Fig. 4.

Nuclear PFKM regulates DNA damage response to ensure genome stability. (A) Subcellular fractionation followed by immunoprecipitation confirms nuclear localization of ectopically expressed PFKM. Cytosolic marker: GAPDH; nuclear marker: Lamin B1 and H3. (B) PFKM accumulates in the nucleus after leptomycin (NESi; 2 nM) treatment of GBM cells. (C) GBM cells with silenced PFKM show higher sensitivity to IR (2 Gy) as assessed by cell proliferation measurement at 72-hour post-IR. (D) PFKM-silencing impairs IR-induced DDR activation (^PATM and ^PNBS1) in GBM cells. Loading control: tubulin (TUB). (E) PLA assay shows PFKM::NBS1 interaction in the nucleus of GBM cells. Fluorescence intensity peaks (right graph) marked the co-localization (proximity) of PFKM and NBS1 along the dotted line across the selected cell. Scale bar = 10 µm. (F) Representative images of comet tails in GBM cells transfected with siCONT or siPFKM. DSBs quantification was determined 48-hour post-IR by measuring comet tail length (~200 tails per condition was measured). Scale bar = 100 µm. (G) Homologous recombination (HR) repair (GFP-positive cells above diagonal line) rates are lower in GBM cells with silenced PFKM. (H) FACS-based quantification of Annexin V-positive cells (%) shows increased apoptosis in GBM cells with silenced PFKM. (I) Microscopy analysis of GBM cells with silenced PFKM shows higher micronuclei (MN) formation using DAPI staining. Quantification was based on ~1000 cells and presented the number of micronuclei per 100 cells. Images of GBM cells with MN (arrows). Scale bar = 5 µm.

Significance is determined by t test (B, C, and F–I), and data are shown as mean ± SEM (*P < .05, **P < .01, ***P < .001).