Fig 1: CPAP interacts with DCDC2 and DYX1C1. (A) CPAP endogenously interacts with DCDC2 and DYX1C1. Endogenous immunoprecipitations using HeLa cell extracts. CPAP was used as a bait to pull down interactors. CEP350 was used as a negative control and CEP152 was used as a positive control for interaction with CPAP. Beads alone were used as a negative control for the IP´s. (B) CPAP and DYX1C1 interaction in brain organoids. CPAP was immunoprecipitated by anti- DYX1C1 in 15 day old brain organoids. Reciprocally, DYX1C1 was pulled down by anti-CPAP. (C) Schematic representation of domain structures of DYX1C1, CPAP and the deletion constructs DYX1C1?TPR, DYX1C1?p23, DYX1C1?DYX. p23 = p23 domain, TPR = tetratricopeptide repeat domain, DYX = DYX domain. (D) CPAP interacts with DYX1C1 via the p23 domain. hTERT-RPE1 cells stably expressing DOX-CPAP-GFP and growing in normal serum conditions were induced with doxycycline and transiently transfected with the indicated constructs. GFP-Trap was used to pull down CPAP-GFP. anti-V5 antibody was used for immunodetection of interactors.
Fig 2: Transcriptional control between DYX1C1/dyx1c1 and DCDC2/dcdc2b.A-D). hTERT-RPE1 cells were transfected with siDYX1C1, siDCDC2 or siCtrl, respectively, RNA was extracted and qRT-PCR was run (n = 3 experiments with 3 replicates, mean ± sem). A, C) Knockdown of DYX1C1 and DCDC2 efficiently reduced the respective transcripts (p-value < 0.0001, t-test). B, D) Expression of DCDC2 and DYX1C1 is reduced after knockdown of siDYX1C1 and siDCDC2, respectively (p-value < 0.05, t-test). E) dcdc2b expression was unchanged in dyx1c1 morphants: RNA of dyx1c1 morphant zebrafish was collected at 1dpf and 2dpf after morpholino injection at 1-cell stage. RT-qPCR analysis shows that dcdc2b expression is unchanged in dyx1c1 morphant embryos compared to control embryos (n = 3, mean ± sem, p-value (1dpf) = 0.2597, p-value (2dpf) = 0.4521), t-test). dpf = days post fertilization
Fig 3: Co-occurrence of DYX1C1 and CPAP at the subcellular level. A) DYX1C1 and CPAP co-occur around the centrosome. Confocal immunofluorescence images of endogenous protein in hTERT-RPE1 cells grown in normal serum conditions. B)-C) DCDC2 co-occurs with CPAP depending on cell cycle stage. Nuclei are stained with DRAQ5. Scale bar = 10 µm
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