Fig. 3: ATF3 could interact with ZNFTR and the promoter of ZNF24.

A ATF3 showed potential mutual interaction with ZNFTR and the promoter of ZNF24 through intersecting from the catRAPID and JASPAR database. B RNA interaction profile showed the putative binding position between ZNFTR and ATF3. C RIP assay was performed to verify the interaction between ATF3 and ZNFTR with anti-ATF3 antibody in BxPC-3 and PANC-1 cells. The co-precipitated RNAs were detected by qRT-PCR. Anti-SNRNP70 rabbit polyclonal antibody co-precipitated with U1 snRNA was used as a positive control. The qRT-PCR products were separated by 2% agarose gel electrophoresis. D The RNA-pulldown assay was conducted to demonstrate the interaction between ATF3 and ZNFTR by biotinylated ZNFTR probe in BxPC-3/PANC-1 cells. Then the pulldown protein was detected by immunoblot assay with anti-ATF3 and anti-HuR antibody. RNA from the 3′ untranslated-region (UTR) of the androgen receptor (AR), involving UC-rich HuR binding areas, was used as a positive control. E Biotinylated ZNFTR full length, antisense, and part of it were conducted, and RNA-pulldown assay was performed to verify the region of ZNFTR interacted with ATF3. F The schematic diagram exhibited two predicted binding sites between ATF3 and promoter of ZNF24. G ChIP assay was conducted to verify the interaction between ATF3 and promoter of ZNF24. The qRT-PCR products were separated by 2% agarose gel electrophoresis. H The vectors containing the wild type (WT) or three mutants (MUT) of ATF3 binding sites were co-transfected with empty vector or pcDNA-ATF3 in BxPC-3 and PANC-1 cells to perform luciferase reporter assay. WT: wild type, MUT-1: Site1 mutated, MUT-2: Site2 mutated, MUT-3: Both Site1 and Site2 mutated. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as *P < 0.05 and **P < 0.01. n.s means the difference was not significant.