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. 2022 Nov 14;33(21-22):1197–1212. doi: 10.1089/hum.2022.109

Figure 4.

Figure 4.

Rescue of fibrotic phenotype in an siRNA-screening assay in human hepatic stellate cells, using AAV9-SLRSPPS. (a) An expression construct encoding human TGFBR1 under the control of a CMV promoter was codon-usage optimized (“codon-opt”) to escape targeting by the anti-TGFBR1 siRNA pool (siTGFBR1) and packaged into AAV-SLRSPPS. Green letters indicate mismatches between the four siRNAs contained in the pool and the codon-optimized TGFBR1 sequence contained in the AAV expression construct. Hepatic stellate cells were then reversely transfected with siRNA controls or siTGFBR1 and simultaneously transduced by adding different amounts (1,000, 3,000, 10,000, or 30,000 vg/cell) of either AAV-GFP or noncoding “AAV-stuffer” controls or “AAV-TGFBR1-codon-opt.” Collagen-I expression was induced by TGFβ1 (10 ng/mL) and visualized 3 days later by (b) immunocytochemistry and (c) quantified using a high-content image analysis script detecting nuclei and collagen fibrils. The dashed background indicates collagen-I control levels, defined by the range observed for siCtrl ± SD under AAV-stuffer conditions, for visual guidance. n = 8 wells/condition, mean ± SD. ***p < 0.001 as indicated. ###p < 0.001 relative to AAV-stuffer. siRNA, small interfering RNA.