Fig 1: (A) Experimental design showing the experimental timeline of exposures in the dams and offspring. Exposures in the dams (indicated by red arrows) occurred across 2 weeks of preconception, then pregnancy and lactation. Time points of birth, weaning and behavior testing of the offspring are shown in post-natal days (PND). (B) The one-carbon metabolism pathway in cells showing the folate cycle and methionine cycle. Key enzymes: dihydrofolate reductase (DHFR); serine hydroxymethyltransferase 1 (SHMT1); methylene-tetrahydrofolate reductase (MTHFR); methionine synthase reductase (MTRR); methylene-tetrahydrofolate dehydrogenase 1 (MTHFD1); folate receptor α (FOLR1). Co-Factors and metabolites: dihydrofolate (DHF); tetrahydrofolate (THF); 5,10-methyltetrahydrofolate (5,10-me THF); 5-methyltetrahydrofolate (5-me THF); S-adenosylmethionine (SAM).
Fig 2: MTHFD2 expressions are elevated in the glutamine-deprived cells and tumor core of GBM patients. See also Additional File 2: Supplemental Fig. 3. a A schematic showing the enzymes involved in one-carbon metabolism that were targeted in this study. PSAT1; phosphoserine aminotransferase 1, SHMT1 and 2; serine hydroxymethyl transferase 1 and 2, and MTHFD1 and 2; methylenetetrahydrofolate dehydrogenase 1 and 2. MTHFD1L; monofunctional tetrahydrofolate synthase, mitochondrial b mRNA levels of PSAT1, SHMT1 and 2, MTHFD1 and 2, and MTHFD1L in U87 and T98 GBM cells which were grown with or without glutamine for 48 h. Data represent the mean ± SEM of three independent experiments (statistically significant with *p < 0.05, **p < 0.01). c Immunoblot analysis of PSAT1, SHMT1 and 2, MTHFD1 and 2 staining in central tumors (T) and normal brain tissues (N) around tumor edge obtained at tumor resection from 6 patients with GBM. d Representative immunohistochemical images of MTHFD2 in central tumors obtained from a GBM patient. Tissue was counterstained with hematoxylin. Scale bar upper 200 μm, lower 100 μm arrow; pseudopalisading asterisk; necrosis
Fig 3: KLF5 modulates epigenetic modifiers: MTHFD1 and NCAPD2. A Strategy to narrow down candidate effector genes of KLF5 showing that potential KLF5 targets contained 147 distant negative genes from the CRISPR screen data and that six genes, including KLF5, were identified to be commonly downregulated following KLF5 knockdown (q < 0.001 in response to both shRNAs), using the two different shRNAs in A13Lg. B Heatmap of the relative mRNA expression level of the candidate effector genes in KLF5 knockdown A13Lg cells with shKLF5−5 or shKLF5−7 compared to that of control A13Lg cells with the corresponding scrambled RNAs. *** q < 0.001, and **** q < 0.0001. C qRT-PCR analysis showing relative mRNA expression levels as mean ± sem(n = 3). The one-tailed p-value was determined by unpaired t-test, using the ⊿Ct-GAPDH values compared to the corresponding shCtrl. NS p > 0.05, *p < 0.05, **p < 0.01 D, E, F. Western blotting analysis of KLF5, NCAPD2, MTHFD1, and GAPDH as loading control in A13Lg cells with (D) KLF5 knockdown (shKLF5-5 and −7), (E) NCAPD2 knockdown (shNCAPD2−2, and −3), (F) MTHFD1 knockdown (shMTHFD1−3 and −4) and control (shCtrl-5), using extracted histone proteins. G, H Western blotting analysis of histone modifications in (G) NCAPD2 knockdown (shNCAPD2−2 and −3) or (H) MTHFD1 knockdown (shMTHFD1−3 and −4) and control (shCtrl-5) A13 cells, using their extracted histone proteins
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