identify the secreted gene in Casp8-silenced mouse hepatocyte cell line

Hepatocyte caspase-8 is elevated in MASH and promotes fibrosis independently of apoptosis changes. To investigate how hepatocyte caspase-8 might drive liver fibrosis progression in MASH, we focused on activated hepatic stellate cells (HSCs), which are the primary source of collagen-producing myofibroblasts and central players in MASH fibrosis. To assess a potential link between hepatocyte caspase-8 and HSC activation, we used an ex vivo model where primary murine HSCs were cultured with conditioned media from siCasp8-treated or control primary hepatocytes. Supporting our hypothesis, HSC activation markers were reduced in HSCs exposed to conditioned media from Casp8-silenced AML12 hepatocytes compared to controls. To pinpoint caspase-8-dependent secretory proteins that could activate HSCs, we conducted RNAseq on these cells, identifying secretory genes reduced in Casp8-silenced AML12 hepatocytes. Overall design: Scrambled control siRNA and Casp8 siRNAs were transfected into AML12 cells using Lipofectamine RNAiMAX (Life Technologies) at a final concentration of 40 nM siRNA in 24-well plates, following the manufacturer's instructions. After 48 hours, the cells were collected for RNA sequencing. Gene expression profiling was performed using RNA-seq data from four biological replicates. Comparative analysis of gene expression focused on differences between scrambled (scr) control and siCasp8-treated AML12 hepatocyte cells.