Fig. 2: MAT2B regulates MAT2A in an NADP⁺-dependent manner.

A MAT2A increased two days after NADK over-expression. The U2OS cells with or without NADK overexpression were collected daily for 4 days after virus infection. The assay was repeated twice with similar results. B The total level of NADP(H) increased two days after NADK over-expression. The contents of individual compounds in empty-vector transfected U2OS cells, as determined by mass spectrometry, were normalized as 1. The data were represented by the mean ± SEM (N = 3 independent replicates). The p value was determined by two-sided unpaired t-test. C NADK overexpression increased the interaction between MAT2B and MAT2A. MAT2B was purified by FLAG IP. HEK293T cells transfected with empty vectors were used as negative controls. Proteins from input and IP samples were analyzed by Western blotting using the indicated antibodies. To avoid the changes of MAT2A, cells were collected one day after NADK over-expression. The assay was repeated twice with similar results. D Depletion of NADK reduced the protein level of MAT2A. U2OS cells with or without NADK depletion were collected daily for 5 days after virus infection. The assay was repeated twice with similar results. E The total level of NADP(H) decreased after NADK depletion. The contents of individual compounds in NT shRNA treated U2OS cells, as determined by mass spectrometry, were normalized as 1. The data were represented by the mean ± SEM (N = 3 independent replicates). The p value was determined by two-sided unpaired t-test. F Western blotting results showing the protein levels of MAT2A in WT MAT2B and MAT2BΔ35-41 rescued U2OS cell lines with NADK depletion. The assay was repeated three times with similar results. G Depletion of NADK reduced the interaction between MAT2B and MAT2A. To avoid the changes of MAT2A and MAT2B, cells were collected one day after the depletion of NADK. Proteins from input and IP samples were analyzed by Western blotting using the indicated antibodies. The assay was repeated twice with similar results. H NADK2 depletion increased the protein level of MAT2A. Two independent shRNAs were used to deplete NADK2 in U2OS cells. The assay was repeated three times with similar results. I The contents of NADP(H) outside mitochondria increased after Nadk2 was knocked down. The contents of individual compounds in NT shRNA treated U2OS cells, as determined by mass spectrometry, were normalized as 1. The data were represented by the mean ± SEM (N = 3 independent replicates). The p value was determined by two-sided unpaired t-test. J The overexpression of mitoTPNOX decreased the protein level of MAT2A in U2OS cells. The assay was repeated four times with similar results. K Overexpression of mitoTPNOX decreased the contents of NADP⁺ outside mitochondria. The contents of individual compounds in empty-vector transfected U2OS cells, as determined by mass spectrometry, were normalized as 1. The data were represented by the mean ± SEM (N = 3 independent replicates). The p value was determined by two-sided unpaired t-test. L NADP⁺ incubation increased MAT2A in WT U2OS cells but not in Mat2b KO U2OS cells. 0, 5, and 10 mM NADP⁺ were transfected into cells for 48 h and cell extracts were analyzed by Western blotting using the indicated antibodies. The assay was repeated twice with similar results.