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 Oct 10;216(12):2778–2799. doi: 10.1084/jem.20190147

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

© 2019 Lam et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 4. — p.R186C is associated with defects in proliferation, migration, and formation of actin-based structures. (A) Proliferation of BM CD34⁺ cells from Pt 1 in response to stem cell factor (SCF/KITLG) or a cytokine mixture (MIX; n = 1). (B) Proliferation of primary fibroblasts from Pt 1 (n = 3) and Pt 2 (n = 3) at indicated time points of culture, and NIH-3T3 cells transiently expressing WT or mutant CDC42 or an empty vector (EV). (C) Migration assays of primary fibroblasts from Pt 1 (n = 3), transfected NIH-3T3 cells (n = 3; *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with Sidak’s multiple comparisons), purified BM CD34⁺ sorted cells (n = 1), PBMCs (n = 2), and a YTS CRISPR/Cas9-modified cell line (n = 3; ****, P < 0.0001, unpaired two-tailed t test; mean ± SEM for CD34⁺/PBMCs and mean ± SD for NK cells, NIH-3T3 cell line, and primary fibroblasts). Migration was assayed using a wound-healing assay on primary fibroblasts and transfected NIH-3T3 cells and directed migration toward chemoattractant CXCL12 in BM CD34⁺ cells, PBMCs, and YTS NK cells. Decreased directed migration of all tested cell types was observed. FI, fold increase of migratory cells. (D) Cytoskeletal rearrangements of cells expressing CDC42^R186C. Multipolarization and filopodia in primary fibroblasts from Pt 1 (n = 3) compared with fibroblasts from an HD. Immunofluorescence staining of CDC42 (red), F-actin (green), and nuclei (blue) in cells stimulated 3 h with 20% serum. In HD fibroblasts, CDC42 localizes to the front leading edge, whereas in Pt 1 fibroblasts, the protein mostly localizes to the perinuclear area. On the right of the panel, quantification of multipolar cells and number of cells with short and long filopodia length are shown. Cells bearing multiple F-actin flat protruding edges (butterfly shaped) were considered multipolar. While HD fibroblasts polarize showing a front (leading edge), a large proportion of Pt 1 fibroblasts show multiple protruding edges. Filopodia twofold longer than nuclei diameter were considered long filopodia. Filopodial length and number were notably increased in fibroblasts, suggesting a disruption of CDC42-dependent actin architecture. Scale bar, 20 µm (applicable to all images shown). (E) Filopodia dynamics of the YTS cells on an activating CD18/CD28 surface. Filopodia were imaged using SIM-TIRF microscopy with representative images showing decreased filopodia count (mean ± SD, n = 3; **, P < 0.01, unpaired two-tailed t test with Welch’s correction) in cells expressing CDC42^R186C. Scale bar, 10 µm (applicable to all images shown). n.s., not significant.