Fig 1: IPMK and SF1 depletion suppresses PH domain accumulation and IPMK is required for ATR recruitment. a–d Western blots showing effective knock-down or knock-out with or without rescue of selected phosphoinositide kinases: PIP5K1A (siRNA), PIP5K1B (shRNA), PIK3CB (CRISPR-Cas9) and IPMK (siRNA) together with e–h the quantified accumulation magnitude of Btk-PH and PLCd-PH after background subtraction in designated protein depleted backgrounds (n = 15 for all cases). In panel d&h, the cells were rescued with either siRNA-resistant and flag-tagged wild type human IPMK (siRes2-3xFlag-hIPMK-WT) or its kinase dead mutant (siRes2-3xFlag-hIPMK-D144N). Numbers labeled next to the blots indicated the molecular weight of the ladder. i Western blot showing the effective depletion and rescue of SF1 by shRNA and an shRNA-resistant Myc-DDK-tagged mouse SF1 construct (shResA-mSF1-WT-Myc-DDK). The overpression of SF1 rescue gave two separate bands, likely with and without the Myc-DDK tag. j The quantified accumulation magnitude of Btk-PH and PLCd-PH with background subtraction in SF1 depleted and rescued conditions. k Schematic depicting the SF1-PIP2 complex as a substrate of IPMK but not classical PI3K while both kinases phosphorylate PIP2 in vitro. l Confocal montage of transiently expressed SF1-EGFP upon laser microirradiation. m, n ATR and ?H2AX immunofluorescence micrographs of U2OS nucleus upon laser microirradiation over time for control and IPMK-depleted conditions, respectively. o Normalized quantification comparing the magnitude of recruitment of ATR (n = 20), ATM (n = 13), DNA-PKcs (n = 14) and ?H2AX (n = 47) within ROIs in control and IPMK-depleted conditions. Please refer to Supplementary Fig. 4 for the representative images of ATM and DNA-PKcs staining. p Transient depletion of IPMK in U2OS cells and the q transient depletion of SF1 in MEF cells both demonstrated a suppressed phosphorylation of Chk1 upon global UV irradiation. Data are representative of three independent experiments. in all panels. Dashed circles indicate the ROI for laser microirradiation. Error bars represent mean ± s.e.m in all panels. Student’s t-test, **p < 0.01; ***p < 0.001. n.s. represents not significant. Scale bars are 10 µm for all panels