Fig. 6: IscU2 promotes α-KG catabolism and subsequent DNA 5mC-dependent PDAC cell proliferation and tumor growth in mice.

a Cell proliferation of the PaTu-8988t cells with or without DM-α-KG (7 mM) treatment (means ± SEM, n = 3). b Determination of the cell cycle stage of the PaTu-8988t cells treated with or without DM-α-KG (7 mM) for 48 h (means ± SEM, n = 3). c The PaTu-8988t cells transfected with a control siRNA or IscU2 siRNA were plated for 48 h before determination of the cell cycle stage (means ± SEM, n = 3). d, e The PaTu-8988t cells were transfected with a control siRNA, IscU2 siRNA, and TET3 siRNA (d), or IscU2 siRNA in combination with 100 μM octyl-(R)-2HG (e). The cells were plated for 4 days before measurement of cell proliferation (means ± SEM, n = 3). f BXPC3, PaTu-8988t, and PANC-1 cells were seeded in 12-well plates at a density of 2 × 10⁴ cells/well, respectively. After 48 h of culture, cells were treated with different concentrations of decitabine and cultured for another 48 h. A relative value was obtained by comparing decitabine-treated cells with decitabine-free cells at final time point. (means ± SEM, n = 3). g PaTu-8988t cells transfected with a control siRNA or IscU2 siRNA were seeded in 12-well plates at a density of 2 × 10⁴ cells/well. After 48 h of culture, the cells were treated with different concentrations of decitabine and cultured for another 48 h. A relative value was obtained by comparing decitabine-treated cells with decitabine-free cells at final time point. (means ± SEM, n = 3). h–j The PaTu-8988t cells (5 × 10⁶) transfected with control siRNA, IscU2 siRNA, and TET3 siRNA were subcutaneously injected into the flank regions of athymic nude mice. When the tumors (1 week after subcutaneous injection) were established, the volume of the tumors were measured every two days (h). And the tumor weights (i) and α-KG levels (j) were measured after 4 weeks of tumor growth (means ± SEM, n ≥ 5). k–m PaTu-8988t cells (5 × 10⁶) with or without overexpression of IscU2 were injected into the flank regions of athymic nude mice. When the tumors were established, these mice were randomly divided into two groups and intraperitoneally injected with dimethyl α-KG (DM-α-KG) (600 mg/kg) or PBS daily. The mice were examined for 4 weeks of tumor growth after injection. The tumor volumes were calculated (k). The tumor weights (l) and the α-KG levels (m) were measured. (means ± SEM, n ≥ 6). n Immunohistochemical analyses of tumor samples were performed with an anti-DNA 5mC antibody. Representative images are shown (left panel). The 5mC levels in the tumor samples were determined (means ± SEM, n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test, ns not significant.