ESRP2 Knockdown in Huh-7 Cells

The Huh-7 cell line (300156, CLS, Eppelheim, Germany) was cultured in Dulbecco’s Modified Eagle’s Media (DMEM) high glucose (D6429, Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were cultured in a humidified chamber at a constant 37 °C and 5% O₂. The knockdown of ESRP2 expression was performed using PepMute siRNA Transfection Reagent (SL100566, SignaGen Laboratories, Rockville, MD, USA). Cells were seeded at a density of 200.000 cells/well on a 6-well plate, where they reached approximate 50% confluence 24 h after seeding. Cells were transfected using the protocol provided by the manufacturer with 30 nM concentration of siRNA concentration. The cell medium was changed 24 h after transfection with complete medium, and cells were cultured for an additional 48 h before RNA extraction. siRNA sequences used for silencing were: siRNA-ESRP2 5′-GACUUAAUCCUCCUAGUUUtt-3′ [68 (link)] and siRNA-negative 5′-AGGUAGUGUAAUCGCCUUGtt-3′ (Non Specific Control 47% GC, Eurofins Genomics, Ebersberg, Germany) (siMAX siRNA, Eurofins Genomics, Ebersberg, Germany). Silencing was performed in three independent biological triplicates.