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. 2021 Jul 1;2021:9965099. doi: 10.1155/2021/9965099

Figure 3.

Figure 3

KDM1A binds to MEF2D, reduces its methylation, and leads to increased expression of PD-L1 in HCC cells. (a) The expression profile of MEF2D according to the clinical data of HCC patients from TCGA database. (b) 293T cells transiently expressed HA-MEF2D and Flag-KDM1A. HA-MEF2D was immunoprecipitated with anti-K267me, followed by immunoblotting with anti-HA and anti-Flag. (c) Top: proteins immunoprecipitated with anti-K267me were analyzed using anti-MEF2D in L-O2 cells and SMMC7721 cells. Bottom: protein expression in whole cell lysates of L-O2 cells and SMMC7721 cells. (d) mRNA expression of MEF2D in L-O2 cells and SMMC7721 cells. (e) Top: proteins immunoprecipitated with anti-K267me were analyzed using anti-MEF2D in SMMC7721KDM1A-/- cells with or without KDM1A overexpression. Bottom: protein expression in whole cell lysates of SMMC7721KDM1A-/- cells with or without KDM1A overexpression. (f) Scheme and sequences of the CD274 promoter reporter vectors. (g) Luciferase activity of CD274 promoter reporter vectors in wild-type SMMC7721 cells, SMMC7721KDM1A-/- cells, SMMC7721MEF2D-/- cells, and wild-type SMMC7721 cells treated with different concentrations of ORY-1001. (h) Scheme of the gene type after the MEF2D knockout in SMMC7721 cells. (i) ChIP-qPCR analysis of the PD-L1 promoter binding with MEF2D in wild-type SMMC7721 cells, SMMC7721KDM1A-/- cells, SMMC7721KDM1A-/- cells overexpressing KDM1A, and SMMC7721KDM1A-/- cells overexpressing KDM1A and treating with 300 mM ORY-1001. (j, k) mRNA level (j) and protein level (k) of PD-L1 and KDM1A in SMMC7721MEF2D-/- cells transfected with the si-NC, si-KDM1A, empty plasmid (vehicle), and KDM1A overexpression plasmid for 72 h. (l, m) mRNA level (l) and protein level (m) of PD-L1 and KDM1A in SMMC7721MEF2D-/-+MEF2D-WT cells and SMMC7721MEF2D-/-+MEF2D-4KR cells transfected with the si-NC, si-KDM1A, vehicle, and KDM1A overexpression plasmid for 72 h. (n) Top: proteins immunoprecipitated with anti-K267me were analyzed using anti-MEF2D. Bottom: protein expression in whole cell lysates. SMMC7721KDM1A-/- cells, SMMC7721MEF2D-/- cells, and wild-type SMMC7721 cells treated with different concentrations of ORY-1001. (o) FACS results for PD-L1 expression in wild-type SMMC7721 cells treated with different concentrations of ORY-1001. (p) The results of RGA reflecting the strength of T cell-mediated antitumor immunity in wild-type SMMC7721 cells, SMMC7721KDM1A-/- cells, SMMC7721MEF2D-/- cells, and wild-type SMMC7721 cells treated with different concentrations of ORY-1001. (q–s) The results of the cytotoxic rate, IFN-γ level, and IL-2 level in wild-type SMMC7721 cells, SMMC7721KDM1A-/- cells, SMMC7721MEF2D-/- cells, and wild-type SMMC7721 cells treated with different concentrations of ORY-1001. The data are presented as the means ± SD. n = 3 experiments in (a–o). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.01.