Gapdh sirna

The siRNAs were purchased from GenePharma Corporation (Shanghai, China). The sequences are as follows: siNC: 5′-UUCUCCGAACGUGUCACGUTT-3′; GAPDH siRNA:5′-UGACCUCAACUACAUGGUUTT-3′; galectin-3 siRNA:5′-GCUCACUUGUUGCAGUACATT-3′; HPMECs were seeded into 10 cm culture dishes and incubated for 24 h. Then, the 70–80% confluent HPMECs were transfected with negative control group—siNC, positive control group—GAPDH siRNA and target galectin-3 siRNA using Lipofectamine 3000 (Thermo Fisher, USA) according to the manufacturer’s instructions. The final concentration of the siRNA solution was 50 nM.

GAPDH siRNA (Thermo Scientific, Loughborough, UK; sequences used were sense 5′ UGG UUU ACA UGU UCC AAU AUU 3′, antisense 5′ Phosphate U AUU GGA ACA UGU AAA ACC UU 3′) was mixed in RNAse-free water with varying concentrations of either SMoC, PySSCH₂CH₂CO-RRRR-NH₂, 9 (R4SSPy) or PySSCH₂CH₂CO-RRRRRRRR-NH₂, 10, (R8SSPy), (Peptide Synthetics, Ltd, UK) in the molar ratios of 1/1, 1/5, 1/10, 1/20 and 1/50. The final siRNA concentration was 17 μm in 100 μL of water (1.7 nmol) in each case. The complexes were incubated for 30 min at room temperature. The samples were diluted with 2× RNA loading dye (Fermentas, now Thermo Scientific) containing ethidium bromide (composition 95% formamide, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 0.025% ethidium bromide, 0.5 mm EDTA) and analyzed by gel electrophoresis on a 1% agarose gel prepared using 10× MOPS buffer [0.4 m MOPS (pH 7), 0.1 m sodium acetate, 0.01 m EDTA (pH 8)], loading 25 μL of sample into each well. The gel was run at 5V/cm till the blue dye had migrated 2/3 of the gel. The gels were analyzed by UV illumination and the ethidium bromide bands quantified using the ImageJ software (NIH, Bethesda, MD, USA). The EtBr intensities were plotted, a dose–response curve fitted and EC₅₀ values calculated using the Origin software (OriginLab, Northampton, MD, USA).

Cholesterol was purchased from Sigma Aldrich, while distearoyl-sn-glycerol-3-phosphocholine (DSPC) and PEG2000-DMG were obtained from Avanti Polar Lipids. Dual HeLa and Caco-2 cells were purchased from American Type Culture Collection (Manassas, VA). Porcine pepsin, pancreatin, bile salts, and mucin type II were obtained from Sigma (St Louis, MO). Dulbecco’s Modified Eagles Media (DMEM), trypsin, penicillin/streptomyocin, phosphate buffered saline (PBS), fetal bovine serum (FBS), and GAPDH siRNA were purchased from Thermo Fisher (Waltham, MA). Corning BioCoat HTS 1 µm porous support Transwell plates, basal seeding medium (BSM), Corning EnteroSTIM enterocyte differentiation medium (EDM), and MITO + serum extender were obtained from VWR (Radnor, PA). Anti-firefly luciferase siRNA (siLuc) was purchased from Dharmacon (Lafayette, CO).