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Santa Cruz Biotechnology mthfd1 a 8
Mthfd1 A 8, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mthfd1
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mthfd1
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology anti mthfd1
shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, <t>MTHFD1</t> ( A ), as well as DNA repair protein MLH1, and p53 expressions ( B ) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in . The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion ( B). These results may account in part for the stable isotopic tracer experiments using L- [3- 13 C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3- 13 C]-serine were significantly higher in shMTHFR compared to Neg and WT . In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability ( A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus ( B, C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.
Anti Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA"

Article Title: MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22179392

shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, MTHFD1 ( A ), as well as DNA repair protein MLH1, and p53 expressions ( B ) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in . The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion ( B). These results may account in part for the stable isotopic tracer experiments using L- [3- 13 C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3- 13 C]-serine were significantly higher in shMTHFR compared to Neg and WT . In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability ( A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus ( B, C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.
Figure Legend Snippet: shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, MTHFD1 ( A ), as well as DNA repair protein MLH1, and p53 expressions ( B ) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in . The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion ( B). These results may account in part for the stable isotopic tracer experiments using L- [3- 13 C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3- 13 C]-serine were significantly higher in shMTHFR compared to Neg and WT . In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability ( A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus ( B, C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.

Techniques Used: Expressing

shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS 1 protein expression 2 in HepG2 cells.
Figure Legend Snippet: shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS 1 protein expression 2 in HepG2 cells.

Techniques Used: Expressing

MTHFR knockdown by shRNA can protect DNA under folate deficiency. Lower MTHFR is associated with increased proportional cell populations in the G2/M phase, increased cytosol, and nuclear SHMT1/DHFR/TYMS protein expression under folate deficiency. shMTHFR assisted purine synthesis in HepG2 cells under folate deficiency and such impacts were amplified after folinate supplementation. shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency that can protect cells from folate depletion induced micronuclei and uracil misincorporation. Abbreviations: MTHFD1, methylenetetrahydrofolate dehydrogenase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; GNMT, glycine N-methyltransferase; MTR, methionine synthase; MAT, S -adenosylmethionine synthase; SAHH, S-adenosylhomocysteinehydrolase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; THF, tetrahydrofolate; dUMP, deoxyuridine monophosphate; DHF, dihydrofolate; hMLH1: Human Mut L homologue-1 (hMLH1).
Figure Legend Snippet: MTHFR knockdown by shRNA can protect DNA under folate deficiency. Lower MTHFR is associated with increased proportional cell populations in the G2/M phase, increased cytosol, and nuclear SHMT1/DHFR/TYMS protein expression under folate deficiency. shMTHFR assisted purine synthesis in HepG2 cells under folate deficiency and such impacts were amplified after folinate supplementation. shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency that can protect cells from folate depletion induced micronuclei and uracil misincorporation. Abbreviations: MTHFD1, methylenetetrahydrofolate dehydrogenase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; GNMT, glycine N-methyltransferase; MTR, methionine synthase; MAT, S -adenosylmethionine synthase; SAHH, S-adenosylhomocysteinehydrolase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; THF, tetrahydrofolate; dUMP, deoxyuridine monophosphate; DHF, dihydrofolate; hMLH1: Human Mut L homologue-1 (hMLH1).

Techniques Used: shRNA, Expressing, Amplification


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Santa Cruz Biotechnology sc 271412
Sc 271412, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mthfd1
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mthfd1
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mthfd1
G3P is an intrinsic proliferation-suppressive metabolite via targeting ENO1 and <t>MTHFD1.</t> (a) Intracellular concentrations of G3P in HCT116 cells treated with solvent control, NaF (3 mM) and GNE140 (10 μM) for 24 hr determined by targeted LC/MS-MS (n = 3). (b) Accumulative G3P upon direct supplementation of 0.5 mM G3P for 4 hr in HCT116 cells (n = 3). (c) Growth curve of HCT116 cells under G3P administration (n = 5). (d) Colony formation assay validates G3P-induced suppressive proliferation in HCT116 cells. (e) S-plot of TRPs shows top-ranked target proteins of G3P that displayed marked accessibility changes following G3P administration. (f) Thermal shift assay validated the ENO1-G3P engagement in HCT116 cell lysates. (g) Volcano plot of the TRAP-identified G3P TRPs in recombinant ENO1. Accessibility of the TRP carrying Lys330/Lys335 exhibited the most dramatic change after G3P incubation. (h) The ENO1 TRPs of G3P identified by TRAP are highlighted (PDB: 3B97). TRPs spanning residue 328-343, 407-426 and 254-269 are color-coded in blue, yellow and purple. (i) Binding affinities of G3P to the wide type (WT) and the K330E mutant ENO1 measured by SPR. (j) Stability changes of the WT and K330E mutant ENO1 induced by G3P incubation examined by nanoDSF. (k) Conservation analysis of K330 of ENO1 among several species. (l) Enzyme activity assay showed G3P inhibited the activity of WT ENO1 yet weakly to the K335E mutant ENO1 (n = 3). (m) Proliferation of the control and ENO1 knockdown cells under G3P treatment (n = 5). (n) Homology modeling of MTHFD1 indicates the TRP of G3P is located in proximity to the ATP binding site and thus the interaction likely interferes with the 10-formyl-THF synthetase reaction. (o) Multi-target regulatory network of G3P uncovered by TRAP. (p) DrugBank and non-DrugBank fraction of the identified glycolytic targetome. (q) NAMPT enzymatic assay suggests synergistic inhibition by FBP with FK866. (r) TRAP analysis reveals the TRP (blue, cartoon) of FBP located in proximity to FK866 (orange, stick) displayed reduced accessibility in response to FBP (n = 3), suggesting a FBP binding-promoted interaction between FK866 and NAMPT (PDB: 2GVJ). For (a)-(c) and (l)-(m), data represent mean ± SEM. For (a, b, m), *p < 0.05, **p < 0.01, ***p < 0.001, n.s. non-significant, Student’s t-test.
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Chemoproteomics Maps Glycolytic Targetome in Cancer Cells"

Article Title: Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

Journal: bioRxiv

doi: 10.1101/2020.11.18.387670

G3P is an intrinsic proliferation-suppressive metabolite via targeting ENO1 and MTHFD1. (a) Intracellular concentrations of G3P in HCT116 cells treated with solvent control, NaF (3 mM) and GNE140 (10 μM) for 24 hr determined by targeted LC/MS-MS (n = 3). (b) Accumulative G3P upon direct supplementation of 0.5 mM G3P for 4 hr in HCT116 cells (n = 3). (c) Growth curve of HCT116 cells under G3P administration (n = 5). (d) Colony formation assay validates G3P-induced suppressive proliferation in HCT116 cells. (e) S-plot of TRPs shows top-ranked target proteins of G3P that displayed marked accessibility changes following G3P administration. (f) Thermal shift assay validated the ENO1-G3P engagement in HCT116 cell lysates. (g) Volcano plot of the TRAP-identified G3P TRPs in recombinant ENO1. Accessibility of the TRP carrying Lys330/Lys335 exhibited the most dramatic change after G3P incubation. (h) The ENO1 TRPs of G3P identified by TRAP are highlighted (PDB: 3B97). TRPs spanning residue 328-343, 407-426 and 254-269 are color-coded in blue, yellow and purple. (i) Binding affinities of G3P to the wide type (WT) and the K330E mutant ENO1 measured by SPR. (j) Stability changes of the WT and K330E mutant ENO1 induced by G3P incubation examined by nanoDSF. (k) Conservation analysis of K330 of ENO1 among several species. (l) Enzyme activity assay showed G3P inhibited the activity of WT ENO1 yet weakly to the K335E mutant ENO1 (n = 3). (m) Proliferation of the control and ENO1 knockdown cells under G3P treatment (n = 5). (n) Homology modeling of MTHFD1 indicates the TRP of G3P is located in proximity to the ATP binding site and thus the interaction likely interferes with the 10-formyl-THF synthetase reaction. (o) Multi-target regulatory network of G3P uncovered by TRAP. (p) DrugBank and non-DrugBank fraction of the identified glycolytic targetome. (q) NAMPT enzymatic assay suggests synergistic inhibition by FBP with FK866. (r) TRAP analysis reveals the TRP (blue, cartoon) of FBP located in proximity to FK866 (orange, stick) displayed reduced accessibility in response to FBP (n = 3), suggesting a FBP binding-promoted interaction between FK866 and NAMPT (PDB: 2GVJ). For (a)-(c) and (l)-(m), data represent mean ± SEM. For (a, b, m), *p < 0.05, **p < 0.01, ***p < 0.001, n.s. non-significant, Student’s t-test.
Figure Legend Snippet: G3P is an intrinsic proliferation-suppressive metabolite via targeting ENO1 and MTHFD1. (a) Intracellular concentrations of G3P in HCT116 cells treated with solvent control, NaF (3 mM) and GNE140 (10 μM) for 24 hr determined by targeted LC/MS-MS (n = 3). (b) Accumulative G3P upon direct supplementation of 0.5 mM G3P for 4 hr in HCT116 cells (n = 3). (c) Growth curve of HCT116 cells under G3P administration (n = 5). (d) Colony formation assay validates G3P-induced suppressive proliferation in HCT116 cells. (e) S-plot of TRPs shows top-ranked target proteins of G3P that displayed marked accessibility changes following G3P administration. (f) Thermal shift assay validated the ENO1-G3P engagement in HCT116 cell lysates. (g) Volcano plot of the TRAP-identified G3P TRPs in recombinant ENO1. Accessibility of the TRP carrying Lys330/Lys335 exhibited the most dramatic change after G3P incubation. (h) The ENO1 TRPs of G3P identified by TRAP are highlighted (PDB: 3B97). TRPs spanning residue 328-343, 407-426 and 254-269 are color-coded in blue, yellow and purple. (i) Binding affinities of G3P to the wide type (WT) and the K330E mutant ENO1 measured by SPR. (j) Stability changes of the WT and K330E mutant ENO1 induced by G3P incubation examined by nanoDSF. (k) Conservation analysis of K330 of ENO1 among several species. (l) Enzyme activity assay showed G3P inhibited the activity of WT ENO1 yet weakly to the K335E mutant ENO1 (n = 3). (m) Proliferation of the control and ENO1 knockdown cells under G3P treatment (n = 5). (n) Homology modeling of MTHFD1 indicates the TRP of G3P is located in proximity to the ATP binding site and thus the interaction likely interferes with the 10-formyl-THF synthetase reaction. (o) Multi-target regulatory network of G3P uncovered by TRAP. (p) DrugBank and non-DrugBank fraction of the identified glycolytic targetome. (q) NAMPT enzymatic assay suggests synergistic inhibition by FBP with FK866. (r) TRAP analysis reveals the TRP (blue, cartoon) of FBP located in proximity to FK866 (orange, stick) displayed reduced accessibility in response to FBP (n = 3), suggesting a FBP binding-promoted interaction between FK866 and NAMPT (PDB: 2GVJ). For (a)-(c) and (l)-(m), data represent mean ± SEM. For (a, b, m), *p < 0.05, **p < 0.01, ***p < 0.001, n.s. non-significant, Student’s t-test.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Colony Assay, Thermal Shift Assay, Recombinant, Incubation, Binding Assay, Mutagenesis, Nano Differential Scanning Fluorimetry, Enzyme Activity Assay, Activity Assay, Enzymatic Assay, Inhibition


Structured Review

Santa Cruz Biotechnology mthfd1
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mthfd1
a , Schematic overview of the gene-trap based genetic screen. b , Representative panels of the applied FACS-sorting strategy showing non-infected (upper panel) and gene-trap infected (lower panel) REDS1 cells; infected double-positive (GFP + /RFP + ) cells (shown in red: 0.01%) were sorted. c , Circos plot illustrating the results from the gene-trap screen by genomic location (outside ring), number of independent inactivating integrations (bubble size) and significance (distance from center). P values were calculated by one-sided Fisher's exact test of insertions over an unselected control data set adjusted for false discovery rate (FDR) using Benjamini-Hochberg procedure. The screen was performed in three biologically independent experiments. d , Western blot showing <t>MTHFD1</t> protein levels after downregulation with the indicated shRNAs in REDS1 cells. Numbers indicate the percentage of MTHFD1 protein remaining, tubulin was used as a loading control. The experiment was repeated three times with similar results. e , Quantification of RFP + cells from live-cell imaging of REDS1 cells treated with MTHFD1 shRNA. Two biological replicates were done for each experimental condition. f , Representative live-cell images of MTHFD1 knock-down in REDS1 cells. Scale bar 100 μm.
Mthfd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mthfd1/product/Santa Cruz Biotechnology
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mthfd1 - by Bioz Stars, 2023-12
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1) Product Images from "MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation"

Article Title: MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

Journal: Nature genetics

doi: 10.1038/s41588-019-0413-z

a , Schematic overview of the gene-trap based genetic screen. b , Representative panels of the applied FACS-sorting strategy showing non-infected (upper panel) and gene-trap infected (lower panel) REDS1 cells; infected double-positive (GFP + /RFP + ) cells (shown in red: 0.01%) were sorted. c , Circos plot illustrating the results from the gene-trap screen by genomic location (outside ring), number of independent inactivating integrations (bubble size) and significance (distance from center). P values were calculated by one-sided Fisher's exact test of insertions over an unselected control data set adjusted for false discovery rate (FDR) using Benjamini-Hochberg procedure. The screen was performed in three biologically independent experiments. d , Western blot showing MTHFD1 protein levels after downregulation with the indicated shRNAs in REDS1 cells. Numbers indicate the percentage of MTHFD1 protein remaining, tubulin was used as a loading control. The experiment was repeated three times with similar results. e , Quantification of RFP + cells from live-cell imaging of REDS1 cells treated with MTHFD1 shRNA. Two biological replicates were done for each experimental condition. f , Representative live-cell images of MTHFD1 knock-down in REDS1 cells. Scale bar 100 μm.
Figure Legend Snippet: a , Schematic overview of the gene-trap based genetic screen. b , Representative panels of the applied FACS-sorting strategy showing non-infected (upper panel) and gene-trap infected (lower panel) REDS1 cells; infected double-positive (GFP + /RFP + ) cells (shown in red: 0.01%) were sorted. c , Circos plot illustrating the results from the gene-trap screen by genomic location (outside ring), number of independent inactivating integrations (bubble size) and significance (distance from center). P values were calculated by one-sided Fisher's exact test of insertions over an unselected control data set adjusted for false discovery rate (FDR) using Benjamini-Hochberg procedure. The screen was performed in three biologically independent experiments. d , Western blot showing MTHFD1 protein levels after downregulation with the indicated shRNAs in REDS1 cells. Numbers indicate the percentage of MTHFD1 protein remaining, tubulin was used as a loading control. The experiment was repeated three times with similar results. e , Quantification of RFP + cells from live-cell imaging of REDS1 cells treated with MTHFD1 shRNA. Two biological replicates were done for each experimental condition. f , Representative live-cell images of MTHFD1 knock-down in REDS1 cells. Scale bar 100 μm.

Techniques Used: Infection, Western Blot, Live Cell Imaging, shRNA

a , BRD4 interactomes in MEG-01, K-562, MV4-11and MOLM-13 cell lines. Proteins are represented as circles, colors indicate the number of cell lines in which a particular interacting protein was detected. b , Western blot confirmation of the BRD4-MTHFD1 interaction in leukemia cell lines. The experiment was repeated twice with similar results. c , Upper panel: Western blot following nuclear vs cytoplasmic fractionation in HAP1, KBM7 and HEK293T cell lines. RCC1 was used as nuclear loading control while tubulin was used as cytosolic loading control. Lower panel: Western blot following MTHFD1 pull-down in the different cell fractions. The experiment was repeated three times with similar results. d , Western blot performed on chromatin-associated protein samples extracted from HAP1 cells treated with the indicated compounds for 2 h (dBET1: 0.5 μM; dBET6: 0.5 μM; MTX: 1 μM) or 24 h (dBET1: 0.5 μM; dBET6: 0.05 μM; MTX: 1 μM). H2B was used as loading control. The experiment was repeated three times with similar results. e , Western blot for nuclear vs cytoplasmic protein levels in HAP1 cells treated for 24 h as above. The experiment was repeated twice with similar results. f , Western blot from chromatin fractions of MEG-01, K-562, MV4-11 and MOLM-13 cells treated with dBET6 for 2 h. The experiment was repeated twice with similar results. g , Immunofluorescence images of HeLa cells treated with the indicated compounds and stained for MTHFD1, BRD4, and DAPI (small inserts). Scale bar 10 μm.
Figure Legend Snippet: a , BRD4 interactomes in MEG-01, K-562, MV4-11and MOLM-13 cell lines. Proteins are represented as circles, colors indicate the number of cell lines in which a particular interacting protein was detected. b , Western blot confirmation of the BRD4-MTHFD1 interaction in leukemia cell lines. The experiment was repeated twice with similar results. c , Upper panel: Western blot following nuclear vs cytoplasmic fractionation in HAP1, KBM7 and HEK293T cell lines. RCC1 was used as nuclear loading control while tubulin was used as cytosolic loading control. Lower panel: Western blot following MTHFD1 pull-down in the different cell fractions. The experiment was repeated three times with similar results. d , Western blot performed on chromatin-associated protein samples extracted from HAP1 cells treated with the indicated compounds for 2 h (dBET1: 0.5 μM; dBET6: 0.5 μM; MTX: 1 μM) or 24 h (dBET1: 0.5 μM; dBET6: 0.05 μM; MTX: 1 μM). H2B was used as loading control. The experiment was repeated three times with similar results. e , Western blot for nuclear vs cytoplasmic protein levels in HAP1 cells treated for 24 h as above. The experiment was repeated twice with similar results. f , Western blot from chromatin fractions of MEG-01, K-562, MV4-11 and MOLM-13 cells treated with dBET6 for 2 h. The experiment was repeated twice with similar results. g , Immunofluorescence images of HeLa cells treated with the indicated compounds and stained for MTHFD1, BRD4, and DAPI (small inserts). Scale bar 10 μm.

Techniques Used: Western Blot, Fractionation, Immunofluorescence, Staining

a , Validation of MTHFD1 knock-out HAP1 cell lines. The experiment was repeated three times with similar results. b , Representative genome browser view of BRD4, MTHFD1, and H3K27ac binding in the promoters of TFAP4 (left) and KEAP1 (right). All ChIP tracks were normalized to 1X genome coverage. All the IPs were performed in biological duplicate. Specifically for MTHFD1 knock-out cells, MTHFD1 KO_1 and MTHFD1 KO_3 were used as independent biological replicates. c , Enrichment of BRD4 and MTHFD1 ChIP signal. Peaks were sorted by total abundance and data represent merged replicates normalized to 1× coverage. d , Principal component analysis of RNA-seq data of two MTHFD1 knock-out clones and of WT HAP1 cells treated with 0.1 μM dBET6, 1 μM ( S )-JQ1, 1 μM MTX, shRNAs targeting BRD4 or MTHFD1. Equal amount of DMSO, or non-targeting hairpins were used as respective control conditions and two biological replicates were performed for each experimental condition. e , Heatmap of relative transcription changes in HAP1 cells compared to respective control cells. f , Integration of ChIP-seq and RNA-seq data in HAP1 cells. BRD4 and MTHFD1 binding at sites associated with genes which are significantly up- or down-regulated upon knockdown of BRD4 and/or MTHFD1 and in MTHFD1 knock-out cells compared to HAP1 WT cells. Values represent estimated factor abundance normalized by matched IgG signal and equality of distributions was assessed with with a one-sided Mann–Whitney U test. Boxplot boxes represent interquartile range with center on median, and whiskers represent values 1.5× outside the respective interquartile range.
Figure Legend Snippet: a , Validation of MTHFD1 knock-out HAP1 cell lines. The experiment was repeated three times with similar results. b , Representative genome browser view of BRD4, MTHFD1, and H3K27ac binding in the promoters of TFAP4 (left) and KEAP1 (right). All ChIP tracks were normalized to 1X genome coverage. All the IPs were performed in biological duplicate. Specifically for MTHFD1 knock-out cells, MTHFD1 KO_1 and MTHFD1 KO_3 were used as independent biological replicates. c , Enrichment of BRD4 and MTHFD1 ChIP signal. Peaks were sorted by total abundance and data represent merged replicates normalized to 1× coverage. d , Principal component analysis of RNA-seq data of two MTHFD1 knock-out clones and of WT HAP1 cells treated with 0.1 μM dBET6, 1 μM ( S )-JQ1, 1 μM MTX, shRNAs targeting BRD4 or MTHFD1. Equal amount of DMSO, or non-targeting hairpins were used as respective control conditions and two biological replicates were performed for each experimental condition. e , Heatmap of relative transcription changes in HAP1 cells compared to respective control cells. f , Integration of ChIP-seq and RNA-seq data in HAP1 cells. BRD4 and MTHFD1 binding at sites associated with genes which are significantly up- or down-regulated upon knockdown of BRD4 and/or MTHFD1 and in MTHFD1 knock-out cells compared to HAP1 WT cells. Values represent estimated factor abundance normalized by matched IgG signal and equality of distributions was assessed with with a one-sided Mann–Whitney U test. Boxplot boxes represent interquartile range with center on median, and whiskers represent values 1.5× outside the respective interquartile range.

Techniques Used: Knock-Out, Binding Assay, RNA Sequencing Assay, Clone Assay, ChIP-sequencing, MANN-WHITNEY

a , Representation of the folate pathway. Enzyme names are reported inside the geometric shapes, connecting the different metabolites. Enzymes that were found associated with chromatin in HAP1 and K-562 cells by mass spectrometry analysis are indicated in red and blue, respectively. Two biological replicates were done. b , Western blot for folate pathway enzymes in the cytoplasmic (C) and chromatin (Ch) fractions of HAP1 cells. The experiment was repeated twice with similar results. c , Recombinant enzyme assays for MTHFD1 activity to convert THF and formate to 5,10-methenyl-THF and vice versa in the presence or absence of full-length BRD4 or its first bromodomain. Mean ± SD from n = 2 independent samples. d , Scatter plot representing metabolite changes in the pyrimidine, purine and methionine biosynthetic pathways upon downregulation of BRD4 or MTHFD1 by shRNA. Two biological replicates were done for each experimental condition. r -value indicates the Pearson correlation coefficient. e , Incorporation of labeled formate into RNA. HAP1 WT and MTHFD1 knock-out cells were treated with 13 C-labeled formate for 24 h, followed by RNA extraction and LC-MS-MS analysis of nucleotides for the 13 C/ 12 C ratio. Two biological replicates were performed for each experimental condition. f , Incorporation of labeled formate into RNA using the same procedure with MTHFD1 knock-out cells transiently transfected with full-length MTHFD1, or the protein with either a nuclear localization signal (NLS) or a nuclear export signal (NES). Percent of control is calculated considering the 13 C incorporation in HAP1 WT and MTHFD1 knock-out respectively as 100% and 0. Two biological replicates were performed for each experimental condition.
Figure Legend Snippet: a , Representation of the folate pathway. Enzyme names are reported inside the geometric shapes, connecting the different metabolites. Enzymes that were found associated with chromatin in HAP1 and K-562 cells by mass spectrometry analysis are indicated in red and blue, respectively. Two biological replicates were done. b , Western blot for folate pathway enzymes in the cytoplasmic (C) and chromatin (Ch) fractions of HAP1 cells. The experiment was repeated twice with similar results. c , Recombinant enzyme assays for MTHFD1 activity to convert THF and formate to 5,10-methenyl-THF and vice versa in the presence or absence of full-length BRD4 or its first bromodomain. Mean ± SD from n = 2 independent samples. d , Scatter plot representing metabolite changes in the pyrimidine, purine and methionine biosynthetic pathways upon downregulation of BRD4 or MTHFD1 by shRNA. Two biological replicates were done for each experimental condition. r -value indicates the Pearson correlation coefficient. e , Incorporation of labeled formate into RNA. HAP1 WT and MTHFD1 knock-out cells were treated with 13 C-labeled formate for 24 h, followed by RNA extraction and LC-MS-MS analysis of nucleotides for the 13 C/ 12 C ratio. Two biological replicates were performed for each experimental condition. f , Incorporation of labeled formate into RNA using the same procedure with MTHFD1 knock-out cells transiently transfected with full-length MTHFD1, or the protein with either a nuclear localization signal (NLS) or a nuclear export signal (NES). Percent of control is calculated considering the 13 C incorporation in HAP1 WT and MTHFD1 knock-out respectively as 100% and 0. Two biological replicates were performed for each experimental condition.

Techniques Used: Mass Spectrometry, Western Blot, Recombinant, Activity Assay, shRNA, Labeling, Knock-Out, RNA Extraction, Liquid Chromatography with Mass Spectroscopy, Transfection

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    shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, <t>MTHFD1</t> ( A ), as well as DNA repair protein MLH1, and p53 expressions ( B ) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in . The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion ( B). These results may account in part for the stable isotopic tracer experiments using L- [3- 13 C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3- 13 C]-serine were significantly higher in shMTHFR compared to Neg and WT . In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability ( A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus ( B, C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.
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    shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, MTHFD1 ( A ), as well as DNA repair protein MLH1, and p53 expressions ( B ) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in . The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion ( B). These results may account in part for the stable isotopic tracer experiments using L- [3- 13 C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3- 13 C]-serine were significantly higher in shMTHFR compared to Neg and WT . In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability ( A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus ( B, C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.

    Journal: International Journal of Molecular Sciences

    Article Title: MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA

    doi: 10.3390/ijms22179392

    Figure Lengend Snippet: shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, MTHFD1 ( A ), as well as DNA repair protein MLH1, and p53 expressions ( B ) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in . The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion ( B). These results may account in part for the stable isotopic tracer experiments using L- [3- 13 C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3- 13 C]-serine were significantly higher in shMTHFR compared to Neg and WT . In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability ( A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus ( B, C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.

    Article Snippet: Thirty micrograms of proteins (from folate depletion experiments) or 16 µg of proteins (from folate repletion experiments) were separated by SDS-PAGE (12% gel) and subsequently transferred to a polyvinylidene difluoride membrane and blotted with primary antibodies, including anti-Thymidylate synthase (1:1000; sc-376161; Santa Cruz, Dallas, TX, USA), anti-DHFR (1:1000; ab133546; Abcam, Cambridge, non-metropolitan county, UK, Cambridgeshire), anti-a-Tubulin (1:5000; NB100-690; Novus, Littleton, CO, USA), anti-MTHFD1 (1:500; sc-134732; Santa Cruz, Dallas, TX, USA), anti-MLH1 (1:1000; sc-271978; Santa Cruz, Dallas, TX, USA), anti-SHMT1 (1:1000; #80715; Cell Signaling, Danvers, MA, USA, Essex), anti-Actin (1:5000; NB600-501; Novus, Littleton, CO, USA), anti–Lamin A/C (1:1000; GTX101127; GeneTex, Irvine, CA, USA, Orange), anti-p53 (1:1000; #9282; Cell Signaling, Danvers, MA, USA, Essex), followed by incubation with HRP-conjugated polyclonal secondary antibody (1:2000; ab6721; Abcam, Cambridge, non-metropolitan county, UK).

    Techniques: Expressing

    shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS 1 protein expression 2 in HepG2 cells.

    Journal: International Journal of Molecular Sciences

    Article Title: MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA

    doi: 10.3390/ijms22179392

    Figure Lengend Snippet: shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS 1 protein expression 2 in HepG2 cells.

    Article Snippet: Thirty micrograms of proteins (from folate depletion experiments) or 16 µg of proteins (from folate repletion experiments) were separated by SDS-PAGE (12% gel) and subsequently transferred to a polyvinylidene difluoride membrane and blotted with primary antibodies, including anti-Thymidylate synthase (1:1000; sc-376161; Santa Cruz, Dallas, TX, USA), anti-DHFR (1:1000; ab133546; Abcam, Cambridge, non-metropolitan county, UK, Cambridgeshire), anti-a-Tubulin (1:5000; NB100-690; Novus, Littleton, CO, USA), anti-MTHFD1 (1:500; sc-134732; Santa Cruz, Dallas, TX, USA), anti-MLH1 (1:1000; sc-271978; Santa Cruz, Dallas, TX, USA), anti-SHMT1 (1:1000; #80715; Cell Signaling, Danvers, MA, USA, Essex), anti-Actin (1:5000; NB600-501; Novus, Littleton, CO, USA), anti–Lamin A/C (1:1000; GTX101127; GeneTex, Irvine, CA, USA, Orange), anti-p53 (1:1000; #9282; Cell Signaling, Danvers, MA, USA, Essex), followed by incubation with HRP-conjugated polyclonal secondary antibody (1:2000; ab6721; Abcam, Cambridge, non-metropolitan county, UK).

    Techniques: Expressing

    MTHFR knockdown by shRNA can protect DNA under folate deficiency. Lower MTHFR is associated with increased proportional cell populations in the G2/M phase, increased cytosol, and nuclear SHMT1/DHFR/TYMS protein expression under folate deficiency. shMTHFR assisted purine synthesis in HepG2 cells under folate deficiency and such impacts were amplified after folinate supplementation. shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency that can protect cells from folate depletion induced micronuclei and uracil misincorporation. Abbreviations: MTHFD1, methylenetetrahydrofolate dehydrogenase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; GNMT, glycine N-methyltransferase; MTR, methionine synthase; MAT, S -adenosylmethionine synthase; SAHH, S-adenosylhomocysteinehydrolase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; THF, tetrahydrofolate; dUMP, deoxyuridine monophosphate; DHF, dihydrofolate; hMLH1: Human Mut L homologue-1 (hMLH1).

    Journal: International Journal of Molecular Sciences

    Article Title: MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA

    doi: 10.3390/ijms22179392

    Figure Lengend Snippet: MTHFR knockdown by shRNA can protect DNA under folate deficiency. Lower MTHFR is associated with increased proportional cell populations in the G2/M phase, increased cytosol, and nuclear SHMT1/DHFR/TYMS protein expression under folate deficiency. shMTHFR assisted purine synthesis in HepG2 cells under folate deficiency and such impacts were amplified after folinate supplementation. shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency that can protect cells from folate depletion induced micronuclei and uracil misincorporation. Abbreviations: MTHFD1, methylenetetrahydrofolate dehydrogenase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; GNMT, glycine N-methyltransferase; MTR, methionine synthase; MAT, S -adenosylmethionine synthase; SAHH, S-adenosylhomocysteinehydrolase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; THF, tetrahydrofolate; dUMP, deoxyuridine monophosphate; DHF, dihydrofolate; hMLH1: Human Mut L homologue-1 (hMLH1).

    Article Snippet: Thirty micrograms of proteins (from folate depletion experiments) or 16 µg of proteins (from folate repletion experiments) were separated by SDS-PAGE (12% gel) and subsequently transferred to a polyvinylidene difluoride membrane and blotted with primary antibodies, including anti-Thymidylate synthase (1:1000; sc-376161; Santa Cruz, Dallas, TX, USA), anti-DHFR (1:1000; ab133546; Abcam, Cambridge, non-metropolitan county, UK, Cambridgeshire), anti-a-Tubulin (1:5000; NB100-690; Novus, Littleton, CO, USA), anti-MTHFD1 (1:500; sc-134732; Santa Cruz, Dallas, TX, USA), anti-MLH1 (1:1000; sc-271978; Santa Cruz, Dallas, TX, USA), anti-SHMT1 (1:1000; #80715; Cell Signaling, Danvers, MA, USA, Essex), anti-Actin (1:5000; NB600-501; Novus, Littleton, CO, USA), anti–Lamin A/C (1:1000; GTX101127; GeneTex, Irvine, CA, USA, Orange), anti-p53 (1:1000; #9282; Cell Signaling, Danvers, MA, USA, Essex), followed by incubation with HRP-conjugated polyclonal secondary antibody (1:2000; ab6721; Abcam, Cambridge, non-metropolitan county, UK).

    Techniques: shRNA, Expressing, Amplification