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    Addgene inc protein expression vector pet28a mcherry cna35
    (A) Fluorescent image of collagen fiber alignment and elongated aggregates expressing the cell membrane marker GFP-HRasC20. Collagen fibers were labeled with <t>mCherry-CNA35</t> peptide (magenta). (B) Cartoon of symmetry ratio of aggregates (i) and gel coherency (ii). (C) Scatter plot of aggregate symmetry ratio and collagen fiber coherency. (n = 75 aggregates). (D) Regional analysis of collagen coherency around aggregates. Cartoon illustrates the approach: for elongating aggregates coherency was measured in regions of interest (ROIs) placed both at the tips of elongations and proximate to their non-rounded areas. For rounded aggregates, ROIs were placed orthogonally. Regional differences in coherency were measured as the fold difference, measured around rounded aggregates (n = 19 aggregates) and elongated aggregates (n = 57 aggregates). Elongated aggregates were defined as symmetry ratio > 1.5. Double headed arrow: elongating axis. (E) Fold difference of collagen fiber coherency around aggregates measured at early stages of elongation (first 3 days of culture) subdivided based on symmetry ratio (n = 98 aggregates). (F) Fluorescent images of collagen fibers labeled with mCherry-CNA35 (magenta) and aggregates expressing GFP-HRasC20 cultured for 8 days with mitomycin C or aphidicolin. (G) Coherency of collagen fibers surrounding the aggregates treated with mitomycin C and aphidicolin. (n = 40 aggregates). (H) Fluorescent images of collagen fiber alignment with aggregates expressing GFP-HRasC20 treated with blebbistatin or Y-27632 for 8 days. Collagen fibrils were labeled with CNA35-mCherry (magenta). (I) Symmetry ratio of aggregates cultured for 8 days with or without blebbistatin or Y-27632. (n = 88 aggregates). (J) Distribution of collagen fiber orientation surrounding elongated aggregates (n = 22 aggregates) or non-elongated aggregates (n = 13 aggregates). (K) Difference between elongation axis of aggregates and average angle of collagen fibers in non-elongating area or elongating area. (n = 57 aggregates). All data are means ± SEM, ns, not significant, *P<0.05, **P<0.01, ***P<0.001. Data in (D, E, G, I) were analyzed with one-way ANOVA Tukey’s multiple comparisons test. Data in (K) were analyzed by unpaired Student’s t -test. The following figure supplements are available for : Figure supplement 1. Immunofluorescent staining of ECM proteins in the aggregates. Figure supplement 2. Effect of Rac1 GEF inhibitor on MCF10A aggregate elongation.
    Protein Expression Vector Pet28a Mcherry Cna35, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein expression vector pet28a mcherry cna35/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein expression vector pet28a mcherry cna35 - by Bioz Stars, 2024-07
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    86
    Thermo Fisher finnigan surveyor 61607 system
    (A) Fluorescent image of collagen fiber alignment and elongated aggregates expressing the cell membrane marker GFP-HRasC20. Collagen fibers were labeled with <t>mCherry-CNA35</t> peptide (magenta). (B) Cartoon of symmetry ratio of aggregates (i) and gel coherency (ii). (C) Scatter plot of aggregate symmetry ratio and collagen fiber coherency. (n = 75 aggregates). (D) Regional analysis of collagen coherency around aggregates. Cartoon illustrates the approach: for elongating aggregates coherency was measured in regions of interest (ROIs) placed both at the tips of elongations and proximate to their non-rounded areas. For rounded aggregates, ROIs were placed orthogonally. Regional differences in coherency were measured as the fold difference, measured around rounded aggregates (n = 19 aggregates) and elongated aggregates (n = 57 aggregates). Elongated aggregates were defined as symmetry ratio > 1.5. Double headed arrow: elongating axis. (E) Fold difference of collagen fiber coherency around aggregates measured at early stages of elongation (first 3 days of culture) subdivided based on symmetry ratio (n = 98 aggregates). (F) Fluorescent images of collagen fibers labeled with mCherry-CNA35 (magenta) and aggregates expressing GFP-HRasC20 cultured for 8 days with mitomycin C or aphidicolin. (G) Coherency of collagen fibers surrounding the aggregates treated with mitomycin C and aphidicolin. (n = 40 aggregates). (H) Fluorescent images of collagen fiber alignment with aggregates expressing GFP-HRasC20 treated with blebbistatin or Y-27632 for 8 days. Collagen fibrils were labeled with CNA35-mCherry (magenta). (I) Symmetry ratio of aggregates cultured for 8 days with or without blebbistatin or Y-27632. (n = 88 aggregates). (J) Distribution of collagen fiber orientation surrounding elongated aggregates (n = 22 aggregates) or non-elongated aggregates (n = 13 aggregates). (K) Difference between elongation axis of aggregates and average angle of collagen fibers in non-elongating area or elongating area. (n = 57 aggregates). All data are means ± SEM, ns, not significant, *P<0.05, **P<0.01, ***P<0.001. Data in (D, E, G, I) were analyzed with one-way ANOVA Tukey’s multiple comparisons test. Data in (K) were analyzed by unpaired Student’s t -test. The following figure supplements are available for : Figure supplement 1. Immunofluorescent staining of ECM proteins in the aggregates. Figure supplement 2. Effect of Rac1 GEF inhibitor on MCF10A aggregate elongation.
    Finnigan Surveyor 61607 System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/finnigan surveyor 61607 system/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    finnigan surveyor 61607 system - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    Image Search Results


    (A) Fluorescent image of collagen fiber alignment and elongated aggregates expressing the cell membrane marker GFP-HRasC20. Collagen fibers were labeled with mCherry-CNA35 peptide (magenta). (B) Cartoon of symmetry ratio of aggregates (i) and gel coherency (ii). (C) Scatter plot of aggregate symmetry ratio and collagen fiber coherency. (n = 75 aggregates). (D) Regional analysis of collagen coherency around aggregates. Cartoon illustrates the approach: for elongating aggregates coherency was measured in regions of interest (ROIs) placed both at the tips of elongations and proximate to their non-rounded areas. For rounded aggregates, ROIs were placed orthogonally. Regional differences in coherency were measured as the fold difference, measured around rounded aggregates (n = 19 aggregates) and elongated aggregates (n = 57 aggregates). Elongated aggregates were defined as symmetry ratio > 1.5. Double headed arrow: elongating axis. (E) Fold difference of collagen fiber coherency around aggregates measured at early stages of elongation (first 3 days of culture) subdivided based on symmetry ratio (n = 98 aggregates). (F) Fluorescent images of collagen fibers labeled with mCherry-CNA35 (magenta) and aggregates expressing GFP-HRasC20 cultured for 8 days with mitomycin C or aphidicolin. (G) Coherency of collagen fibers surrounding the aggregates treated with mitomycin C and aphidicolin. (n = 40 aggregates). (H) Fluorescent images of collagen fiber alignment with aggregates expressing GFP-HRasC20 treated with blebbistatin or Y-27632 for 8 days. Collagen fibrils were labeled with CNA35-mCherry (magenta). (I) Symmetry ratio of aggregates cultured for 8 days with or without blebbistatin or Y-27632. (n = 88 aggregates). (J) Distribution of collagen fiber orientation surrounding elongated aggregates (n = 22 aggregates) or non-elongated aggregates (n = 13 aggregates). (K) Difference between elongation axis of aggregates and average angle of collagen fibers in non-elongating area or elongating area. (n = 57 aggregates). All data are means ± SEM, ns, not significant, *P<0.05, **P<0.01, ***P<0.001. Data in (D, E, G, I) were analyzed with one-way ANOVA Tukey’s multiple comparisons test. Data in (K) were analyzed by unpaired Student’s t -test. The following figure supplements are available for : Figure supplement 1. Immunofluorescent staining of ECM proteins in the aggregates. Figure supplement 2. Effect of Rac1 GEF inhibitor on MCF10A aggregate elongation.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) Fluorescent image of collagen fiber alignment and elongated aggregates expressing the cell membrane marker GFP-HRasC20. Collagen fibers were labeled with mCherry-CNA35 peptide (magenta). (B) Cartoon of symmetry ratio of aggregates (i) and gel coherency (ii). (C) Scatter plot of aggregate symmetry ratio and collagen fiber coherency. (n = 75 aggregates). (D) Regional analysis of collagen coherency around aggregates. Cartoon illustrates the approach: for elongating aggregates coherency was measured in regions of interest (ROIs) placed both at the tips of elongations and proximate to their non-rounded areas. For rounded aggregates, ROIs were placed orthogonally. Regional differences in coherency were measured as the fold difference, measured around rounded aggregates (n = 19 aggregates) and elongated aggregates (n = 57 aggregates). Elongated aggregates were defined as symmetry ratio > 1.5. Double headed arrow: elongating axis. (E) Fold difference of collagen fiber coherency around aggregates measured at early stages of elongation (first 3 days of culture) subdivided based on symmetry ratio (n = 98 aggregates). (F) Fluorescent images of collagen fibers labeled with mCherry-CNA35 (magenta) and aggregates expressing GFP-HRasC20 cultured for 8 days with mitomycin C or aphidicolin. (G) Coherency of collagen fibers surrounding the aggregates treated with mitomycin C and aphidicolin. (n = 40 aggregates). (H) Fluorescent images of collagen fiber alignment with aggregates expressing GFP-HRasC20 treated with blebbistatin or Y-27632 for 8 days. Collagen fibrils were labeled with CNA35-mCherry (magenta). (I) Symmetry ratio of aggregates cultured for 8 days with or without blebbistatin or Y-27632. (n = 88 aggregates). (J) Distribution of collagen fiber orientation surrounding elongated aggregates (n = 22 aggregates) or non-elongated aggregates (n = 13 aggregates). (K) Difference between elongation axis of aggregates and average angle of collagen fibers in non-elongating area or elongating area. (n = 57 aggregates). All data are means ± SEM, ns, not significant, *P<0.05, **P<0.01, ***P<0.001. Data in (D, E, G, I) were analyzed with one-way ANOVA Tukey’s multiple comparisons test. Data in (K) were analyzed by unpaired Student’s t -test. The following figure supplements are available for : Figure supplement 1. Immunofluorescent staining of ECM proteins in the aggregates. Figure supplement 2. Effect of Rac1 GEF inhibitor on MCF10A aggregate elongation.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Expressing, Marker, Labeling, Cell Culture, Staining

    (A) Fluorescent images of collagen fiber alignment with aggregates expressing GFP- HRasC20 treated with NSC23766 for 8 days. Collagen fibers were labeled with mCherry-CNA35 (magenta). (B) Speed of cell movement based on nuclear tracking in rounded and elongated aggregates treated with NSC23766. (n = 82 movies) (C) Effect of delayed inhibition of proliferation on aggregate elongation. Aggregates were cultured for 3 days before treatment with NSC23766. Data are fold change of elongation in control and drug-treated cultures (n = 40 aggregates). All data are means ± SEM; ns, not significant, *P<0.05, **P<0.01. Data in (B,C) were analyzed with one-way ANOVA Tukey’s multiple comparisons test. Data in (D) was analyzed by unpaired Student’s t -test.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) Fluorescent images of collagen fiber alignment with aggregates expressing GFP- HRasC20 treated with NSC23766 for 8 days. Collagen fibers were labeled with mCherry-CNA35 (magenta). (B) Speed of cell movement based on nuclear tracking in rounded and elongated aggregates treated with NSC23766. (n = 82 movies) (C) Effect of delayed inhibition of proliferation on aggregate elongation. Aggregates were cultured for 3 days before treatment with NSC23766. Data are fold change of elongation in control and drug-treated cultures (n = 40 aggregates). All data are means ± SEM; ns, not significant, *P<0.05, **P<0.01. Data in (B,C) were analyzed with one-way ANOVA Tukey’s multiple comparisons test. Data in (D) was analyzed by unpaired Student’s t -test.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Expressing, Labeling, Inhibition, Cell Culture

    (A) Second harmonic generation (SHG) images of collagen fibers in the gel with or without stretching and incubated for 7 days after re-embedding in gel. Double head arrow: stretching axis. (B) Coherency of collagen fiber in the gel with or without stretching. (n = 24 positions in multiple gels). (C) Distribution of collagen fiber orientation in the gel with or without stretching. 0° is defined as the axis of stretch. (N = 3 independent experiments). (D) SHG images of collagen fiber floated for 7 days with or without after stretching. Double head arrow: stretching axis. (E) Coherency of collagen fiber in 7 days floated gel with or without stretching. (n = 46 positions in multiple gels). (F) Distribution of collagen fiber orientation in gels that had been allowed to float (7 days) with or without prior stretching. (N = 3 independent experiments). (G) Time lapse images of aggregates embedded in stretched gel. Double head arrow: stretching axis. (H) Initiation time of aggregate elongation in the gel with or without stretching. (-Stretch: n = 25 aggregates, +Stretch: n = 71 aggregates). (I) Symmetry ratio of aggregates in the early phase of culture (0-3 days) with or without stretching. (-Stretch, day 0: n = 12, day 1: n = 12, day 2: n = 24, day 3: n = 24, +Stretch, day 0: n = 21, day 1: n = 32, day 2: n = 17, day 3: n = 22). (J) Distribution of elongation axes of aggregates in the gel with or without stretching. (- Stretch: n = 112 aggregates, +Stretch: n = 115 aggregates). (K) Difference between elongating axis of aggregates and average angle of collagen fibers in the gel with or without stretching. (- Stretch: n = 21 aggregates, +Stretch: n = 102 aggregates). (L) Fluorescence image of aggregates cultured for 5 days after gel stretching and co-stained with phalloidin (green) and DAPI (blue) in stretched gel. Collagen fibrils were labeled with CNA35-mCherry (magenta). (M) Coherency of collagen fibers surrounding elongated aggregates in the gel with or without stretching. (- Stretch: n = 21 aggregates, +Stretch: n = 102 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (B, E, H, K, M) were analyzed by unpaired Student’s t -test. Data in (I) was analyzed with one-way ANOVA Tukey’s multiple comparisons test. The following figure supplements are available for : Figure supplement 1. External gel stretching aligns collagen fiber. Figure supplement 2. MCF10A aggregates elongate along the gel stretching axis.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) Second harmonic generation (SHG) images of collagen fibers in the gel with or without stretching and incubated for 7 days after re-embedding in gel. Double head arrow: stretching axis. (B) Coherency of collagen fiber in the gel with or without stretching. (n = 24 positions in multiple gels). (C) Distribution of collagen fiber orientation in the gel with or without stretching. 0° is defined as the axis of stretch. (N = 3 independent experiments). (D) SHG images of collagen fiber floated for 7 days with or without after stretching. Double head arrow: stretching axis. (E) Coherency of collagen fiber in 7 days floated gel with or without stretching. (n = 46 positions in multiple gels). (F) Distribution of collagen fiber orientation in gels that had been allowed to float (7 days) with or without prior stretching. (N = 3 independent experiments). (G) Time lapse images of aggregates embedded in stretched gel. Double head arrow: stretching axis. (H) Initiation time of aggregate elongation in the gel with or without stretching. (-Stretch: n = 25 aggregates, +Stretch: n = 71 aggregates). (I) Symmetry ratio of aggregates in the early phase of culture (0-3 days) with or without stretching. (-Stretch, day 0: n = 12, day 1: n = 12, day 2: n = 24, day 3: n = 24, +Stretch, day 0: n = 21, day 1: n = 32, day 2: n = 17, day 3: n = 22). (J) Distribution of elongation axes of aggregates in the gel with or without stretching. (- Stretch: n = 112 aggregates, +Stretch: n = 115 aggregates). (K) Difference between elongating axis of aggregates and average angle of collagen fibers in the gel with or without stretching. (- Stretch: n = 21 aggregates, +Stretch: n = 102 aggregates). (L) Fluorescence image of aggregates cultured for 5 days after gel stretching and co-stained with phalloidin (green) and DAPI (blue) in stretched gel. Collagen fibrils were labeled with CNA35-mCherry (magenta). (M) Coherency of collagen fibers surrounding elongated aggregates in the gel with or without stretching. (- Stretch: n = 21 aggregates, +Stretch: n = 102 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (B, E, H, K, M) were analyzed by unpaired Student’s t -test. Data in (I) was analyzed with one-way ANOVA Tukey’s multiple comparisons test. The following figure supplements are available for : Figure supplement 1. External gel stretching aligns collagen fiber. Figure supplement 2. MCF10A aggregates elongate along the gel stretching axis.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Incubation, Fluorescence, Cell Culture, Staining, Labeling

    (A) MCF10A aggregates in the gel after 4 hours stretching. Aggregate was co- stained with Phalloidin (green) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (B) Symmetry ratio of aggregates with or without gel stretching for 4 hours (n = 274 aggregates). (C) Schematic image of measurement of elongation angle. (D) The average angle of elongated aggregates in non-stretched gel and stretched gel. (n = 227 aggregates). (E) MCF10A aggregates cultured for 1 day in non-stretched and stretched gels. Aggregates were co-stained with anti-fibronectin antibody (green) and DAPI (blue). Collagen fibrils were labeled with CNA35-mCherry (red). All data are means ± SEM; ns, not significant, ***P<0.001. Data were analyzed by unpaired Student’s t -test.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) MCF10A aggregates in the gel after 4 hours stretching. Aggregate was co- stained with Phalloidin (green) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (B) Symmetry ratio of aggregates with or without gel stretching for 4 hours (n = 274 aggregates). (C) Schematic image of measurement of elongation angle. (D) The average angle of elongated aggregates in non-stretched gel and stretched gel. (n = 227 aggregates). (E) MCF10A aggregates cultured for 1 day in non-stretched and stretched gels. Aggregates were co-stained with anti-fibronectin antibody (green) and DAPI (blue). Collagen fibrils were labeled with CNA35-mCherry (red). All data are means ± SEM; ns, not significant, ***P<0.001. Data were analyzed by unpaired Student’s t -test.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Staining, Labeling, Cell Culture

    (A) Time course of cell proliferation within aggregates in control gels (-stretch) or after stretching (+ stretch). Data are percentage of cells that were Ki67 positive (n = 206 aggregates). (B,C) Length (B) and (C) symmetry ratio of aggregates in stretched gel incubated with mitomycin C or aphidicolin for 8 days. (n = 214 aggregates) (D) Fluorescence images of elongated aggregates cultured for 7 days in the re-embedded or floated gel after stretching. Aggregates were co-stained with anti-Ki67 antibody (green), Phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (E) Percentage of Ki67 positive cells in the aggregates cultured for 7 days in the re-embedded or floated gel after stretching. (n = 56 aggregates). (F) Percentage of Ki67 positive cells in the elongating area of aggregates in the re-embedded or floated gel. (n = 47 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (E, F) were analyzed by unpaired Student’s t -test. Data in (A, B, C) were analyzed with one-way ANOVA Tukey’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) Time course of cell proliferation within aggregates in control gels (-stretch) or after stretching (+ stretch). Data are percentage of cells that were Ki67 positive (n = 206 aggregates). (B,C) Length (B) and (C) symmetry ratio of aggregates in stretched gel incubated with mitomycin C or aphidicolin for 8 days. (n = 214 aggregates) (D) Fluorescence images of elongated aggregates cultured for 7 days in the re-embedded or floated gel after stretching. Aggregates were co-stained with anti-Ki67 antibody (green), Phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (E) Percentage of Ki67 positive cells in the aggregates cultured for 7 days in the re-embedded or floated gel after stretching. (n = 56 aggregates). (F) Percentage of Ki67 positive cells in the elongating area of aggregates in the re-embedded or floated gel. (n = 47 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (E, F) were analyzed by unpaired Student’s t -test. Data in (A, B, C) were analyzed with one-way ANOVA Tukey’s multiple comparisons test.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Incubation, Fluorescence, Cell Culture, Staining, Labeling

    (A) Fluorescent images of aggregates expressing ERK/KTR-mClover biosensor cultured for 7 days and stained with phalloidin (magenta) and DAPI (blue). (B) Percentage of ERK active cells in elongating areas and non-elongating areas of the aggregates. (n = 30 aggregates). (C) Percentage of ERK active cells at the surface in elongating and non-elongating areas of aggregates. (n = 30 aggregates). (D) Fluorescent images of aggregates cultured for 7 days treated with FR180207 after gel stretching. Aggregates were co-stained with anti-Ki67 antibody (green), Phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (E) Percentage of Ki67-positive cells in aggregates incubated with FR180207 for 7days after stretching. (n = 24 aggregates). (F) Effect of inhibiting ERK on stretch-induced aggregate elongation. Proportion of elongated aggregates in stretched gel incubated with FR180207 for 3 days and 7 days. (N = 3 independent experiments). (G) Length and (H) symmetry ratio of elongated aggregates incubated with FR180207 for 7 days. (n = 166 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (B, C, E,-H) were analyzed by unpaired Student’s t -test. The following figure supplement is available for : Figure supplement 1. ERK biosensor and YAP1 localization in MCF10A cells.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) Fluorescent images of aggregates expressing ERK/KTR-mClover biosensor cultured for 7 days and stained with phalloidin (magenta) and DAPI (blue). (B) Percentage of ERK active cells in elongating areas and non-elongating areas of the aggregates. (n = 30 aggregates). (C) Percentage of ERK active cells at the surface in elongating and non-elongating areas of aggregates. (n = 30 aggregates). (D) Fluorescent images of aggregates cultured for 7 days treated with FR180207 after gel stretching. Aggregates were co-stained with anti-Ki67 antibody (green), Phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (E) Percentage of Ki67-positive cells in aggregates incubated with FR180207 for 7days after stretching. (n = 24 aggregates). (F) Effect of inhibiting ERK on stretch-induced aggregate elongation. Proportion of elongated aggregates in stretched gel incubated with FR180207 for 3 days and 7 days. (N = 3 independent experiments). (G) Length and (H) symmetry ratio of elongated aggregates incubated with FR180207 for 7 days. (n = 166 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (B, C, E,-H) were analyzed by unpaired Student’s t -test. The following figure supplement is available for : Figure supplement 1. ERK biosensor and YAP1 localization in MCF10A cells.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Expressing, Cell Culture, Staining, Labeling, Incubation

    (A) Schematic of potential integrin-ERK pathway that mediates the effect of collagen polarization on cell proliferation. (B) Immunoblot of integrin α2, β1 and ERK1/2 protein levels in MCF10A cell lysate. (C) Fluorescent images of aggregates cultured for 7 days treated with AIIB2 antibody after gel stretching. Aggregates were co-stained with anti-Ki67 antibody (green), Phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (D) Percentage of Ki67 positive cells in the aggregates incubated with AIIB2 antibody for 7 days after stretching the gels. (n = 35 aggregates). (E) Proportion of elongated aggregates in stretched gel incubated with AIIB2 antibody for 3 days and 7 days. (N = 3 independent experiments). (F) Length and (G) symmetry ratio of elongated aggregates incubated with AIIB2 antibody for 7 days. (n = 134 aggregates). (H) Fluorescent images of aggregates expressed with ERK/KTR-mClover and incubated with IgG (control) or AIIB2 antibody for 2 days after gel stretching. Aggregates were co-stained with phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (I) Percentage of ERK active cells in aggregates incubated with AIIB2 antibody for 2 days or 7 days in stretched gel. (n = 78 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (D-G, I) were analyzed by unpaired Student’s t -test.

    Journal: bioRxiv

    Article Title: Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

    doi: 10.1101/2021.02.28.433274

    Figure Lengend Snippet: (A) Schematic of potential integrin-ERK pathway that mediates the effect of collagen polarization on cell proliferation. (B) Immunoblot of integrin α2, β1 and ERK1/2 protein levels in MCF10A cell lysate. (C) Fluorescent images of aggregates cultured for 7 days treated with AIIB2 antibody after gel stretching. Aggregates were co-stained with anti-Ki67 antibody (green), Phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (D) Percentage of Ki67 positive cells in the aggregates incubated with AIIB2 antibody for 7 days after stretching the gels. (n = 35 aggregates). (E) Proportion of elongated aggregates in stretched gel incubated with AIIB2 antibody for 3 days and 7 days. (N = 3 independent experiments). (F) Length and (G) symmetry ratio of elongated aggregates incubated with AIIB2 antibody for 7 days. (n = 134 aggregates). (H) Fluorescent images of aggregates expressed with ERK/KTR-mClover and incubated with IgG (control) or AIIB2 antibody for 2 days after gel stretching. Aggregates were co-stained with phalloidin (red) and DAPI (blue). Collagen fibers were labeled with mCherry-CNA35 (magenta). (I) Percentage of ERK active cells in aggregates incubated with AIIB2 antibody for 2 days or 7 days in stretched gel. (n = 78 aggregates). All data are means ± SEM; ns, not significant, **P<0.01, ***P<0.001. Data in (D-G, I) were analyzed by unpaired Student’s t -test.

    Article Snippet: Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607).

    Techniques: Western Blot, Cell Culture, Staining, Labeling, Incubation