Figure 6.

Binding of lncNEAT1 and NPM1-SP1 complex inhibits MAN1A1 transcription

(A) Schematic summary of biotin-labeled lncNEAT1 pull-down protocol.

(B) LC-MS/MS analysis of lncNEAT1 binding proteins by Venn diagram: lncNEAT1 pull-down protein in HESCs and HESCs-ID (left); transcription-associated proteins and nuclear proteins in upregulated HESCs-ID (right).

(C) GO analysis of 47 intersecting proteins.

(D) Protein-protein interaction network analysis of lncNEAT1-binding proteins associated with decidualization and transcription factors of MAN1A1 by STRING database.

(E) lncNEAT1 pull-down analysis of NPM1 (nucleophosmin) expression in HESCs after transfection with lncNEAT1-siRNA or lncNEAT1-cDNA.

(F) RIP (RNA-binding protein immunoprecipitation) analysis of lncNEAT1 levels in HESCs after transfection with NPM1-siRNA.

(G) Demonstration of molecular docking of lncNEAT1 and NPM1. Yellow dashed line: hydrogen bonds.

(H) SP1 binding motif sequence logo from JASPAR database.

(I) ChIP (Chromatin immunoprecipitation) analysis of the binding site between SP1 and the promoter of MAN1A1.

(J and M) Western blot analysis of MAN1A1 expression in HESCs after transfection with SP1-siRNA or NPM1-siRNA.

(K) Molecular docking of NPM1 and SP1. Yellow dashed line: hydrogen bonds; pink dashed line: salt bridges.

(L) Immunoprecipitation analysis of NPM1 and SP1 in HESCs transfected with scramble or NPM1 siRNA. IP: protein pulled down with an anti-NPM1 antibody. IB: detection of SP1 expression by antibody.

(N) ChIP analysis of SP1 and the MAN1A1 promoter binding in HESCs after transfection with lncNEAT1-siRNA and lncNEAT1-cDNA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.