Fig. 4: PSD3 downregulation lowered intracellular neutral fat content in primary human hepatocytes cultured in 2D and 3D spheroids.
From: PSD3 downregulation confers protection against fatty liver disease
Downregulation of endogenous PSD3 expression using siRNA in primary human hepatocytes cultured in 2D and 3D. For 2D culture, after attachment of cells in collagen-coated plates, cells were incubated with regular growth medium supplemented with 10 µM OA and transfected with negative control SCR siRNA or PSD3 siRNA for 48 h. a,b, Intracellular neutral fat content was visualized by ORO staining and quantified by Biopix iQ software v.2.3.1 in primary human hepatocytes carrying (a) the 186L allele (n = 5) or (b) the 186T allele (n = 4). Average PSD3 downregulation efficiency was ~80% as evaluated by quantitative retro transcription PCR analysed by the 2−ΔΔCt method and western blotting for both donor types. For 3D culture of primary human hepatocytes, spheroids were generated by seeding 2,000 cells per well in a 96-well round-bottom flask, along with transfection mix in 100 µl of medium. For the generation of 186T allele spheroids, 5 nM of FMK-Z-VAD was added to support spheroid formation. After 24 h, additional growth medium was added to give a total volume of 200 µl per well. Fifty per cent of the total media was replenished with fresh media every 48 h. After 7 days of formation, spheroids were collected and 8-µM sections were subjected to ORO staining to visualize intracellular neutral fat content. c,d, Nuclei were stained with 4,6-diamidino-2-phenylindole and ORO staining was quantified by Image J, normalized to number of nuclei of primary human hepatocyte spheroids carrying (c) the 186L allele (n = 4) and (d) the 186T allele (n = 4). Average gene knockdown efficiency was ~50–60% as evaluated by quantitative retro transcription PCR analysed by the 2−ΔΔCt method for both donor types. Cellular ATP levels (marker of viability) remained stable between the negative control SCR and PSD3 siRNA groups. Data are shown as mean ± s.d. of the reported independent experiments. Two-sided P values were calculated by Mann–Whitney non-parametric test comparing SCR siRNA versus PSD3 siRNA. RU, relative units; CNX, calnexin.
