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. 2023 Oct 16;19(16):5218–5232. doi: 10.7150/ijbs.85656

Figure 6.

Figure 6

Lactylation of CENPA enhanced the transactivation of target genes in HCC. (A) Lactylation of CENPA in HEK293T cells was confirmed by IP method. HEK293T cells transfected with Flag-CENPA, treated with PBS or 25 mM L-lactic acid for 24h. (B) Lactylation of CENPA in Huh7 and MHCC-97H cells was confirmed by IP method. Huh7 and MHCC-97H cells transfected with Flag-CENPA, treated with PBS or 25 mM 2-DG for 24h. (C) Flag-CENPA (WT), Flag-CENPA (K124R) were transfected into HEK293T cells for 24 h. Then the indicated cells were treated with PBS or L-lactic acid for 24 h. (D) Flag-NC, Flag-CENPA (WT), Flag-CENPA (K124R) were transfected into Huh7 and MHCC-97H cells for 24 h. Then cells were treated with L-lactic acid for 24 h. (E) MHCC-97H vector and CENPA overexpression cells were treated with PBS or 25mM 2-DG for 24 h. Western blotting was used to detect the expression level of YY1, CCND1 and NRP2. (F) MHCC-97H vector and CENPA overexpression cells were treated with PBS or 25mM 2-DG for 24 h. qRT-PCR was used to detect the expression level of YY1, CCND1 and NRP2. (G) MHCC-97H vector and CENPA overexpression cells were treated with PBS or 25mM 2-DG for 24 h. Luciferase assay identified CENPA promotes the transcription activity of YY1, CCND1 and NRP2. (H) Flag-NC, Flag-CENPA (WT), Flag-CENPA (K124R) were transfected into Huh7 cells for 24 h. Then cells were treated with 25mM L-lactic acid for 24 h. ChIP-qPCR assays demonstrated the binding of Flag-CENPA and Flag-CENPA (K124R) at the promoter regions of YY1, CCND1 and NRP2. (I) After treated with PBS or 25mM L-lactic acid for 24 h, cellular fraction of MHCC-97H was performed to detect the abundance of CENPA and YY1 in cytoplasm and chromatin. (J) Representative tumor images and tumor volumes after treated with PBS or 25mM 2-DG for one month in the orthotopic MHCC-97H cell model. (K) Lactylation level of CENPA in MHCC-97H tumor tissues after treated with PBS or 25 mM 2-DG. (L) Expression of YY1, CCND1 and NRP2 in MHCC-97H tumor tissues after treated with PBS or 25 mM 2-DG. The statistical significance was determined by Student's two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001.