Fig 2: SIRT2 inhibitor has an anti-tumor effect on CCA in vivo.(A) The levels of cMYC, p-PDHA1, and SIRT2 proteins in tumors and adjacent normal tissues from 6 donors were detected by western blotting. (B) HuCCT1 cells were injected subcutaneously into the flanks of nude mice. When tumors were palpable, DMSO (control) or Tm were administered. Tumors were collected and photographed. Scale bars, 1 cm. (C) Tumor volumes of mice were measured. (D) The body weights of mice were compared among the groups. (E & F) cMYC, p-PDHA1, PHGDH, PSAT1 and PSPH protein levels in xenograft tissues from control and Tm group were detected by staining and Western blotting. Data represent mean ± SEM, n = 3. *P < .05 and **P < .01.

Fig 3: P4HB interacts with PHGDH and destabilizes PHGDH by increasing its ubiquitination level. (a) Identification of P4HB‐interacting proteins. Proteins extracted from GA muscle of YES2‐bearing mice were incubated with Protein A/G Sepharose beads conjugated with anti‐P4HB antibody, subjected to SDS‐PAGE and visualized by silver staining. The band indicated by the red arrow was subjected to mass spectrometry. (b) Endogenous PHGDH detected by the co‐immunoprecipitation assay with anti‐P4HB antibody in C2C12 myoblasts. The immunoglobulin G (IgG) was used as the control group. (c) Flag‐P4HB plasmid was transfected into C2C12 myoblast, and exogenous Flag‐tag was detected by the co‐immunoprecipitation assay with anti‐PHGDH antibody. (d) Immunofluorescence showed that endogenous PHGDH and P4HB colocalized in C2C12 myoblasts. Scale bars, 30 μm. (e) In vitro GST pull‐down assay to verify the interaction between P4HB and PHGDH. C2C12 myoblasts were transfected with GST alone or GST‐PHGDH. Retrieved proteins were evaluated with immunoblotting. GST‐immunocomplexes were boiled and subjected to immunoblotting analysis using P4HB antibody. (f) Left panel: A schematic diagram shows the structural domains of P4HB. Right panel: The control vector, Flag‐tagged wild type or truncated mutants of P4HB were transfected into C2C12 myoblasts. The cell lysates were subjected to the co‐immunoprecipitation assay by anti‐FLAG M2 Magnetic Beads and PHGDH expression was analyzed by immunoblotting. (g) P4HB plasmid was transfected into C2C12 myoblasts for 48 h and mRNA levels of P4HB and PHGDH were analyzed by RT‐qPCR assay (top panel). The protein levels of P4HB and PHGDH were analyzed by immunoblotting (bottom panel). (h) C2C12 myoblasts transfected with P4HB plasmid were treated with 100 μg/ml CHX and harvested at the indicated time point. The cell lysates were subjected to immunoblotting and PHGDH expression was quantified by ImageJ software. (i) HA‐Ub and V5‐PHGDH plasmid were co‐transfected with Flag‐P4HB plasmid in C2C12 myoblasts. After 36 h, cells were treated with 10 μM MG132 for 6 h. Anti‐V5 antibody was used to immunoprecipitate with exdogenous P4HB. Its ubiquitination level was assessed by anti‐HA antibody. 5% input of cell lysates was used to analyze the expression of P4HB and PHGDH

Fig 4: Purine depletion triggers cell elongation and an EMT transcriptional program through an increase in cellular serine levels.a Representative images of actin cytoskeleton staining with Phalloidin (green) and nuclei (blue) from A375 cells treated with MTX (200 nM, 24 h) in the presence or absence of inosine (50 µM, 24 h). Quantification of cell length from seven independent cells is presented relative to vehicle control. b As in (a), but from A375 cells that were treated with LTX (800 nM, 24 h) or AG2037 (100 nM, 24 h). c Relative mRNA expression of the indicated EMT markers upon treatment with MTX (200 nM, 24 h) in the presence or absence of inosine (50 µM, 24 h). d As in (a), but from A375 cells that were treated with MTX (200 nM, 24 h) in the presence or absence of PHGDH inhibitor (PHGDHi, BI-4924, 15 µM, 24 h). e Relative mRNA expression of the indicated EMT markers upon treatment with MTX (200 nM) in the presence or absence of PHGDH inhibitor (PHGDHi, BI-4924, 15 µM, 24 h). f Relative mRNA expression of the indicated EMT markers as in c, but from wild-type or ΔPHGDH A375 cells treated with either vehicle (DMSO) or MTX (200 nM, 24 h). g As in (a), but from A375 cells that were treated with MTX (200 nM, 24 h) in the presence or absence of TEPP-46 (100 µM, 24 h). h As in (a), but from A375 cells that were treated with serine (400 nM, 24 h) or Formate (2 mM, 24 h) in normal DMEM media. i Relative mRNA expression of the indicated EMT markers upon treatment with serine (400 nM, 24 h) or Formate (2 mM, 24 h) in normal DMEM media. a–i Data are presented as the mean ± s.d from n = 3 (c, e, f, i) or n = 7 (a, b, d, g, h) of biologically independent samples. a–i One-way ANOVA, Turkey’s post-hoc test, multiple comparison *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. a, b, d, g, h) Scale bars are indicated. Data are representative of n = 2 (c, e, f, i) or n = 3 (a, b, d, g, h) independent experiments. Source data and exact p-values are provided as a Source Data file.

Fig 5: Overexpression of PHGDH but not exogenous serine supplementation reversed the effects of AIL on OS cells. (A, B) Overexpression efficiency of PHGDH was verified by qRT-PCR and Western blot assay. (C) OS cells were assigned to five groups: negative control (NC) treated with DMSO (DMSO + NC), NC treated with AIL (AIL + NC), PHGDH overexpression treated with AIL (AIL + PHGDH), NC treated with AIL and serine (AIL + NC + Serine), PHGDH overexpression treated with AIL and serine (AIL + PHGDH + Serine). The viability ratio of Saos-2 and U-2OS cells in the AIL + PHGDH group and AIL + PHGDH + Serine group but not in the AIL + NC + Serine group was significantly higher than that in the AIL + NC group (** P < 0.01, *** P < 0.001 compared with the DMSO + NC group, ### P < 0.001 compared with the AIL + NC group). (D) The wound healing assay revealed that overexpression of PHGDH but not exogenous serine supplementation reversed the suppressive effect of AIL on the migration of OS cells. (E) The Transwell assay demonstrated that overexpression of PHGDH but not exogenous serine supplementation reversed the suppressive effect of AIL on OS cell invasion. (F) Comparisons of the migration rates and relative invasion rates between the NC group and the other groups are shown (*** P < 0.001, ns P > 0.05).