Levels of COX-2 mRNA (A) and protein (B) were measured by RT-qPCR and Western blotting in control-scrambled siRNA, siRNA-CTCF, and siRNA-lncRNA transfected HMECs 72 hr post transfection. (C) Schematic representation of the monocyte-macrophage system used in this study. The human monocyte cell line, U937, was differentiated to macrophages with PMA and induced to express high levels of COX-2 by LPS stimulation. (D and E) COX-2 mRNA (D) and PACER (E) levels were measured by RT-qPCR in U937 lines carrying stably integrated control shRNA or shRNA-PACER expressing constructs before and after differentiation into macrophages. Values were normalized using 18S rRNA, TBP, and ß-globin genes. (F) COX-2 protein expression analyzed by Western blotting in the same experiment. (G and H) COX-2 ncRNA and mRNA levels were measured in a time course of LPS stimulation in control U937 (black bars) and PACER knockdown U937 macrophages (white bars). Values are relative to maximal COX-2 expression in control U937 cells after 6 hr LPS stimulation. (I) COX-2 protein expression analyzed by Western blotting in the same experiment. TBP served as a control. (J) Subcellular localization of COX-2 mRNA, PACER lncRNA and controls: nuclear intronic RNA and exclusively cytoplasmic actin mRNA. Relative levels in each fraction were measured by RT-qPCR and plotted such that they add up to 100%. CH, chromatin-bound fraction, N, nucleoplasm, CE, cytoplasm.