cacl2 powder  (Millipore)


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    Millipore cacl2 powder
    Cacl2 Powder, supplied by Millipore, 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/cacl2 powder/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cacl2 powder - by Bioz Stars, 2020-09
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    Sublimation:

    Article Title: Mechanistic Insights of BHT-Mg-Catalyzed Ethylene Phosphate’s Coordination Ring-Opening Polymerization: DFT Modeling and Experimental Data
    Article Snippet: .. CH2 Cl2 was washed with aqueous Na2 CO3 , stirred with CaCl2 powder, refluxed over CaH2 for 8 h and distilled. l -lactide and rac -lactide (Sigma-Aldrich/Merck, Darmstadt, Germany) were purified by recrystallization and subsequent sublimation. ε-Caprolactone was distilled prior to use under argon over CaH2 . .. Methyl ethylene phosphate (MeOEP) [ ] and [(BHT)Mg(μ-OBn)(THF)]2 ( Mg1 ) [ ] were synthesized according to the literature procedures.

    Recrystallization:

    Article Title: Mechanistic Insights of BHT-Mg-Catalyzed Ethylene Phosphate’s Coordination Ring-Opening Polymerization: DFT Modeling and Experimental Data
    Article Snippet: .. CH2 Cl2 was washed with aqueous Na2 CO3 , stirred with CaCl2 powder, refluxed over CaH2 for 8 h and distilled. l -lactide and rac -lactide (Sigma-Aldrich/Merck, Darmstadt, Germany) were purified by recrystallization and subsequent sublimation. ε-Caprolactone was distilled prior to use under argon over CaH2 . .. Methyl ethylene phosphate (MeOEP) [ ] and [(BHT)Mg(μ-OBn)(THF)]2 ( Mg1 ) [ ] were synthesized according to the literature procedures.

    Purification:

    Article Title: Mechanistic Insights of BHT-Mg-Catalyzed Ethylene Phosphate’s Coordination Ring-Opening Polymerization: DFT Modeling and Experimental Data
    Article Snippet: .. CH2 Cl2 was washed with aqueous Na2 CO3 , stirred with CaCl2 powder, refluxed over CaH2 for 8 h and distilled. l -lactide and rac -lactide (Sigma-Aldrich/Merck, Darmstadt, Germany) were purified by recrystallization and subsequent sublimation. ε-Caprolactone was distilled prior to use under argon over CaH2 . .. Methyl ethylene phosphate (MeOEP) [ ] and [(BHT)Mg(μ-OBn)(THF)]2 ( Mg1 ) [ ] were synthesized according to the literature procedures.

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    Millipore human keratinocytes
    ROS-reducing effect of resveratrol on human <t>keratinocytes</t> following PM treatment. ( A ) Confocal microscopic images of the DCF-DA assay (ROS; green) ( B ) Relative intracellular ROS fluorescence intensity. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p
    Human Keratinocytes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human keratinocytes/product/Millipore
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    human keratinocytes - by Bioz Stars, 2020-09
    99/100 stars
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    ROS-reducing effect of resveratrol on human keratinocytes following PM treatment. ( A ) Confocal microscopic images of the DCF-DA assay (ROS; green) ( B ) Relative intracellular ROS fluorescence intensity. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Particulate Matter-Induced Inflammatory Responses in Human Keratinocytes

    doi: 10.3390/ijms21103446

    Figure Lengend Snippet: ROS-reducing effect of resveratrol on human keratinocytes following PM treatment. ( A ) Confocal microscopic images of the DCF-DA assay (ROS; green) ( B ) Relative intracellular ROS fluorescence intensity. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Article Snippet: In conclusion, in the present study, we found that resveratrol inhibits the PM-induced inflammatory response in human keratinocytes by inhibiting the AhR/ROS/MAPK pathway.

    Techniques: Fluorescence, Standard Deviation

    Effect of resveratrol on the production of proinflammatory cytokines in human keratinocytes following PM treatment. ( A ) Protein levels of MMP-1, ( B ) MMP-9, and ( C ) IL-8 were determined using ELISA. Cells were pretreated with resveratrol (1 μM) for 6 h and subsequently incubated with PM 10 (25 μg/cm 2 ) for 24 h. Keratinocyte culture supernatants were collected and used for further analyses. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Particulate Matter-Induced Inflammatory Responses in Human Keratinocytes

    doi: 10.3390/ijms21103446

    Figure Lengend Snippet: Effect of resveratrol on the production of proinflammatory cytokines in human keratinocytes following PM treatment. ( A ) Protein levels of MMP-1, ( B ) MMP-9, and ( C ) IL-8 were determined using ELISA. Cells were pretreated with resveratrol (1 μM) for 6 h and subsequently incubated with PM 10 (25 μg/cm 2 ) for 24 h. Keratinocyte culture supernatants were collected and used for further analyses. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Article Snippet: In conclusion, in the present study, we found that resveratrol inhibits the PM-induced inflammatory response in human keratinocytes by inhibiting the AhR/ROS/MAPK pathway.

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Standard Deviation

    Effects of particulate matter and resveratrol on human keratinocyte viability. Viability of keratinocytes treated with ( A ) PM 10 (0, 1.25 μg/mL, 3 μg/mL, 6 μg/mL, 12 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, or 200 μg/cm 2 ) or ( B ) resveratrol (0, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 50 μg/mL, or 100 μM) for 24h measured using the water-soluble tetrazolium salt assay. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). The ratio of surviving cells is relative to the untreated control. PM, particulate matter.

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Particulate Matter-Induced Inflammatory Responses in Human Keratinocytes

    doi: 10.3390/ijms21103446

    Figure Lengend Snippet: Effects of particulate matter and resveratrol on human keratinocyte viability. Viability of keratinocytes treated with ( A ) PM 10 (0, 1.25 μg/mL, 3 μg/mL, 6 μg/mL, 12 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, or 200 μg/cm 2 ) or ( B ) resveratrol (0, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 50 μg/mL, or 100 μM) for 24h measured using the water-soluble tetrazolium salt assay. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). The ratio of surviving cells is relative to the untreated control. PM, particulate matter.

    Article Snippet: In conclusion, in the present study, we found that resveratrol inhibits the PM-induced inflammatory response in human keratinocytes by inhibiting the AhR/ROS/MAPK pathway.

    Techniques: WST Salt Assay, Standard Deviation

    Effect of resveratrol on AhR expression in human keratinocytes following PM treatment. ( A ) Confocal microscopic images of AhR expressions in keratinocytes (AhR; green, nucleus; blue). ( B ) Relative AhR intensity. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Particulate Matter-Induced Inflammatory Responses in Human Keratinocytes

    doi: 10.3390/ijms21103446

    Figure Lengend Snippet: Effect of resveratrol on AhR expression in human keratinocytes following PM treatment. ( A ) Confocal microscopic images of AhR expressions in keratinocytes (AhR; green, nucleus; blue). ( B ) Relative AhR intensity. Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Article Snippet: In conclusion, in the present study, we found that resveratrol inhibits the PM-induced inflammatory response in human keratinocytes by inhibiting the AhR/ROS/MAPK pathway.

    Techniques: Expressing, Standard Deviation

    Effects of resveratrol on transcription factor activator protein-1 components and p38 MAPK phosphorylation in human keratinocytes following PM treatment. ( A ) Protein levels of JNK and ( B ) p38 MAPK were analyzed by Western blotting and densitometry. Equal amounts of protein loading were confirmed using JNK, p38, and actin antibodies. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Particulate Matter-Induced Inflammatory Responses in Human Keratinocytes

    doi: 10.3390/ijms21103446

    Figure Lengend Snippet: Effects of resveratrol on transcription factor activator protein-1 components and p38 MAPK phosphorylation in human keratinocytes following PM treatment. ( A ) Protein levels of JNK and ( B ) p38 MAPK were analyzed by Western blotting and densitometry. Equal amounts of protein loading were confirmed using JNK, p38, and actin antibodies. * p

    Article Snippet: In conclusion, in the present study, we found that resveratrol inhibits the PM-induced inflammatory response in human keratinocytes by inhibiting the AhR/ROS/MAPK pathway.

    Techniques: Western Blot

    Effect of resveratrol on PM-induced COX-2 expression and PGE2 generation in human keratinocytes. ( A ) The protein expression of COX-2 determined by Western blot and densitometry. Equal amounts of protein loading were verified using an anti-actin antibody, ( B ) PGE2 generation measured by ELISA, Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Particulate Matter-Induced Inflammatory Responses in Human Keratinocytes

    doi: 10.3390/ijms21103446

    Figure Lengend Snippet: Effect of resveratrol on PM-induced COX-2 expression and PGE2 generation in human keratinocytes. ( A ) The protein expression of COX-2 determined by Western blot and densitometry. Equal amounts of protein loading were verified using an anti-actin antibody, ( B ) PGE2 generation measured by ELISA, Data are presented as the mean ± standard deviation of three independent experiments ( n = 3). * p

    Article Snippet: In conclusion, in the present study, we found that resveratrol inhibits the PM-induced inflammatory response in human keratinocytes by inhibiting the AhR/ROS/MAPK pathway.

    Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

    The vinculin/α-catenin cytoskeletal scaffold is essential for telophase reorientation and afadin accumulation in AJs. ( A ) Cumulative frequency distribution of division angles from E16.5 live imaging experiments of Ctnna1 912 RFP+ and wild-type littermates; n values indicate cells imaged for each group. ( B ) Division orientation at anaphase onset (ϕ) and one hour later (θ) for Ctnna1 912 knockdown and wild-type littermates, plotted from data in ( A ). Ctnna1 912 RFP+ cells show no obvious correction pattern. ( C ) E16.5 Ctnna1 912 RFP+ mitotic cell (pHH3+, white), showing normal apical localization of LGN (green). ( D ) Quantification of LGN recruitment patterns in pHH3+ cells from E16.5 Ctnna1 912 , Vcl 3466 , and Vcl 2803 embryos (RFP+ cells), with age-matched littermate controls for each cohort. ( E ) Orientation of LGN crescents from indicated groups. Knockdown of Vcl or Ctnna1 does not significantly alter the tendency of LGN to localize apically. ( F ) Primary mouse keratinocytes after 8 h Ca 2+ shift—labeled with afadin (red), E-cadherin (green) and actin (phalloidin, grey)—which accumulate in linear bands at cell-cell junctions in Scramble control cells. Yellow boxed region shown at high magnification below; n indicates junctions evaluated. Vcl and Ctnna1 knockdown cells show defects in linear actin accumulation as well as afadin recruitment to AJs, quantified in ( G ). Scale bars, 5μm ( C ), 20μm ( F ). P values determined by Kolmogorov-Smirnov test ( A ), χ 2 ( D ), or student’s unpaired t-test ( E,G ). * P

    Journal: bioRxiv

    Article Title: Telophase correction refines division orientation in stratified epithelia

    doi: 10.1101/668244

    Figure Lengend Snippet: The vinculin/α-catenin cytoskeletal scaffold is essential for telophase reorientation and afadin accumulation in AJs. ( A ) Cumulative frequency distribution of division angles from E16.5 live imaging experiments of Ctnna1 912 RFP+ and wild-type littermates; n values indicate cells imaged for each group. ( B ) Division orientation at anaphase onset (ϕ) and one hour later (θ) for Ctnna1 912 knockdown and wild-type littermates, plotted from data in ( A ). Ctnna1 912 RFP+ cells show no obvious correction pattern. ( C ) E16.5 Ctnna1 912 RFP+ mitotic cell (pHH3+, white), showing normal apical localization of LGN (green). ( D ) Quantification of LGN recruitment patterns in pHH3+ cells from E16.5 Ctnna1 912 , Vcl 3466 , and Vcl 2803 embryos (RFP+ cells), with age-matched littermate controls for each cohort. ( E ) Orientation of LGN crescents from indicated groups. Knockdown of Vcl or Ctnna1 does not significantly alter the tendency of LGN to localize apically. ( F ) Primary mouse keratinocytes after 8 h Ca 2+ shift—labeled with afadin (red), E-cadherin (green) and actin (phalloidin, grey)—which accumulate in linear bands at cell-cell junctions in Scramble control cells. Yellow boxed region shown at high magnification below; n indicates junctions evaluated. Vcl and Ctnna1 knockdown cells show defects in linear actin accumulation as well as afadin recruitment to AJs, quantified in ( G ). Scale bars, 5μm ( C ), 20μm ( F ). P values determined by Kolmogorov-Smirnov test ( A ), χ 2 ( D ), or student’s unpaired t-test ( E,G ). * P

    Article Snippet: For knockdown screening, primary keratinocytes were seeded at a density of ~150,000 cells per well into 6-well plates and grown to ~80% confluency in E-Low calcium medium and infected with an MOI of ~1.

    Techniques: Imaging, Labeling

    Related to Figure 6 | Afadin loss-of-function division orientation phenotypes are polarity and LGN-independent ( A ) Immunostaining of E16.5 interphase cell with Par3 (red). ( B ) Quantification of Par3 radial fluorescent intensity. Apical accumulation is reduced by afadin knockout via K14-Cre ( Afdn cKO ) or lentiviral Cre-RFP; n indicates interphase cells from 2-3 independent embryos. ( C ) E16.5 sagittal sections show that centrosomes (green) localize to the apical cortex of interphase cells in both Afdn cKO basal progenitors and Afdn fl / fl controls. ( D ) Quantification of centrosome radial position in basal keratinocytes; n indicates interphase cells from 2 independent embryos. ( E ) LGN (red) localization patterns in mitotic (green) basal keratinocytes. ( F ) Quantification of LGN rate of recruitment, binned by genotype. LGN localizes to the apical cortex in ~50% of mitoses (black/red), is absent in ~45% (grey/pink), and “other” in the remaining ~5% (white); n indicates mitotic cells from 2-3 independent embryos. ( G ) Quantification of LGN radial fluorescence intensity in E16.5 mitotic cells (as in B ); n indicates LGN + mitoses from 2-3 independent embryos. ( H ) Dynactin (red) localization in LGN + mitoses is unaffected by afadin knockout. ( I ) Afdn knockdown increases radial deviation between LGN (green) and the division plane (red line) ( J ) Quantification of LGN radial localization in telophase (survivin+) mitoses. LGN apical bias is unaffected by α-catenin or vinculin knockdown. ( K ) Costaining for LGN (red), Gαi3 (grey), and mInsc-YFP (green) in E17.5 CD1 embryos infected with mInsc-YFP demonstrates a high degree of colocalization (yellow arrows). Fluorescence intensity linescan across LGN crescent is displayed in the right panel. ( L-M ) Costaining of LGN (green) and afadin (red) in metaphase ( L ) and telophase ( M ) mitoses from E16.5 Afdn fl / fl epidermis. Fluorescence intensity linescan across LGN crescent is displayed in the right panel. Regions of colocalization (yellow arrows) are just as prevalent as regions lacking signal overlap (black arrows). Scale bars, 5μm. P values determined by student’s t-test or Mann-Whitney test depending on tests of normality ( D,J ) or χ 2 ( F ). n.s. not significant (P > 0.05).

    Journal: bioRxiv

    Article Title: Telophase correction refines division orientation in stratified epithelia

    doi: 10.1101/668244

    Figure Lengend Snippet: Related to Figure 6 | Afadin loss-of-function division orientation phenotypes are polarity and LGN-independent ( A ) Immunostaining of E16.5 interphase cell with Par3 (red). ( B ) Quantification of Par3 radial fluorescent intensity. Apical accumulation is reduced by afadin knockout via K14-Cre ( Afdn cKO ) or lentiviral Cre-RFP; n indicates interphase cells from 2-3 independent embryos. ( C ) E16.5 sagittal sections show that centrosomes (green) localize to the apical cortex of interphase cells in both Afdn cKO basal progenitors and Afdn fl / fl controls. ( D ) Quantification of centrosome radial position in basal keratinocytes; n indicates interphase cells from 2 independent embryos. ( E ) LGN (red) localization patterns in mitotic (green) basal keratinocytes. ( F ) Quantification of LGN rate of recruitment, binned by genotype. LGN localizes to the apical cortex in ~50% of mitoses (black/red), is absent in ~45% (grey/pink), and “other” in the remaining ~5% (white); n indicates mitotic cells from 2-3 independent embryos. ( G ) Quantification of LGN radial fluorescence intensity in E16.5 mitotic cells (as in B ); n indicates LGN + mitoses from 2-3 independent embryos. ( H ) Dynactin (red) localization in LGN + mitoses is unaffected by afadin knockout. ( I ) Afdn knockdown increases radial deviation between LGN (green) and the division plane (red line) ( J ) Quantification of LGN radial localization in telophase (survivin+) mitoses. LGN apical bias is unaffected by α-catenin or vinculin knockdown. ( K ) Costaining for LGN (red), Gαi3 (grey), and mInsc-YFP (green) in E17.5 CD1 embryos infected with mInsc-YFP demonstrates a high degree of colocalization (yellow arrows). Fluorescence intensity linescan across LGN crescent is displayed in the right panel. ( L-M ) Costaining of LGN (green) and afadin (red) in metaphase ( L ) and telophase ( M ) mitoses from E16.5 Afdn fl / fl epidermis. Fluorescence intensity linescan across LGN crescent is displayed in the right panel. Regions of colocalization (yellow arrows) are just as prevalent as regions lacking signal overlap (black arrows). Scale bars, 5μm. P values determined by student’s t-test or Mann-Whitney test depending on tests of normality ( D,J ) or χ 2 ( F ). n.s. not significant (P > 0.05).

    Article Snippet: For knockdown screening, primary keratinocytes were seeded at a density of ~150,000 cells per well into 6-well plates and grown to ~80% confluency in E-Low calcium medium and infected with an MOI of ~1.

    Techniques: Immunostaining, Knock-Out, Fluorescence, Infection, MANN-WHITNEY

    Related to Figure 5 | Multiple models of afadin loss-of-function demonstrate telophase division orientation defects ( A-B ) Afadin (green) and E-cadherin (red) immunostaining in E16.5 sections. ( A ) Mosaic region of Afdn 2711 H2B-RFP (top panel) or Cre-RFP (in Afdn fl / fl embryo; bottom panel) lentiviral transduction. Region of high transduction (red line) demonstrates efficient loss of junctional afadin signal, spared in region of low transduction (white line). ( B ) E16.5 Afdn fl / fl controls (left) with conditional deletion mediated by K14-Cre ( cKO ). ( C ) RFP-negative (top) and RFP+ (bottom) telophase cells from mosaic Afdn 2711 epidermis, identified by survivin expression (green). ( D ) Cumulative frequency distributions of telophase division angles from E16.5 sagittal sections, or wholemounts (far right panel). Afdn knock-down or knockout results in randomized division orientation at telophase, regardless of Cre driver used or analysis method; n indicates number of observed divisions from 3-4 independent embryos. Note, fewer perpendicular divisions are observed in wholemounts compared to sections, likely due to 1) the relative difficulty of detecting perpendicular divisions compared to planar divisions in wholemounts, and 2) the likelihood of undercounting planar-mediolateral divisions in sagittal sections. ( E ) Timelines of division orientation at 5-minute intervals from movies of Afdn 2711 RFP-negative (left) and RFP+ (right) for 15 representative cells per group. Telophase reorientation establishes bimodal distribution within ~30 minutes in RFP-control cells that enter anaphase at oblique angles, while RFP+ cells fail to demonstrate any sorting behavior over a full hour following anaphase onset. ( F ) Radial correction (ϕ - θ) for Afdn 2711 RFP-negative (black/grey) and RFP+ (red/pink) plotted versus initial anaphase orientation (ϕ). Afadin knockdown cells are less responsive to the orienting function of basal contacts compared to RFP-negative controls. ( F ) Basal contact maintenance over a 1 h interval post-anaphase for Afdn 2711 RFP-negative (black) and RFP+ (red) cells. Afdn knockdown apical daughter cells, particularly those that enter anaphase at oblique angles, fail to maintain basal contact through mitosis. ( G-H ) Timecourse of junction formation in wild-type Afdn fl / fl primary keratinocytes 30 min ( G ) and 8 h ( H ) after the addition of 1.5 mM Ca2+, stained for E-cad (green), afadin (red), and phalloidin (grey). Between early and late timepoints, junctions transform from punctate to linear and actin becomes tightly associated with the junction. ( I ) Quantification of E-cad continuity along junction length in Afdn fl / fl primary keratinocytes at 30 min and 8 h timepoints. ( J-K ) Scramble ( J ) and Afadin knockdown ( Afdn 2711 ; K ) keratinocytes after 8 h Ca 2+ shift, stained same as in ( G-H ). ( L ) Quantification of E-cad continuity in Scramble and Afdn 2711 as in ( I ). Scale bars, 20µm ( A-B,G-H,J-K ), 5µm ( C ). ** P

    Journal: bioRxiv

    Article Title: Telophase correction refines division orientation in stratified epithelia

    doi: 10.1101/668244

    Figure Lengend Snippet: Related to Figure 5 | Multiple models of afadin loss-of-function demonstrate telophase division orientation defects ( A-B ) Afadin (green) and E-cadherin (red) immunostaining in E16.5 sections. ( A ) Mosaic region of Afdn 2711 H2B-RFP (top panel) or Cre-RFP (in Afdn fl / fl embryo; bottom panel) lentiviral transduction. Region of high transduction (red line) demonstrates efficient loss of junctional afadin signal, spared in region of low transduction (white line). ( B ) E16.5 Afdn fl / fl controls (left) with conditional deletion mediated by K14-Cre ( cKO ). ( C ) RFP-negative (top) and RFP+ (bottom) telophase cells from mosaic Afdn 2711 epidermis, identified by survivin expression (green). ( D ) Cumulative frequency distributions of telophase division angles from E16.5 sagittal sections, or wholemounts (far right panel). Afdn knock-down or knockout results in randomized division orientation at telophase, regardless of Cre driver used or analysis method; n indicates number of observed divisions from 3-4 independent embryos. Note, fewer perpendicular divisions are observed in wholemounts compared to sections, likely due to 1) the relative difficulty of detecting perpendicular divisions compared to planar divisions in wholemounts, and 2) the likelihood of undercounting planar-mediolateral divisions in sagittal sections. ( E ) Timelines of division orientation at 5-minute intervals from movies of Afdn 2711 RFP-negative (left) and RFP+ (right) for 15 representative cells per group. Telophase reorientation establishes bimodal distribution within ~30 minutes in RFP-control cells that enter anaphase at oblique angles, while RFP+ cells fail to demonstrate any sorting behavior over a full hour following anaphase onset. ( F ) Radial correction (ϕ - θ) for Afdn 2711 RFP-negative (black/grey) and RFP+ (red/pink) plotted versus initial anaphase orientation (ϕ). Afadin knockdown cells are less responsive to the orienting function of basal contacts compared to RFP-negative controls. ( F ) Basal contact maintenance over a 1 h interval post-anaphase for Afdn 2711 RFP-negative (black) and RFP+ (red) cells. Afdn knockdown apical daughter cells, particularly those that enter anaphase at oblique angles, fail to maintain basal contact through mitosis. ( G-H ) Timecourse of junction formation in wild-type Afdn fl / fl primary keratinocytes 30 min ( G ) and 8 h ( H ) after the addition of 1.5 mM Ca2+, stained for E-cad (green), afadin (red), and phalloidin (grey). Between early and late timepoints, junctions transform from punctate to linear and actin becomes tightly associated with the junction. ( I ) Quantification of E-cad continuity along junction length in Afdn fl / fl primary keratinocytes at 30 min and 8 h timepoints. ( J-K ) Scramble ( J ) and Afadin knockdown ( Afdn 2711 ; K ) keratinocytes after 8 h Ca 2+ shift, stained same as in ( G-H ). ( L ) Quantification of E-cad continuity in Scramble and Afdn 2711 as in ( I ). Scale bars, 20µm ( A-B,G-H,J-K ), 5µm ( C ). ** P

    Article Snippet: For knockdown screening, primary keratinocytes were seeded at a density of ~150,000 cells per well into 6-well plates and grown to ~80% confluency in E-Low calcium medium and infected with an MOI of ~1.

    Techniques: Immunostaining, Transduction, Expressing, Knock-Out, Staining

    The cell-adhesion/cytoskeletal scaffolding protein vinculin is required for telophase reorientation. ( A ) Cumulative frequency distribution of telophase division angles in Vcl 3466 H2B-RFP+ (red), RFP-negative (grey), and littermate controls (WT; black); n indicates number of divisions observed from 3-4 independent embryos. Note bimodal distribution observed in wild-type littermates and RFP-negative cells, in contrast to random distribution of Vcl 2803 RFP+ cells. Vcl 2803 RFP+ cells ( Supplementary Fig. 3B ) show a similar phenotype. ( B ) Movie stills of Vcl 3466 RFP+ mitotic cell, annotated as in Fig. 1e . While the presence of basal contact (open arrowhead) would predict planar correction, this division remains oblique when reevaluated 1 h later. ( C ) Cumulative frequency distribution of division orientation at anaphase onset (ϕ) and 1 h post-anaphase (θ) for RFP+ and RFP-negative populations, from movies of Vcl 3466 mosaic tissue; n indicates divisions from 4 embryos imaged in 3 separate sessions. ( D ) Quantification of telophase reorientation (θ - ϕ) for Vcl 2803 RFP+ cells and RFP-controls. ( E ) Data from ( C ) depicting orientation at anaphase onset (ϕ) and 1 h later (θ) for RFP-negative and RFP+ cells. RFP-negative controls sort anaphase orientation (ϕ) into bimodal distribution within 1 h (θ) in a basal-contact dependent manner; Vcl 3466 RFP+ cells display minimal change, or correct irrespective of basal contact. ( F ) Cartoon representation of tension-sensitivity model of AJ assembly. In the absence of tension, α-catenin exists in an autoinhibited closed conformation, masking the α18 epitope. In the presence of actin-mediated tension, α-catenin opens, exposing the α18 epitope and vinculin binding domain. ( G ) Stable primary murine keratinocytes cell lines grown in the presence of high (1.5 mM) Ca 2+ for 8 h form nascent cell-cell adhesions, stained for total α-catenin (green); open, “tensile” α-catenin (α18, red); and vinculin (grey). Single junction magnifications (yellow dashed region) shown below, suggest that vinculin knockdown results in a reduced α18:α-catenin ratio, quantified in ( H ). High magnification single channel images of boxed region shown below; n indicates number of junctions evaluated. Scale bars, 10 μm ( B ), 20 μm ( G ). * P

    Journal: bioRxiv

    Article Title: Telophase correction refines division orientation in stratified epithelia

    doi: 10.1101/668244

    Figure Lengend Snippet: The cell-adhesion/cytoskeletal scaffolding protein vinculin is required for telophase reorientation. ( A ) Cumulative frequency distribution of telophase division angles in Vcl 3466 H2B-RFP+ (red), RFP-negative (grey), and littermate controls (WT; black); n indicates number of divisions observed from 3-4 independent embryos. Note bimodal distribution observed in wild-type littermates and RFP-negative cells, in contrast to random distribution of Vcl 2803 RFP+ cells. Vcl 2803 RFP+ cells ( Supplementary Fig. 3B ) show a similar phenotype. ( B ) Movie stills of Vcl 3466 RFP+ mitotic cell, annotated as in Fig. 1e . While the presence of basal contact (open arrowhead) would predict planar correction, this division remains oblique when reevaluated 1 h later. ( C ) Cumulative frequency distribution of division orientation at anaphase onset (ϕ) and 1 h post-anaphase (θ) for RFP+ and RFP-negative populations, from movies of Vcl 3466 mosaic tissue; n indicates divisions from 4 embryos imaged in 3 separate sessions. ( D ) Quantification of telophase reorientation (θ - ϕ) for Vcl 2803 RFP+ cells and RFP-controls. ( E ) Data from ( C ) depicting orientation at anaphase onset (ϕ) and 1 h later (θ) for RFP-negative and RFP+ cells. RFP-negative controls sort anaphase orientation (ϕ) into bimodal distribution within 1 h (θ) in a basal-contact dependent manner; Vcl 3466 RFP+ cells display minimal change, or correct irrespective of basal contact. ( F ) Cartoon representation of tension-sensitivity model of AJ assembly. In the absence of tension, α-catenin exists in an autoinhibited closed conformation, masking the α18 epitope. In the presence of actin-mediated tension, α-catenin opens, exposing the α18 epitope and vinculin binding domain. ( G ) Stable primary murine keratinocytes cell lines grown in the presence of high (1.5 mM) Ca 2+ for 8 h form nascent cell-cell adhesions, stained for total α-catenin (green); open, “tensile” α-catenin (α18, red); and vinculin (grey). Single junction magnifications (yellow dashed region) shown below, suggest that vinculin knockdown results in a reduced α18:α-catenin ratio, quantified in ( H ). High magnification single channel images of boxed region shown below; n indicates number of junctions evaluated. Scale bars, 10 μm ( B ), 20 μm ( G ). * P

    Article Snippet: For knockdown screening, primary keratinocytes were seeded at a density of ~150,000 cells per well into 6-well plates and grown to ~80% confluency in E-Low calcium medium and infected with an MOI of ~1.

    Techniques: Scaffolding, Binding Assay, Staining

    Afadin is essential for telophase correction and basal contact maintenance (A) Cumulative frequency distribution of telophase division angles from E16.5 epidermis. Afdn knockdown (red) results in random telophase orientation, while uninfected RFP-negative (grey) and littermate (black) controls display a bimodal distribution; n indicates cells analyzed from 3-6 embryos. (B-E) Live imaging of E16.5 Afdn 2711 explants. (B) An obliquely-oriented Afdn 2711 RFP+ cell fails to reorient, while losing basal contact (open arrowhead). (C) Cumulative frequency distributions of division orientation from E16.5 live imaging of Afdn 2711 RFP+ and wild-type littermates; n indicates observed divisions from 3 embryos imaged in 2 separate sessions. (D) Division orientation at anaphase onset (ϕ) and one hour later (θ) for Afdn 2711 RFP+ and RFP-negative cells, plotted from data in (C) . RFP-negative controls correct into a bimodal distribution, while RFP+ cells reorient randomly. (E) Afdn fl / fl primary keratinocytes mosaically infected with Cre-RFP (red) after 8 h 1.5 mM Ca 2+ shift, stained for E-cadherin (green), afadin (red), and phalloidin (grey). Junctions between two uninfected cells (WT:WT) show linear morphology with consistent E-cadherin (green), afadin (red) and phalloidin (grey) labeling. In contrast, junctions between two infected cells are punctate, with less junction-associated phalloidin. (F) Quantification of E-cad continuity along junction length, as in Fig. 4J . (G) Quantification of fluorescence intensity of actin (phalloidin) measured by orthogonal linescans. Phalloidin is decentralized in KO:KO junctions (red) compared to WT:WT (black;. n indicates junctions evaluated. (H) Primary keratinocytes derived from Afdn fl / fl K14-Cre+ embryos ( Afdn cKO ) and Cre-negative littermates after 8 h Ca 2+ shift and stained for α18 (red), α-E-catenin (green), and vinculin (grey). (I) Quantification of vinculin:total α-catenin and (J) α18:total α-catenin fluorescent ratios; n indicates junctions analyzed. (K) Cumulative frequency distribution of E16.5 telophase division angles in Afdn fl / fl , Afdn cKO , and Afdn cKO + Vcl 3466 H2B-RFP epidermis. Vinculin knockdown does not exacerbate Afdn knockout phenotype. Scale bars, 10μm (B) , 20μm (E,H) . P values determined by Kolmogorov-Smirnov test (A,C,K) , student’s unpaired t-test (F,I,J) . * P

    Journal: bioRxiv

    Article Title: Telophase correction refines division orientation in stratified epithelia

    doi: 10.1101/668244

    Figure Lengend Snippet: Afadin is essential for telophase correction and basal contact maintenance (A) Cumulative frequency distribution of telophase division angles from E16.5 epidermis. Afdn knockdown (red) results in random telophase orientation, while uninfected RFP-negative (grey) and littermate (black) controls display a bimodal distribution; n indicates cells analyzed from 3-6 embryos. (B-E) Live imaging of E16.5 Afdn 2711 explants. (B) An obliquely-oriented Afdn 2711 RFP+ cell fails to reorient, while losing basal contact (open arrowhead). (C) Cumulative frequency distributions of division orientation from E16.5 live imaging of Afdn 2711 RFP+ and wild-type littermates; n indicates observed divisions from 3 embryos imaged in 2 separate sessions. (D) Division orientation at anaphase onset (ϕ) and one hour later (θ) for Afdn 2711 RFP+ and RFP-negative cells, plotted from data in (C) . RFP-negative controls correct into a bimodal distribution, while RFP+ cells reorient randomly. (E) Afdn fl / fl primary keratinocytes mosaically infected with Cre-RFP (red) after 8 h 1.5 mM Ca 2+ shift, stained for E-cadherin (green), afadin (red), and phalloidin (grey). Junctions between two uninfected cells (WT:WT) show linear morphology with consistent E-cadherin (green), afadin (red) and phalloidin (grey) labeling. In contrast, junctions between two infected cells are punctate, with less junction-associated phalloidin. (F) Quantification of E-cad continuity along junction length, as in Fig. 4J . (G) Quantification of fluorescence intensity of actin (phalloidin) measured by orthogonal linescans. Phalloidin is decentralized in KO:KO junctions (red) compared to WT:WT (black;. n indicates junctions evaluated. (H) Primary keratinocytes derived from Afdn fl / fl K14-Cre+ embryos ( Afdn cKO ) and Cre-negative littermates after 8 h Ca 2+ shift and stained for α18 (red), α-E-catenin (green), and vinculin (grey). (I) Quantification of vinculin:total α-catenin and (J) α18:total α-catenin fluorescent ratios; n indicates junctions analyzed. (K) Cumulative frequency distribution of E16.5 telophase division angles in Afdn fl / fl , Afdn cKO , and Afdn cKO + Vcl 3466 H2B-RFP epidermis. Vinculin knockdown does not exacerbate Afdn knockout phenotype. Scale bars, 10μm (B) , 20μm (E,H) . P values determined by Kolmogorov-Smirnov test (A,C,K) , student’s unpaired t-test (F,I,J) . * P

    Article Snippet: For knockdown screening, primary keratinocytes were seeded at a density of ~150,000 cells per well into 6-well plates and grown to ~80% confluency in E-Low calcium medium and infected with an MOI of ~1.

    Techniques: Imaging, Infection, Staining, Labeling, Fluorescence, Derivative Assay, Knock-Out