Fig 2: CBX4 increases PHGDH transcription via recruiting GCN5 to the PHGDH promoter.A ChIP-qPCR assays were performed using anti-CBX4 or IgG, with primer pairs targeting the PHGDH promoter in A549 cells. B Genome browser view of the H2AK119ub signal on the PHGDH gene obtained by ChIP-seq in A549 cells. C-D Immunoprecipitation assays were carried out with anti-CBX4 (C) or anti-GCN5 (D), followed by immunoblotting analysis in A549 cells. ChIP-qPCR was performed using anti-GCN5 (E) and anti-H3K27ac (F) with primer pairs targeting the PHGDH promoter in A549 cells expressing control shRNA and shCBX4-1. For figures A, E, and F, each bar represents the mean ± SD for n = 3; *P < 0.05, **P < 0.01 versus control (Student’s t-test). G The sketch map presents the dual role of CBX4 in transcriptional regulation and LUAD progression.

Fig 3: CBX4 promotes the proliferation of LUAD via transcriptional activation of PHGDH.A Western blot analysis of the expression of PHGDH, CBX4, and β-actin in A549 cells expressing control or CBX4 shRNAs. The levels of a-KG (B) and NADPH (C) in the cell lysate from A549 cells expressing control or CBX4 shRNAs were detected by HPLC and spectrophotometry, respectively. D Western blotting of cell lysates from A549 cells expressing control or PHGDH shRNAs, using PHGDH and β-actin antibodies. E MTT assays were performed in A549 cells expressing control or PHGDH shRNAs. F Colony formation assays were conducted in A549 cells expressing control or PHGDH shRNAs. The number of colonies was counted. G EdU-incorporation assays were performed in indicated cells, and the percentage of EdU-positive cells was calculated. Scale bar, 100 μm. For figures B, C and E–G, each bar represents the mean ± SD for n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus control (Student’s t-test). H A549 cells stably expressing control shRNA or CBX4 shRNA-1 were transfected with Flag-PHGDH expression constructs or empty vectors. The mRNAs were extracted, and subjected to real-time quantitative RT-PCR assays. I MTT assays were performed in the indicated cells. J EdU-incorporation assays were performed in the indicated cells. Scale bars, 100 μm. For figures H–J, each bar represents the mean ± SD for n = 3; *P < 0.05, ***P < 0.001 (one-way ANOVA followed by Bonferroni post-hoc test or Tambane’s T2 post-hoc test).

Fig 4: The serine metabolic pathway is downregulated in macrophages. (A) Levels of serine metabolic pathway metabolites in MDA-MB-231 cells after conditioned medium treatment. (B, C) Levels of metabolites of the PPP and 1-carbon metabolism, determined via mass spectrometry. All metabolite levels were normalized to the control. Bar graphs represent the mean ± SD of experimental triplicates (t-test, *P < 0.05, **P < 0.01, ***P < 0.001). (D) Relative mRNA expression of SSP metabolic pathways, assessed with qRT-PCR. (E, F) Relative mRNA expression and protein expression levels of PHGDH in various macrophages. (G) CIBERSORT immune infiltration analysis of primary breast cancer tumor samples from TCGA (n = 1,097). The colors in each cell represent the correlation of the serine metabolic pathway with various immune cell infiltration levels. Red indicates positive correlation, blue indicates negative correlation, and white indicates a correlation coefficient of 0 or a correlation coefficient that is not statistically significant.

Fig 5: Schematic overview of the molecular mechanism through which PHGDH regulates macrophage function. During the polarization of M0 macrophages into TAMs, PHGDH, the initial rate-limiting enzyme in endogenous serine synthesis, translocates to the nucleus. PHGDH exhibits low expression in TAMs but high expression under normal conditions. In the nucleus, PHGDH interacts with the transcription factor STAT3, thereby suppressing transcription of the key metabolic regulators GLUD1 and GLS2. The downregulation of GLUD1 and GLS2 decreases glutaminolysis and α-ketoglutarate (α-KG) synthesis, thereby inhibiting Jmjd3-dependent epigenetic reprogramming of M2 genes30,36.