Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Mel92.1 and MP41 cells were treated with DMSO, UNC0642 (1 μM), or A366 (1 μM) for 48 h and analyzed for the indicated proteins using immunoblotting. ACTINB was used as a loading control. ( B ) Mel92.1 and MP41 cells expressing either nonspecific (NS) shRNA or TGF-β1 shRNAs were analyzed for the indicated proteins using immunoblotting. ACTINB was used as a loading control. ( C ) Mel92.1 and MP41 cells expressing either an empty vector or V5-tagged TGF-β1 ORF were treated with DMSO or UNC0642 (1 μM) for 48 h and analyzed for the indicated protein using immunoblotting. ACTINB was used as a loading control. ( D ) Mel92.1 and MP41 cells expressing either NS shRNA, ULBP3 shRNAs, or MICB shRNAs were analyzed for the indicated proteins using immunoblotting. ACTINB was used as a loading control. ( E ) Mel92.1 and MP41 cells expressing either NS shRNA or MICB shRNAs were treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) for 48 h and were analyzed for NK cell-mediated cytotoxicity using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( F ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h followed by treated NK cells were used for NK cell-mediated cytotoxicity assay against Mel92.1 Cells. Relative NK cell-mediated cytotoxicity under indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( G ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and were analyzed for IFN-γ mRNA levels using RT-qPCR. Relative IFN-γ mRNA levels are presented under the indicated conditions. ACTINB was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( H ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for IFN-γ protein levels using ELISA. IFN-γ protein levels are presented under the indicated conditions. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( I ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for EOMES and T-Bet protein levels using immunoblotting. ACTINB was used as a loading control. ( J ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for NCR3 and NKG2D protein using immunoblotting. ACTINB was used as a loading control. ( K ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for CD69, CD49a, CD103, CD56, and CXCR6 proteins using immunoblotting. ACTINB was used as a loading control. All quantitative data are shown as the mean ± SEM. .

Article Snippet: CD103 (Diluted at 1:1000 for WB) , Novus Biologicals , NBP3-03545 RRID: AB_3532135.

Techniques: Western Blot, Control, Expressing, shRNA, Plasmid Preparation, LDH Cytotoxicity Assay, Two Tailed Test, Cytotoxicity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay