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. 2018 Apr 25;9:858. doi: 10.3389/fimmu.2018.00858

Figure 5.

Figure 5

D4476-induced keratin changes did not induce Dsg3 internalization. HaCaT-CK5 keratinocytes were transfected with pDest-mDsg3-mCherry, incubated with D4476 or DMSO and followed up by three-dimensional confocal time-lapse microscopy (A). D4476 caused keratin retraction after 60 min (arrows) but did not lead to internalization of Dsg3. Subsequent addition of PV2-IgG caused Dsg3 membrane depletion (arrowheads). Bar represents 10 µm (n = 4 cells from 4 independent experiments, *p < 0.05). (B) Immunostaining of HaCaT-CK5 keratinocytes and Dsg3. Keratin changes occurred under D4476 treatment (arrows) and signs of Dsg3 fragmentation and internalization were visible in response to PV1-IgG treatment only (arrowheads). Images shown are representative for three to four independent experiments. Bar represents 10 µm. (C) Streptavidin pulldown of biotinylated Dsg3 in HaCaT-CK5 cells incubated with DMSO or D4476 for 4 h as a control and 1 h incubation followed by a 3 h PV2-IgG incubation. Dsg3 membrane levels were densitometrically assessed (n = 3–5, *p < 0.05 vs. respective control condition). (D) Dsg3-clustering was analyzed in 1 h live cell imaging sequence of DMSO or D4476 incubation. Clustering was detected using a bar of 10 µm applied linearly on the membrane (n = 4, *p < 0.05 vs. DMSO at respective time point). Bar represents 10 µm.