LDB1 promotes PU.1 expression and chromosomal looping between PU.1 gene regulatory elements. (A) Western blot confirming reduced LDB1 expression upon induction of LDB1 knockdown in 2 THP-1 lines carrying different LDB1 shRNA constructs in comparison with THP-1 cells harboring the SC4 shRNA. Cells were Dox treated for 72 hours. (B) PU.1 expression was downregulated upon LDB1 knockdown as seen by western blotting with an anti-PU.1 antibody. VCP served as loading control, and the molecular protein mass is indicated (A-B). (C-D) 4C-seq profiles displaying spatial contacts of the PU.1 URE (URE VP) VP within the PU.1 SubTAD in induced THP-1 cells carrying Dox-responsive LDB1 shRNA or SC4 shRNA constructs. (C) Cells were Dox treated for a total of 36 hours, whereby PMA and VD3 were added 12 hours prior to harvest to trigger differentiation. (D) Cells were first treated with PMA + VD3 for an initial 24 hours followed by a treatment with PMA + VD3 and Dox for additional 48 hours. Green arrows at the top indicate the direction of VP interaction with the PU.1 promoter. Green arrowheads at the bottom represent position of the PU.1 promoter (PP). Dashed blue lines represent heights of the promoter interacting with the used URE VP. Each plot (C-D) is representative of at least 2 independent shRNAs against LDB1.