Fig. 4

TROJAN associated with the NKRF protein in ER+ breast cancer. a Schematic diagram of the RNA pull-down assay combined with quantitative proteomics. b RIP assay and the subsequent qRT-PCR assay. The relative quantification of TROJAN in MCF7 RNA-protein complexes immunoprecipitated with IgG or antibodies against four potential interacting proteins (TAF15, HNRPK, NKRF and PTBP3). n = 3 independent experiments. c Confocal RNA fluorescence ISH and immunofluorescence images showing the colocalization of TROJAN and NKRF in MCF7 cells. Scale bar: 10 μm. n = 3 independent experiments. d Schematic representation of Flag-tagged full-length human NKRF and NKRF deletion mutants. e The anti-Flag Western blot image showing NKRF full-length or deletion mutant expression in HEK293T cells. f RIP assay and the subsequent qRT-PCR assay performed in HEK293T cells ectopically expressing the control vector (Vec), full-length Flag-tagged ZMYND8 and ZMYND8 deletion mutants. The relative quantification of TROJAN expression in RNA-protein complexes immunoprecipitated with a Flag antibody. n = 3 independent experiments. g, h Luciferase reporter assay detecting the activity of the NF-κB pathway during NKRF over expression or TROJAN knockdown with or without TNFα treatment (20 ng/mL for 6 h). n = 3 independent experiments. i In vitro growth of MCF7 cells expressing TROJAN and/or NKRF shRNA. n = 3 independent experiments. j Western blot images showing the interaction of RELA with NKRF during TROJAN knockdown. n = 3 independent experiments. Unpaired t test, **p < 0.01, ***p < 0.001 and NS: not significant