Male C57BL/6 mice (Jackson Laboratories, USA) were housed in the animal facility of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. Animals were kept under controlled environmental conditions, in particular in a room with ventilation (16-18 air changes/ hour), relative humidity (45-65 %), controlled temperature (20-24 °C) and light/dark cycle 12:12, and were given water and balanced diet (Nuvital Nutrientes LTDA, Brazil) ad libitum. In the first set of experiments, mice were starved 15-16 hours prior to administration of PAR or 0.9 % sodium chloride (SAL) solution. PAR was dissolved in saline solution, slightly heated and injected (30-37 °C) intraperitoneally at 300 mg/kg body weight. After injection, mice regained free access to food. Mice were euthanized 24 hours following PAR overdosing by exsanguination during sampling under isoflurane-induced anesthesia. Another group of male C57BL/6 mice were administered corn oil (OIL) or CCL4 at a gradually increased dose for 8 weeks. Animals received 3 intraperitoneally injections weekly. The initial dose of CCL4 was 0.25 mg/kg body weight, and there were 0.25 mg weekly increments to the utmost dose of 1.25 mg/kg body weight. Mice were euthanized after completion of OIL or CCL4 treatment by exsanguination during sampling under isoflurane-induced anesthesia. Mouse blood, collected by cardiac puncture, was drawn into a heparinized syringe and centrifuged for 10 minutes at 1503×g, and serum was stored at -20 °C. Livers were snap-frozen in liquid nitrogen with storage at -80 °C. Both studies have been approved by the Committee on Bioethics of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil and all animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”.

Alanine aminotransferase (ALT) (IU/L), aspartate aminotransferase (AST) (IU/L), alkaline phosphatase (ALP) (IU/L) and conjugated bilirubin (mg/dL) values were measured with an automated spectrophotometric Labmax 240 analyzer (Labtest Diagnostica, Brazil) or an automated bench-top dry chemistry analyzer (IDEXX Laboratories Ltd, UK) after appropriate dilution of collected serum samples.

Immunoblot analysis of liver tissue was performed as previously described with slight modifications (Willebrords et al., 2016[38]). In short, liver tissue was homogenized in radioimmunoprecipitation assay buffer containing Halt Protease Inhibitor Cocktail (Thermo Scientific, USA). Homogenates were centrifuged at 14000×g for 20 minutes at 4 °C. Protein concentrations in supernatants were determined using a commercial Pierce BCA Protein Assay kit (Thermo Scientific, USA). Protein lysates of 25 µg were resolved on sodium dodecyl sulphate polyacrylamide stain-free gels (Bio-Rad, USA) under reducing conditions. Separated proteins were transferred to nitrocellulose membranes (Bio-Rad, USA) and blocking was performed with 5 % milk in Tris-buffered saline solution containing 0.1 % Tween 20. Membranes were incubated overnight at 4 °C with primary antibody directed against E-cadherin, β-catenin, γ-catenin, Cx26, Cx32, Cx43, zonula occludens 2 and claudin 1 (Supplementary Table 1) followed by incubation for 1 hour at room temperature with a secondary HRP-conjugated goat anti-rabbit IgG antibody (1:1000) (Dako, USA). Detection of the proteins was carried out by means of enhanced chemiluminescence. For semi-quantification purposes, a normalization method based on total protein loading was used. Stain-free technology introduces the binding of a trihalo compound to tryptophan residues present in the sample. This allows visualization of total protein signals with low background and overcomes the drawbacks of housekeeping proteins (Diller et al., 2021[6]). All cell junction proteins were normalized against total protein loading and expressed as relative alterations compared to SAL-treated and OIL-treated animals for the acute and chronic liver disease model, respectively.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis of liver tissue was performed as explained elsewhere (Maes et al., 2016[24]). Briefly, total RNA was isolated from liver tissue using a GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma, USA) and the On-Column DNase Digestion Set (Sigma, USA) according to the manufacturer's instructions. A NanoDrop ND-100 Spectrophotometer (Thermo Scientific, USA) was used to control purity and quantify RNA. Next, 2 µg RNA was reversely transcribed into cDNA with an iScript cDNA Synthesis Kit (Bio-Rad, USA) using an iCycler iQ (Bio-Rad, USA) followed by cDNA purification using a GenElute PCR Clean-Up Kit (Sigma, USA). cDNA products were quantitatively amplified using Taqman probes and primers specific for target and reference genes (Thermo Scientific, USA) (Supplementary Table 2). After running the samples in duplicate in a QuantStudio PCR System (Thermo Scientific, USA), the system's software was used to calculate quantification cycles. Stable reference genes for normalization were determined by geNorm using the qbase⁺ software (Biogazelle, Belgium) (Vandesompele et al., 2002[35]). The resulting ΔCq values of the test samples were normalized to those of the calibrator samples, yielding ΔΔCq values. Relative alterations (fold change) in RNA levels were calculated according to the 2^(-ΔΔCq) formula (Livak and Schmittgen, 2001[19]). Results were expressed as fold change of ΔΔCq values obtained from SAL-treated and OIL-treated mice.

All data were expressed as mean ± standard deviation (SD). The number of biological replicates (n) and technical replicates (N) are specified for each analysis in the figure legends. Results were statistically processed by 2-tailed unpaired student t-tests and Welch's correction using GraphPad Prism7 software, with probability (p) values of less than or equal to 0.05 considered as significant.

Overdosing mice with the analgesic drug PAR results in excessive accumulation of N-acetyl-p-benzoquinone imine (NAPQI). This triggers the formation of NAPQI-protein adducts, which in turn leads to mitochondrial dysfunction and nuclear DNA damage. Consequently, an overdose of PAR causes massive necrotic hepatic cell death and inflammation within 24 hours (Maes et al., 2016[23]). Measuring serum ALT levels is still considered as the most relevant clinical biomarker to assess hepatocellular injury, including PAR-evoked insults (Maes et al., 2016[23]). PAR overdosing indeed increased serum levels of ALT and AST after 24 hours (^***p ≤ 0.001) (Supplementary Figure 1). Protein and mRNA levels of E-cadherin, β-catenin, γ-catenin, Cx26, Cx32, Cx43, zonula occludens 2 and claudin 1 were measured by immunoblot and RT-qPCR analysis, respectively. Whereas protein levels of β-catenin (^***p ≤ 0.001) and γ-catenin (^*p ≤ 0.05) (Figure 1A(Fig. 1)) decreased, mRNA quantities of E-cadherin increased (^*p ≤ 0.05) (Figure 2(Fig. 2)). Hepatic protein expression levels of Cx26 (^*p ≤ 0.05) and Cx32 (^**p ≤ 0.01) (Figure 1B(Fig. 1)) were negatively affected. The effect of PAR overdosing was not mirrored at transcriptional level as no shifts in mRNA levels of Cx26, Cx32 and Cx43 were observed (Supplementary Figure 2). While liver protein levels of zonula occludens 2 (^*p ≤ 0.05) (Figure 1C(Fig. 1)) declined, mRNA levels of zonula occludens 2 (^*p ≤ 0.05) and claudin 1 (^*p ≤ 0.05) (Figure 2(Fig. 2)) increased following PAR overdosing. Protein levels of E-cadherin, Cx43 and claudin 1 were unaffected upon PAR overdosing (Figure 1A-C(Fig. 1)).

Following biotransformation of CCL4, the noxious trichloromethyl radical is generated in the liver. Trichloromethyl radical triggers various reactions involving free radicals and lipid peroxidation, which burgeons into the onset of cell death, inflammation and liver fibrosis (Yanguas et al., 2016[42]). CCL4 is widely used to trigger liver fibrosis in mice and repeated administration of CCL4 evokes chronic liver disease between 2 and 6 months (Ghallab et al., 2019[9]). Induction of chronic liver disease was evidenced by increased serum levels of ALT (^****p ≤ 0.0001), AST (^***p ≤ 0.001), total bilirubin (^*p ≤ 0.05), and conjugated bilirubin (^****p ≤ 0.0001) after 8 weeks (Supplementary Figure 3). Protein expression levels of E-cadherin (^*p ≤ 0.05) and β-catenin (^***p ≤ 0.001) (Figure 3A(Fig. 3)) were increased in mouse liver tissue following repeated CCL4 treatment, yet without changes in mRNA profiles (Supplementary Figure 4). mRNA (Supplementary Figure 4) and protein levels (Figure 3A(Fig. 3)) of γ-catenin were not altered upon CCL4 treatment. Hepatic Cx26 (^*p ≤ 0.05), Cx32 (^*p ≤ 0.05) and Cx43 (^*p ≤ 0.05) (Figure 3B(Fig. 3)) protein levels were increased. Here again, these changes were not reflected at the transcriptional level (Supplementary Figure 4). Protein levels (^***p ≤ 0.001) (Figure 3C(Fig. 3)) and mRNA quantities of claudin 1 (^*p ≤ 0.05) (Figure 4(Fig. 4)) were positively affected upon CCL4 treatment. No shifts in mRNA (Supplementary Figure 4) and protein levels (Figure 3C(Fig. 3)) of zonula occludens 2 were observed.

The authors wish to thank Miss Fien Haenen and Mr. Arne Loosen for their dedicated technical assistance. This work was financially supported by the Methusalem program of the Flemish Government and the University Hospital of the Vrije Universiteit Brussel-Belgium (Scientific Fund Willy Gepts).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.