Fig. 3.

PFKM-KIF11 interaction is critical for invasion and mitosis in glioblastoma. (A) Pathway enrichment analysis using genes downregulated (log₂fc ≥ 2 and adjusted P ≤ .05) after PFKM-silencing in T10 and T4121 GBM cells. (B) Confocal microscopy confirms ectopically expressed PFKM-GFP to localize to mitotic spindle. Nuclei counterstained by DAPI. Scale bar = 3 µm. (C) Immunoblot analysis of T4121 cells shows increased PFKM level in mitosis. Asynchronous cells (AS); cells synchronized at G₂ phase, released and collected at indicated time points. ^PH3 as mitotic marker. Loading control: GAPDH. (D) Immunoprecipitation of PFKM followed by immunoblot analysis confirms its interaction with KIF11 in the cytosolic fraction (Cyto) and not in the nuclear fraction (Nuc). (E) GBM cells with silenced PFKM show decreased KIF11 protein expression. Loading control: GAPDH. (F) MG132 (10 µM; proteasomal inhibitor) treatment (6 hours) restores KIF11 protein levels in GBM cells with silenced PFKM. Loading control: GAPDH. (G) PFKM loss in GBM cells leads to a mitotic catastrophe. Tubulin, H3Ser10 (red). Nuclei were counterstained with DAPI. Scale bar = 5 µm. (H) Ectopic PFKM expression rescues KIF11 degradation in PFKM-silenced GBM cells. Loading control: GAPDH. (I) Ectopic PFKM expression restores invasion capacity of PFKM-silenced GBM cells. Scale bar = 100 µm. (J) Ectopic PFKM expression rescues mitotic phenotype in PFKM-silenced GBM cells as shown by FACS analysis.

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