Fig. 6: EZH2-92aa indirectly represses the NKG2D ligand ULBP1 by stabilizing EZH2.

a Relative RNA levels of ULBPs (ULBP1-6) in circEZH2 stable KD MES28 GSCs. Scramble vs sh-circEZH2, circEZH2 P = 1.68e−04, ULBP1 P = 2.49e−04, ULBP4 P = 8.36e−04, ULBP6 P = 6.42e−05. b Relative RNA and protein levels of circEZH2/EZH2-92aa and EZH2 were measured in MES28 and GSC23 GSCs transfected with circEZH2-siRNAs or control scrambled siRNAs. H3K27me3 protein levels were also determined. MES28, scramble vs si-circEZH2-1, P = 1.87e−08, si-circEZH2-2, P = 9.90e−09; GSC23, scramble vs si-circEZH2-1, P = 6.00e−05, si-circEZH2-2, P = 3.25e−05. c Left, half-life of EZH2 in circEZH2-siRNA-transfected MES28 GSCs. Right, quantitative analysis of the immunoblotting data by greyscale analysis at the indicated time points. CHX = cycloheximide. Scramble vs sh-circEZH2, 3 h P = 0.0025, 6 h P = 0.0015, 9 h P = 0.0069. d Protein level of EZH2 in circEZH2-siRNA-transfected MES28 and GSC23 GSCs after MG132 treatment (20 μM) for 6 h. e 293T cells were transfected with EZH2-92aa-3xflag, and total cell lysates were subjected to immunoprecipitation using anti-Flag and anti-FBXW7 antibodies, followed by immunoblotting with an anti-Flag or anti-FBXW7 antibody. f EZH2-92aa-3xFlag and FBXW7-6xHis were transfected into 293T cells as indicated in combination with Ub-HA. After treatment with MG132 (20 μM) for 6 h, total cell lysates were subjected to immunoprecipitation using an anti-EZH2 antibody, followed by immunoblotting with an anti-HA antibody. g MES28 and GSC23 circEZH2 stable KD or control cells were analysed for H3K27me3 levels in the ULBP1 promoter using a ChIP assay. Relative fold changes compared with IgG are shown. Scramble anti-H3K27me3 vs sh-circEZH2 anti-H3K27me3, MES28 P = 3.07e−04, GSC23 1.15e−04. h Relative RNA levels of ULBP1 in MES28 and GSC23 GSCs transfected with scrambled shRNA control, sh-circEZH2 or sh-circEZH2 together with the EZH2 OV plasmid. Scramble vs sh-circEZH2, MES28 P = 3.25e−05, GSC23 P = 0.0067; sh-circEZH2 vs sh-circEZH2+OV-EZH2, MES29 P = 2.65e−04, GSC23 P = 0.0157. i Cytotoxicity of NK cells after coculture with MES28 GSCs with stable circEZH2 KD, stable circEZH2 KD plus NKG2D block (10 μg/ml). Sh-circEZH2 vs sh-circEZH2 plus block, NK-92MI, 1:2 P = 0.0481, 1:5 P = 0.0147, 1:10 P = 0.0092; NK #1, 1:2 P = 0.0238, 1:5 P = 0.0155, 1:10 P = 0.0097; NK #2, 1:2 P = 0.0231, 1:5 P = 0.0086, 1:10 P = 0.0087. j Left panel, images showing remaining live MES28 GSCs with indicated modifications after coculture with NK cells using calcein AM staining. NKG2D block (10 μg/ml) was added in the indicated groups. Scale bar, 50 μm. Right panel, quantification of the remaining live GSCs. Sh-circEZH2 vs sh-circEZH2 plus block, NK-92MI, P = 0.0051; NK #1, P = 0.0081; NK #2, P = 0.0059. The data are presented as the mean ± SD from three independent experiments. Unpaired two-tailed Student’s t test was used to determine the significance of differences between the indicated groups where applicable. ns, nonsignificant, *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source data file.