Fig 1: PHGDH/cMyc axis drives CXCL1/IL8 expression. A–D) RNA‐sequencing analyses were performed using PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Gene Ontology (GO) enrichment analyses of the differentially expressed genes were presented (A). GSEA enrichment plot of the KEGG pathway NOD‐like receptor‐related gene was shown in (B). The correlation of all NOD‐like receptor‐related gene expression with the phgdh expression status was displayed by the ranking metric score. A positive score indicates a correlation with the rPHGDH‐dACT and a negative score indicates a correlation with rPHGDH‐WT; The red indicates a gene that contributes most to the enrichment result and the blue indicates a gene that contributes less (C). The total differentially expressed genes (FC>2 or FC<0.5; p value <0.05) were displayed using a volcano plot. FC, fold change of rPHGDH‐dACT compared to rPHGDH‐WT (D). E) In PHGDH‐depleted PLC/PRF/5 cells with rPHGDH‐WT or rPHGDH‐dACT or further depletion of c‐Myc, qRT‐PCR validated the top up‐regulated genes from (C) (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3). F) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium culturing PHGDH‐depleted PLC/PRF/5 cells, which were rescued with rPHGDH‐WT or rPHGDH‐dACT (mean ± SD, two‐tailed Student's t‐test, n = 3). G) Immunoblotting analysis of CXCL1/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. H) Co‐IP analysis of cMyc, p300, and PHGDH was performed using PLC/PRF/5 cells expressing WT or K148R mutant Myc. Antibody against cMyc was used to enrich the cMyc‐associated complex. Immunoblotting analysis of PHGDH, p300, cMyc, and AF9 was performed using the indicated antibodies. I) qRT‐PCR validated CXCL1/IL8 and IL1B genes using PLC/PRF/5 cells expressing WT or K148R mutant Myc (mean ± SD, two‐tailed Student's t‐test, n = 3). J) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium culturing PLC/PRF/5 cells expressing WT or K148R mutant Myc (mean ± SD, two‐tailed Student's t‐test, n = 3). K) Immunoblotting analysis of CXCL1/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. L) ChIP analysis of PHGDH, cMyc, p300, RNA Pol II (Pol 2), AF9, and H3Kac on CXCL1 gene promoter was performed using indicated cells. IgG was used as a blank control (mean ± SD, two‐tailed Student's t‐test, n = 3).
Fig 2: The PHGDH ACT domain is required for forming PHGDH/p300/cMyc/AF9 axis and regulates cMyc transactivation. A) Schematic diagram of human PHGDH protein and its five domains. aa, amino acid. B) HA‐tagged cMyc and FLAG‐tagged PHGDH (including WT and five domain truncates) were transiently transfected into HEK293T cells. Co‐IP was performed with an antibody against FLAG. Antibodies against HA and FLAG were used to detect the association between cMyc and PHGDH. HA was used as an input control. dSB1, SB1 domain depletion; dNB, NB domain depletion; dSB2, SB2 domain depletion; dASB, ASB domain depletion; dACT, ACT domain depletion. C) GST‐tagged ACT domain and His‐tagged cMyc protein were purified from Escherichia coli, and a GST pull‐down assay was performed by incubating both the recombinant proteins together. GST was used as a blank control. Antibodies against His and GST were used to detect the association between cMyc and the ACT domain. D) cMyc transcriptional activity was measured by the Dual‐Luciferase Reporter Assay System according to the manual using PHGDH‐depleted PLC/PRF/5 and Hep3B cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Cells expressing shNT were used as control. rPHGDH‐dACT, shRNA‐resistant PHGDH‐dACT (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3). E) Chromatin immunoprecipitation (ChIP) analysis of cMyc enrichment on promoters of five canonical cMyc targets was performed using indicated cells. IgG was used as a blank control (mean ± SD, two‐tailed Student's t‐test, n = 3). F) qRT‐PCR assay of the five genes from (E) (mean ± SD, two‐tailed Student's t‐test, n = 3). G) Co‐IP analysis of p300, cMyc, and PHGDH was performed using PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Antibody against p300 was used to enrich p300 associated complex. Immunoblotting analysis of PHGDH, cMyc, and p300 was performed using the indicated antibodies. H) p300 was transiently depleted by specific siRNA in PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Immunoblotting analysis of cMyc‐AcK148, cMyc, p300, and PHGDH was performed using the indicated antibodies. NC, negative control. I) Co‐IP analysis of cMyc, p300, and PHGDH was performed using PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Antibody against cMyc was used to enrich the cMyc‐associated complex. Immunoblotting analysis of PHGDH, p300, cMyc, and AF9 was performed using the indicated antibodies.
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