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. 2019 May 9;44:502–515. doi: 10.1016/j.ebiom.2019.04.062

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© 2019 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 3 — Cell-ECM interactions are disturbed with enhanced focal adhesions after 1 mM 4-PBA treatment in NHK and EBS cells.

A For the keratinocyte adhesion assay, the cells were coated on collagen I or left on untreated plates. We refer to the adhesion of the untreated cells on collagen I as 100% adhesion. After 4-PBA treatment, the adhesion of NHK appeared unchanged, whereas significant effects were found in 4-PBA-treated KRT14mut and less in KRT5mut cells [for KRT14mut + *P = .0411 and for KRT5mut + P = .3267, student's t-test].

B Reduction of the fraction of adherent cells in NHK and EBS cells in the spinning disk assay after treatment with 1 mM 4-PBA [NHK+ P = .4296; KRT14mut + P = .0652; KRT5mut + P = .6818]. The effect of 4-PBA on adhesiveness is significant, with NHK and EBS cells taken together (All cells vs all cells+: *P = .0001, student's t-test).

C The focal adhesion-associated molecule integrin β1 (green) was stained and found greatly enhanced after 1 mM 4-PBA treatment especially in control and KRT5-mutated cells. Scale: 200 μm.

D 4-PBA treatment resulted in altered staining of the epidermal F-actin cytoskeleton in control and EBS keratinocytes, whereas the total amount of F-actin was unchanged, as shown by immunoblotting, suggesting altered distribution of the actin filaments in the cells [for KRT14mut + P = .9092 and for KRT5mut + P = .1796, student's t-test]. Scale bar: 50 μm. The IF stainings were done in triplicates with at least 2 different cell lines for each group, before and after 4-PBA treatment. Shown are representative results from one experiment.

E Immunoblotting for laminin-332 revealed increased amounts in cell lysates after 1 mM 4-PBA treatment. Bar graphs on the right show expression of laminin-332 normalized to tubulin as percentage of expression in untreated cells. Values represent mean ± S.E.M., paired t-test was used; for NHK P = .6097, for KRT14mut P = .4084 and for KRT5mut P = .6291. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)