Fig. 5.

Bupivacaine phenocopies PFKM loss to potentiate the effect of bevacizumab. (A) Pathway enrichment analysis using overlapping up- and downregulated genes in GBM cells with silenced PFKM and treated with bupivacaine (BUPI) (log₂fc ≥ 2 and adjusted P ≤ .05) de-regulated mitosis and cell division among the top 10 scoring hits. (B) GBM cells show higher sensitivity to bupivacaine compared to normal human astrocytes (NHA). GI₅₀ was assessed at 72-hour post-treatment. (C) Bupivacaine treatment (24 hours) of GBM cells decreases PFKM and KIF11 protein expression. Loading control: GAPDH. (D) Bupivacaine treatment (24 hours) impairs GBM cell invasion. Quantification was performed by normalizing to 0 hours. Scale bar = 100 µm. (E) Bupivacaine treatment of GBM cells lowers mitotic index (% of ^pH3-positive cells). Nuclei were counterstained with DAPI. (F) Bupivacaine treatment (24 hours) of GBM cells impairs IR-induced DDR activation (^PATM and ^PNBS1) Loading control: TUB. (G) Comet assay shows increased DSB formation in GBM cells treated with bupivacaine for 24 hours (~200 tails per condition was measured). (H) Bupivacaine treatment (24 hours) of GBM cells increases micronuclei (MN) formation. MN quantification was based on ~1000 cell count and presented as a number of identified MN per 100 cells. (I) FACS-based quantification of Annexin V-positive cells (%) shows higher apoptotic rate in GBM cells treated with bupivacaine for 24 hours.

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