opa1* = 476, s-Opa1* = 318, Opa1-KO =302. For e: N wild-type and Opa1- OE = 20, l-Opa1* = 27, s-Opa1* = 32, Opa1-KO = 13. For f: N wild-type and Opa1-OE = 20, l-Opa1* = 27, s-Opa1* = 30, Opa1-KO = 13. Scale bar = 200 nm.
    Figure Legend Snippet: (a) Gallery of representative mitochondrial morphology observed by TEM for indicated MEF cell lines. Matrix phenotype is indicated. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Graph bar representing the relative proportion of mitochondrial matrix density based on four categories: empty, uneven, dark and normal in indicated MEF cell lines. (d) Violin graphs plotting mitochondrial area, (e) number of mitochondria per cell and (f) mitochondrial area in µm 2 per cell in MEF cell lines included in this study. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001. For b: N wild-type, Opa1-OE and Opa1-KO = 301, l-Opa1* = 531, s-Opa1* = 359. For c: N wild-type and Opa1-OE = 20, l-Opa1* = 33, s-Opa1* = 32, Opa1-KO = 13. For d: N wild- type = 536, Opa1-OE = 337, l-Opa1* = 476, s-Opa1* = 318, Opa1-KO =302. For e: N wild-type and Opa1- OE = 20, l-Opa1* = 27, s-Opa1* = 32, Opa1-KO = 13. For f: N wild-type and Opa1-OE = 20, l-Opa1* = 27, s-Opa1* = 30, Opa1-KO = 13. Scale bar = 200 nm.

    Techniques Used: MANN-WHITNEY

    (a) (Top) Summed, projected central slices of cryo- electron tomograms visualizing mitochondria with stacking cristae characteristics, supported by 3D representations consisting of their sub compartments (bottom) in indicated MEF cell lines. (b) Graph bar representing percentage of mitochondria with stacking cristae formation in each MEF cell line. N: wild- type = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.
    Figure Legend Snippet: (a) (Top) Summed, projected central slices of cryo- electron tomograms visualizing mitochondria with stacking cristae characteristics, supported by 3D representations consisting of their sub compartments (bottom) in indicated MEF cell lines. (b) Graph bar representing percentage of mitochondria with stacking cristae formation in each MEF cell line. N: wild- type = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.

    Techniques Used:

    (a) Graph bars showing the proportions of straight, tilted, and disconnected crista and (b) of lamellar, tubular, globular and unusual crista observed in indicated MEF lines. (c) Measured cristae length and (d) cristae width across cell lines. (e) (Top rows) Computational slices of straight, tilted, disconnected, globular and tubular crista across cell lines with zipping, pinching and tight regions indicated (arrows) in 3D renderings (bottom rows) from cryo-electron tomograms. n.a.: not applicable. (f) Histograms of crista widths across cell conditions (see Methods). (g) Subtomogram averages of mitochondrial cristae membranes with the average width indicated. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, ***p<0.001, ****p<0.000. For a, b: N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. For c, d: N = 50 for all cell lines. Scale bar = 200 nm.
    Figure Legend Snippet: (a) Graph bars showing the proportions of straight, tilted, and disconnected crista and (b) of lamellar, tubular, globular and unusual crista observed in indicated MEF lines. (c) Measured cristae length and (d) cristae width across cell lines. (e) (Top rows) Computational slices of straight, tilted, disconnected, globular and tubular crista across cell lines with zipping, pinching and tight regions indicated (arrows) in 3D renderings (bottom rows) from cryo-electron tomograms. n.a.: not applicable. (f) Histograms of crista widths across cell conditions (see Methods). (g) Subtomogram averages of mitochondrial cristae membranes with the average width indicated. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, ***p<0.001, ****p<0.000. For a, b: N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. For c, d: N = 50 for all cell lines. Scale bar = 200 nm.

    Techniques Used: MANN-WHITNEY

    (a) Graph bar representing the relative proportion of unusual cristae morphology observed in indicated MEF cell lines. Unusual cristae were categorized into vesicular, zipped, ring, split, amorphous, straight-across, pinched and loop. N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (b) Summed, projected central slices of cryo-electron tomograms showing examples of unusual cristae in mitochondria across cell lines in 2D (top) and 3D (bottom). Loop, ring, straight-across, pinched, vesicular, and amorphous cristae are shown. Scale bar = 200 nm.
    Figure Legend Snippet: (a) Graph bar representing the relative proportion of unusual cristae morphology observed in indicated MEF cell lines. Unusual cristae were categorized into vesicular, zipped, ring, split, amorphous, straight-across, pinched and loop. N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (b) Summed, projected central slices of cryo-electron tomograms showing examples of unusual cristae in mitochondria across cell lines in 2D (top) and 3D (bottom). Loop, ring, straight-across, pinched, vesicular, and amorphous cristae are shown. Scale bar = 200 nm.

    Techniques Used:

    (a) Violin graphs plotting the percentage of multi-junction cristae per mitochondrion in indicated MEF cell lines. (b) Graph bar representing percentage of multi-junction cristae categorized into straight-across and loop morphology in each MEF cell line. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.0001. For a: N wild-type = 18, Opa1- OE =5, l-Opa1* = 30, s-Opa1* = 16, Opa1-KO = 3. For b: N wild-type = 26, Opa1-OE =9, l-Opa1* = 79, s-Opa1* = 29, Opa1-KO = 4.
    Figure Legend Snippet: (a) Violin graphs plotting the percentage of multi-junction cristae per mitochondrion in indicated MEF cell lines. (b) Graph bar representing percentage of multi-junction cristae categorized into straight-across and loop morphology in each MEF cell line. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.0001. For a: N wild-type = 18, Opa1- OE =5, l-Opa1* = 30, s-Opa1* = 16, Opa1-KO = 3. For b: N wild-type = 26, Opa1-OE =9, l-Opa1* = 79, s-Opa1* = 29, Opa1-KO = 4.

    Techniques Used: MANN-WHITNEY

    BH3 profiling of (a) WT, l-Opa1*, s-Opa1*, Opa1- KO and (b) WT along with Opa1-OE MEF cell lines for sensitizer BIM BH3 and PUMA. N = 3-4 biological replicates. Significance of difference is tested relative to wild-type using the Holm-Sidak’s multiple comparison test; **p<0.01. (c) MEF cell lines were treated with indicated agents for 24h and apoptosis was detected by Annexin V positivity staining. N = minimum 4 biological replicates.
    Figure Legend Snippet: BH3 profiling of (a) WT, l-Opa1*, s-Opa1*, Opa1- KO and (b) WT along with Opa1-OE MEF cell lines for sensitizer BIM BH3 and PUMA. N = 3-4 biological replicates. Significance of difference is tested relative to wild-type using the Holm-Sidak’s multiple comparison test; **p<0.01. (c) MEF cell lines were treated with indicated agents for 24h and apoptosis was detected by Annexin V positivity staining. N = minimum 4 biological replicates.

    Techniques Used: Staining

    Assessment of cell viability by Annexin V staining in MEF cell lines after treatment with the indicated compounds for (a) 24 hours, (b) 48 hours and (c) 72 hours. N = minimum 4 biological replicates.
    Figure Legend Snippet: Assessment of cell viability by Annexin V staining in MEF cell lines after treatment with the indicated compounds for (a) 24 hours, (b) 48 hours and (c) 72 hours. N = minimum 4 biological replicates.

    Techniques Used: Staining

    mouse embryonic fibroblasts mefs  (ATCC)


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    ATCC mouse embryonic fibroblasts mefs
    Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic <t>murine</t> <t>embryonic</t> fibroblasts. Co-culture of PBMC with non-engineered <t>MEFs</t> was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.
    Mouse Embryonic Fibroblasts Mefs, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse embryonic fibroblasts mefs - by Bioz Stars, 2023-01
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    1) Product Images from "Analyzing trogocytosis of T lymphocytes by flow cytometry and confocal microscopy"

    Article Title: Analyzing trogocytosis of T lymphocytes by flow cytometry and confocal microscopy

    Journal: STAR Protocols

    doi: 10.1016/j.xpro.2022.102013

    Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic murine embryonic fibroblasts. Co-culture of PBMC with non-engineered MEFs was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.
    Figure Legend Snippet: Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic murine embryonic fibroblasts. Co-culture of PBMC with non-engineered MEFs was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.

    Techniques Used: Co-Culture Assay, Transgenic Assay, Negative Control, Expressing


    Figure Legend Snippet:

    Techniques Used: Staining, Recombinant, Modification, Software, Cell Culture, Microscopy, Flow Cytometry, Fluorescence

    mouse embryonic fibroblasts mef  (ATCC)


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    ATCC mouse embryonic fibroblasts mef
    Mouse Embryonic Fibroblasts Mef, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse embryonic fibroblasts mef - by Bioz Stars, 2023-01
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    mef cells  (ATCC)


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    ATCC mef cells
    (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in <t>MEF</t> <t>cells</t> within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.
    Mef Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A SNX1–SNX2–VAPB partnership regulates endosomal membrane rewiring in response to nutritional stress"

    Article Title: A SNX1–SNX2–VAPB partnership regulates endosomal membrane rewiring in response to nutritional stress

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202201652

    (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in MEF cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.
    Figure Legend Snippet: (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in MEF cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.

    Techniques Used: Expressing, Staining, Imaging

    mefs  (ATCC)


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    ATCC mefs
    Mefs, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse embryonic fibroblasts mefs  (ATCC)


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    ATCC mouse embryonic fibroblasts mefs
    a , Left panel: sequence comparison of hAtg3 C-terminal residues 262 to 277 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 265 to 277. b , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its mutants (n = 3). CL is the control without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. c , Left panel: sequence comparison of hAtg3 C-terminal residues 291 to 300 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 291 to 300. d , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its mutants (n = 3). CL is the control without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. e , Representative immunoblot (n = 4 <t>blots).</t> <t>Atg3</t> knockout (Atg3 -/- ) mouse embryonic fibroblasts <t>(MEFs)</t> stably expressing mCherry EV (empty vector), mCherry-hAtg3 WT (wildtype), mCherry-hAtg3 F296L , or mCherry-hAtg3 F296S mutant were cultured in complete media (CM) with 100 nM bafilomycin A1 (BafA1 to block LC3-II degradation) for 3 hrs and subjected to immunoblotting with the indicated antibodies. f , Quantitative analysis of the relative LC3B–II level (n = 4 blots) in in vivo LC3B lipidation experiments. Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. Data are presented as mean ± SD. P values: **** P < 0.0001; ns, not significant.
    Mouse Embryonic Fibroblasts Mefs, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Translating Membrane Geometry into Protein Function: Multifaceted Membrane Interactions of Human Atg3 Promote LC3-Phosphatidylethanolamine Conjugation during Autophagy"

    Article Title: Translating Membrane Geometry into Protein Function: Multifaceted Membrane Interactions of Human Atg3 Promote LC3-Phosphatidylethanolamine Conjugation during Autophagy

    Journal: bioRxiv

    doi: 10.1101/2022.12.23.521840

    a , Left panel: sequence comparison of hAtg3 C-terminal residues 262 to 277 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 265 to 277. b , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its mutants (n = 3). CL is the control without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. c , Left panel: sequence comparison of hAtg3 C-terminal residues 291 to 300 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 291 to 300. d , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its mutants (n = 3). CL is the control without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. e , Representative immunoblot (n = 4 blots). Atg3 knockout (Atg3 -/- ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry EV (empty vector), mCherry-hAtg3 WT (wildtype), mCherry-hAtg3 F296L , or mCherry-hAtg3 F296S mutant were cultured in complete media (CM) with 100 nM bafilomycin A1 (BafA1 to block LC3-II degradation) for 3 hrs and subjected to immunoblotting with the indicated antibodies. f , Quantitative analysis of the relative LC3B–II level (n = 4 blots) in in vivo LC3B lipidation experiments. Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. Data are presented as mean ± SD. P values: **** P < 0.0001; ns, not significant.
    Figure Legend Snippet: a , Left panel: sequence comparison of hAtg3 C-terminal residues 262 to 277 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 265 to 277. b , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its mutants (n = 3). CL is the control without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. c , Left panel: sequence comparison of hAtg3 C-terminal residues 291 to 300 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 291 to 300. d , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its mutants (n = 3). CL is the control without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. e , Representative immunoblot (n = 4 blots). Atg3 knockout (Atg3 -/- ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry EV (empty vector), mCherry-hAtg3 WT (wildtype), mCherry-hAtg3 F296L , or mCherry-hAtg3 F296S mutant were cultured in complete media (CM) with 100 nM bafilomycin A1 (BafA1 to block LC3-II degradation) for 3 hrs and subjected to immunoblotting with the indicated antibodies. f , Quantitative analysis of the relative LC3B–II level (n = 4 blots) in in vivo LC3B lipidation experiments. Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. Data are presented as mean ± SD. P values: **** P < 0.0001; ns, not significant.

    Techniques Used: Sequencing, SDS Page, Western Blot, Knock-Out, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Cell Culture, Blocking Assay, In Vivo

    a , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its H266L and H266F mutants under different pHs (n = 3). CL is the control without liposomes. Asterisk indicates the degradation of LC3B in the presence of ATP. b , Plot of LC3B-PE formation at 1.5 hr for hAtg3 and its H266L and H266F mutants against pHs. Data are presented as mean ± SD. Quantification of conjugation reactions were obtained from three separate measurements (n = 3). c , Representative immunoblot (n = 5 blots). Atg3 knockout (Atg3 -/- ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry EV (empty vector), mCherry-hAtg3 WT (wildtype), mCherry-hAtg3 H266F , mCherry-hAtg3 H266K , or mCherry-hAtg3 H266L mutant were cultured in complete media (CM) with 100 nM bafilomycin A1 (BafA1 to block LC3-II degradation) for 3 hrs and subjected to immunoblotting with the indicated antibodies. d , Quantitative analysis of the relative LC3B-II level (n = 5 blots) in in vivo LC3B lipidation experiments. Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. Data are presented as mean ± SD. P values: **** P < 0.0001; ns, not significant.
    Figure Legend Snippet: a , Representative SDS-PAGE gel images of time dependent formation of LC3B-PE for hAtg3 and its H266L and H266F mutants under different pHs (n = 3). CL is the control without liposomes. Asterisk indicates the degradation of LC3B in the presence of ATP. b , Plot of LC3B-PE formation at 1.5 hr for hAtg3 and its H266L and H266F mutants against pHs. Data are presented as mean ± SD. Quantification of conjugation reactions were obtained from three separate measurements (n = 3). c , Representative immunoblot (n = 5 blots). Atg3 knockout (Atg3 -/- ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry EV (empty vector), mCherry-hAtg3 WT (wildtype), mCherry-hAtg3 H266F , mCherry-hAtg3 H266K , or mCherry-hAtg3 H266L mutant were cultured in complete media (CM) with 100 nM bafilomycin A1 (BafA1 to block LC3-II degradation) for 3 hrs and subjected to immunoblotting with the indicated antibodies. d , Quantitative analysis of the relative LC3B-II level (n = 5 blots) in in vivo LC3B lipidation experiments. Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. Data are presented as mean ± SD. P values: **** P < 0.0001; ns, not significant.

    Techniques Used: SDS Page, Conjugation Assay, Western Blot, Knock-Out, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Cell Culture, Blocking Assay, In Vivo

    mefs  (ATCC)


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    ATCC mefs
    Mefs, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse embryonic fibroblast mef cells  (ATCC)


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    ATCC mouse embryonic fibroblast mef cells
    Mouse Embryonic Fibroblast Mef Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse embryonic fibroblasts mef  (ATCC)


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    ATCC mouse embryonic fibroblasts mef
    <t>NFAT5-deficient</t> <t>MEF</t> cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.
    Mouse Embryonic Fibroblasts Mef, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase"

    Article Title: Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007036

    NFAT5-deficient MEF cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.
    Figure Legend Snippet: NFAT5-deficient MEF cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.

    Techniques Used: Construct, Cell Culture, Activity Assay

    NFAT5-deficient MEF were cotransfected with a constant amount of the ORE-Luc reporter plus different concentrations of expression vectors (1.5 to 6 µg of DNA/9.4 cm 2 -well) encoding wild-type NFAT5a (FL5), a constitutively nuclear mutant (FL5 AED ) or an inactive DNA binding mutant (FL5 DB1 ), and then were cultured in isotonic medium (290 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of 1.5 µg of FL5 in isotonic medium (arbitrary value of 1). Results are the mean±SEM of four independent experiments (* p<0.05).
    Figure Legend Snippet: NFAT5-deficient MEF were cotransfected with a constant amount of the ORE-Luc reporter plus different concentrations of expression vectors (1.5 to 6 µg of DNA/9.4 cm 2 -well) encoding wild-type NFAT5a (FL5), a constitutively nuclear mutant (FL5 AED ) or an inactive DNA binding mutant (FL5 DB1 ), and then were cultured in isotonic medium (290 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of 1.5 µg of FL5 in isotonic medium (arbitrary value of 1). Results are the mean±SEM of four independent experiments (* p<0.05).

    Techniques Used: Expressing, Mutagenesis, Binding Assay, Cell Culture, Activity Assay, Construct

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    ATCC mef feeder layer
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    ATCC mef cell lines
    (a) (Top) Western blot detection <t>of</t> <t>Opa1</t> forms in indicated <t>MEF</t> cell lines using Opa1 antibody. (Bottom) Actin was used as loading control. (b) Genetic schematic and cartoon depictions of Opa1 forms present in MEF cell lines used in this study.
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    ATCC mouse embryonic fibroblasts mefs
    Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic <t>murine</t> <t>embryonic</t> fibroblasts. Co-culture of PBMC with non-engineered <t>MEFs</t> was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.
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    ATCC mouse embryonic fibroblasts mef
    Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic <t>murine</t> <t>embryonic</t> fibroblasts. Co-culture of PBMC with non-engineered <t>MEFs</t> was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.
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    ATCC mef cells
    (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in <t>MEF</t> <t>cells</t> within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.
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    mefs  (ATCC)
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    ATCC mefs
    (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in <t>MEF</t> <t>cells</t> within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.
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    ATCC mouse embryonic fibroblast mef cells
    (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in <t>MEF</t> <t>cells</t> within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.
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    (a) (Top) Western blot detection of Opa1 forms in indicated MEF cell lines using Opa1 antibody. (Bottom) Actin was used as loading control. (b) Genetic schematic and cartoon depictions of Opa1 forms present in MEF cell lines used in this study.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: (a) (Top) Western blot detection of Opa1 forms in indicated MEF cell lines using Opa1 antibody. (Bottom) Actin was used as loading control. (b) Genetic schematic and cartoon depictions of Opa1 forms present in MEF cell lines used in this study.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: Western Blot

    Mitochondria with distinguishable inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) visualized by cryo-ET. (a) (Right) Summed, projected central slices of cryo-electron tomograms visualizing representative mitochondria in indicated MEF lines. (Left) Three-dimensional (3D) rendering of segmented membranes with mitochondria shown across Z slices. Green and yellow surfaces indicate OMM and IMM, respectively. (Bottom left) Schematic of Opa1 forms present in respective cell lines. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Plot of mitochondria size (µm 2 ) observed in cryo-electron tomograms in MEF lines. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, **p<0.0, ****p< 0.0001. N: wild-type = 57, Opa1-OE = 17, l- Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: Mitochondria with distinguishable inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) visualized by cryo-ET. (a) (Right) Summed, projected central slices of cryo-electron tomograms visualizing representative mitochondria in indicated MEF lines. (Left) Three-dimensional (3D) rendering of segmented membranes with mitochondria shown across Z slices. Green and yellow surfaces indicate OMM and IMM, respectively. (Bottom left) Schematic of Opa1 forms present in respective cell lines. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Plot of mitochondria size (µm 2 ) observed in cryo-electron tomograms in MEF lines. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, **p<0.0, ****p< 0.0001. N: wild-type = 57, Opa1-OE = 17, l- Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: Tomography, MANN-WHITNEY

    Representative images of mitochondrial morphology in indicated MEF cell lines labeled with MitoTracker TM Deep Red FM. Insets show magnified view of regions indicated with dashed boxes. Scale bar = 10 µm. Inset scale bar = 5 µm.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: Representative images of mitochondrial morphology in indicated MEF cell lines labeled with MitoTracker TM Deep Red FM. Insets show magnified view of regions indicated with dashed boxes. Scale bar = 10 µm. Inset scale bar = 5 µm.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: Labeling

    (a) Gallery of representative mitochondrial morphology observed by TEM for indicated MEF cell lines. Matrix phenotype is indicated. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Graph bar representing the relative proportion of mitochondrial matrix density based on four categories: empty, uneven, dark and normal in indicated MEF cell lines. (d) Violin graphs plotting mitochondrial area, (e) number of mitochondria per cell and (f) mitochondrial area in µm 2 per cell in MEF cell lines included in this study. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001. For b: N wild-type, Opa1-OE and Opa1-KO = 301, l-Opa1* = 531, s-Opa1* = 359. For c: N wild-type and Opa1-OE = 20, l-Opa1* = 33, s-Opa1* = 32, Opa1-KO = 13. For d: N wild- type = 536, Opa1-OE = 337, l-Opa1* = 476, s-Opa1* = 318, Opa1-KO =302. For e: N wild-type and Opa1- OE = 20, l-Opa1* = 27, s-Opa1* = 32, Opa1-KO = 13. For f: N wild-type and Opa1-OE = 20, l-Opa1* = 27, s-Opa1* = 30, Opa1-KO = 13. Scale bar = 200 nm.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: (a) Gallery of representative mitochondrial morphology observed by TEM for indicated MEF cell lines. Matrix phenotype is indicated. (b) Graph bar representing the relative proportion of different mitochondrial shapes observed. (c) Graph bar representing the relative proportion of mitochondrial matrix density based on four categories: empty, uneven, dark and normal in indicated MEF cell lines. (d) Violin graphs plotting mitochondrial area, (e) number of mitochondria per cell and (f) mitochondrial area in µm 2 per cell in MEF cell lines included in this study. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001. For b: N wild-type, Opa1-OE and Opa1-KO = 301, l-Opa1* = 531, s-Opa1* = 359. For c: N wild-type and Opa1-OE = 20, l-Opa1* = 33, s-Opa1* = 32, Opa1-KO = 13. For d: N wild- type = 536, Opa1-OE = 337, l-Opa1* = 476, s-Opa1* = 318, Opa1-KO =302. For e: N wild-type and Opa1- OE = 20, l-Opa1* = 27, s-Opa1* = 32, Opa1-KO = 13. For f: N wild-type and Opa1-OE = 20, l-Opa1* = 27, s-Opa1* = 30, Opa1-KO = 13. Scale bar = 200 nm.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: MANN-WHITNEY

    (a) (Top) Summed, projected central slices of cryo- electron tomograms visualizing mitochondria with stacking cristae characteristics, supported by 3D representations consisting of their sub compartments (bottom) in indicated MEF cell lines. (b) Graph bar representing percentage of mitochondria with stacking cristae formation in each MEF cell line. N: wild- type = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: (a) (Top) Summed, projected central slices of cryo- electron tomograms visualizing mitochondria with stacking cristae characteristics, supported by 3D representations consisting of their sub compartments (bottom) in indicated MEF cell lines. (b) Graph bar representing percentage of mitochondria with stacking cristae formation in each MEF cell line. N: wild- type = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Scale bar = 200 nm.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques:

    (a) Graph bars showing the proportions of straight, tilted, and disconnected crista and (b) of lamellar, tubular, globular and unusual crista observed in indicated MEF lines. (c) Measured cristae length and (d) cristae width across cell lines. (e) (Top rows) Computational slices of straight, tilted, disconnected, globular and tubular crista across cell lines with zipping, pinching and tight regions indicated (arrows) in 3D renderings (bottom rows) from cryo-electron tomograms. n.a.: not applicable. (f) Histograms of crista widths across cell conditions (see Methods). (g) Subtomogram averages of mitochondrial cristae membranes with the average width indicated. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, ***p<0.001, ****p<0.000. For a, b: N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. For c, d: N = 50 for all cell lines. Scale bar = 200 nm.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: (a) Graph bars showing the proportions of straight, tilted, and disconnected crista and (b) of lamellar, tubular, globular and unusual crista observed in indicated MEF lines. (c) Measured cristae length and (d) cristae width across cell lines. (e) (Top rows) Computational slices of straight, tilted, disconnected, globular and tubular crista across cell lines with zipping, pinching and tight regions indicated (arrows) in 3D renderings (bottom rows) from cryo-electron tomograms. n.a.: not applicable. (f) Histograms of crista widths across cell conditions (see Methods). (g) Subtomogram averages of mitochondrial cristae membranes with the average width indicated. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney test; *p<0.05, ***p<0.001, ****p<0.000. For a, b: N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. For c, d: N = 50 for all cell lines. Scale bar = 200 nm.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: MANN-WHITNEY

    (a) Graph bar representing the relative proportion of unusual cristae morphology observed in indicated MEF cell lines. Unusual cristae were categorized into vesicular, zipped, ring, split, amorphous, straight-across, pinched and loop. N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (b) Summed, projected central slices of cryo-electron tomograms showing examples of unusual cristae in mitochondria across cell lines in 2D (top) and 3D (bottom). Loop, ring, straight-across, pinched, vesicular, and amorphous cristae are shown. Scale bar = 200 nm.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: (a) Graph bar representing the relative proportion of unusual cristae morphology observed in indicated MEF cell lines. Unusual cristae were categorized into vesicular, zipped, ring, split, amorphous, straight-across, pinched and loop. N wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (b) Summed, projected central slices of cryo-electron tomograms showing examples of unusual cristae in mitochondria across cell lines in 2D (top) and 3D (bottom). Loop, ring, straight-across, pinched, vesicular, and amorphous cristae are shown. Scale bar = 200 nm.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques:

    (a) Violin graphs plotting the percentage of multi-junction cristae per mitochondrion in indicated MEF cell lines. (b) Graph bar representing percentage of multi-junction cristae categorized into straight-across and loop morphology in each MEF cell line. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.0001. For a: N wild-type = 18, Opa1- OE =5, l-Opa1* = 30, s-Opa1* = 16, Opa1-KO = 3. For b: N wild-type = 26, Opa1-OE =9, l-Opa1* = 79, s-Opa1* = 29, Opa1-KO = 4.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: (a) Violin graphs plotting the percentage of multi-junction cristae per mitochondrion in indicated MEF cell lines. (b) Graph bar representing percentage of multi-junction cristae categorized into straight-across and loop morphology in each MEF cell line. Violin graphs show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann Whitney; *p<0.0001. For a: N wild-type = 18, Opa1- OE =5, l-Opa1* = 30, s-Opa1* = 16, Opa1-KO = 3. For b: N wild-type = 26, Opa1-OE =9, l-Opa1* = 79, s-Opa1* = 29, Opa1-KO = 4.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: MANN-WHITNEY

    BH3 profiling of (a) WT, l-Opa1*, s-Opa1*, Opa1- KO and (b) WT along with Opa1-OE MEF cell lines for sensitizer BIM BH3 and PUMA. N = 3-4 biological replicates. Significance of difference is tested relative to wild-type using the Holm-Sidak’s multiple comparison test; **p<0.01. (c) MEF cell lines were treated with indicated agents for 24h and apoptosis was detected by Annexin V positivity staining. N = minimum 4 biological replicates.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: BH3 profiling of (a) WT, l-Opa1*, s-Opa1*, Opa1- KO and (b) WT along with Opa1-OE MEF cell lines for sensitizer BIM BH3 and PUMA. N = 3-4 biological replicates. Significance of difference is tested relative to wild-type using the Holm-Sidak’s multiple comparison test; **p<0.01. (c) MEF cell lines were treated with indicated agents for 24h and apoptosis was detected by Annexin V positivity staining. N = minimum 4 biological replicates.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: Staining

    Assessment of cell viability by Annexin V staining in MEF cell lines after treatment with the indicated compounds for (a) 24 hours, (b) 48 hours and (c) 72 hours. N = minimum 4 biological replicates.

    Journal: bioRxiv

    Article Title: In situ architecture of Opa1-dependent mitochondrial cristae remodeling

    doi: 10.1101/2023.01.16.524176

    Figure Lengend Snippet: Assessment of cell viability by Annexin V staining in MEF cell lines after treatment with the indicated compounds for (a) 24 hours, (b) 48 hours and (c) 72 hours. N = minimum 4 biological replicates.

    Article Snippet: Wildtype and Opa1 knock-out MEF cell lines were purchased from ATCC.

    Techniques: Staining

    Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic murine embryonic fibroblasts. Co-culture of PBMC with non-engineered MEFs was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.

    Journal: STAR Protocols

    Article Title: Analyzing trogocytosis of T lymphocytes by flow cytometry and confocal microscopy

    doi: 10.1016/j.xpro.2022.102013

    Figure Lengend Snippet: Exemplary gating strategies for primary human T cells and murine T cell line (A) Trogocytosis by activated primary human CD4 + T cells after 12 h co-culture of PBMC with CD80-mScarlet transgenic murine embryonic fibroblasts. Co-culture of PBMC with non-engineered MEFs was used as negative control. (B) Trogocytosis of CTLA4-transgenic murine 58 αβ T cells after 2 h co-culture with CD80-TagRFP transgenic CHO cells. 58 αβ T cells lacking expression of CTLA4 and CD28 were used as negative control.

    Article Snippet: Mouse embryonic fibroblasts (MEFs) , ATCC , CRL-2907.

    Techniques: Co-Culture Assay, Transgenic Assay, Negative Control, Expressing

    Journal: STAR Protocols

    Article Title: Analyzing trogocytosis of T lymphocytes by flow cytometry and confocal microscopy

    doi: 10.1016/j.xpro.2022.102013

    Figure Lengend Snippet:

    Article Snippet: Mouse embryonic fibroblasts (MEFs) , ATCC , CRL-2907.

    Techniques: Staining, Recombinant, Modification, Software, Cell Culture, Microscopy, Flow Cytometry, Fluorescence

    (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in MEF cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.

    Journal: Life Science Alliance

    Article Title: A SNX1–SNX2–VAPB partnership regulates endosomal membrane rewiring in response to nutritional stress

    doi: 10.26508/lsa.202201652

    Figure Lengend Snippet: (A) HeLa cells were grown under control and starvation for 1 h (stv. 1 h) conditions with transient expression of SNX1-GFP (+SNX1-GFP versus untransfected cells), fixed and stained with anti-EEA1 antibody (red channel) and DAPI (blue channel). Magnified areas show EEA1 endosomes in depicted conditions. Scale bars = 5 μm. (A, B) Quantification of the percentage of EEA1 vesicle roundness parameter (per cell) in conditions depicted in (A). Means ± SD, from three independent experiments; ns, not significant; * P < 0.05 and **** P < 0.0001 in ordinary one-way ANOVA. (C) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (C, D) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (C). Each connected line represents one spatial frame, * P < 0.05 and **** P < 0.0001 in RM one-way ANOVA. (E) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in MEF cells within the same cell and the same spatial frame under control condition and starvation (stv.) for 2 and 5 min. Scale bars = 2 μm. (E, F) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (E). Each connected line represents one spatial frame, **** P < 0.0001 in RM one-way ANOVA. (G) Stills of live imaging showing the tubulation of SNX1-positive endosomal structures in HeLa cells within the same cell and the same spatial frame under control condition and rapamycin 200 nM (rapa.) for 2 and 5 min. Scale bars = 2 μm. (G, H) Quantification of the number of SNX1-GFP tubules per 100 μm 2 area in condition depicted in (G). Each connected line represents one spatial frame, * P < 0.05 and *** P < 0.001 in RM one-way ANOVA.

    Article Snippet: MEF cells from ATCC were grown in DMEM (41966; Gibco) supplemented with 20% FCS at 37°C and 5% CO 2 .

    Techniques: Expressing, Staining, Imaging