Fig 2: MBGRP3 cells are sensitive to pharmacological inhibition of the de novo SGP in a MYC-dependent fashion. (A) Dose–response curves following PHDGH inhibition mediated by NCT-503 treatment in shMYC1 isogenic cell lines ± Dox. Respective IC50 values are indicated. Confidence intervals D425med shMYC1 (− Dox) = 11.5–19.9 µM, D425med shMYC1 (+ Dox) = 32–57.4 µM. HDMB03 shMYC1 (− Dox) = 19.8–27.9 µM, HDMB03 shMYC1 (+ Dox) = 36.7–52.5 µM D283med shMYC1 (− Dox) = 6.7–13.6 µM, D283med shMYC1 (+ Dox) = 30.8–56.5 µM. Data represents the mean of 5 independent experiments ± SEM (B) Quantification of PHGDH activity following NCT-503 treatment in shMYC ± Dox conditions. Data expressed as mean ± SEM of 3 biological replicates. * P < .05, ** P < .01, *** P < .001. (C) Immunoblot analysis of MYC protein expression in MYC-amplified/gained MBGRP3 and non-amplified non-MBGRP3 cells. β-actin was used as a loading control. (D) Comparison of IC50 values of MYC-amplified MBGRP3 cells versus non-amplified non-MBGRP3 to PHGDH inhibitors, NCT-503 and CBR5884. Boxplot representation of IC50 values from 3 MBGRP3 and 2 non-MBGRP3 cell lines. IC50s derived from 3 independent experiments. The median with upper and lower quartiles are shown as appropriate (E). Representative images of clonogenic assays in D425med, D458med, D283med, and HDMB03 cell lines following 10-day treatment with 25 µM and 50 µM NCT-503. Quantifications represent means of 3 biological replicates ± SEM. Significance determined by one-way ANOVA. * P < .05, ** P < .01, *** P < .001, **** P < .0001. (F) Effect of NCT-503 treatment on colony formation on D425med MYC isogenic cells ± Dox treatment. Representative images of the clonogenic assay in D425med shMYC 1 cell ± Dox following 10-day treatment with 12.5 and 25 µM NCT-503 treatment. Colony numbers are shown normalized to their respective untreated controls. Data represents means of 3 biological replicates ± SEM. Significance determined by one-way ANOVA. *** P < .001, **** P < .0001. (G) Cell cycle analysis using propidium iodide staining following 72 hours of NCT-503 treatment. Data represents mean ± SEM from 3 biological replicates. Significance was tested using a two-way ANOVA for each cell line. * P < .05, ** P < .01. *** P < .001, **** P < .0001

Fig 3: CSFV inhibits the activity of the PHGDH protein and its acetylation modification. (A) Immunohistochemical identification of PHGDH protein expression after CSFV infection of tissues. (B) qPCR detection of PHGDH mRNA transcript levels after CSFV infection of tissues. (C) Western blot detection of PHGDH protein expression in CSFV-infected PK-15 cells and 3D4/2. (D) qPCR detection of PHGDH mRNA transcript levels in CSFV-infected PK-15 cells and 3D4/2. (E) Western blot detection of the effect of CSFV infection on the acetylation level of cells. (F) IP experiments were performed with Ace-lys as a specific antibody to determine the binding of PHGDH to Ace-lys. (G) The binding of PHGDH to Ace-lys was determined in IP experiments using PHGDH as a specific antibody. (H) Characterization of CSFV effects on PHGDH acetylation by IP assay. (I) Characterization of CSFV effects on PHGDH acetylation by the co-IP assay.

Fig 4: PHGDH enhances IFN-β signaling via the mitochondria–MAVS–IRF3 axis. (A) PHGDH localization to mitochondria induced by viral infection. PHGDH and mitochondrial membrane protein VDAC1 co-localization in CSFV- and poly(I:C)-stimulated cells was visualized by laser confocal microscopy. (B) PHGDH silencing impairs mitochondrial membrane potential. PHGDH-knockdown PK-15 cells were treated with JC-1. Mitochondrial membrane potential was assessed by the JC-1 fluorescent probe, and mitochondrial depolarization was determined by the ratio of red to green fluorescence intensity. (C) PHGDH silencing enhances mitochondrial division. PHGDH-inhibited cells showed increased mitochondrial length and division by laser confocal microscopy. (D) PHGDH overexpression augments the mitochondrial number. Mitochondrial morphological changes induced by PHGDH and its mutants were observed by confocal laser microscopy. (E) PK-15 cells were transfected with the indicated plasmid for 24 h. co-IP and western blot analysis with specified antibodies. (F) PHGDH-mediated IFN-β secretion reduced by MAVS knockout. Error bars indicate the mean (±SD) of three independent experiments. ****P < 0.0001 (t-tests). (G and H) IRF3 expression in the nucleus of PHGDH-silenced or overexpressing PK-15 cells was measured by karyoplasmic separation. (I) PHGDH silencing prevents IRF3 nuclear translocation. IRF3 localization in PHGDH-silent cells was visualized by confocal laser microscopy. (J) PHGDH overexpression facilitates IRF3 nuclear translocation. IRF3 localization in PHGDH-overexpressing or mutant PK-15 cells was visualized by confocal laser microscopy.

Fig 5: PHGDH-mediated serine metabolism inhibits viral proliferation. (A, B, and C) PHGDH silencing enhances CSFV replication. CSFV titer (A), mRNA (B), and viral protein Npro (C) in PHGDH-silenced cells were measured by IFA, qPCR, and western blotting. (D, E, and F) PHGDH overexpression suppresses CSFV replication. CSFV titer (D), mRNA (E), and viral protein Npro (F) in PHGDH-overexpressing cells were assessed at different time points. (G) Effect of exogenous deficiency of serine on CSFV protein expression. (H) Effect of exogenous deficiency of serine on CSFV copy numbers. (I) Molecular mechanisms by which CSFV infections modulate immunity to serine metabolism. Error bars indicate the mean (±SD) of three independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001 (t-tests or two-way ANOVA).