Figure 4. SERPINB5 rescues cell–cell... | Rockefeller University Press

Figure 4.

$SERPINB5 rescues cell–cell adhesion and cytoskeletal rearrangements caused by DPM1 loss. (a and b) Dispase-based dissociation assays with sgDPM1 HaCaT keratinocytes overexpressing SERPINB3, SERPINB4, and SERPINB5 (N = 3). Each dot represents one biological replicate. One-way ANOVA, Dunnett’s multiple comparison test. (c) Images of DSP immunostainings in sgDPM1 and SERPIN overexpressing HaCaT keratinocytes. Panel shows representative of three biological replicates. Scale bar: 10 µm distance. (d) Quantification of the number of DSP puncta on the cell membrane normalized to the length of plasma membrane (µm). Each dot represents individual cells analyzed from three independent biological replicates. One-way ANOVA, Dunnett’s multiple comparison test. (e–g) Keratin staining depicted by pan-cytokeratin in sgNT1, DPM1 KD, and DPM1 KD overexpressing SERPINB5-GFP in HaCaT keratinocytes. Analysis done by measuring the keratin intensity across cell junctions (A.U.) over a distance of 10 µm. Peak width calculated between the two highest points from the distribution plot profile graph. Scale bar: 10 µm distance. 30 individual cells were quantified from three independent biological replicates. One-way ANOVA, Dunnett’s multiple comparison test. (h–j) F-actin staining by phalloidin in sgNT1, DPM1 KD, and DPM1 KD overexpressing SERPINB5-GFP in HaCaT keratinocytes. Scale bar: 10 µm distance. Analysis done by measuring the phalloidin intensity across cell junctions (A.U.) over a distance of 10 µm. The width of the peaks was calculated at baseline levels from distribution plot profile graphs. 30 individual cells were quantified from three independent biological replicates. One-way ANOVA, Dunnett’s multiple comparison test. (k and l) Kymographs and mobile fraction analysis derived from FRAP assays used to measure DSP stability at cell–cell contact sites (N = 3) in sgDPM1 and sgDPM1 SERPINB5 overexpressing cells. Scale bar on the y-axis = 1 µm. Each dot represents individual contact sites from three biological replicates. Unpaired Student’s t test. (m) Graph shows cellular elasticity, indicated by Young’s modulus (kPa). Each dot represents single cells from three biological replicates. Unpaired Student’s t test. (n and o) Dispase-based intercellular adhesion assays showing reduced cell–cell adhesion upon KD of SERPINB5 in HaCaT keratinocytes (N = 4). Unpaired Student’s t test. (p and q) Images and analysis of DSP at the cell membrane upon loss of SERPINB5 (N = 3). Each dot represents single cells from three biological replicates. Scale bar: 10 µm distance. Unpaired Student’s t test.$

SERPINB5 rescues cell–cell adhesion and cytoskeletal rearrangements caused by DPM1 loss. (a and b) Dispase-based dissociation assays with sgDPM1 HaCaT keratinocytes overexpressing SERPINB3, SERPINB4, and SERPINB5 (N = 3). Each dot represents one biological replicate. One-way ANOVA, Dunnett’s multiple comparison test. (c) Images of DSP immunostainings in sgDPM1 and SERPIN overexpressing HaCaT keratinocytes. Panel shows representative of three biological replicates. Scale bar: 10 µm distance. (d) Quantification of the number of DSP puncta on the cell membrane normalized to the length of plasma membrane (µm). Each dot represents individual cells analyzed from three independent biological replicates. One-way ANOVA, Dunnett’s multiple comparison test. (e–g) Keratin staining depicted by pan-cytokeratin in sgNT1, DPM1 KD, and DPM1 KD overexpressing SERPINB5-GFP in HaCaT keratinocytes. Analysis done by measuring the keratin intensity across cell junctions (A.U.) over a distance of 10 µm. Peak width calculated between the two highest points from the distribution plot profile graph. Scale bar: 10 µm distance. 30 individual cells were quantified from three independent biological replicates. One-way ANOVA, Dunnett’s multiple comparison test. (h–j) F-actin staining by phalloidin in sgNT1, DPM1 KD, and DPM1 KD overexpressing SERPINB5-GFP in HaCaT keratinocytes. Scale bar: 10 µm distance. Analysis done by measuring the phalloidin intensity across cell junctions (A.U.) over a distance of 10 µm. The width of the peaks was calculated at baseline levels from distribution plot profile graphs. 30 individual cells were quantified from three independent biological replicates. One-way ANOVA, Dunnett’s multiple comparison test. (k and l) Kymographs and mobile fraction analysis derived from FRAP assays used to measure DSP stability at cell–cell contact sites (N = 3) in sgDPM1 and sgDPM1 SERPINB5 overexpressing cells. Scale bar on the y-axis = 1 µm. Each dot represents individual contact sites from three biological replicates. Unpaired Student’s t test. (m) Graph shows cellular elasticity, indicated by Young’s modulus (kPa). Each dot represents single cells from three biological replicates. Unpaired Student’s t test. (n and o) Dispase-based intercellular adhesion assays showing reduced cell–cell adhesion upon KD of SERPINB5 in HaCaT keratinocytes (N = 4). Unpaired Student’s t test. (p and q) Images and analysis of DSP at the cell membrane upon loss of SERPINB5 (N = 3). Each dot represents single cells from three biological replicates. Scale bar: 10 µm distance. Unpaired Student’s t test.

This Feature Is Available To Subscribers Only