Breakdown of the PKR-ATF4 or PERK-ATF4 signaling. (A) In silico knock-out of PKR and PERK. The essential model includes PKR, PERK and Ca2+ pathways as the downstream responders to palmitate stimulation. In silico simulation is performed on the essential model and compared with a model wherein either PKR or PERK is knocked-out. (B) and (C) Real-time quantitative RT-PCR analysis: (B) Knockdown of PKR and PERK gene expression and (C) knockdown of PACT gene expression. HepG2 cells were grown and transfected with siRNA of PKR, PERK, and PACT or negative control siRNA for 24 hr as described in the Methods. The cells were incubated for 3 and 6 hr in media containing BSA or BSA-complexed palmitate. The total mRNA were extracted and transcribed into cDNA. mRNA expression levels were quantified by real-time quantitative RT-PCR analysis and displayed as fold-changes as compared to the control samples treated with BSA in the bar graph. Data are presented as mean ± SD from three independent experiments: *p < 0.05 and **p < 0.01 vs the control at each time point. A line indicates comparison between the 2 bars connected by the line.