X tremegene 9 transfection reagent kit

HeLa cells were seeded in 24-well plates at 9 × 10⁴ cells/well in triplicate per condition. The cells were transfected with 250 ng of pGL3-basic or human VCAM1 promoter, with or without 40 ng of SOX18, SOX17, SOX7, and SOX18^RaOp for 24 h. Transfections were performed using the X-tremeGENE 9 Transfection Reagent kit (Roche) as per manufacturer's instructions, with a DNA to X-tremeGENE 9 ratio of 1 μg to 3 μl. After 24 h, the cells were washed twice in PBS and harvested using the luciferase assay reporter system, according to the manufacturer's instructions. Firefly luciferase activity was determined and normalised to protein concentration to control for cell number, pGL3-basic activity subtracted from the VCAM promoter activity, and then made relative to the +VCAM +SOX18 condition, which was set to 1. These values were plotted into GraphPad Prism (version 9.0.0), with the mean ± s.e.m. shown. Statistical significance for pairwise comparisons was assessed using ANOVA, multiple comparisons were corrected using the Šidák correction.

HeLa cells were seeded in 3 × 15 cm dishes per condition at 2.65 × 10⁶ cells/dish, and then transfected for 24 h with the following combination of plasmids: (i) 15 μg pcDNA3.1 glomyc-SOX18 and 15 μg untagged-SOX18; (ii) 15 μg pcDNA myc-SOX18^RaOp and 15 μg untagged-SOX18^RaOp; (iii) 15 μg pcDNA3.1 glomyc-SOX18 and 15 μg untagged-SOX18^RaOp. Transfections were performed using the X-tremeGENE 9 Transfection Reagent kit (Roche) as per manufacturer's instructions, with a DNA to X-tremeGENE 9 ratio of 1 μg to 3 μl.
Cells were then fixed by adding 2 ml of molecular biology grade formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, 50 mM HEPES) in the cells' existing media (20 ml) for 15 min at room temperature with agitation. Fixing was quenched by adding 1.155 ml of 2.5 M glycine solution for 5 min at room temperature. Cells were then scraped and pelleted at 800 g for 10 min at 4°C. Pellets were washed with 10 ml ice-cold PBS, pooled, and then centrifuged at 800 g for 10 min at 4°C. Resuspension of pellets was performed in 10 ml ice-cold PBS supplemented with cOmplete protease inhibitor (Roche), and then centrifuged at 800 g for 10 min at 4°C. Supernatant were removed completely, and the resultant pellets were snap-frozen in dry ice, and then stored at −80°C until analysis.

HeLa cells were seeded at a density of 155 000 cells in 29 mm (D29-20-1.5-N, Cellvis) or 35 mm (P35G-1.5-20-C, Matek) glass coverslip dishes coated with 1% gelatin 24 h prior to transfection. Transfections were performed using the X-tremeGENE 9 Transfection Reagent kit (Roche) to introduce 1–2 μg of plasmid DNA as per manufacturer's instructions, using FluoroBrite DMEM (Gibco) supplemented with 1% GlutaMAX (Gibco) as the low serum transfection media. Cells were incubated at 37°C with 5% CO₂ for 24 h prior to imaging.