Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 1 Characterization of ARHGAP29 knockdown keratinocytes. (A) Western blot for ARHGAP29 (A29, top panel) and Ponceau red staining of the same membrane (bottom; one representative of three experiments) of keratinocytes transduced with CRISPR scrambled (sc), CRISPR ARHGAP29 (#1, #2), shRNA scramble, shRNA ARHGAP29 (#2, #3), and sh#3 transduced with ARHGAP29 (+A29) or GFP (+GFP). (B) Quantification of ARHGAP29 protein levels. Values are the means (N = 3) ± SEM, **p < .01 and ***p < .001 following ordinary one-way ANOVA test with Tukey's multiple comparisons post-hoc test (only relevant comparisons are shown). (C) Representative phase contrast micrographs of scramble, CRISPR knockdown, shRNA knockdown (sh#3) and shRNA knockdown keratinocytes transduced with ARHGAP29-GFP (sh#3 + A29), all grown in KSFM. Scale bar = 100 μm. (D) Quantification of keratinocyte area under the conditions shown in C. Boxes display the 25–75th percentiles where the line represents the median and whiskers display the minimum to maximum values, *p < .05, ***p < .001 and ****p < .0001 following Kruskal–Wallis test with Dunn's multiple comparisons post-hoc test (only relevant comparisons are shown). N = 200–400 cells per group. ns, non-significant.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: Knockdown, Western Blot, Staining, Membrane, Transduction, CRISPR, shRNA

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 3 ARHGAP29 promotes keratinocyte proliferation. (A) Quantification of population doubling time. Values are the means ± SEM, *p < .05 following Brown–Forsythe test and Welch ANOVA test with Dunnett's T3 multiple comparisons post-hoc test. N = 6–8 per group. (B) Representative images of colony forming efficiency dishes with CRISPR scramble (CRISPRsc), CRISPR ARHGAP29 (CRISPR#1), shRNA scramble (shSc) and shRNA ARHGAP29 (sh#3) keratinocytes. (C) Quantification of colony area for all cell lines (including those represented in B). N = 752–1639 per group. Values are the means ± SEM, ****p < .0001 following Kruskal–Wallis test with Dunn's multiple comparisons post-hoc test (only comparisons to scrambled are shown). (D) Quantification of number of cells per colony for all cell lines (N = 9–12). Values are the means ± SEM, **p < .01, ****p < .0001 following ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test (only comparisons to scrambled are shown). (E) Quantification of number of cells per colony area (“cell density”) for all cell lines (N = 9–12). Values are the means ± SEM, *p < .05, ****p < .0001 following ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test. Only comparisons to scrambled are shown.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: CRISPR, shRNA

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 5 ARHGAP29 promotes collective cell migration. (A) Phase contrast micrographs of in vitro scratch wounds in confluent monolayers of shRNA scramble (shSc), shRNA ARHGAP29 (sh#3), and sh#3 transduced with ARHGAP29 (sh#3 + A29) keratinocytes grown in DMEM:HAM. Scale bar = 100 μm. T0 = 0 h after scratch and T 12 = 12 h after scratch. (B) Quantifications of the percentage of scratch closure over a 12-h period in all CRISPR and shRNA cell lines compared to their respective scrambled controls. Values are the means ± SEM, *p < .05 and **p < .01 after two-way ANOVA with Tukey's multiple comparisons post-hoc test. N = 6 per group.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: Migration, In Vitro, shRNA, Transduction, CRISPR

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 6 ARHGAP29 is present in embryonic, but not in adult keratinocytes, and is upregulated following wounding. (A)– (F) Immunofluorescent staining for ARHGAP29 (cyan) of murine wild-type skin of an unwounded E14.5 embryo (A), E18.5 embryo (B), adult (C), and of a 2-day (D), 4-day (E), and 7-day (F) wound. Nuclear DNA is stained with Hoechst (magenta). Scale bar = 50 μm; yellow arrow heads indicate the leading edge of the epidermis in open wounds and white dotted lines indicate the epidermal–dermal junction. HF, hair follicle. One representative image of N = 3 per time point. (G) Western blot analysis for ARHGAP29 and GAPDH protein levels of E14.5, E17.5 and adult unwounded skin extracts. (H) Quantification of ARHGAP29 levels shown in panel G normalized to GAPDH (loading) and to E14.5 values used as the reference developmental time point. Values are the means ± SEM, *p < .05 and after ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test. N = 3 per group. (I) Quantification of ARHGAP29 levels (using immunofluorescent signal) in keratinocytes at different time points during wound healing. Values are the means ± SEM, *p < .05 and **p < .01 after ordinary one-way ANOVA with Dunnett's multiple comparisons post-hoc test. N = 3 per group.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: Staining, Western Blot

Journal: Cancer Genomics & Proteomics

Article Title: ARHGAP29 Is Involved in Increased Invasiveness of Tamoxifen-resistant Breast Cancer Cells and its Expression Levels Correlate With Clinical Tumor Parameters of Breast Cancer Patients

doi: 10.21873/cgp.20454

Figure Lengend Snippet: Information on tissue of origin, pathology, grading, staging, TNM classification and assessment of ARHGAP29 expression. (–) not expressed, (+) slightly expressed, (++) moderately expressed and (+++) strongly expressed.

Article Snippet: Primary antibodies against ARHGAP29 1:2000 (#NBP1-05989, Novus Biologicals, Centennial, CO, USA), RhoC 1:2,000 (#GTX100546, GeneTex, Irvine, CA, USA), pAKT1 1:1,000 (#9271, Cell Signaling, Danvers, MA, USA), and GAPDH 1:2,000 (#5174S, Cell Signaling) were used.

Techniques: Expressing

Journal: Cancer Genomics & Proteomics

Article Title: ARHGAP29 Is Involved in Increased Invasiveness of Tamoxifen-resistant Breast Cancer Cells and its Expression Levels Correlate With Clinical Tumor Parameters of Breast Cancer Patients

doi: 10.21873/cgp.20454

Figure Lengend Snippet: Information on tissue of origin, pathology, grading, staging, TNM classification and assessment of ARHGAP29 expression. (–) not expressed, (+) slightly expressed, (++) moderately expressed and (+++) strongly expressed.

Article Snippet: Antigens were retrieved by incubation with 0.01 M citrate buffer (pH 6.0) in a microwave (700 W) for 5 min. Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide solution for 6 min. After washing in PBS, the slides were treated with polyclonal rabbit anti-human ARHGAP29 receptor antiserum (#NBP1-05989, Novus Biologicals, Centennial, CO, USA) in a 1:50 dilution in 1% BSA in 10 mM Tris, pH 8.0, 500 mM NaCl and 0.1% Tween-20 (TBST) over night.

Techniques: Expressing