Fig. 5

MNX1-AS1 enhances PKM2 nuclear translocation in response to EGF. a HepG2 cells were infected with lentiviruses containing control shRNA (sh-ctrl) or shRNA targeting MNX1-AS1. Fluorescence in situ hybridization of MNX1-AS1 followed by immunofluorescence staining of PKM2 and importin α5. Cell nuclei were decorated by DAPI. b and c Co-immunoprecipitation analysis conducted in HepG2 cells bearing either sh-ctrl or sh-MNX1-AS1 lentiviruses. Cell lysates were immunoprecipitated by antibodies against PKM2 (C) or importin α5 (D) with potential interactions determined by Western blotting. d Mammalian two-hybrid assays were used to measure interactions between PKM2 and importin α5 in the presence and absence of endogenous MNX1-AS1. The indicated combinations of pBIND/pACT plasmids were transfected into HepG2 cells stably expressing either sh-ctrl or sh-MNX1-AS1 before measuring relative luciferase activity (top; a proxy for the interaction between proteins). Western blotting verified the presence of the transfected fusion proteins (bottom). e-h Huh7 cells infected with sh-ctrl or sh-PKM2 lentiviruses were subjected to infection of pCDH control or pCDH-MNX1-AS1 lentiviruses. ECAR measurements using Seahorse XF assays (E and F), Extracellular lactate production (G) and Glucose uptake (H) were measured. The glycolysis rate were calculated as: (Maximum ECAR before Oligomycin injection)-(Last ECAR before Glucose injection). i-k HepG2 cells were infected with lentiviruses containing sh-ctrl or sh-MNX1-AS1. Cells were with and without EGF (100 ng/ml) treatment before subcellular fractions. Nucleus PKM2 were assessed by Western blot analysis (I). Cells from (I) were subsequently subjected to immunofluorescence staining of PKM2 (J), and nucleus PKM2 was measured using ZEN imaging software (K). A, B, C, I and J are represent of three independent experiments. D-H and K are mean ± SD; n = 3 independent experiments, two-way ANOVA with Bonferroni’s multiple comparison post-test, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001