Fig. 4: KIMAT1 directly interacts with DHX9 and NPM1.

a Schematic representation of RAP-MS. RBP = RNA-binding protein. b KIMAT1 pull-down with biotinylated antisense probes after UV crosslinking followed by immunoblotting analysis showing that DHX9 and NPM1 are direct KIMAT1 binding partners. A sample treated with RNase A or pull-down of the housekeeping gene UBC were used as negative controls. c Immunoblotting showing DHX9 and NPM1 pull-down efficiency for the experiment in d. d KIMAT1 is enriched by CLIP assay using DHX9 or NPM1 specific antibodies. e, f DHX9 and NPM1 sequential immunofluorescence (green) and KIMAT1 smFISH (Red) in different lung adenocarcinoma cell lines. Scale bar, 75 μm. g Schematic representation of human KIMAT1, its antisense (AS) and various deletion constructs generated to detect DHX9 or NPM1 binding regions in KIMAT1 sequence. Fragment sizes were confirmed by PCR (bottom panel), and binding of each fragment to DHX9 or NPM1 determined via pull-down of biotinylated-labeled RNA fragments and immunoblotting (IB) (top panels) with the indicated antibodies. BR binding region; nt nucleotide. h RNA immunoprecipitation assay. H1299 cells were transfected with FLAG-tagged full-length DHX9 (FL), or DHX9 without the two double-stranded RNA-binding domains (dsRBD Del) or Empty vector (NT), crosslinked and subjected to immunoprecipitation using FLAG-specific antibody. (Top) DHX9-bound KIMAT1 was analyzed by qPCR. (Bottom) Immunoblotting showing immunoprecipitation’s efficiency. i RNA immunoprecipitation assay. H1299 cells were transfected with FLAG-tagged full-length NPM1 (FL), or NPM1 without the RNA-binding domain (DRBD Del) or Empty vector (NT), crosslinked and subjected to immunoprecipitation with a FLAG-specific antibody. (Top) NPM1-bound KIMAT1 was analyzed by qPCR. (Bottom) Immunoblotting showing immunoprecipitation’s efficiency. j Immunoblotting showing DHX9 KO and NPM1 KO in two different CRISPR/Cas9 clones. k 3D invasion assay and quantification of the spheroid area in the two DHX9 and NPM1 CRISPR/Cas9 clones in j compared to control cells (Cas9 only). Scale bar, 500 μm. l immunoblotting showing downregulation of DHX9 and NPM1 upon transfection of H1299 cells with two KIMAT1 GpRs. a, b, e, f, g Representative results or images of two biological replicates. Mean ± S.D (n = 3). p values were calculated by two-tailed Student’s t test.