1. Plakoglobin but Not Desmoplakin Regulates Keratinocyte Cohesion via Modulation of p38MAPK Signaling 
Volker Spindler, Carina Dehner, Stefan Hübner, Jens Waschke 
Journal of Investigative Dermatology 
Volume 134, Issue 6, Pages (June 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Pemphigus autoantibodies disrupted the distribution of plakoglobin (Pg). (a) A 24-hour incubation of AK23 or pemphigus vulgaris (PV)-IgG reduced cell cohesion as detected by dispase-based dissociation assays. n=7, *P<0.05 versus control. (b) AK23 and PV-IgG disrupted the linear staining of Pg at the membrane and, minor pronounced, of desmoplakin (DP). Bar=20 μm. Panel shows representative images from three independent experiments. (c) Immunoblots of whole-cell lysates demonstrate that AK23 and PV-IgG reduce Pg and desmoglein 3 (Dsg3) protein content, but not that of DP (n=5, *P<0.05 vs. phosphate-buffered saline (PBS)). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Silencing of plakoglobin (Pg) induced loss of cell cohesion and p38 mitogen-activated protein kinase (p38MAPK) activation. (a) Silencing of either Pg (siPg) or desmoplakin (siDP) reduced intercellular adhesion as detected by dispase-based dissociation assays. n=6, *P<0.05 versus nontarget siRNA (siNT) phosphate-buffered saline (PBS); #P<0.05. (b) Silencing of Pg but not of DP led to increased p38MAPK phosphorylation detected by western blots of whole-cell lysates. n=4, *P<0.05 versus siNT. (c) A 30-minute AK23 or pemphigus vulgaris (PV)-IgG treatment did not further increase p38MAPK activation induced by Pg silencing as indicated by immunoblots of whole-cell lysates. n=3, *P<0.05 versus siNT.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Loss of cell cohesion and keratin filament retraction induced by plakoglobin (Pg) silencing was blocked by p38 mitogen-activated protein kinase (p38MAPK) inhibition. (a) Cell dissociation by Pg depletion (siPg) was blocked by the p38MAPK-specific inhibitor SB202190, whereas p38MAPK inhibition had no effect in desmoplakin (siDP)-depleted cells. SB blocked the effect of AK23 independent of Pg and DP expression state. n=6, *P<0.05. (b) Pg silencing (siPg) induced keratin filament retraction (arrows), which was prevented by SB (arrowheads). Bar=20 μm. Panel shows representative images from three independent experiments. (c) Knockdown of DP (siDP) did not induce pronounced keratin filament collapse (arrows) and SB had no effect (arrowheads). Bar=20 μm. Panel shows representative images from three independent experiments. siNT, nontarget siRNA.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Extranuclear plakoglobin (Pg) ameliorated autoantibody-induced cell dissociation, p38 mitogen-activated protein kinase (p38MAPK) activation, and keratin filament retraction. (a) Expression of a Pg mutant with predominant localization in the nucleus (nuclear localization sequence (NLS)-Pg) was sufficient to disrupt HaCaT monolayer integrity. In contrast, extranuclear Pg (nuclear export sequence (NES)-Pg) or wild-type Pg (Pg-WT) reduced AK23-mediated cell dissociation. n=10, *P<0.05 versus respective phosphate-buffered saline (PBS) condition; #P<0.05 versus green fluorescent protein (GFP) AK23, §P<0.05. (b) Expression of Pg-WT or NES-Pg reduced the amount of active p38MAPK as detected by immunoblots of whole-cell lysates. n=5, *P<0.05 versus GFP. (c) Under conditions of Pg depletion via small interfering RNA (siRNA) targeting of the 3′UTR, keratin retraction was evident, which was still present in GFP- or NLS-Pg-expressing cells (arrows in first and third line). In contrast, in cells expressing Pg-WT and NES-Pg, keratin filament retraction was reduced (arrowheads in second and fourth line). Bar=10 μm. Panel shows representative images from three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Extranuclear plakoglobin (Pg) blocked desmoglein 3 (Dsg3) depletion by pemphigus autoantibodies and tethered p38 mitogen-activated protein kinase (p38MAPK) to the desmosome. (a) Expression of nuclear export sequence (NES)-Pg prevented Dsg3 depletion induced by pemphigus vulgaris (PV)-IgG in both the cytoskeleton-bound (Triton X-100 insoluble) and membrane/cytosol (Triton X-100 soluble) pools after 1 hour. n=5, *P<0.05 versus respective phosphate-buffered saline (PBS) green fluorescent protein (GFP) condition; #P<0.05. (b) Immunoprecipitation (IP) of desmoplakin (DP) from whole-cell lysates revealed that p38MAPK and phospho-p38MAPK form a complex with DP and Pg. Silencing of Pg by shRNA (shPg) reduced the amount of p38MAPK and phospho-p38MAPK present in this complex. n=4, *P<0.05 versus non-target shRNA (shNT). (c) p38MAPK activation was detectable after 1 hour of PV-IgG incubation in the non-cytoskeletal fraction. n=4, *P<0.05 versus respective phosphate-buffered saline (PBS) condition.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions