PU.1 is dispensable for stabilization of the PU.1 SubTAD. (A) PU.1 western blot with an antibody against PU.1 (top) or VCP (bottom), as loading control, of THP-1 cells carrying either the SC4 or the PU.1 shRNA. The cells were first treated with PMA and VD3 for 48 hours, and then Dox was added for another 24 hours, again in the presence of PMA and VD3. The total differentiation time was therefore 72 hours, and the PU.1 knockdown was induced late during differentiation. The molecular protein mass is indicated. (B-C) 4C-seq profiles displaying spatial contacts of the PU.1 promoter (PP VP; B) or the URE (URE VP; C) VP within the SubTAD in induced THP-1 cells carrying Dox-responsive shRNA constructs against PU.1 or the SC4 control. The cells were Dox and PMA + VD3 treated as in panel A. Green arrows at the top indicate the direction of VP interaction with the URE or the PU.1 promoter, respectively. Green arrowheads at the bottom represent position of the PU.1 promoter (PP) and the URE. Dashed blue lines represent heights of the URE or promoter interacting with the used VP.