Article Figures & Data
Figures
- Figure 1. TPX2 is lactylated at K249 in hepatocellular carcinoma (HCC) tumour tissues.
(A) Venn diagram illustrating the intersection between the four cohorts. Cohort 1 shows the lactylproteins in HCC tumour tissues from lactylation-modified proteomics data from published papers (Yang et al, 2023). Cohort 2 shows the cell cycle regulators defined by the KEGG database. Cohort 3 shows the genes that were negatively correlated with PFS in HCC patients from TCGA database (P < 0.01). Cohort 4 shows the genes with increased mRNA expression in HCC tumour tissues compared with those in tumour-adjacent noncancerous liver tissues from TCGA database (fold change > 2). The histogram shows the fold changes in the mRNA levels (tumour/normal) of the 20 intersection genes. (B) Western blot analysis of the lactylation of TPX2 in HepG2 cells after immunoprecipitation (IP) assays with anti-TPX2. (C) Western blot analysis of the lactylation of TPX2 in HepG2 cells after IP assays with anti-Pan-Kla. Blot bands were quantified by ImageJ, and the fraction of lactylated TPX2 was represented by the ratio of the immunoprecipitated TPX2 band to the corresponding Input TPX2 band. (D, E) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2 overexpression using IP samples as indicated. HepG2 cells were treated with lactate (25 mM) or GSK2837808A (75 μM) for 24 h. (F) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2 overexpression and lactate dehydrogenase A knockdown using IP samples as indicated. (G) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2WT or Flag-TPX2K249R overexpression using IP samples as indicated. (H) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2WT or Flag-TPX2K249R overexpression using IP samples as indicated. HepG2 cells were treated with GSK2837808A (75 μM) for 24 h. (I) Western blot analysis of the lactylation of TPX2 in the indicated cell lines. Blot bands were quantified by ImageJ, and the fraction of lactylated TPX2 was represented by the ratio of the Kla band to the immunoprecipitated TPX2 band. (J) Western blot analysis of the lactylation of TPX2 in tumour-adjacent noncancerous liver tissues (Normal) and liver cancer tumour tissues (Tumour) from the YAP5SA-induced HCC mouse model using IP samples as indicated. Blot bands were quantified by ImageJ, and the fraction of lactylated TPX2 was represented by the ratio of the immunoprecipitated TPX2 blot band to the corresponding Input TPX2 blot band.
Source data are available for this figure.
Source Data for Figure 1[LSA-2024-02978_SdataF1.pdf]
- Figure S1. TPX2 is lactylated at K249 in hepatocellular carcinoma tumour tissues.
(A) mRNA levels of TPX2 in tumour-adjacent noncancerous liver tissues (Normal) and liver cancer tissues (Tumour) from the Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn/). (B) Kaplan‒Meier analysis of overall survival with log-rank tests for low versus high expression of TPX2 genes in liver cancer patients from the Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn/). (C, D) Western blot analysis of the lactylation of TPX2 in HepG2 or Hep3B cells with Flag-EV and Flag-TPX2 overexpression using IP samples as indicated. (E) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2 overexpression using IP samples as indicated. HepG2 cells were treated with oxamate (20 mM) for 24 h. (F) Western blot analysis of the lactylation of TPX2 in HEK293T cells with Flag-TPX2 overexpression using IP samples as indicated. HEK293T cells were treated with lactate (25 mM) or oxamate (20 mM) for 24 h. (G) Western blot analysis of the lactylation of TPX2 in HEK293T cells using IP samples as indicated. HEK293T cells were treated with GSK2837808A (75 μM) for 24 h. (H) Western blot analysis of the lactylation of TPX2 in HEK293T cells with Flag-TPX2 overexpression and lactate dehydrogenase A knockdown using IP samples as indicated. (I) Conservation analysis of the lysine residues at position 249 in different species. The potential lactylation modification sites were searched from lactylation-modified proteomics data of hepatocellular carcinoma tumour tissues from published papers (Yang et al, 2023). (J) Western blot analysis of the lactylation of TPX2 in Hep3B cells with Flag-TPX2WT or Flag-TPX2K249R overexpression using IP samples as indicated. (K) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2WT or Flag-TPX2K249R overexpression using IP samples as indicated. HepG2 cells were treated with oxamate (20 mM) for 24 h. (L) Western blot analysis of the acetylation levels of TPX2 in HepG2 cells with Flag-TPX2WT or Flag-TPX2K249R overexpression using IP samples as indicated.
- Figure 2. TPX2 is lactylated by CBP and delactylated by HDAC1.
(A) Western blot analysis of the lactylation of TPX2 in HEK293T cells with Flag-TPX2 and lactylase overexpression using IP samples as indicated. HA blots corresponding to different regions of the filter are shown at the same exposure. Blot bands were quantified by ImageJ, and the fraction of lactylated TPX2 was represented by the ratio of the Kla band to the immunoprecipitated Flag-TPX2 band. (B) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2 overexpression and CBP knockdown using IP samples as indicated. (C) IP assay showing the protein interaction between CBP and TPX2 in HepG2 cells. (D) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2WT or Flag-TPX2K249R overexpression and CBP knockdown using IP samples as indicated. (E) Western blot analysis of the lactylation of TPX2 in HEK293T cells with Flag-TPX2 and delactylase overexpression using IP samples as indicated. Blot bands were quantified by ImageJ, and the fraction of lactylated TPX2 was represented by the ratio of the Kla band to the immunoprecipitated Flag-TPX2 band. (F) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2 and HA-HDAC1 overexpression using IP samples as indicated. (G) IP assay showing the protein interaction between HDAC1 and TPX2 in HepG2 cells. (H) Western blot analysis of the lactylation of TPX2 in HepG2 cells with Flag-TPX2WT or Flag-TPX2K249R and HA-HDAC1 overexpression using IP samples as indicated.
Source data are available for this figure.
Source Data for Figure 2[LSA-2024-02978_SdataF2.pdf]
- Figure S2. TPX2 interacts with CBP and HDAC1.
(A) In vitro TPX2 lactylation assay. The Flag-TPX2 proteins were incubated with HA-CBP proteins, which were purified from HEK293T cells in the presence or absence of lactyl-CoA. TPX2 lactylation was analysed by Western blotting using anti-Kla. (B) IP assay showing the protein interaction between CBP and TPX2 in HepG2 cells. (C) Co-IP assay showing the protein interaction between CBP and TPX2 in HEK293T cells with Flag-CBP and HA-TPX2 overexpression. (D) Representative immunofluorescence staining for TPX2 and CBP in Hep3B cells. The nucleus was stained with DAPI. Colocalization analysis of immunofluorescence images was quantitated by the colocalization plugin. Scare bar, 10 μm. (E) IP assay showing the protein interaction between HDAC1 and TPX2 in HepG2 cells. (F) Co-IP assay showing the protein interaction between HDAC1 and TPX2 in HEK293T cells with Flag-HDAC1 and HA-TPX2 overexpression. (G) Representative immunofluorescence staining for TPX2 and HDAC1 in Hep3B cells. The nucleus was stained with DAPI. Colocalization analysis of immunofluorescence images was quantitated by the colocalization plugin. Scare bar, 10 μm.
- Figure 3. TPX2 lactylation promotes tumour growth in vitro and in vivo.
(A, B) Cell growth analysis of HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. Western blotting was used to determine the protein levels of TPX2 in HepG2 cells with the indicated genotypes. (C) Cell growth analysis of HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with indicated genotypes were treated with GSK2837808A (50 μM) for 4 d. (D) Tumour growth analysis of HepG2 xenografts with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. The cells with indicated genotypes (5 × 106 cells per mouse) were subcutaneously injected into nude mice (n = 5 for each group). Tumour sizes were measured starting 12 d after inoculation. (E) Tumour weights of HepG2 xenografts with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. Images of the tumours are shown at the top. (F) Western blot analysis of the protein levels of TPX2 in HepG2 xenograft tumours of the indicated genotypes. (B, C, D, E) Data information: statistical significance was determined by two-way ANOVA (B, C, D, E), ns, not significant, *P < 0.05.
Source data are available for this figure.
Source Data for Figure 3.1[LSA-2024-02978_SdataF3.1_F4.1_F5.xlsx]
Source Data for Figure 3.2[LSA-2024-02978_SdataF3.2.pdf]
- Figure S3. TPX2 lactylation promotes tumour growth in vitro and in vivo.
(A, B) Cell growth analysis of HepG2 cells with TPX2 knockdown. Western blotting was used to determine the protein levels of TPX2 in HepG2 cells with the indicated genotypes. (C, D) Cell growth analysis of Hep3B cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. Western blotting was used to determine the protein levels of TPX2 in HepG2 cells with the indicated genotypes. (E) IC50 values for GSK2837808A in cytotoxicity assays in parental HepG2 cells. Parental HepG2 cells were treated with various drug concentrations as indicated for 4 d to assess the cytotoxic activity of GSK2837808A. (B, D) Data information: statistical significance was determined by two-way ANOVA (B, D), ns, not significant, *P < 0.05.
- Figure S4. TPX2 lactylation is necessary for cell cycle regulation by increasing Aurora kinase A (AURKA) phosphorylation.
(A) Western blot analysis of the lactylation of TPX2 in Hep3B cells during cell cycle. Hep3B cells were synchronized using a double-thymidine block, and then, cells were collected immediately (G1 phase) or released to complete DMEM containing 10% FBS for 6 h (S phase) or 10 h (G2/M phase). Blot bands were quantified by ImageJ, and the fraction of lactylated TPX2 was represented by the ratio of the Kla band to the immunoprecipitated TPX2 band. (B) Flow cytometric analysis of the cell cycle distribution of Hep3B cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. The data are presented as the mean ± SEM of three independent experiments. (C) Western blot analysis of AURKA T288 phosphorylation in Hep3B cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. (D) IC50 values for alisertib in cytotoxicity assays in parental HepG2 cells. Parental HepG2 cells were treated with various drug concentrations as indicated for 4 d to assess the cytotoxic activity of alisertib. (E) Co-IP assay showing the protein interaction between AURKA and TPX2WT or TPX2K249R in HepG2 cells with GFP-AURKA and Flag-TPX2WT or Flag-TPX2K249R re-expression. (F) Representative immunofluorescence staining for TPX2 and AURKA at the interphase and the mitotic phase in Hep3B cells with endogenous TPX2 knockdown and mCherry-TPX2WT or mCherry-TPX2K249R re-expression. The nucleus was stained with DAPI. Scare bar, 10 μm.
- Figure 4. TPX2 lactylation is necessary for cell cycle regulation by increasing Aurora kinase A (AURKA) phosphorylation.
(A) Flow cytometric analysis of the cell cycle distribution of HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or TPX2K249R re-expression. The data are presented as the mean ± SEM of three independent experiments, n = 3. (B) Flow cytometric analysis of the cell cycle distribution of HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with the indicated genotypes were treated with GSK2837808A (75 μM) for 24 h. The data are presented as the mean ± SEM of three independent experiments, n = 3. (C) Western blot analysis of AURKA T288 phosphorylation in HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. (D) Western blot analysis of AURKA T288 phosphorylation in HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with indicated genotypes were treated with GSK2837808A (75 μM) for 24 h. (E) Western blot analysis of AURKA T288 phosphorylation in HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with the indicated genotypes were treated with alisertib (1 μM) for 24 h. (F) Flow cytometric analysis of the cell cycle distribution of HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with the indicated genotypes were treated with alisertib (1 μM) for 24 h. The data are presented as the mean ± SEM of three independent experiments, n = 3. (G) Cell growth analysis of HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with the indicated genotypes were treated with alisertib (5 μM) for 4 d. (H) Co-IP assay showing the protein interaction between AURKA and TPX2WT or TPX2K249R in HepG2 cells with GFP-AURKA and Flag-TPX2WT or Flag-TPX2K249R overexpression. (I) Co-IP assay showing the protein interaction between AURKA and PP1. HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression were infected with lentiviruses carrying Flag-AURKA and HA-PP1 plasmids. (J) Western blot analysis of AURKA T288 phosphorylation in HepG2 cells with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression. HepG2 cells with the indicated genotypes were infected with lentiviruses carrying HA-PP1 plasmids. (A, B, F, G) Data information: statistical significance was determined by two-way ANOVA (A, B, F, G), ns, not significant, *P < 0.05.
Source data are available for this figure.
Source Data for Figure 4.1[LSA-2024-02978_SdataF3.1_F4.1_F5.xlsx]
Source Data for Figure 4.2[LSA-2024-02978_SdataF4.2.pdf]
- Figure 5. Inhibition of the lactate/TPX2 lactylation/Aurora kinase A axis suppresses in vivo hepatocellular carcinoma tumour growth.
(A) Tumour growth analysis of HepG2 xenografts with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression subjected to the indicated treatments. The cells with the indicated genotypes (5 × 106 cells per mouse) were subcutaneously injected into nude mice (n = 5 for each group). When the average tumour size reached ∼50–100 mm3, GSK2837808A (6 mg/kg) or alisertib (30 mg/kg) was intraperitoneally injected every 3 d. Tumour sizes were measured starting at 12 d after inoculation. (B) Tumour weights of HepG2 xenografts with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression subjected to the indicated treatments. Images of tumours are shown at the top. (C) Schematic cartoon showing that lactate-induced TPX2 lactylation is required for cell cycle regulation and hepatocellular carcinoma progression by protecting Aurora kinase A from PP1-mediated dephosphorylation to maintain its activation. (A, B) Data information: statistical significance was determined by two-way ANOVA (A, B), ns, not significant, *P < 0.05.
Source data are available for this figure.
Source Data for Figure 5[LSA-2024-02978_SdataF3.1_F4.1_F5.xlsx]
- Figure S5. Inhibition of the lactate/TPX2 lactylation/Aurora kinase A axis suppresses in vivo hepatocellular carcinoma tumour growth.
Western blot analysis of the protein levels of TPX2 in HepG2 xenograft tumours with endogenous TPX2 knockdown and Flag-TPX2WT or Flag-TPX2K249R re-expression.
Supplementary Materials
Table S1. Reagents and tools table.
Table S2. Knockdown plasmid table.
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