Journal: Oncology reports

Article Title: ARHGAP29 expression may be a novel prognostic factor of cell proliferation and invasion in prostate cancer.

doi: 10.3892/or.2020.7811

Figure Lengend Snippet: Figure 1. AR, YAP, ARHGAP29, and F‑actin expression in prostate cancer cell lines. (A) mRNA levels of ARHGAP29 in prostate cancer cell lines (22Rv1, LNCaP, DU145 and PC‑3). Experiments were performed in triplicate. The vertical axis of the graph is presented on a logarithmic scale. The results are expressed as the mean ± SD. *P<0.01 compared with PC‑3 cells. (B) Western blotting of the expression of various proteins in prostate cancer cell lines. (C) Densitometric analysis of B (relative protein expression to GAPDH). There was an inverse association between ARHGAP29 and F‑actin expression, but no clear association between the expression of ARHGAP29 and that of other proteins. The results are expressed as the mean ± SD (at least three independent experiments). *P<0.05, **P<0.01 compared with PC‑3 cells. AR, androgen receptor; YAP, yes‑associated protein; ARHGAP29, Rho GTPase‑activating protein 29.

Article Snippet: A mammalian expression of HA tagged ARHGAP29 (#104154) was purchased from Addgene, Inc.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: ARHGAP29 expression may be a novel prognostic factor of cell proliferation and invasion in prostate cancer.

doi: 10.3892/or.2020.7811

Figure Lengend Snippet: Figure 2. Effects of downregulation or upregulation of ARHGAP29 in prostate cancer cell lines (PC‑3, LNCaP and DU145) on the mRNA and protein expres- sion. (A) Quantitative comparison of ARHGAP29 expression by RT‑qPCR in prostate cancer cell lines with downregulation or upregulation of ARHGAP29 compared with the control. Experiments were performed in triplicate. The results are expressed as the mean ± SD. *P<0.01 compared with si‑NC (PC‑3 cells) or pcDNA empty vector (mock; LNCaP and DU145 cells). In LNCaP and DU145, the vertical axis of the graph is presented on a logarithmic scale. (B) Western blotting of the expression of various proteins in prostate cancer cell lines. (C) Densitometric analysis of B (relative protein expression to GAPDH). The protein expression was only slightly altered in ARHGAP29‑upregulated LNCaP and DU145 cells compared with ARHGAP29‑downregulated PC‑3 cells. The results are expressed as the mean ± SD (at least three independent experiments). *P<0.01 compared with si‑NC (PC‑3 cells) or pcDNA empty vector (mock; LNCaP and DU145 cells). (D) The ratio of phosphorylated protein to non‑phosphorylated protein (YAP and cofilin). The results are expressed as the mean ± SD (at least three independent experiments). *P<0.05, **P<0.01 compared with si‑NC (PC‑3 cells) or pcDNA empty vector (mock; LNCaP and DU145 cells). ARHGAP29, Rho GTPase‑activating protein 29.

Article Snippet: A mammalian expression of HA tagged ARHGAP29 (#104154) was purchased from Addgene, Inc.

Techniques: Comparison, Expressing, Control, Plasmid Preparation, Western Blot

Journal: Oncology reports

Article Title: ARHGAP29 expression may be a novel prognostic factor of cell proliferation and invasion in prostate cancer.

doi: 10.3892/or.2020.7811

Figure Lengend Snippet: Figure 3. Effects of downregulation or upregulation of ARHGAP29 in prostate cancer cell lines (PC‑3, LNCaP and DU145) on cell viability and invasion. (A) MTS assay results. Cell viability was significantly decreased in ARHGAP29‑downregulated PC‑3 cells compared with control cells (si‑NC). In contrast, cell viability was significantly increased in ARHGAP29‑upregulated LNCaP and DU145 cells compared with control cells (mock). Experiments were per- formed in triplicate. The results are expressed as the mean ± SD. *P<0.05, **P<0.01 compared with si‑NC (PC‑3 cells) or pcDNA empty vector (mock; LNCaP and DU145 cells). (B) Invasion assay results. The number of invasive PC‑3 cells was significantly decreased after knocking down ARHGAP29. In contrast, the number of invasive LNCaP and DU145 cells was significantly increased after upregulation of ARHGAP29. Experiments were performed in triplicate. The results are expressed as the mean ± SD. *P<0.01 compared with si‑NC (PC‑3 cells) or pcDNA empty vector (mock; LNCaP and DU145 cells). ARHGAP29, Rho GTPase‑activating protein 29.

Article Snippet: A mammalian expression of HA tagged ARHGAP29 (#104154) was purchased from Addgene, Inc.

Techniques: MTS Assay, Control, Plasmid Preparation, Invasion Assay

Journal: Oncology reports

Article Title: ARHGAP29 expression may be a novel prognostic factor of cell proliferation and invasion in prostate cancer.

doi: 10.3892/or.2020.7811

Figure Lengend Snippet: Figure 4. Identification of other cell motility‑related genes after knockdown of ARHGAP29. (A) The 84 genes related to cell motility are presented and analysis of the results was performed using the web‑based software ‘RT2 Profiler PCR Array’ Data Analysis version 3.5. All of genes involved in the array are presented. (B) A heatmap is presented. Numerous genes were downregulated after knockdown of ARHGAP29 in PC3 cells. A1‑G12 corresponds to A1‑G12 of A. In the heatmap the data was displayed in a grid where each row represents a gene included in the commercial array. The color and intensity of the boxes represent changes of gene expression. For example, red represents upregulated genes and blue represents downregulated genes. (C) PCR Array of cell motility genes in PC‑3 cells before and after downregulation of ARHGAP29. The graph shows the relative log value between cell motility gene expression in PC‑3 cells before and after downregulation of ARHGAP29 (vertical axis, after downregulation of ARHGAP29; horizontal axis, before downregulation of ARHGAP29). The diagonal line in the center of the graph shows equal level of gene expression in PC‑3 cells before and after downregulation of ARHGAP29. The upper diagonal lines indicate that the level of gene expression after downregulation was three times higher than that of before downregulation. The lower diagonal line indicates that the level of gene expression after downregulation was one third times lower than that of before downregulation. The mRNA level of MMP‑2 was significantly downregulated. (D) Quantitative comparison of MMP‑2 by RT‑qPCR between si‑NC and si‑ARHGAP29 PC‑3 transfectants. MMP‑2 mRNA expression was suppressed after downregulation of ARHGAP29. Experiments were performed in triplicate. The results represent the mean ± SD. *P<0.01 compared with si‑NC. ARHGAP29, Rho GTPase‑activating protein 29.

Article Snippet: A mammalian expression of HA tagged ARHGAP29 (#104154) was purchased from Addgene, Inc.

Techniques: Knockdown, Software, Gene Expression, Comparison, Expressing

Journal: Oncology reports

Article Title: ARHGAP29 expression may be a novel prognostic factor of cell proliferation and invasion in prostate cancer.

doi: 10.3892/or.2020.7811

Figure Lengend Snippet: Figure 5. Association between the expression levels of YAP and ARHGAP29 and the prognosis of prostate cancer patients. (A) Histology (H&E staining) and IHC staining of YAP or ARHGAP29. Protein expression patterns were different between YAP and ARHGAP29. YAP was heterogeneously stained. Scale bars represent 200 µM. (B) Association of the D’Amico risk classification with the expression of YAP and ARHGAP29. When comparing three groups, one‑way ANOVA (inside the black frame) followed by Tukey‑Kramer test were used. *P<0.05. YAP was unrelated to the risk classification, but ARHGAP29 was signifi- cantly associated with the risk classification. The diamond indicates the mean (long horizontal line) and 95% confidence interval of the H‑score. (C) ROC curve of YAP, ARHGAP29 and both (AUC: 0.5971, 0.6216 and 0.6400, respectively). Both proteins had low AUC scores as prognostic markers. (D) Kaplan‑Meier plot of biochemical PFS stratified by the expression levels of YAP, ARHGAP29 and both. For each protein, high expression was associated with a poor prognosis of prostate cancer patients. Furthermore, the group with high expression of both YAP and ARHGAP29 had the worst prognosis. PFS was compared by a log‑rank test. P<0.05 was considered to indicate a statistically significant difference. YAP, yes‑associated protein; ARHGAP29, Rho GTPase‑activating protein 29; ROC, receiver operating characteristic; AUC, area under the curve; PFS, progression‑free survival; NS, not significant.

Article Snippet: A mammalian expression of HA tagged ARHGAP29 (#104154) was purchased from Addgene, Inc.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Acta pharmaceutica Sinica. B

Article Title: EHMT2 promotes tumorigenesis in GNAQ/11 -mutant uveal melanoma via ARHGAP29-mediated RhoA pathway.

doi: 10.1016/j.apsb.2023.12.002

Figure Lengend Snippet: Figure 5 ARHGAP29 is a target gene of EHMT2. (A) Genomic snapshot showing EHMT2 enrichment at the ARHGAP29 promoter. (B) A diagram representing the predicted promoter of ARHGAP29 and the designed site of primers. (C) ChIP-qPCR shows EHMT2, H3K9me2 and RNA polymerase II distribution at the ARHGAP29 promoter in PIG1 and OMM1. (D) ChIP-qPCR is done in OMM1 and UMC0631-treated (2 mmol/L, 72 h) OMM1 for H3K9me2 and RNA polymerase II enrichment. (E, F) ARHGAP29 mRNA and protein levels upon BIX01294/ UNC0631 treatment (2 mmol/L, 72 h), as well as in sgScr and sgEHMT2 cells, are detected by qPCR and WB in OMM2.3 and OMM1 cells. (G, H) The expression level of ARHGAP29 is detected in one PIG1 and six UM cells and quantified with ImageJ. Quantification of data is shown in bar graphs. (I) The expression level of ARHGAP29 mRNA is detected in eight cases of UM, as well as two control choroid tissues, via qPCR. GAPDH is used as the loading control. n Z 3 independent experiments for (CeG, I), *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The slides were incubated with primary antibodies overnight at 4 C, including EHMT2 (Invitrogen, PA5-78347), RhoA-GTP (NewEast Biotechnology, 26904), Ki67 (Cell Signaling, 9449), ARHGAP29 (Santa Cruz, sc-365554).

Techniques: ChIP-qPCR, Expressing, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: EHMT2 promotes tumorigenesis in GNAQ/11 -mutant uveal melanoma via ARHGAP29-mediated RhoA pathway.

doi: 10.1016/j.apsb.2023.12.002

Figure Lengend Snippet: Figure 6 ARHGAP29 regulates RhoA pathway and cell motility. (A) 92.1 and OMM2.3 cells are transfected with ARHGAP29-overexpressing plasmid. Expression of ARHGAP29, as well as p-YAP, YAP, and CTGF, is analyzed. (B) RhoA activity is analyzed in OMM2.3 with and without ARHGAP29 overexpression. (C) Representative IF images of 92.1 and OMM2.3 showing the change in YAP localization after ARHGAP29 overexpression. (D) Relative quantification of IF assay. Wound healing assay (E) and Transwell assay (F) is done for motility. Quantification of data is shown in bar graphs. GAPDH is used as the loading control. n Z 3 independent experiments for (AeC, E, F), *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The slides were incubated with primary antibodies overnight at 4 C, including EHMT2 (Invitrogen, PA5-78347), RhoA-GTP (NewEast Biotechnology, 26904), Ki67 (Cell Signaling, 9449), ARHGAP29 (Santa Cruz, sc-365554).

Techniques: Transfection, Plasmid Preparation, Expressing, Activity Assay, Over Expression, Wound Healing Assay, Transwell Assay, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: EHMT2 promotes tumorigenesis in GNAQ/11 -mutant uveal melanoma via ARHGAP29-mediated RhoA pathway.

doi: 10.1016/j.apsb.2023.12.002

Figure Lengend Snippet: Figure 8 EHMT2 combined with MEK/ERK inhibition impairs UM growth in vivo. Nude mice are injected orthotopically with 92.1 cells transfected with luciferase. After 2 weeks, mice are treated with either vehicle DMSO, binimetinib (3 mg/kg, qd), ulixertinib (50 mg/kg, qd), UNC0631 (5 mg/kg, qd), or in combination. The tumor bioluminescent signal (A) and quantification (B) of 92.1 in orthotopic xenografts are recorded 21 days after treatment. (C) Tumors are collected after 21-day treatment. (D) The weight of the eyes is measured 21 days after treatment. (E) KaplaneMeier survival plot for xenograft mice in six groups. Representative images of H&E staining (F), as well as Ki67, TUNEL, ARHGAP29 and RhoA-GTP expression determined by IF. (G) PDX models are established by planting UM tissues subcutaneously in nude mice. Mice are treated with either vehicle DMSO, binimetinib (3 mg/kg, qd), ulixertinib (50 mg/kg, qd), UNC0631 (5 mg/kg, qd), or in combination. Tumors are harvested after 21-day treatment. Representative images of H&E staining (H), as well as Ki67, TUNEL, ARHGAP29 and RhoA-GTP levels assessed by IF in the tumor tissues. n Z 6 mice in each group for (A, G), *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The slides were incubated with primary antibodies overnight at 4 C, including EHMT2 (Invitrogen, PA5-78347), RhoA-GTP (NewEast Biotechnology, 26904), Ki67 (Cell Signaling, 9449), ARHGAP29 (Santa Cruz, sc-365554).

Techniques: Inhibition, In Vivo, Injection, Transfection, Luciferase, Staining, TUNEL Assay, Expressing

Journal: Acta pharmaceutica Sinica. B

Article Title: EHMT2 promotes tumorigenesis in GNAQ/11 -mutant uveal melanoma via ARHGAP29-mediated RhoA pathway.

doi: 10.1016/j.apsb.2023.12.002

Figure Lengend Snippet: Figure 9 A schematic picture for this study. A schematic picture of the mechanism of how EHMT2i targets GNAQ/11-mutant UM. Gaq/11/Trio/ RhoA/YAP pathway is continuously activated in UM. EHMT2, together with H3K9me2, could bind with the promoter region of the ARHGAP29 gene and inhibit its transcription, resulting in the continuous activation of RhoA. By using an EHMT2 inhibitor or stably knocking down EHMT2, the expression of ARHGAP29 can be rescued. Then, RhoA turns from the activated state (RhoA-GTP) to inactivated state (RhoA-GDP), thus blocking the downstream YAP pathway, to inhibit the progression of UM. EHMT2 combined with MEKi/ERKi is a novel therapeutic approach for UM.

Article Snippet: The slides were incubated with primary antibodies overnight at 4 C, including EHMT2 (Invitrogen, PA5-78347), RhoA-GTP (NewEast Biotechnology, 26904), Ki67 (Cell Signaling, 9449), ARHGAP29 (Santa Cruz, sc-365554).

Techniques: Mutagenesis, Activation Assay, Stable Transfection, Expressing, Blocking Assay

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