Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/).

PMC Copyright notice

Fig. 1 — Keratin aggregates in EBS keratinocytes at resting state and after stress: rescue by 4-PBA.

A Analysis of the subcellular localisation of plectin and keratin 5 as shown by immunofluorescence staining of control normal human keratinocytes (NHK), keratinocytes from patients with mutations in the keratin 14 gene (KRT14mut) and keratinocytes from patients with mutations in the keratin 5 gene (KRT5mut) with anti-plectin [Alexa555 (Cy3; red)] and keratin 5 [Alexa488 (GFP; green)] antibodies. Yellow arrows depict cells with aggregates of their keratin IF network. Scale bar: 50 μm.

B The upper panel shows quantification of the keratinocytes with aggregates. The NHKs show no aggregates at resting state, whereas they are significantly more in KRT14mut than KRT5mut cells (for NHK versus KRT14mut ***P: 0.0001, for NHK versus KRT5mut *P: 0.0211 and for KRT5mut versus KRT14mut ***P: 0.0004, all done with student's t-test). In the lower panel the percentage of cells before (NHK, KRT14mut, KRT5mut) and after use of 4-PBA (NHK+, KRT14mut+, KRT5mut+) is shown. The data are presented as mean ± SEM. Intermediate filament (IF) aggregates are significantly less in cells treated with 1 mM 4-PBA [KRT14mut (***P < .0001, student's t-test) and KRT5mut + cells (***P < .0001, student's t-test)], irrespective of the treatment time (24 or 48 h).

C Immunofluorescence staining for keratin 5 of EBS cells upon ocadaic treatment (OA) and upon heat-shock (HS). In the 4 subimages, different KRT5mut and KRT14mut cells are shown, highlighting the different appearances of the cells. The well-spread keratin IF network, as seen in NHK in A, forms thick bundles of filaments in several EBS keratinocytes, which seem to collapse towards the nucleus (yellow arrows). On the left quantification graph, the percentage of keratinocytes with aggregates before (NHK OA, KRT14mut OA, KRT5mut OA) and after use of 4-PBA (NHK OA+, KRT14mut OA+, KRT5mut OA+) are shown. The experiments were done in triplicates; the average of the three experiments is shown. The use of 4-PBA significantly reduces the number of cells with aggregates after OA treatment [NHK-treated cells (NHK OA+), KRT14mutated-treated cells (KRT14mut +) and KRT5mutated-treated cells (KRT5mut+): for all ***P < .0001, student's t-test]. EBS keratinocytes treated with HS (KRT14mut HS and KRT5mut HS) showed significant induction of aggregate formation [for both KRT14mut vs KRT14mut HS and KRT5mut vs KRT5mut HS ***P < .0001, student's t-test], which was reversed after 4-PBA use [KRT14mut HS+ and KRT5mut HS+ ***P < .0001, student's t-test]. Scale bar: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)