Figure 1: Disruption of hepatic architecture in HepaRG cells following 24 hours APAP treatment (a–c).

Characteristic hepatocyte (H)/cholangiocyte (Ch) in vivo-like hepatic cord phenotype in control cultures is progressively lost with increasing APAP concentration. This is demonstrated in phase-contrast images (a), with (b) Transferrin co-localization (green; Hoechst nuclear counter-stain, blue), a highly-specific marker of mature hepatocytes, and (c) Strong F-actin/Phalloidin staining (yellow) reveals distinct pericanalicular-cytoskeletal/TJ-associated F-actin bands (red arrows) in control cells and with decreased staining at low-dose (5 mM) APAP (arrowheads); and progressively less intense staining at higher APAP dose; consistent with a direct effect of APAP/and or APAP reactive metabolites on actin structures. (d) Confocal microscopy demonstrated reduced intensity (green immunofluorescence staining) of the hepatic tight junction-associated structural protein ZO-1, with increasing APAP. The ‘chicken wire’ network of delineated by ZO-1, progressively decreases, as the hepatic cord phenotype is compromised. (e) Representative TEM images: Left panel shows typical ultrastructure in untreated controls, showing numerous mitochondria (Mt) with tight junctions (arrows), which seal bile canalicular lumen (BC) formed between two adjacent hepatocytes. At 5 mM APAP, although discrete TJ structures were not visible, an ‘electron-dense’ perimeter surrounded hepatocytes (red arrowheads), adjacent cholangiocytes (Ch) are shown. At 10–20 mM APAP, TJs were not present, with cells commonly exhibiting necrotic (NH) or apoptotic appearance, with Type-I blebbing, and pseudomembranous structures (whorls) representing autophagolysomes; altered mitochondria shape was evident, although retaining some dense mitochondrial granules (DMGs). Scale bars: 100 μm (a–c); 50 μm (d); and as indicated in TEM micrographs. Nucleus (N), nucleolus (nu).