Skip to main content
. 2022 Feb 20;12(2):e654. doi: 10.1002/ctm2.654

FIGURE 7.

FIGURE 7

CCCTC‐binding factor (CTCF) binds directly to PTGS2 antisense NF‐κB1 complex‐mediated expression regulator RNA (PACERR) and recruits E1A binding protein p300 (EP300) to the promoter region of prostaglandin‐endoperoxide synthase 2 (PTGS2) in a PACERR‐dependent manner. (A) RNA immunoprecipitation (RIP) was performed using a CTCF‐specific antibody. Eluted CTCF‐binding RNAs were reverse transcribed, and quantitative polymerase chain reaction (qPCR) was performed with primers specific for PACERR. Normal rabbit IgG (IgG) was used as a negative control. Data are shown as the results from three independent experiments. (B) Imaging of RNA pull‐down experiment followed by silver staining. WB validation of CTCF proteins pulled down with PACERR is shown at the bottom. (C) Overview of CTCF domains and mutant control. The predicted RNA‐protein binding sites of PACERR and CTCF are shown at the bottom. (D) THP‐1 cells were infected with negative control‐Flag lentivirus or lentiviral virus encoding Flag‐tagged CTCF transcripts with or without the predicted RNA binding region (CTCF‐Flag or CTCF‐Mutant‐Flag) and stimulated into tumour‐associated macrophage (TAM) cell models before RIP assays. Whole‐cell lysates were subjected to immunoprecipitation with the indicated antibodies. Eluted RNAs were reverse transcribed, and qPCR was performed with primers specific for PACERR. Normal rabbit IgG was used as a negative control. (E) The results of co‐immunoprecipitation (Co‐IP) in THP‐1‐derived TAMs. Normal rabbit IgG was used as a negative control. (F) Association of EP300 with the promoter region of PTGS2 analysed by ChIP‐qPCR in THP‐1‐derived TAMs (shNC/ shCTCF/ shPACERR/ shCTCF & shPACERR). (G) Association of H3K27ac with the promoter region of PTGS2 and PACERR in THP‐1‐derived TAMs (shNC/ sh1 EP300/ sh2 EP300) analysed by ChIP‐qPCR. (H) Western blot analysis of PTGS2 protein expression in THP‐1‐derived TAMs after EP300 knockdown. (J) qPCR analysis of PACERR RNA level in THP‐1 derived TAMs after EP300 knockdown. The image is representative of three independent experiments. shNC means that cells were transfected in negative control plasmids. * p < .05; ** p < .01; *** p < .001; **** p < .0001. “ns” means no statistically significance