Journal: bioRxiv

Article Title: Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

doi: 10.1101/2025.03.16.643506

Figure Lengend Snippet: (A) Schematic overview of the folate cycle and cellular purine synthesis pathway. Corresponding folate intermediates are generated by the enzymatic functions of the MTHFD1 synthetase (S), cyclohydrolase (C) and dehydrogenase (D) domains. 10-CHO-THF is used for the synthesis of IMP via the purine de novo synthesis pathway. Alternatively, IMP can be synthesized through the salvage pathway in presence of exogenous adenosine. The schematic domain structure of MTHFD1 is shown below with sites of K56R and K386E mutations that ablate the enzymatic reactions of each respective domain. (B) Normalized cell counts of WT, MTHFD1 KO , MTHFD1 386E , and MTHFD1 K56R cells grown for 72 h in media containing dialyzed serum (DIA) or dialyzed serum supplemented with 50 µM adenosine (ADE), normalized to the respective clone grown in media containing full serum (FULL), (n=3 biological replicates, mean ± SD, *p<0.05). (C) Normalized cell counts of WT and MTHFD1 K56R cells at indicated concentrations of adenosine. Cells were grown for 72 h media containing dialyzed serum (DIA) or dialyzed serum supplemented with respective adenosine concentrations and normalized to the lowest adenosine concentrations. (n=2 biological replicates, mean ± SD). (D) Normalized cell counts of MTHFD1 K56R cells cultured at indicated media conditions and with supplemented nucleotides and precursors (50 µM). (E) Representative cell cycle plots for WT and MTHFD1 mutant HAP1 cells in DIA and ADE conditions. (F) Representative images of γH2A.X and RPA2 staining of MTHFD1 K56R cells with adenosine supplementation. Scale bar is 10 µm. (G) Growth assays of fibroblasts derived from MTHFD1 deficiency patients in FULL, DIA and ADE (n=3 biological replicates, Mean ± s.d, *p<0.05). (H) Schematic overview of the genetic folate trap model.

Article Snippet: The fixed cells were then blocked with 5% BSA, diluted in PBS, for 1 hour and then incubated overnight with anti-γH2A.X antibody (1:500, 9718S, Cell Signalling Technology) or rabbit anti-RPA2 antibody (1:500, HPA026306, Atlas Antibodies) at 4°C.

Techniques: Generated, Synthesized, Cell Culture, Mutagenesis, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

doi: 10.1101/2025.03.16.643506

Figure Lengend Snippet: (A) Quantification of the percentage of the depicted cell lines in specific cell cycle states. Cells were pretreated in FULL, DIA, or ADE for 24 h. (n=3 biological replicates, mean ± SD). (B) Quantification of the average number of γH2A.X nuclear foci and the integrated intensity (size and brightness) of RPA2 foci of HAP1 WT and MTHFD1 K56R cells grown in FULL, DIA, or ADE media (n=3 biological replicates, mean ± s.d., *p<0.05).

Article Snippet: The fixed cells were then blocked with 5% BSA, diluted in PBS, for 1 hour and then incubated overnight with anti-γH2A.X antibody (1:500, 9718S, Cell Signalling Technology) or rabbit anti-RPA2 antibody (1:500, HPA026306, Atlas Antibodies) at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

doi: 10.1101/2025.03.16.643506

Figure Lengend Snippet: (A) Schematic overview of the genetic screening strategy. (B) Results of the genome-wide KO screen as performed in a), highlighting significantly depleted genes (HPRT1, NUDT5, MTHFD1). (C) Model of folate trap rescue by MTHFD1 knocked-out in MTHFD1 K56R cells. (D) Normalized viability of MTHFD1 K56R HPRT1 KO and MTHFD1 K56R NUDT5 KO cells cultured in media containing dialyzed serum (DIA) or the same media supplemented with 50 µM adenosine (ADE). Viability was measured after 72 h and normalized to respective DIA conditions. (n=2, mean ± s.d., two-way ANOVA, ****p<0.0001). (E) Representative images of γH2A.X and RPA2 staining of MTHFD1 K56R , MTHFD1 K56R HPRT1 KO and MTHFD1 K56R NUDT5 KO cells after being cultured in DIA or ADE for 72 h. Spots/nuclei values were normalized to DIA conditions. Scale bar is 50 µm.

Article Snippet: The fixed cells were then blocked with 5% BSA, diluted in PBS, for 1 hour and then incubated overnight with anti-γH2A.X antibody (1:500, 9718S, Cell Signalling Technology) or rabbit anti-RPA2 antibody (1:500, HPA026306, Atlas Antibodies) at 4°C.

Techniques: Genome Wide, Cell Culture, Staining

Journal: bioRxiv

Article Title: Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

doi: 10.1101/2025.03.16.643506

Figure Lengend Snippet: (A) Normalized cell counts of MTHFD1 K56R and MTHFD1 K56R NUDT5 KO cells treated for 72 h in ADE and 10 µM NUDT5 inhibitor TH5427 alone and in combination. (B) Chemical structure of the NUDT5 targeting PROTAC dNUDT5. Representative Western blot of dose-dependent dNUDT5 effects on NUDT5 protein levels after 20 h treatment in WT cells. (C) Dose-dependent effects of dNUDT5 on the growth of WT and MTHFD1 K56R cells cultivated in FULL or DIA for 72 h. (D) Representative images of RPA2-foci formation of MTHFD1 K56R RPA2-RFP (intron tagged) cells at indicated media conditions and times.

Article Snippet: The fixed cells were then blocked with 5% BSA, diluted in PBS, for 1 hour and then incubated overnight with anti-γH2A.X antibody (1:500, 9718S, Cell Signalling Technology) or rabbit anti-RPA2 antibody (1:500, HPA026306, Atlas Antibodies) at 4°C.

Techniques: Western Blot

Journal: International Journal of Biological Sciences

Article Title: Mycotoxin zearalenone exposure impairs genomic stability of swine follicular granulosa cells in vitro

doi: 10.7150/ijbs.23898

Figure Lengend Snippet: Western blot analysis of the protein expression levels of DNA damage response (DDR) - related proteins in porcine granulosa cells treated with different concentrations of ZEA. GAPDH and ACTIN were used as protein loading controls. ZEA increased RAD51 protein levels while decreasing XRCC4, TP53, USP1, RPA2 and CCND protein levels. Compared to the control group, * indicated significant differences ( P < 0.05), ** indicated extremely significant differences ( P < 0.01). All experiments were repeated at least three times.

Article Snippet: Then the membranes were blocked with 5 % BSA for 1 h, and then were incubated with primary antibodies: anti-GAPDH (ImmunoWay, YM3040, USA), anti-RAD51 (BBI, D262104), anti-XRCC4 (BBI, D151845), anti-TP53 (BBI, D120082), anti-RPA2 (BBI, D123054), anti-USP1 (BBI, D123511), anti-CCND1 (BBI, D220509), anti-ACTIN (Abcam, ab8226, USA) at the concentration of 1.0 μg/ml overnight at 4 °C.

Techniques: Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: Mycotoxin zearalenone exposure impairs genomic stability of swine follicular granulosa cells in vitro

doi: 10.7150/ijbs.23898

Figure Lengend Snippet: Effect of the estrogen recepter antagonist, Tamoxifen, on DDR in porcine granulosa cells treated with different concentrations of ZEA (10 μM and 30 μM) for 24 h. Western blot analysis of the expression levels of DDR-related proteins (RAD51, USP1, XRCC4 and RPA2). ACTIN was used as a protein loading control. All experiments were repeated at least three times.

Article Snippet: Then the membranes were blocked with 5 % BSA for 1 h, and then were incubated with primary antibodies: anti-GAPDH (ImmunoWay, YM3040, USA), anti-RAD51 (BBI, D262104), anti-XRCC4 (BBI, D151845), anti-TP53 (BBI, D120082), anti-RPA2 (BBI, D123054), anti-USP1 (BBI, D123511), anti-CCND1 (BBI, D220509), anti-ACTIN (Abcam, ab8226, USA) at the concentration of 1.0 μg/ml overnight at 4 °C.

Techniques: Western Blot, Expressing