Fig. 8: Cancer-associated STN1 mutations impair STN1 phosphorylation and fork stability. | Nature Communications

Fig. 8: Cancer-associated STN1 mutations impair STN1 phosphorylation and fork stability.

From: CaMKK2 and CHK1 phosphorylate human STN1 in response to replication stress to protect stalled forks from aberrant resection

Fig. 8: Cancer-associated STN1 mutations impair STN1 phosphorylation and fork stability.

A Western blot of S96 phosphorylation in HeLa cells expressing RNAi-resistant Myc-WT-STN1, Myc-S96V, Myc-S96A, Myc-E95G with concurrent depletion of endogenous STN1. Cells were treated with 4 mM HU for 3 h or 2 μM A23187 for 1 h. Images are cropped from the same blot. Representative blots from three independent experiments are shown. B Western blot of S96 phosphorylation in HeLa and U2OS cells expressing Myc-WT-STN1, Myc-E95G, or Myc-E95G/S96D. Cells were treated with 4 mM HU for 3 h. Representative blots from three independent experiments are shown. C DNA fiber assay in HeLa cells expressing RNAi-resistant WT-STN1, S96A, S96V, and E95G with concurrent depletion of endogenous STN1. Two independent experiments were performed and results from one experiment are shown. P: One-way ANOVA. Red line: mean. n = 200 fibers were measured per sample in each experiment. D Model. Fork stalling leads to the increase of intracellular calcium concentration and accumulation of ssDNA, activating the CaMKK2 and ATR pathways, respectively. Both CaMKK2 and ATR-CHK1 phosphorylate STN1 to promote CST access to stalled forks, which facilitates RAD51 recruitment to stalled forks and blocks MRE11-mediated NSD. Source data are provided in the Source Data file. The model was created with BioRender.com.

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