Journal: Frontiers in Genetics

Article Title: Targeted re-sequencing on 1p22 among non-syndromic orofacial clefts from Han Chinese population

doi: 10.3389/fgene.2022.947126

Figure Lengend Snippet: (A) Sanger sequencing results of the de novo harmful rare variant in ARHGAP29 . Sequence chromatograms indicate the heterozygous variant (NM_004815.3, NP_004806.3; c.1652G>C, p.R551T). The red letter and box emphasize the cross-species conservation of the altered amino acid. (B, C) Western blot and RT-qPCR analysis of the ARHGAP29 expression in HEK-293T cells 48 h after plasmid transfection. The results are presented as mean values with standard deviation (SD) normalized to GAPDH , and there were three biological replicates, *** p < 0.001.

Article Snippet: Subsequently, protein samples were separated by electrophoresis in agarose gels and transferred onto PVDF membranes, which were then blocked by 5% milk for 1 h and incubated with rabbit anti-human Arhgap29 antibody (Novus Biologicals, United States) at 4°C overnight, followed by incubation with anti-rabbit antibody (Proteintech, China) at room temperature for 1 h. At last, proteins were visualized by ECL substrate (Epizyme, China).

Techniques: Sequencing, Variant Assay, Western Blot, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Standard Deviation

Journal: Frontiers in Genetics

Article Title: Targeted re-sequencing on 1p22 among non-syndromic orofacial clefts from Han Chinese population

doi: 10.3389/fgene.2022.947126

Figure Lengend Snippet: Results of RNA sequencing on Arhgap29 R553T/R553T and wild-type mice. (A) Volcanic maps of differential expression genes. (B) GO analysis of DEGs. All the shown GO terms were significantly enriched with Q-value less than 0.05.

Article Snippet: Subsequently, protein samples were separated by electrophoresis in agarose gels and transferred onto PVDF membranes, which were then blocked by 5% milk for 1 h and incubated with rabbit anti-human Arhgap29 antibody (Novus Biologicals, United States) at 4°C overnight, followed by incubation with anti-rabbit antibody (Proteintech, China) at room temperature for 1 h. At last, proteins were visualized by ECL substrate (Epizyme, China).

Techniques: RNA Sequencing, Quantitative Proteomics

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 immunofluorescence: Phalloidin-TRITC (Sigma-Aldrich, St Louis, MO; catalog #P1951) was used at 1/10,000; rabbit polyclonal against phospho-Myosin regulatory light chain 2 at position Ser19 (Cell Signaling, Danvers, MA; catalog #3671) was used at 1/100 (in Figure 2, used with Alexa Fluor 488 goat anti-mouse); rabbit polyclonal against ARHGAP29 (Novus Biologicals, catalog #NBP1-05989) was used at 1/150; mouse monoclonal against Keratin 14 (Santa Cruz Biotechnology, catalog #sc-53253) was used at 1/20; Alexa Fluor 488 goat anti-mouse and antirabbit IgG (Invitrogen, Waltham, MA; catalog #A-11001 (mouse) and A-11008 (rabbit)) and Alexa Fluor 568 goat anti-mouse and anti-rabbit IgG (Invitrogen, catalog #A11004 (mouse) and A-11011 (rabbit)) were used at 1/200.

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 immunofluorescence: Phalloidin-TRITC (Sigma-Aldrich, St Louis, MO; catalog #P1951) was used at 1/10,000; rabbit polyclonal against phospho-Myosin regulatory light chain 2 at position Ser19 (Cell Signaling, Danvers, MA; catalog #3671) was used at 1/100 (in Figure 2, used with Alexa Fluor 488 goat anti-mouse); rabbit polyclonal against ARHGAP29 (Novus Biologicals, catalog #NBP1-05989) was used at 1/150; mouse monoclonal against Keratin 14 (Santa Cruz Biotechnology, catalog #sc-53253) was used at 1/20; Alexa Fluor 488 goat anti-mouse and antirabbit IgG (Invitrogen, Waltham, MA; catalog #A-11001 (mouse) and A-11008 (rabbit)) and Alexa Fluor 568 goat anti-mouse and anti-rabbit IgG (Invitrogen, catalog #A11004 (mouse) and A-11011 (rabbit)) were used at 1/200.

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 immunofluorescence: Phalloidin-TRITC (Sigma-Aldrich, St Louis, MO; catalog #P1951) was used at 1/10,000; rabbit polyclonal against phospho-Myosin regulatory light chain 2 at position Ser19 (Cell Signaling, Danvers, MA; catalog #3671) was used at 1/100 (in Figure 2, used with Alexa Fluor 488 goat anti-mouse); rabbit polyclonal against ARHGAP29 (Novus Biologicals, catalog #NBP1-05989) was used at 1/150; mouse monoclonal against Keratin 14 (Santa Cruz Biotechnology, catalog #sc-53253) was used at 1/20; Alexa Fluor 488 goat anti-mouse and antirabbit IgG (Invitrogen, Waltham, MA; catalog #A-11001 (mouse) and A-11008 (rabbit)) and Alexa Fluor 568 goat anti-mouse and anti-rabbit IgG (Invitrogen, catalog #A11004 (mouse) and A-11011 (rabbit)) were used at 1/200.

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 immunofluorescence: Phalloidin-TRITC (Sigma-Aldrich, St Louis, MO; catalog #P1951) was used at 1/10,000; rabbit polyclonal against phospho-Myosin regulatory light chain 2 at position Ser19 (Cell Signaling, Danvers, MA; catalog #3671) was used at 1/100 (in Figure 2, used with Alexa Fluor 488 goat anti-mouse); rabbit polyclonal against ARHGAP29 (Novus Biologicals, catalog #NBP1-05989) was used at 1/150; mouse monoclonal against Keratin 14 (Santa Cruz Biotechnology, catalog #sc-53253) was used at 1/20; Alexa Fluor 488 goat anti-mouse and antirabbit IgG (Invitrogen, Waltham, MA; catalog #A-11001 (mouse) and A-11008 (rabbit)) and Alexa Fluor 568 goat anti-mouse and anti-rabbit IgG (Invitrogen, catalog #A11004 (mouse) and A-11011 (rabbit)) were used at 1/200.

Techniques: Staining, Western Blot

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 2. Gene expression analysis un- covers dysregulation of members of the ARHGAP gene family in U251 cells, and sta- ble silencing of ARHGAP12 and ARHGAP29 in U251 cells exerts distinct cytoskeletal re- arrangements (A and B) Representative immunofluorescence images of ARHGAP12 and ARHGAP29 expres- sion, untreated and after exposure to the GSK-3 inhibitor BIO (A), with quantification by total fluo- rescence (B). Student’s t test, *p < 0.05. Scale bar: 10 mm. Data presented as mean ± SEM. (C) Measurement of cellular localization showed loss of nuclear expression of both ARHGAP12 and ARHGAP29 following exposure to BIO. (D) Representative bright-field micrographs of collagen-embedded U251 spheroids immuno- stained for either ARHGAP12 or ARHGAP29 (brown) and counterstained with hematoxylin. Cytoplasmic labeling of ARHGAP12 in the spheroid core became more pronounced after BIO treatment (black arrowheads). For ARHGAP29, cytoplasmic and membranous labeling was noted, especially on the spheroid periphery and on migratory cells, which was reduced after treatment with BIO (red arrowheads). Scale bar: 50 mm. Data presented as median. (E) Stable gene silencing of ARHGAP12 (A12 kd) and ARHGAP29 (A29 kd) in U251 cells was confirmed by western blot. (F) Representative immunofluorescence of U251 cells with stable ARHGAP12 and ARHGAP29 kd showing morphological changes and cytoskeletal rearrangement in U251 cells in 2D monolayers. Scale bar: 100 mm. (G) Time-lapse microscopy of U251 cells with kd of the 2 different ARHGAPs showed distinct cellular morphological characteristics compared to control cells. Scale bar: 200 mm. (H) In 3D spheroid assays, over 72 h, shorter cell protrusions consisting of rounded cells for the ARHGAP29 kd and protrusions consisting of in- terconnected, elongated cells became evident. Scale bar: 100 mm. (I) 3D invasion assays highlight cellular features and morphological changes of migrating cells after ARHGHAP29 and ARHGAP12 kd. Scale bar: 200 mm.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Gene Expression, Expressing, Staining, Labeling, Western Blot, Time-lapse Microscopy, Control

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 3. Stable silencing of ARHGAP12 or ARHGAP29 induces changes in the number of appendages emanating from spheroids and distance traveled away from spheroids by individual migratory cells Using Cloudbuster software,26 ARHGAP12 and ARHGAP29 kd cell spheroids were analyzed at time point 0 and at 48 h. (A) In U87 cells, ARHGAP29 kd spheroids showed reduced length of protrusions in comparison to ARHGAP12 (one-way ANOVA, p = 0.0002) but no difference in the number of extensions after 48 h (one-way ANOVA, p > 0.05). (B) For U251, a similar effect was seen after ARHGAP12 kd in the length of cellular protrusions compared to ARHGAP29 kd (p = 0.0016) after 48 h. Data presented as median, interquartile range. (C) Representative reconstructed spheroids and migratory cells treated with a non-target control, ARHGAP29 kd, or ARHGAP12 kd. Insets high- light a selected region of an individual spheroid with visible extensions and individual cells (white arrowheads) with (left to right) a non-target (NT) control spheroid, ARHGAP29 kd, and ARHGAP12 kd.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Software, Comparison, Control

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 4. ARHGAP transcription is regu- lated in part by GSK-3 signaling via b-cate- nin translocation (A) Immunofluorescence labeling with various markers (ARHGAP12, ARHGAP29, b-catenin, and CD44) of U251 cells treated with the GSK-3 in- hibitor BIO. ICG001 and inhibitor combination re- veals that ARHGAP12 and ARHGAP29 protein levels are altered after treatment with BIO and not affected by treatment with ICG001 alone or the combination treatment (n = 3/group). Scale bar: 200 mm. (B and C) Significant differences (mean ± SEM) in (B) CD44 expression and (C) b-catenin expression in cell populations treated with the GSK-3 inhibitor BIO, ICG001, or ICG001 in combination with the GSK-3 inhibitor BIO were observed (n = 3). Data presented as mean ± SEM. (D) Representative images from time-lapse mi- croscopy of U251 cells, showing changes in the position of cells over a 24-h period in untreated, BIO-treated, ICG001-treated, and combination treatment groups. Scale bar: 200 mm (E) Representative plots demonstrating the nega- tive effect of the GSK inhibitor BIO on cell migra- tion, with no effect of the inhibitor ICG001 and no effect after combination treatment. (F and G) Significant differences (mean ± SEM) in (F) ARHGAP12 and (G) ARHGAP29 expression in cell populations treated with the GSK-3 inhibitor BIO, ICG001, or ICG001 in combination with the GSK-3 inhibitor BIO were observed (n = 3). Data presented as mean ± SEM. Post hoc Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Translocation Assay, Labeling, Expressing

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 6. Intracranially injected ARHGAP12 and ARHGAP29 kd cells induce tumors with altered morphological features (A–C) Representative mouse brain tissue sections of intracranial tumors from non-target control, ARHGAP12 kd, and ARHGAP29 kd cells with immunohisto- chemical staining (brown) and corresponding column graphs for expression of the following cell markers: (A) vimentin; (B) cleaved caspase-3 (CC3), an apoptosis marker; and (C) Ki67, expressed in proliferating cell nuclei. Black arrowheads indicate different morphological features of the tumor margin. Vimentin scale bar: 150 mm; CC3 and Ki67 scale bars: 75 mm. Data presented as mean ± SEM. (D and E) Both (D) N-cadherin and (E) E-cadherin were strongly expressed on tumor cells in the control group, with a significant loss of N-cadherin expression in ARHGAP12 kd tumors. Scale bar: 75 mm; post hoc Dunnett’s test. Data presented as median, interquartile range, and range.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Injection, Control, Staining, Expressing, Marker

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 7. The ARHGAPs regulate glioma cell migration via a novel GSK-3 signaling pathway Targeting GSK-3 activity with a small-molecule inhibitor (1) prevents b-catenin degradation by ubiquitination and (2) promotes b-catenin translocation to the nucleus, where it acts as a transcription factor. Transcription of ARHGAP12 and prevention of transcription of ARHGAP29 lead to changes in Src signaling and/or phosphorylation status of RhoA and Rac1 with (3) concomi- tant adoption of a less aggressive, ameboid phenotype in migratory cells. The phenotypic change in migrating cells may affect recurrence after surgery in patients.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Migration, Activity Assay, Ubiquitin Proteomics, Translocation Assay, Phospho-proteomics

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: Identification of RCC antigen PARG1 by SEREX and expression of PARG1 mRNA in normal kidney, RCC tissues, and RCC cell lines. (A) Presence of anti-PARG1 IgG from sera of RCC was detected by Western blot analysis with recombinant His-tagged PARG1 protein. Detection samples are shown in red. (B) Frequent detection of anti-PARG1 IgG in sera from patients with RCC was evaluated by ELISA. ELISA was done with the recombinant PARG1 protein. The horizontal line indicates the cutoff value for positivity (OD = 0.032: the average absorbance of the healthy individuals plus 2 SD). Positive sera were found in 13 of 24 (54.2%) patients with RCC but not in healthy donors. (C) Expression of PARG1 mRNA in normal kidney and RCC tissue in the same RCC patient sample was detected by qPCR analysis. GAPDH mRNA expression was used as an internal control. (D) Expression of PARG1 in human RCC cell lines was detected by qPCR analysis. GAPDH was used as an internal control. HEK293T was used as control sample for this assay.

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: Expression of PARG1 and survival analysis in patients with RCC. (A) Representative immunohistochemical analysis of PARG1 protein in paraffin-embedded tissues. (a) Normal proximal tubules (magnification, ×40), (b) RCC tissues (level 1), (c) RCC tissues (level 2), (d) RCC tissues (level 3), (e) pancreatic metastasis region, (f) lymph node metastasis legion, (g) microvascular invasion (magnification, ×10), and (h) microvascular invasion (high magnification, ×40). (B) RCC tissues were stained with anti–Ki-67 Ab, and Ki-67–positive cells (indicated by arrows) in high-powered field (HPF; magnification, ×40) were counted. The right graph shows the number of Ki-67–positive cells in each PARG1 expression level. (C) Kaplan-Meier overall survival curve with respect to low expression level ( n = 51) and high expression level ( n = 23) of PARG1. Five-year survival rate; P = .035. (D) Kaplan-Meier recurrence-free survival curve with respect to low expression level ( n = 40) and high expression level ( n = 13) of PARG1 in N0M0 patients with RCC. Five-year recurrence-free survival rate; P = .0084.

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: Correlation between PARG1 Expression and Clinicopathological Features in RCC Patients

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Expressing

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: Correlation between Clinicopathological Features and Overall Survival in 74 RCC Patients

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques:

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: High PARG1 Expression Is an Independent Factor Correlating with 53 RCC Recurrence in N0M0 Patients

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Expressing

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: PARG1 was involved in cell proliferation and cell cycle progression through regulation of p53 and p21 Cip1/Waf1 in RCC cell lines. (A) Decrease of PARG1 mRNA and protein was observed after 2 days of incubation with two PARG1-specific siRNAs (si#2 and si#3) in SW839 and 769-p, whereas increase of PARG1 mRNA and protein was observed after 2 days of incubation with pcDNA3.1-PARG1 vector in HEK293T. (B) The inhibition of cell proliferation of SW839 and 769-p after 3 days of incubation with PARG1 siRNAs was observed in WST-1 assay (left graph) or trypan blue cell count (right graph); however, cell proliferation of HEK293T was increased by transfection with pcDNA3.1-PARG1. ** P < .01; data are presented as the mean ± SD of three independent experiments. (C) Cell cycle analysis confirmed that treating SW839 and 769-p cells with PARG1 siRNA blocked the cell cycle in G1 phase at day 3 after transfection. (D) PARG1 siRNA upregulated p53, p-p53(Ser15), and p21 Cip1/Waf1 protein expression by Western blotting in SW839 and 769-p. GAPDH was used as control. Representative results from three independent experiments (C, D).

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Incubation, Plasmid Preparation, Inhibition, WST-1 Assay, Cell Counting, Transfection, Cell Cycle Assay, Expressing, Western Blot, Control

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: The role of PARG1 involved in cell invasion and migration through inhibition of RhoA activity. (A) Cell invasion ability was evaluated by Matrigel invasion assay in RCC cell lines and HEK293T. Invasion ability was decreased in PARG1 siRNA-transfected SW839 and 769-p cells, but invasion ability was increased in PARG1 expression vector–transfected HEK293T cells. * P < .05, ** P < .01; data are presented as the mean ± SD of three independent experiments. (B) Cell migration ability was performed by wound healing assay. Migration ability was decreased in PARG1 siRNA-transfected SW839 and 769-p cells at 10 hours of incubation; however, migration ability was increased in PARG1 expression vector–transfected HEK293T cells at 24 hours of incubation. * P < .05; data are presented as the mean ± SD of three independent experiments. (C) Effect of PARG1 on RhoA activity was determined using RhoA activation kit (pull-down assay and Western blotting). PARG1 siRNAs induced RhoA-GTP in RCC cell lines, but PARG1 expression vector reduced RhoA-GTP in HEK293T cells. (D) Scramble and PARG1 siRNAs-transfected SW839 cells were stained with PARG1 (FITC), F-actin (Texas red), and DAPI. Downregulation of PARG1 by siRNA induced actin stress fiber formation. Representative results from three independent experiments (C, D).

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Migration, Inhibition, Activity Assay, Invasion Assay, Transfection, Expressing, Plasmid Preparation, Wound Healing Assay, Incubation, Activation Assay, Pull Down Assay, Western Blot, Staining

Journal: Translational Oncology

Article Title: Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1 1 2

doi: 10.1016/j.tranon.2016.12.004

Figure Lengend Snippet: PARG1 promoted cell proliferation and invasion through inhibition of RhoA-ROCK signaling. (A) Dependency of cell proliferation ability on RhoA-ROCK signaling was evaluated by WST-1 assay with Rho-ROCK inhibitor Y27632 (1 μM) on PARG1-silenced SW839 RCC cells. Cell proliferation was restored in PARG1 siRNA transfected SW839 cell line by addition of Y27632 at day 2, compared with PBS treatment. * P < .05, ** P < .01; data are presented as the mean ± SD of three independent experiments. (B) Dependency of cell invasion ability on RhoA-ROCK signaling was evaluated by xCELLigence system analysis as described in Materials and Methods with Y27632 (1 μM) on PARG1-silenced SW839 cells by siRNA. PBS was used as control. Cell invasion ability was rescued by treatment with Y27632 in PARG1 siRNA transfected. SW839 cells transfected with scrambled or PARG1 siRNAs were treated with PBS or Y27632. * P < .05, ** P < .01; data are presented as the mean ± SD of three independent experiments. (C) The impact of Rho-ROCK inhibition on expressions of p53, p-p53 (Ser15), and p21 Cip1/Waf1 was evaluated by Western blotting in SW839 cells treated with scrambled or PARG1 siRNAs. Representative results from three independent experiments. PBS was used as control. (D) Schematic representation of the functional role of PARG1 involved in cell proliferation, migration, and invasion through regulation of RhoA-ROCK signaling pathway.

Article Snippet: PARG1 (ARHGAP29 MaxPab polyclonal antibody) Ab was from Abnova (Taiwan, Taipei), p21 Cip1/Waf1 Ab was from Carbioche (Germany), p53 (DO-1) Ab was from Santa Cruz Biotechnology (Delaware Avenue, CA), and phosphor-p53 (ser15) Ab was from Cell Signaling Technology.

Techniques: Inhibition, WST-1 Assay, Transfection, Control, Western Blot, Functional Assay, Migration

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil is a potential Ras-interacting protein. A , coomassie Brilliant Blue staining of flag pulldown. HEK293T cells transfected with plasmid expressing HRas V12 tagged with Flag or with the empty vector (CT) for 24 h, after which cell lysates were collected for affinity-pulldown using Flag M2 agarose as described in and . Affinity-purified proteins were subjected to SDS-PAGE analysis. Arrow indicates the position of Radil or HRas. The single star (∗) indicates IgG heavy-chain band. The double stars (∗∗) indicate IgG light-chain band. B , affinity-purified proteins were subjected to mass spectrometric analysis. Proteins identified with top scores were listed and Radil was highlighted. C , a survey of Radil expression in various tumor cell lines via Western blotting. The arrow indicates the Radil specific bands. The star (∗) denotes nonspecific bands. IgG, immunoglobulin G.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Staining, Transfection, Plasmid Preparation, Expressing, Affinity Purification, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil physically interacts with KRas. A , HEK293T cells transfected with Flag-Radil were lysed, and cell lysates were immunoprecipitated with the anti-Flag antibody or control IgG. Flag immunoprecipitates, along with lysate inputs, were blotted with antibodies for Flag, KRas 4b, and Pan-Ras proteins. B , HEK293T cells were transfected with the plasmid expressing Flag-KRas V12 for 24 h, after which cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Radil and Flag. C , domain structures of full-length Radil (FL) and its mutant without RA domain (RAΔ). D , HEK293T cells transfected with FLAG-Radil FL and RAΔ expression plasmids for 24 h, after which cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for KRas4B, Rap1, and Flag. IgG, immunoglobulin G.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Transfection, Immunoprecipitation, Control, Plasmid Preparation, Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil interacts with active Ras proteins. A , HEK293T cells were transfected with the plasmid expressing Flag-tagged HRas (WT), constitutively active HRas (V12), or dominant-negative HRas (N17) for 24 h, after which cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Radil and FLAG. The amount of Radil interacting with various forms of KRas was quantified via densitometry. Normalized values equal the density of Ras-bound Radil/density of total Radil/fold density of IP Flag. B , computer modeling structures of KRas/Radil interaction with or without GTP binding. C , HEK293T cells were transfected with plasmids expressing either WT or constitutively active forms of Ras (V12) proteins as indicated for 24 h, after which cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Radil and Flag. The amount of Radil interacting with various forms of KRas was quantified via densitometry. Normalized values equal the density of Ras-bound Radil/density of total Radil/Fold density of IP Flag. D , HEK293T cells were transfected with plasmids expressing GFP-KRas and/or Flag-Radil for 24 h, after which cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Flag, GFP, Rap1, and ArhGAP29.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Immunoprecipitation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil positively regulates KRas signaling. A , 293FT cells engineered to express inducible Flag-KRas V12 (293FT/Flag-KRas V12 ) were starved in low-FBS (0.2%) medium for 16 h and then treated with doxycycline (Dox) for various times and cell lysates were blotted for Flag, phospho-MEK, MEK, phospho-ERK, ERK, and actin. Signals of p-MEK and p-ERK were quantified by densitometry. Values presented are from the calculation of density of phospho-MEK (or p-ERK)/density of total MEK (or ERK). B , 293FT/KRas V12 cells were transfected with Radil-specific siRNA (siRadil) and/or nontargeting control pool siRNAs (siControl) for 12 h and then treated with Dox 12 and 24 h. Cell lysates were then blotted for Flag, Radil, phospho-MEK, MEK, and actin. p-MEK signals were quantified by densitometry. The arrow indicates the position of Radil. Values presented are from the calculation of the density of phospho-MEK normalized by the density of total MEK. ∗ indicates nonspecific bands. C , 293FT cells engineered to express inducible Flag-Radil (293FT/Flag-Radil) were treated with Dox for various times as indicated and cell lysates were blotted for Flag, phospho-ERK, ERK, phospho-MEK, MEK, and actin. Signals of p-MEK and p-ERK were quantified by densitometry. Values presented are from the calculation of the density of phospho-MEK (or p-ERK) divided by the density of total MEK (or ERK). D , 293FT/Flag-Radil cells were treated with Dox for various times as indicated. Equal amounts of lysates were subjected to the in vitro pull-down analysis using Raf-RBD, which specifically interacts with GTP-bound Ras (GST-RBD). The amount of GTP-Ras pulled-down was quantified by sensitometry. The proteins subjected to GST pull-down assay were stained with Ponceau S staining. The values presented are derived from the density of active Ras normalized by the density of total Ras (Pan Ras). FBS, fetal bovine serum.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Transfection, Control, In Vitro, Pull Down Assay, Staining, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil mediates growth factor–induced activation of cRaf and MEK. A549 cells were transfected with Radil siRNAs (siRadil) or nontargeting control pool siRNAs (siControl) for 24 h, starved in low (0.2%) FBS medium for 16 h, and then fed 20% serum for various times as indicated. Cell lysates were then blotted for Radil, phospho-cRaf 338, phospho-MEK, and actin. Signals of p-cRaf and p-MEK were quantified. The arrow indicates the position of Radil. The values presented are derived from the density of phospho-form normalized by the density of actin. ∗ indicates nonspecific bands. FBS, fetal bovine serum.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Activation Assay, Transfection, Control, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil positively regulates cell proliferation, migration, and EMT. A , A549 cells transfected with Radil siRNAs (siRadil), KRas siRNAs (siKRas), and combination of Radil and KRas siRNAs (siR+K) for various times as indicated. Control cells were transfected for the same length of time with nontargeting control pool siRNAs (siControl). Cell proliferation was measured as described in . B , A549 cells transfected with Radil siRNAs and/or KRas siRNAs for 24 h. Cell lysates were then blotted for Radil, KRas, E-cadherin, Vimentin, Zeb1, ZO-1, Snail, and actin. Signals of E-cadherin, vimentin, ZEB1, ZO-1, and Snail were quantified by densitometry. The values presented are derived from the density of each protein normalized by the density of actin. C , representative images of transwell invasion assays. A549 cells transfected with Radil siRNAs and/or KRas siRNAs for 24 h were seeded in Matrigel for cell-invasion assay. Cells transfected with nontargeting control pool siRNAs (siControl) were used as control. Scale bar = 200 μm. D , quantification of transwell invasion assays ( top panel ) and total counts of invaded cells in three individual images from transwell inserts ( bottom panel ). Data on cell invasion after various treatments were summarized and quantified. Data represent the mean (±standard deviation, SD) of three independent experiments, each performed in triplicate and are presented relative to control. Error bars indicate SDs. Stars indicate statistical significance at p < 0.05, ∗; and p < 0.01, ∗∗. EMT, epithelial–mesenchymal transition.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Migration, Transfection, Control, Derivative Assay, Invasion Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Radil as a Ras binding partner and putative activator

doi: 10.1016/j.jbc.2021.100314

Figure Lengend Snippet: Radil regulates cell adhesion and actin dynamics. A , A549 cells seeded on chamber slides were transfected with Radil siRNAs (siRadil) and/or KRas siRNAs (siKRas) for 24 h. Control cells were transfected for the same length of time with nontargeting control pool siRNAs (siControl). Cells of various treatments were fixed and stained with the anti-vinculin antibody and phalloidin. Representative images are shown. B , a model that depicts Radil’s function in regulating KRas signaling in response to growth factors during EMT. EMT, epithelial–mesenchymal transition.

Article Snippet: Polyclonal Radil and ArhGap29 antibody were purchased from ProteinTech.

Techniques: Transfection, Control, Staining

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 2. Gene expression analysis un- covers dysregulation of members of the ARHGAP gene family in U251 cells, and sta- ble silencing of ARHGAP12 and ARHGAP29 in U251 cells exerts distinct cytoskeletal re- arrangements (A and B) Representative immunofluorescence images of ARHGAP12 and ARHGAP29 expres- sion, untreated and after exposure to the GSK-3 inhibitor BIO (A), with quantification by total fluo- rescence (B). Student’s t test, *p < 0.05. Scale bar: 10 mm. Data presented as mean ± SEM. (C) Measurement of cellular localization showed loss of nuclear expression of both ARHGAP12 and ARHGAP29 following exposure to BIO. (D) Representative bright-field micrographs of collagen-embedded U251 spheroids immuno- stained for either ARHGAP12 or ARHGAP29 (brown) and counterstained with hematoxylin. Cytoplasmic labeling of ARHGAP12 in the spheroid core became more pronounced after BIO treatment (black arrowheads). For ARHGAP29, cytoplasmic and membranous labeling was noted, especially on the spheroid periphery and on migratory cells, which was reduced after treatment with BIO (red arrowheads). Scale bar: 50 mm. Data presented as median. (E) Stable gene silencing of ARHGAP12 (A12 kd) and ARHGAP29 (A29 kd) in U251 cells was confirmed by western blot. (F) Representative immunofluorescence of U251 cells with stable ARHGAP12 and ARHGAP29 kd showing morphological changes and cytoskeletal rearrangement in U251 cells in 2D monolayers. Scale bar: 100 mm. (G) Time-lapse microscopy of U251 cells with kd of the 2 different ARHGAPs showed distinct cellular morphological characteristics compared to control cells. Scale bar: 200 mm. (H) In 3D spheroid assays, over 72 h, shorter cell protrusions consisting of rounded cells for the ARHGAP29 kd and protrusions consisting of in- terconnected, elongated cells became evident. Scale bar: 100 mm. (I) 3D invasion assays highlight cellular features and morphological changes of migrating cells after ARHGHAP29 and ARHGAP12 kd. Scale bar: 200 mm.

Article Snippet: Used primary antibodies were obtained from Atlas Antibodies (ARHGAP12, Cat # HPA000412; ARHGAP29, Cat # HPA026534), Cell Signaling Technologies (Fyn, Cat # 4023; Lyn, Cat # 2732; E-cadherin, Cat # 3195; N-cadherin, Cat # 13116; Vimentin, Cat # 5741; GFAP, Cat # 3670, beta-catenin, Cat # 9561, Non-Phospho (P) - beta-catenin, Cat # 19807), ThermoFisher (pan-Actin, Cat # PA578715; cSrc, Cat # AHO1152), Abcam (RhoA, Cat # ab86297; Rac1, Cat # ab155938) and incubated at recommendedmanufacturer’s dilutions.

Techniques: Gene Expression, Expressing, Staining, Labeling, Western Blot, Time-lapse Microscopy, Control

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 3. Stable silencing of ARHGAP12 or ARHGAP29 induces changes in the number of appendages emanating from spheroids and distance traveled away from spheroids by individual migratory cells Using Cloudbuster software,26 ARHGAP12 and ARHGAP29 kd cell spheroids were analyzed at time point 0 and at 48 h. (A) In U87 cells, ARHGAP29 kd spheroids showed reduced length of protrusions in comparison to ARHGAP12 (one-way ANOVA, p = 0.0002) but no difference in the number of extensions after 48 h (one-way ANOVA, p > 0.05). (B) For U251, a similar effect was seen after ARHGAP12 kd in the length of cellular protrusions compared to ARHGAP29 kd (p = 0.0016) after 48 h. Data presented as median, interquartile range. (C) Representative reconstructed spheroids and migratory cells treated with a non-target control, ARHGAP29 kd, or ARHGAP12 kd. Insets high- light a selected region of an individual spheroid with visible extensions and individual cells (white arrowheads) with (left to right) a non-target (NT) control spheroid, ARHGAP29 kd, and ARHGAP12 kd.

Article Snippet: Used primary antibodies were obtained from Atlas Antibodies (ARHGAP12, Cat # HPA000412; ARHGAP29, Cat # HPA026534), Cell Signaling Technologies (Fyn, Cat # 4023; Lyn, Cat # 2732; E-cadherin, Cat # 3195; N-cadherin, Cat # 13116; Vimentin, Cat # 5741; GFAP, Cat # 3670, beta-catenin, Cat # 9561, Non-Phospho (P) - beta-catenin, Cat # 19807), ThermoFisher (pan-Actin, Cat # PA578715; cSrc, Cat # AHO1152), Abcam (RhoA, Cat # ab86297; Rac1, Cat # ab155938) and incubated at recommendedmanufacturer’s dilutions.

Techniques: Software, Comparison, Control

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 4. ARHGAP transcription is regu- lated in part by GSK-3 signaling via b-cate- nin translocation (A) Immunofluorescence labeling with various markers (ARHGAP12, ARHGAP29, b-catenin, and CD44) of U251 cells treated with the GSK-3 in- hibitor BIO. ICG001 and inhibitor combination re- veals that ARHGAP12 and ARHGAP29 protein levels are altered after treatment with BIO and not affected by treatment with ICG001 alone or the combination treatment (n = 3/group). Scale bar: 200 mm. (B and C) Significant differences (mean ± SEM) in (B) CD44 expression and (C) b-catenin expression in cell populations treated with the GSK-3 inhibitor BIO, ICG001, or ICG001 in combination with the GSK-3 inhibitor BIO were observed (n = 3). Data presented as mean ± SEM. (D) Representative images from time-lapse mi- croscopy of U251 cells, showing changes in the position of cells over a 24-h period in untreated, BIO-treated, ICG001-treated, and combination treatment groups. Scale bar: 200 mm (E) Representative plots demonstrating the nega- tive effect of the GSK inhibitor BIO on cell migra- tion, with no effect of the inhibitor ICG001 and no effect after combination treatment. (F and G) Significant differences (mean ± SEM) in (F) ARHGAP12 and (G) ARHGAP29 expression in cell populations treated with the GSK-3 inhibitor BIO, ICG001, or ICG001 in combination with the GSK-3 inhibitor BIO were observed (n = 3). Data presented as mean ± SEM. Post hoc Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: Used primary antibodies were obtained from Atlas Antibodies (ARHGAP12, Cat # HPA000412; ARHGAP29, Cat # HPA026534), Cell Signaling Technologies (Fyn, Cat # 4023; Lyn, Cat # 2732; E-cadherin, Cat # 3195; N-cadherin, Cat # 13116; Vimentin, Cat # 5741; GFAP, Cat # 3670, beta-catenin, Cat # 9561, Non-Phospho (P) - beta-catenin, Cat # 19807), ThermoFisher (pan-Actin, Cat # PA578715; cSrc, Cat # AHO1152), Abcam (RhoA, Cat # ab86297; Rac1, Cat # ab155938) and incubated at recommendedmanufacturer’s dilutions.

Techniques: Translocation Assay, Labeling, Expressing

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 6. Intracranially injected ARHGAP12 and ARHGAP29 kd cells induce tumors with altered morphological features (A–C) Representative mouse brain tissue sections of intracranial tumors from non-target control, ARHGAP12 kd, and ARHGAP29 kd cells with immunohisto- chemical staining (brown) and corresponding column graphs for expression of the following cell markers: (A) vimentin; (B) cleaved caspase-3 (CC3), an apoptosis marker; and (C) Ki67, expressed in proliferating cell nuclei. Black arrowheads indicate different morphological features of the tumor margin. Vimentin scale bar: 150 mm; CC3 and Ki67 scale bars: 75 mm. Data presented as mean ± SEM. (D and E) Both (D) N-cadherin and (E) E-cadherin were strongly expressed on tumor cells in the control group, with a significant loss of N-cadherin expression in ARHGAP12 kd tumors. Scale bar: 75 mm; post hoc Dunnett’s test. Data presented as median, interquartile range, and range.

Article Snippet: Used primary antibodies were obtained from Atlas Antibodies (ARHGAP12, Cat # HPA000412; ARHGAP29, Cat # HPA026534), Cell Signaling Technologies (Fyn, Cat # 4023; Lyn, Cat # 2732; E-cadherin, Cat # 3195; N-cadherin, Cat # 13116; Vimentin, Cat # 5741; GFAP, Cat # 3670, beta-catenin, Cat # 9561, Non-Phospho (P) - beta-catenin, Cat # 19807), ThermoFisher (pan-Actin, Cat # PA578715; cSrc, Cat # AHO1152), Abcam (RhoA, Cat # ab86297; Rac1, Cat # ab155938) and incubated at recommendedmanufacturer’s dilutions.

Techniques: Injection, Control, Staining, Expressing, Marker

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 7. The ARHGAPs regulate glioma cell migration via a novel GSK-3 signaling pathway Targeting GSK-3 activity with a small-molecule inhibitor (1) prevents b-catenin degradation by ubiquitination and (2) promotes b-catenin translocation to the nucleus, where it acts as a transcription factor. Transcription of ARHGAP12 and prevention of transcription of ARHGAP29 lead to changes in Src signaling and/or phosphorylation status of RhoA and Rac1 with (3) concomi- tant adoption of a less aggressive, ameboid phenotype in migratory cells. The phenotypic change in migrating cells may affect recurrence after surgery in patients.

Article Snippet: Used primary antibodies were obtained from Atlas Antibodies (ARHGAP12, Cat # HPA000412; ARHGAP29, Cat # HPA026534), Cell Signaling Technologies (Fyn, Cat # 4023; Lyn, Cat # 2732; E-cadherin, Cat # 3195; N-cadherin, Cat # 13116; Vimentin, Cat # 5741; GFAP, Cat # 3670, beta-catenin, Cat # 9561, Non-Phospho (P) - beta-catenin, Cat # 19807), ThermoFisher (pan-Actin, Cat # PA578715; cSrc, Cat # AHO1152), Abcam (RhoA, Cat # ab86297; Rac1, Cat # ab155938) and incubated at recommendedmanufacturer’s dilutions.

Techniques: Migration, Activity Assay, Ubiquitin Proteomics, Translocation Assay, Phospho-proteomics

Journal: JCI Insight

Article Title: Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling

doi: 10.1172/jci.insight.135385

Figure Lengend Snippet: ( A ) Phalloidin labeled F-actin showing a more disorganized actin cytoskeleton in PKD1 cystic cells (OX161) compared with control (UCL93) cells. ( B and C ) The length of actin filaments was significantly reduced and their normal parallel orientation more variable in PKD1 compared with control cells (N = 60 cells; significance determined by 2-tailed Student’s t test). ( D ) 3D-SIM confocal images of phalloidin-stained cells. Actin fibers can be seen predominantly orientated to the base of control UCL93 cells. In contrast, actin fibers were thicker and frequently localized to the apical surface of OX161 cells. Increased stress fibers were also present. Cilia labeled with Arl13b (green, arrows) are shorter. ( E ) Primary cilia were visualized in quiescent control and ADPKD cell lines after serum starvation by immunofluorescence labeling of Arl13b (red) and nuclei (blue). ( F ) Cilia length was significantly reduced in a panel of human PKD1 cystic compared with noncystic cell lines ( n = 8 patient-derived cell lines, N = 250 cells counted; significance determined by 1-way ANOVA corrected (Tukey) for multiple comparison. **** P < 0.0001. ADPKD, autosomal dominant polycystic kidney disease; SIM, structured illumination.

Article Snippet: The following antibodies were used in this study: PC1 (7e12), PC2 (g20), actin, ARHGAP5, ARHGAP29, myc, streptavidin, GST (Santa Cruz Biotechnology), ArhGAP35, MLC and pMLC (Cell Signaling), Flag (Sigma), RhoA, Cdc42 and Rac1 (Cytoskeleton), and Arl13b (Proteintech).

Techniques: Labeling, Control, Staining, Immunofluorescence, Derivative Assay, Comparison

Journal: JCI Insight

Article Title: Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling

doi: 10.1172/jci.insight.135385

Figure Lengend Snippet: ( A ) Cytochalasin D (1 μM, 4 hours) was associated with a significant increase in cilia length in both control (UCL93) and ADPKD (OX161) lines ( n = 4 independent experiments, N = 115 cells). ( B ) The ROCK inhibitor Y-27632 (1 μM, 4 hours) rescued the cilia length defect in the ADPKD (OX161) line but had no effect on cilia length in control (UCL93) cells ( n = 4 independent experiments, N = 264 cells). ( C ) Isogenic PKD1 -null cells were generated by CRISPR/Cas9 in the parental control line, UCL93. PC1-null clones (c1–3) were expanded for study. ( D ) Primary cilia were visualized in quiescent control (UCL93) and PKD1 null lines (PC1KO) after serum starvation by immunofluorescence labeling of Arl13b (green) and nuclei (blue). ( E ) Cilia length was significantly reduced in PKD1 null lines (PC1KO) compared with control (UCL93) cells ( n = 3 independent experiments, N = 67 cells). ( F ) Expression of mCherry-PC1, mCherry-PC1-4211X, or CFP-PC2 in transfected HEK293 cells showing bands of the expected size by immunoblotting for PC1 (7e12) or PC2 (G20). ( G ) Representative images of primary cilia in UCL93 control cells and OX161 cystic cells showing partial rescue of cilia length (Arl13b, green) in cells cotransfected with mCherry-PC1 (red) and CFP-PC2 but not mCherry-PC1-4211X (red) and CFP-PC2. ( H ) Expression of mCherry-PC1 was associated with a significant increase in cilia length compared with mCherry-PC1 4211X or pcDNA3 transfected control OX161 cells ( n = 3 independent experiments, N = 264 cells). **** P < 0.0001. Significance determined by 2-tailed Student’s t test ( A and B ). Significance determined by 1-way ANOVA corrected (Dunnett) for multiple comparison ( E and H ). PC1, polycystin-1; ADPKD, autosomal dominant polycystic kidney disease.

Article Snippet: The following antibodies were used in this study: PC1 (7e12), PC2 (g20), actin, ARHGAP5, ARHGAP29, myc, streptavidin, GST (Santa Cruz Biotechnology), ArhGAP35, MLC and pMLC (Cell Signaling), Flag (Sigma), RhoA, Cdc42 and Rac1 (Cytoskeleton), and Arl13b (Proteintech).

Techniques: Control, Generated, CRISPR, Clone Assay, Immunofluorescence, Labeling, Expressing, Transfection, Western Blot, Comparison

Journal: JCI Insight

Article Title: Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling

doi: 10.1172/jci.insight.135385

Figure Lengend Snippet: ( A – D ) The proliferation index (Ki67) was significantly reduced ( A and B ) and cilia length (Arl13b) significantly increased ( C and D ) in hydroxyfasudil-treated animals (50 cilia per animal). ( E ) Representative images of primary cilia in Pkd1 –/– kidney tissue. DBA lectin–positive collecting duct cysts were stained green and primary cilia (Arl13b) was labeled red. Dotted lined boxes show the region under higher magnification (original magnification, ×1000). Mean cilia length was significantly increased in DBA-positive cysts after hydroxyfasudil treatment ( n = 3 independent experiments, N = 276 cells). *** P < 0.001, **** P < 0.0001. Significance determined by 2-tailed Student’s t test.

Article Snippet: The following antibodies were used in this study: PC1 (7e12), PC2 (g20), actin, ARHGAP5, ARHGAP29, myc, streptavidin, GST (Santa Cruz Biotechnology), ArhGAP35, MLC and pMLC (Cell Signaling), Flag (Sigma), RhoA, Cdc42 and Rac1 (Cytoskeleton), and Arl13b (Proteintech).

Techniques: Staining, Labeling