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