sc 365554  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: General characteristics of Arhgap29 −/− mice. ( A ) Gross image of the E15.5 mice. ( B ) Image of the head of the P0 mice (the black dashed box indicates the mandible). The penetrance of mandibular anomalies in Arhgap29 −/− mice is 30% (3/10). ( C ) Image of the palate of the E17.5 mice. The black dashed line indicates the cleft palate with 31.25% penetrance (5/16). ( D ) Image of the forelimb digits of the E14.5 mice. The black arrow indicates ectrodactyly with 96.67% penetrance (29/30).

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: ( A ) Three-dimensional reconstruction images of micro-CT scanning of WT and Arhgap29 deletion specimens. The white dashed line indicates the outline of the collapsed skull of the Arhgap29 −/− mice. FR, frontal bone; PA, parietal bone; IP, interparietal bone; M, mandible; co, coronal suture; sa, sagittal suture; la, lambdoidal suture. ( B ) The quantitative analysis of the cranium and mandible showed that the average bone surface area, bone volume, and bone mineral content were reduced to varying degrees in both the cranium and mandible of Arhgap29 −/− mice compared with WT mice. BV, bone volume; BS, bone surface; BMC, bone mineral content. All data: *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Micro-CT

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Alcian blue and Alcian blue/alizarin red staining results of cartilage and bone in the craniofacial region and limbs of WT and Arhgap29 −/− mice. ( A ) Alcian blue staining was performed on Meckel’s cartilage during three developmental stages: E13.5, E14.5, and P0. ( B ) Alcian blue/alizarin red staining of cranial and facial bones at P0. ( C ) Alcian blue/alizarin red staining of the mandible at P0. ( D ) Alcian blue/alizarin red staining of the whole skeleton of mice. ( E ) Alcian blue/alizarin red staining of the front limbs of mice; the Arhgap29 −/− mice have ectrodactyly.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Histological characteristics of Meckel’s cartilage. The results of H&E staining of Meckel’s cartilage in E13.5 ( A ), E15.5 ( B ), and E17.5 ( C ) mice, along with Alcian blue staining of Meckel’s cartilage in P0 mice ( D ). ( A – C ) illustrate the coronal section staining results of the mouse head, demonstrating the delayed hypertrophy of Meckel’s chondrocytes in Arhgap29 −/− mice. ( D ) presents the axial section staining results of the mouse head, which reveal the delayed degeneration of Meckel’s cartilage. The red dashed line indicates Meckel’s cartilage (MC); the mandible is labeled as M. Three mice from each group were chosen for every time period.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Experimental results of osteogenesis in mandibular tissue. ( A , B ) Von Kossa staining of mandibular tissue sections from E17.5 mice. ( C ) ALP staining of mandibular tissue sections from E17.5 WT mice. ALP-positive cells (shown in blue) are distributed on the surface of the bone matrix. ( D ) Immunohistochemical staining results of mandibular tissue sections from E17.5 WT mice. Arhgap29 -positive cells (shown in brown-yellow) are located on the surface of the bone matrix. (The red arrow indicates osteoblasts).

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Staining, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Experimental results of osteoclast activity in mouse mandibular tissue. ( A , B ) TRAP staining of mandibles in E17.5 WT and Arhgap29 −/− mice (osteoclasts are stained red, as indicated by the black arrows). ( C ) Staining of osteoclast marker TRAP in mandibles of WT mice at E17.5. ( D ) Immunohistochemical staining of osteoclasts in mandibles of WT mice at E17.5 (with positive cells indicated in brownish-yellow).

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Activity Assay, Staining, Marker, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Analysis of transcriptome sequencing results for E17.5 mandibular tissue. ( A ) Volcano plot of differentially expressed genes. ( B ) GO classification annotation and enrichment analysis. ( C ) KEGG classification annotation and pathway enrichment analysis. ( D ) Heatmap of differentially expressed gene clustering in calcium signaling pathway. ( E ) Heatmap of differentially expressed gene clustering in cell differentiation. ( F ) qPCR validation of calcium signaling pathway-related molecules. ( G ) qPCR validation of cell differentiation-related molecules. (The internal reference gene used was Gapdh , and the relative expression of the Arhgap29 −/− group was calculated based on the gene expression levels of the WT group. Statistical analyses of differences were performed using a t -test. * indicates a statistically significant difference between groups. *** p < 0.001; ** p < 0.01; ns indicates no significant difference.)

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Sequencing, Cell Differentiation, Biomarker Discovery, Expressing, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: In vitro cell experiment results. ( A ) qPCR assay for osteoblast markers in cells 3 days after they were induced to differentiate. ( B , C ) ALP staining of WT and si Arhgap29 cells 7 days after they were induced to differentiate. ( D , E ) ARS staining of WT and si Arhgap29 cells after they were induced to differentiate for 14 days. ( F ) Quantitative analysis of alkaline phosphatase staining in cells ( n = 5). ( G ) Quantitative analysis of alizarin red staining in cells ( n = 5). All data: *** p < 0.001.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: In Vitro, Staining

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