Journal: bioRxiv

Article Title: Desmosomes polarize mechanical signaling to govern epidermal tissue form and function

doi: 10.1101/2020.01.21.914176

Figure Lengend Snippet: A) Maximum projection micrographs of day 9 transwell epidermal equivalent cultures that were treated with either DMSO (Control) or a Rho activator (CN01, 1 unit/mL) at day 7-9 show immunostaining for the tight junction protein ZO1 and staining for nuclei in blue (DAPI) within the indicated cell layers. Bar is 20 μm. B) Maximum projection micrographs of day 9 transwell epidermal equivalent cultures that were treated with a Rho activator (CN01, 1 unit/mL) at day 7-9 show immunostaining for the tight junction protein ZO1, staining for nuclei in blue (DAPI), and F-actin (phalloidin) within the spinous cell layer. Bar is 10 μm. C) Maximum projection micrographs of day 9 transwell epidermal equivalent cultures that were treated with either DMSO (Control) or a Rho activator (CN01, 1 unit/mL) at day 7-9 show staining for nuclei in blue (DAPI) and F-actin (phalloidin) within the indicated cell layers. Bar is 20 μm. D) Quantification of resistance measurements from TEER experiments performed on a time course of transwell epidermal equivalent cultures that were treated with either DMSO or a Rho activator (CN01, 1 unit/mL) at day 7. Data are presented as mean ± SEM. **p=0.0029, paired t test from 6 independent experiments.

Article Snippet: Pharmacological treatments included 1 unit/ml Rho Activator I (CN01; Cytoskeleton, Denver, CO), 1 mM TAK165 (Mubritinib, Selleck Chemicals), dimethyl sulfoxide (DMSO, Sigma-Aldrich), 20 μM PP2 (Sigma-Aldrich), and 60 nM CCG-1423 (Sigma-Aldrich).

Techniques: Control, Immunostaining, Staining

Journal: Nature Communications

Article Title: Mediated nuclear import and export of TAZ and the underlying molecular requirements

doi: 10.1038/s41467-018-07450-0

Figure Lengend Snippet: Regulation of TAZ import by RhoA. a Effect of the RhoA activator RhoII on the nuclear enrichment of 1C-TAZ 4SA F52A (anchorless construct), n = 3. b Nuclear enrichment of 5C-TAZ 4SA upon RhoA activation by RhoII or by co-expression of constitutively active RhoA Q63L along with 2h LMB treatment, n = 6. c RhoA activation increases LMB-induced nuclear accumulation of 5C-TAZ 4SA. LMB time course without and with RhoA stimulation, n = 3. d RhoA activation increase LMB-induced nuclear accumulation of the 5C-TAZ-NLS. LMB time course without and with RhoA stimulation, n = 3. Rel. nuc./cyto. ratio refers to the difference between 5C-TAZ-NLS and 5C. Curves in c and d are fits as in Fig. . e RhoA activity selectively increases nuclear accumulation of the TAZ-NLS. 5C-290–345 and the SV40-NLS construct 5C-R5A were expressed in combination with control vector or RhoA Q63L. Following a 2 h treatment with or without LMB, nuclear accumulation was visually determined, n = 3, 5, or 6 for individual constructs. f RhoA stimulation does not induce LATS-independent TAZ phosphorylation, as detected by band shifts in western blots. Cells expressing 1C-TAZ 4SA were treated with PBS, RhoII or the phosphatase inhibitor okadaic acid (OA) for 6 h and lysates were run on conventional (upper panel) and Phos-tag (lower panel) polyacrylamide gels. After western blot, ectopic proteins were detected with anti-GFP antibody. Uncropped version shown in Supplementary Figure 6 . Data are represented as means ± SD. * p < 0.05, ** p < 0.01; Student’s t -test

Article Snippet: Leptomycin B, Rapamycin, and okadaic acid were purchased from Sigma Aldrich and RhoII activator from Cytoskeleton Inc. Phos-tag gels (Wako Pure Chemical Industries, Ltd.) were ordered from Cedarlane.

Techniques: Construct, Activation Assay, Expressing, Activity Assay, Plasmid Preparation, Western Blot