Journal: 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: Figure 3. 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 immu- noprecipitates, 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: Cells

Article Title: Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells

doi: 10.3390/cells9122616

Figure Lengend Snippet: ( A ) Schematic representation of the overlap of microarray data of 223 genes upregulated by induced epithelial–mesenchymal transition (EMT) and 32 considered Rho GTPase-activating proteins. Microarray data from Ziegler et al. were re-analyzed to show the upregulated expression of selected genes. Gene expression data of MCF-7 wild-type cells and mesenchymal-transformed MCF-7-EMT cells were compared. Among the genes examined were 32 genes that had previously been recognized as Rho GTPase-activating proteins. EMT-induced upregulation in MCF-7-EMT cells was found in 223 genes in comparison to MCF-7 wild-type cells. Only the Rho GTPase-activating protein 29 (ARHGAP29) was found to overlap. The relative change in expression (FC, fold change) of genes under consideration was 1.0–4.53. ( B ) Kaplan-Meier survival analysis of the luminal A breast cancer subtype (Threshold = 8 FPKM; ARHGAP29 low n = 271; ARHGAP29 high n = 209; Statistical test: Log-rank (Mantel-Cox) test; p = 0.0141). ( C ) Evidence of the significantly increased basal expression of ARHGAP29 in MCF-7-EMT cells was found. Basal expression (average expression) of ARHGAP29 in the cell lines MCF-7- and MCF-7-EMT was found. Microarray data from Ziegler et al., 2014 comparing the gene expression of MCF-7 wild-type cells and mesenchymal-transformed MCF-7-EMT cells were examined to investigate ARHGAP29 gene expression. ( D ) Expression of ARHGAP29 in MCF-7, MCF-7-EMT, T-47D, T-47D-EMT, and HCC1806 breast cancer cells. Expression of ARHGAP29 was analyzed by quantitative real-time PCR and normalized to GAPDH expression using at least three biological and technical replicates. Mean ± SEM values are given. Significance was determined with the help of unpaired t-tests; (*) p < 0.05.

Article Snippet: Cells were transiently transfected with siRNA specific to ARHGAP29 (sc-78491; Santa Cruz Biotechnology, Dallas, TX, USA) in OPTI-MEM I medium (Gibco) with siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: Microarray, Expressing, Gene Expression, Transformation Assay, Comparison, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells

doi: 10.3390/cells9122616

Figure Lengend Snippet: ( A ) Detection of reduced Rho GTPase activating protein 29 (ARHGAP29) expression after transient siRNA transfection of MCF-7-EMT, T-47D-EMT, and HCC1806 breast cancer cells. Breast cancer cells were transiently transfected with ARHGAP29-specific siRNA or non-targeting control siRNA (control). The expression of ARHGAP29 was analyzed by quantitative real-time PCR and normalized to GAPDH expression. The effect of downregulation of ARHGAP29 on the invasion of MCF-7-EMT ( B ), T-47D-EMT, ( C ) and HCC1806 ( D ) breast cancer cells was investigated. Breast cancer cells were transiently transfected with ARHGAP29-specific siRNA or non-targeting control siRNA (control). The invasion of transiently transfected breast cancer cells was analyzed after 96 h of coculture with MG63 osteosarcoma cells. The number of invaded cells in the treatment group was calibrated to the number of invaded cells in the control group using at least three biological and technical replicates. Mean ± SEM values are given. Significance was determined with the help of unpaired t -tests; (****) p < 0.0001; (**) p < 0.01; (*) p < 0.05.

Article Snippet: Cells were transiently transfected with siRNA specific to ARHGAP29 (sc-78491; Santa Cruz Biotechnology, Dallas, TX, USA) in OPTI-MEM I medium (Gibco) with siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: Expressing, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells

doi: 10.3390/cells9122616

Figure Lengend Snippet: Effect of the downregulation of Rho GTPase activating protein 29 (ARHGAP29) on the proliferation of MCF-7-EMT ( A ), T-47D-EMT ( B ), and HCC1806 ( C ) breast cancer cells. Breast cancer cells were transiently transfected with ARHGAP29-specific siRNA or non-targeting control siRNA (control). After cell culture for 120 h, transiently transfected breast cancer cells were detached and counted using a Neubauer counting chamber. The number of cells in the treatment group was calibrated to the cell number in the control group using at least three biological and technical replicates. Mean ± SEM values are given. Significance was determined with the help of unpaired t-tests; (**) p < 0.01; (*) p < 0.05.

Article Snippet: Cells were transiently transfected with siRNA specific to ARHGAP29 (sc-78491; Santa Cruz Biotechnology, Dallas, TX, USA) in OPTI-MEM I medium (Gibco) with siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: Transfection, Control, Cell Culture

Journal: Cells

Article Title: Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells

doi: 10.3390/cells9122616

Figure Lengend Snippet: ( A ) Probability of interaction of Rho GTPase activating protein 29 (ARHGAP29) with AKT1, SIRT1, PTPN13, CDC42, MAGEA11, and RHOD. In silico analyses were carried out using the GIANT web server (Genome-wide Integrated Analysis of gene Networks in Tissues, provided by HumanBase, https://hb.flatironinstitute.org/ , last accessed on 26 July 2019). A value of 0.1 was chosen as a minimum confidence interval to investigate interactions. The maximum number of genes considered was seven. The color of the connecting lines between interaction partners reflects possible interactions from 0 (no interaction) to 1 (high probability of interaction). The expression of AKT1 and the pAKT1/AKT1 ratio in MCF-7-EMT ( B ), T-47D-EMT ( C ), and HCC1806 ( D ) breast cancer cells after knock-down of ARHGAP29 expression are shown. Breast cancer cells were transiently transfected with ARHGAP29-specific siRNA or non-targeting control siRNA (control). The expression of AKT1 was determined using Western blot analysis and normalized to GAPDH expression using at least three biological and technical replicates. The pAKT1/AKT1 ratio was analyzed as the quotient of pAKT1 versus AKT1 expression. Representative blots for ARHGAP29, AKT1 and pAKT1 are shown. Mean ± SEM values are given. Significance was determined with the help of unpaired t-tests; (***) p < 0.001; (**) p < 0.01; (*) p < 0.05.

Article Snippet: Cells were transiently transfected with siRNA specific to ARHGAP29 (sc-78491; Santa Cruz Biotechnology, Dallas, TX, USA) in OPTI-MEM I medium (Gibco) with siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: In Silico, Genome Wide, Expressing, Knockdown, Transfection, Control, 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