Fig. 4: HULC interacts with the glycolytic enzyme PKM2.

a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads^® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (***P < 0.001, by two-sided Student’s t test). f The molecular interaction between PKM2 and HULC was to measure the K_d value. g The PK activities of HepG2 cells with HULC knockdown (left panel) or overexpression (right panel) were measured by a PK activity assay kit and compared with the same number of corresponding control cells. Data represent the mean ± s.d. (n = 4 independent experiments, *P < 0.05, by two-sided Student’s t test). h Western blotting of PKM2 in cells with or without crosslinking. In vivo crosslinking was performed by incubating the cells with 1 mM disuccinimidyl suberate (DSS) at room temperature for 30 min. i HULC was knocked down or overexpressed in HepG2 cells. The levels of p-PKM2 (Y¹⁰⁵) and PKM2 in the cell lysates were detected by western blotting. Band intensities were measured by ImageJ. Data represent the mean ± s.d. of triplicate independent experiments (**P < 0.001, ***P < 0.001, by two-sided Student’s t test). Source data are provided as a Source data file.