Fig. 5: EZH2-92aa directly represses the transcription of the NKG2D ligands MICA/B. | Nature Communications

Fig. 5: EZH2-92aa directly represses the transcription of the NKG2D ligands MICA/B.

From: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

Fig. 5

a Left, representative IF image of MES28 GSCs transfected with EZH2-92aa-3×flag and stained with an anti-flag antibody. VA, vector alone. Scale bar, 5 μm. Right, immunoblot of EZH2-92aa-3×Flag or endogenous EZH2-92aa in the indicated cellular fraction of MES28 GSCs transfected with EZH2-92aa-3×Flag or GSC23 GSCs using an anti-Flag antibody or the custom anti-EZH2-92aa antibody. W, whole cell lysate; N, nuclear fraction; C, cytoplasmic fraction. b Left, mRNA levels of MICA/MICB in circEZH2 stable KD MES28 GSCs. Right, immunoblot showing the levels of MICA/MICB in circEZH2 stable KD MES28 GSCs. Scramble vs sh-circEZH2, circEZH2 P = 0.0023, MICA P = 0.0001, MICB P = 0.0046. c, d Luciferase activity driven by MICA/MICB in MES28 GSCs transfected with increasing doses of the EZH2-92aa OV plasmid. MICA, 0 vs 2 P = 0.0078, 0 vs 5 P = 0.0002, 0 vs 10 P = 0.0002; MICB, 0 vs 2 P = 0.0042, 0 vs 5 P = 8.14e−06, 0 vs 10 P = 5.25e−06. e, f Luciferase activity of the MICA/MICB promoter fragments fused to a luciferase reporter gene. MICA, vector 1# vs EZH2-92aa 1# P = 1.49e−04, vector 1# vs EZH2-92aa 2# P = 6.92e−05, vector 1# vs EZH2-92aa 3# P = 2.11e−04; MICB, vector 1# vs EZH2-92aa 1# P = 0.0014, vector 1# vs EZH2-92aa 2# P = 0.0012, vector 1# vs EZH2-92aa 3# P = 0.0003, vector 1# vs EZH2-92aa 4# P = 0.0006. g, h ChIP-qPCR analysis of the binding site of EZH2-92aa-3×Flag in the MICA/MICB promoters in MES28 GSCs transfected with EZH2-92aa-3×Flag. IgG vs Flag, MICA P = 0.0077, MICB P = 0.0161. i, j EMSA was performed using the nuclear extract of MES28 GSCs transfected with EZH2-92aa-3×Flag and 6 specific biotin-labelled MICA/MICB probes. Independent experiments were performed three times with similar results. k, l EMSA was performed using the nuclear extract of MES28 GSCs transfected with EZH2-92aa-3×Flag, biotin-labelled MICA/MICB probes, a 200-fold excess of unlabelled MICA/MICB probes (200× WT competitor), biotin-labelled mutated MICA/MICB probes (200× Mut competitor) and an anti-flag antibody. Independent experiments were performed three times with similar results. m, n Mutants of the EZH2-92aa binding site in the MICA/MICB promoter were constructed. Luciferase activity of the WT or mutated MICA/MICB constructs with EZH2-92aa-3×Flag OV. Ctrl vs 92aa, MICA WT, P = 5.52e−05; MICB WT, P = 1.40e−05. The data are pooled from three independent experiments. The data are presented as the mean ± SD. 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.

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