Fig 1: NIP30 pS228/pS230 is dephosphorylated by CDC25A.a 293T cells were transiently transfected with 0.5 µg, 1 µg, or 2 µg of flag-CDC25A plasmid for 48 h, and the total cell lysates were analyzed by immunoblotting with antibodies against flag, total NIP30, pNIP30Ser228, pNIP30Ser230, p21, and actin. Data are representative of three independent experiments. b REG? WT and knockout 293T cells were transiently transfected with 2 µg flag-CDC25A plasmid for 48 h, and the total cell lysates were analyzed by immunoblotting with indicated antibodies. Data represent mean ± SEM from three independent experiments. c REG? WT and 293T KO cells were transfected with indicated short interfering RNA (-si) against CDC25A or irrelevant siRNA (si control) for 72 h. Cell lysates were subjected to western blot analysis with indicated antibodies. Data represent mean ± SEM from three independent experiments. d REG? WT and 293T KO cells were transiently transfected with 2 µg of flag-CDC25A plasmid for 48 h and then treated with NSC95397 10 µM, 50 µM, 100 µM for 3 h before harvest. Cell lysates were subjected to western blot analysis using indicated antibodies in three independent experiments. Data represent mean ± SEM. e Immunoprecipitated cellular NIP30 was incubated with bacterially purified CDC25A protein for indicated times. The lane labeled “0” was also incubated for 30 min at 30 °C. Data represent mean ± SEM from three independent experiments. f 293T cells were transfected with HA-NIP30 and flag-CDC25A or a flag-control vector for 36 h. Reciprocal interactions between NIP30 and CDC25A were analyzed by immunoprecipitation assays with Flag-M2 agarose beads or HA beads. Data represent mean ± SEM from three independent experiments. Source data are provided in a Source Data file.
Fig 2: NIP30 mimetics enhance chemotherapeutic effects in p53-deficient cells.a Summary of alterations for NIP30 in different cancer types from the TCGA project. The alteration types include mutation (green), amplification (red), deep deletion (blue) and multiple alternations (gray). b NIP30 phosphorylation mimetics enhance anticancer drug efficiency. H1299 cells stably overexpressing WT, 4A, or 4D NIP30 were treated with indicated concentrations of etoposide, cisplatin, 5-FU, doxorubicin for 48 h followed by MTT assays (n = 4). c Xenograft tumors were generated by injecting H1299, and H1299 overexpressing NIP30 4A/4D cells into dorsal flanking sites of nude mice. Two weeks later, mice were treated with cisplatin (5 mg/kg), or doxorubicin (3 mg/kg), or 5-Fluorouracil (20 mg/kg), three times per week i.p. for 2 weeks. All experiments were repeated three times. d Quantitation of the results in panel c. Values are presented as the means ± SEM. *P < 0.05, ***P < 0.001, (one-way ANOVA). P (lane 1, lane 3) = 0.024, P (lane 1, lane 4) = 1.8E-8, P (lane 1, lane 7) = 3.9E-8, P (lane 1, lane 10) = 3.6E-4, P (lane 4, lane 6) = 0.03, P (lane 7, lane 9) = 0.027, P (lane 10, lane 12) = 1.5E-5. e A model depicting the CDC25A–NIP30-REG? pathway in regulation of the cell cycle and DNA damage response. Source data are provided in a Source Data file.
Fig 3: The CDC25A–NIP30-REG? pathway controls p21 following DNA damage.a H1299 REG? shN and shR cells were irradiated with 0, 15, 25, or 35 J/m2 UVB and harvested 12 h later. Cell lysates were analyzed by western blots with indicated antibodies. b H1299 REG? shN and shR cells were treated with siRNA against CDC25A or a control siRNA followed by UVB irradiation. Cell lysates were subjected to western blot with indicated antibodies. c H1299 REG? shN and shR cells were treated with siRNA against NIP30 or a control siRNA followed by UVB irradiation. Cell lysates were subjected to western blot with indicated antibodies. d, e CDC25A–NIP30 action after DNA damage by chemical reagents. H1299 REG? shN and shR cells were treated with 0.2 mM MMS (d) or 10 µg/ml Doxorubicin (e) for the indicated time course. The levels of CDC25A, total NIP30, pNIP30Ser228, pNIP30Ser230, p21, REG?, and ?H2AX were determined by western blot. f In vivo action of the CDC25A–NIP30-REG? pathway. REG?+/+, REG?-/-, P53-/-, and REG?-/-/P53-/- newborn mice were exposed to UV light as described in “Methods”. Skin samples from exposed dorsal side or ventral side (un-exposed control) were examined for proteins indicated. All the experiments were repeated three times. Source data are provided in a Source Data file.
Fig 4: The CDC25A–NIP30-REG? pathway regulates p21 during the cell cycle.a, REG? WT and 293T KO cells were synchronized to the G0/G1, S, and G2/M phases. Cell lysates were subjected to western blot analysis with indicated antibodies. Data are representative of at least three independent experiments. b Synchronized REG? WT and KO 293T cells were treated with NSC95397 for 3 h. Cell lysates were subjected to western blot analysis. Data represent mean ± SEM from three independent experiments. c REG? WT and KO 293T cells were transfected with an siRNA specific for CDC25A or flag-CDC25A for 72 h. Cells synchronized in the S phase were collected for western blot analysis. Data represent mean ± SEM from three independent experiments. d 293T cells were transfected with a short interfering RNA (-si) for NIP30 or an si control for 72 h. Cells arrested at the S phase were collected for western blot analysis in three independent experiments. Data represent mean ± SEM. e REG? WT and KO 293T cells were transfected with indicated short interfering RNA (-si) for NIP30 or NIP30 4A/4D constructs. Cells synchronized at the S phase were collected for western blots in three independent experiments. Data represent mean ± SEM. Source data are provided in a Source Data file.
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