CD53 promotes DREAM complex binding through association with p130 and its phosphatase PP2A. (A) CUT&Tag sequencing of WT and Cd53^−/− naïve BM- and G-CSF-treated splenic HSPCs. p130 read intensities within 100 kb of the transcription start site of the DREAM targets are shown. See supplemental Table 7 for peak counts and DREAM enrichment. The Duolink proximity ligation assay reveals an interaction between (B) CD53-p130 and (C) CD53-PP2A-Aα in WT HSCs, with no signal in Cd53^−/− HSC controls. Representative maximum intensity projection confocal images of Duolink foci (green) and DAPI (blue) are shown, indicating protein-protein interaction within 40 nm, with a single XY DIC image. (D) Representative maximum intensity projection confocal images of the PP2A-Aα-p130 Duolink interaction (green) in WT and Cd53^−/− HSCs at baseline and after G-CSF treatment. Loss of CD53 impairs the interaction between p130 and the PP2A phosphatase structural subunit, PP2A-Aα, (E) quantified as the number of foci per HSC (n = 55-63 cells per group) in 2 independent experiments. (F) Representative maximum intensity projection confocal images of the PP2A-Aα-p130 Duolink interaction (green) in naïve HSCs and HSCs after 4 days of culture in StemSpan, with 100 ng/mL TPO and 100 ng/mL SCF, and (G) quantified as the number of foci per HSC (n = 42-57 cells per group) in 2 independent experiments. Notably, although the number of foci is not an exact count of the total interactions per cell, it provides a relative measure between groups. Error bars represent mean ± SEM. All scale bars are 5 μm. ∗∗P < .01; and ∗∗∗∗P < .0001 by an unpaired Student t test. DAPI, 4',6-diamidino-2-phenylindole.