Fig. 4: CaMKK2 phosphorylates STN1 S96 in response to Ca²⁺ concentration increase and HU treatment.

A GCaMP6s reporter assay showing the increase of intracellular Ca²⁺ concentration in U2OS cells after treatment with HU (4 mM) or APH (5 μg/ml) for 3 h. Scale bar: 10 µm. Quantification of the images was performed using Image J. P: Two-tailed unpaired t-test. Red line: mean. Two biologically independent experiments were performed and n = ~100 cells were analyzed per sample in each experiment. B Western blot of pS96 after Ca²⁺ ionophore (A23187, 2 μM, 1 h) or thapsigargin (1 μM, 1 h) treatment in both HeLa and U2OS cells. Images shown in upper panels were cropped from the same blot. For the phosphatase treatment, cell lysates were treated with 100 units of lambda phosphatase for 30 min at 30 °C. Representative blots from three independent experiments are shown. C Western blot of pS96 in CaMKK2 KO cells after HU treatment. Two CaMKK2 KO HeLa clones were treated with HU (4 mM, 3 h) and pS96 was detected. Representative blots from two independent experiments are shown. D Western blot of pS96 in CaMKK2 KO cells after A23187 treatment. Two CaMKK2 KO HeLa clones were treated with A23187 (2 μM, 1 h). Representative blots from two independent experiments are shown. E CaMKK2 inhibition diminishes pS96. HeLa cells were pretreated with 25 μM CaMKK2i (STO-609) for 30 min followed by 10 mM HU treatment for 3 h or 2 μM A23187 treatment for 1 h. Western blot was performed using whole cell lysate. Representative blots from two independent experiments are shown. F Effect of AMPKα KO on S96 phosphorylation. Two AMPKα KO clones were treated with 4 mM HU for 3 h and pS96 was detected. Representative blots from two independent experiments are shown. G SDS-PAGE showing purified His₆-SUMO-STN1, GST-CaMKK2 and CHK1 proteins used in the in vitro kinase assay. Representative blots from three independent experiments are shown. H In vitro kinase assay. His₆-SUMO-STN1 was incubated with CaMKK2 or CHK1 in the kinase reaction buffer with or without CaMKK2i or CHK1i. STN1 phosphorylation was detected with the anti-pS96 antibody after SDS-PAGE. Three independent experiments were performed and results from one experiment was shown. Representative blots from three independent experiments are shown. Source data are provided in the Source Data file.