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Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC <t>activator</t> <t>(PMA)</t> and a RhoA inhibitor <t>(C3)</t> in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, *** p < 0.001 (ANOVA, Bonferroni).
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Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC <t>activator</t> <t>(PMA)</t> and a RhoA inhibitor <t>(C3)</t> in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, *** p < 0.001 (ANOVA, Bonferroni).
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Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, *** p < 0.001 (ANOVA, Bonferroni).

Journal: Molecular Biology of the Cell

Article Title: Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation

doi: 10.1091/mbc.E14-05-0968

Figure Lengend Snippet: Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, *** p < 0.001 (ANOVA, Bonferroni).

Article Snippet: For inhibition and activation of adenylyl cyclase- and cAMP-dependent EPAC-Rap1, PKCα, and RhoA, media were supplemented with forskolin (100 µM), isoprenaline (10 µM), propranolol (10 µM), 8-CTP-2Me-cAMP (10 µM), ESI-09 (5 µM), PMA (100 nM) (all from Sigma-Aldrich), or C3 transferase-based RhoA inhibitor (1 µM; Cytoskeleton, Denver, CO) and were used with appropriate vehicle control.

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Cell Adhesion Strength Assay