Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

Galectin-9 (Gal9) is known for its cytoplasmic roles in vesicular damage responses, yet its active functional roles in gene delivery remain unclear. Here, we investigated Gal9 dynamics in electrotransfection (ET), a non-viral gene delivery method widely used for plasmid DNA (pDNA) and mRNA transfection. Confocal imaging revealed unexpected nuclear accumulation of Gal9 after pDNA ET. Gal9 colocalized with SC35, a biomarker of nuclear speckles, and immunogold TEM confirmed its enrichment within those electron-dense, subnuclear domains following pDNA ET. Functionally, Gal9 knockdown markedly reduced ET efficiency and reporter gene expression, whereas overexpression enhanced both. The timing and the extent of Gal9-pDNA association varied with delivery method and cargo type, being prominent for DNA but minimal for mRNA. Across different cell types, endogenous Gal9 expression correlated with ET efficiency. These findings revealed previously unrecognized, active functional roles of Gal9 in organizing exogenous DNA within transcriptionally active regions of the nucleus and regulating transgene expression following their non-viral delivery.