3C and ChIRP assays measuring the effect of LOUP on chromatin looping. (A) Schematic diagram illustrating potential 3C interactions between the URE and genomic viewpoints surrounding the PU.1 locus. Included are restriction recognition sites of ApoI used in the assay. -8 kb and -4 kb are distances from the PrPr in kilobases. (B) 3C-qPCR TaqMan probe-based assay comparing cross-linking frequencies at chromatin viewpoints. The U937 cell clone L2a, carrying a LOUP-homozygous indel that does not alter the recognition pattern of ApoI (supplemental Figure 4D), was used to compare with nontargeting control (sgControl, N1). (C) qRT-PCR assay evaluating levels of LOUP RNA and control GAPDH captured by biotinylated LOUP-tiling and LacZ-tiling probes using ChIRP. (D) ChIRP assay assessing LOUP occupancies at the URE, the PrPr, and ACTB promoter. LOUP-tiling oligos were used to capture endogenous LOUP in U937 cells. LacZ-tiling oligos were used as negative control. Error bars indicate SD (n = 3). *P < .05; ****P < .0001. Int, intergenic; n.d., not detectable.