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: Primary antibodies against ARHGAP29 1:2000 (#NBP1-05989, Novus Biologicals, Centennial, CO, USA), AKT1 1:1000 (#9272, Cell Signaling, Danvers, MA, USA), pAKT1 1:1000 (#4058, Cell Signaling), and GAPDH 1:2000 (#5174S, Cell Signaling) were used.

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: Primary antibodies against ARHGAP29 1:2000 (#NBP1-05989, Novus Biologicals, Centennial, CO, USA), AKT1 1:1000 (#9272, Cell Signaling, Danvers, MA, USA), pAKT1 1:1000 (#4058, Cell Signaling), and GAPDH 1:2000 (#5174S, Cell Signaling) were used.

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: Primary antibodies against ARHGAP29 1:2000 (#NBP1-05989, Novus Biologicals, Centennial, CO, USA), AKT1 1:1000 (#9272, Cell Signaling, Danvers, MA, USA), pAKT1 1:1000 (#4058, Cell Signaling), and GAPDH 1:2000 (#5174S, Cell Signaling) were used.

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: Primary antibodies against ARHGAP29 1:2000 (#NBP1-05989, Novus Biologicals, Centennial, CO, USA), AKT1 1:1000 (#9272, Cell Signaling, Danvers, MA, USA), pAKT1 1:1000 (#4058, Cell Signaling), and GAPDH 1:2000 (#5174S, Cell Signaling) were used.

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

Journal: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Primary antibodies used in western blot analysis.

Article Snippet: Rho GTPase activating protein 29 , Rabbit , Polyclonal , 1:2,000 , NBP1-05989 , Novus Biologicals Canada ULC (Oakville, ON, Canada).

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Immunohistochemical staining of ArhGAP29-RhoA/ROCK1 in human endometrium. ArhGAP29 staining in normal human endometrium was more obvious compared with endometrial tissue with severe IUAs; whereas, immunohistochemical staining of RhoA/ROCK1 in human endometrium with severe IUAs was more obvious compared with normal endometrial tissue. Scale bar, 50 µm. IUAs, intrauterine adhesions; ArhGAP29, Rho GTPase activating protein 29; ROCK1, Rho-associated coiled-coil containing protein kinase 1.

Article Snippet: Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.

Techniques: Immunohistochemical staining, Staining

Journal: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Effect of miR-1291 antagomir on ArhGAP29-RhoA/ROCK1 pathway in murine uterine tissue. Immunofluorescence staining for ArhGAP29, RhoA and ROCK1 (green) and nuclear DNA (DAPI, blue) in murine uterine tissue from the IUAs group, miR-1291 antagomir group and the negative control group. Scale bar, 50 µm. ArhGAP29, Rho GTPase activating protein 29; ROCK1, Rho-associated coiled-coil containing protein kinase 1; IUAs, intrauterine adhesions; miR, microRNA.

Article Snippet: Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.

Techniques: Immunofluorescence, Staining, Negative Control

Journal: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Effect of miR-1291 antagomir on ArhGAP29-RhoA/ROCK1 expression. (A) The expression levels of ArhGAP29, RhoA and ROCK1 were examined by reverse transcription-quantitative polymerase chain reaction. (B) Representative western-blot images and (C) densitometry of expression of ArhGAP29, RhoA and ROCK1 following western blotting analysis. ★ P<0.05, ★★ P<0.01 vs. the IUAs group; **P<0.01, ***P<0.001 vs. miR-1291 antagomir group. ArhGAP29, Rho GTPase activating protein 29; ROCK1, Rho-associated coiled-coil containing protein kinase 1; IUAs, intrauterine adhesions; miR, microRNA.

Article Snippet: Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

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