ATP7B protein model construction and analysis
The fold recognition server Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was used to identify the best structural template and domain of ATP7B protein, and molecular graphics were analyzed by Pymol software. ProtParam (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) and Protscale (http://web.expasy.org/protscale) were used to predict the Physico-chemical properties and hydrophobic of amino acid residues in ATP7B wild-type and R778L mutation type protein, respectively. The ATP7B evolutionary conservation scores were computed via the ConSurf webserver (http://consurf.tau.ac.il/38).
Cell culture
HepG2 cells (ATCC: HB-8065) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (Biochrom AG) and 2 mM L-Glutamine (PAA Laboratories GmbH, Pasching, Austria) at 37 °C under 5% CO2 in a humidified incubator. ATP7B R778L HepG2 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, and 2ug/ml puromycin (Invitrogen, Grand Island, NY, USA).
Construction of ATP7B R778L overexpressed vectors
cDNA fragments coding for the N-terminal (ATP7B R778LN) and C-terminal (ATP7B R778LC) halves of mutant human ATP7B R778L (GenBank, NM_000053.3) were obtained by PCR amplification using the ‘primer 1’ for ATP7B R778LN, and ‘primer 2’ for ATP7B R778LC, respectively. The FLAG tag was amplified using the ‘primer 3’. The PCR was run under the following conditions: 98 °C for 3 min, 30 cycles of 98 °C for 30 s, 55 °C for 15 s, and 72 °C for 1 min with a final extension at 72 °C for 10 min. The lentiviral vector pLVX-CMV7-MCS-EF1a-Puro (Supplementary Fig. S1a) was linearized through restriction enzyme digestion by EcoRI-HF and XhoI. The final PCR products composed of ATP7B R778L and FLAG tag were cloned into the pLVX-CMV7-MCS-EF1a-Puro using a seamless cloning reaction system (Sangon Biotech, Shanghai, China) (Supplementary Fig. S1b) and then validated by DNA sequencing.
Establishment of ATP7B R778L mutant stable HepG2 cell line (R778L cells)
The establishment of lentiviral constructs with ATP7B R778L mutation mainly included packaging, concentration, and purification. HEK-293T cells were transfected with plasmid vectors carrying ATP7B R778L and packaging plasmids pCMV-dR8.9 (Addgene, Cambridge, America) and pCMV-VSV-G (Addgene, Cambridge, America). Then, the packaged recombinant lentiviruses were harvested from the supernatant of post-infection cell cultures. The viral load of recombinant lentivirus was quantified in copies/ml by real-time PCR. Then, successfully established ATP7B R778L mutant lentivirus constructs were utilized to infect HepG2 cells seeded in a 6-well plate. The stable HepG2 cells overexpressing R778L ATP7B were obtained by continuous screening with 2 µg/mL puromycin for 2 weeks and checked by PCR and western blot analyses.
Construction of pLVX-hULK1/-hATG16L1-3flag-ZsGreen-Bsd plasmids
The full-length human ULK1 with FLAG tag in C-terminal and EcoRI/SpeI sites in both ends was generated by PCR amplification. The human ULK1 cDNA (GenBank, NM_003565.2) was used as a template, and ‘primer 4’, ‘primer 5’ and ‘primer 6’ were designed to introduce FLAG tag and EcoRI/SpeI sites. Similarly, the ATG16L1 cDNA (GenBank NM_030803.7) was amplified and modified by introducing EcoRI/SpeI sites through PCR using hATG16L1-Flag as a template and the ‘primer 7’, ‘primer 8’ ‘primer 9’ as primers. The modified ULK1 or ATG16L1 DNA and pLVX-ZsGreen-Bsd plasmid (Supplementary Fig. S2a) were digested with restriction enzymes EcoRI and SpeI and ligated by T4 DNA ligase (New England Biolabs, Beijing, China) (Supplementary Fig. S2b, c) to develop pLVX-hULK1/-hATG16L1-3flag-ZsGreen-Bsd plasmids.
Construction of pLVX-shRNA2-Bsd-hATG16L1 plasmid
Human ATG16L1 mRNA (GenBank: NM_030803.7) was employed as the template strand, and an online shRNA design tool was used to obtain the target gene interference sequence (http://rnaidesigner.thermofisher.com/rnaiexpress/sort.do). In this study, we designed three ATG16L1 target sequences to construct the lentiviral shRNAs. ATG16L1 target sequence fragments were synthesized by PCR using the ‘primer 10’, ‘primer 11’, and ‘primer 12’, with BamHI/EcoRI cleavage sites and FLAG tag inserted. Oligonucleotides were annealed, digested, and then inserted between the BamHI and EcoRI restriction sites of the plasmid vector pLVX-shRNA2-Bsd (Supplementary Fig. S2d) to develop pLVX-shRNA2-Bsd-hATG16L1 plasmid.
Establishment of stable R778L HepG2 cell line with hULK1 overexpression, hATG16L1 overexpression, or hATG16L1 knockdown
The lentivirus expression plasmids pLVX-hULK1-3flag-ZsGreen-Bsd/pLVX- hATG16L1-3flag-ZsGreen-Bsd/LVX-shRNA2-Bsd-hATG16L1 were transfected with the packaging plasmids into HEK 293T cells for lentivirus generation. Lentiviruses were harvested at 24 and 48 h post-infection, centrifuged to remove cell debris, and filtered through 0.45 μm cellulose acetate filters. The ATP7B R778L HepG2 cells were subcultured at 5 × 105 cells per well into six-well tissue culture plates. After 24 h culture, cells were infected with recombinant lentivirus at a multiplicity of infection (MOI) of 50.
Cell viability assay
HepG2 cells were seeded into a 96-well plate. After treatment, the medium was carefully discarded, and 10 μL of Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was subsequently added to each well and incubated at 37 °C for 20 min. Finally, the absorbance was measured at 450 nm using a microplate spectrophotometer (Bio-Rad), and the cell viability was calculated.
RNA isolation and RT-qPCR
Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, 1 μg RNA was reverse transcribed into cDNA using the AMV retrotranscriptase system (TaKaRa, Dalian, Liaoning, China). qPCR reactions were run in triplicate on an ABI StepOne Plus System (Thermo Fisher Scientific) using SYBR Green reaction mix (TaKaRa, Dalian, Liaoning, China). The primers were designed by Primer Version 0.4.0 and listed in Supplementary Table 1. The relative expression of the target gene was calculated and normalized to the expression of the reference gene Gapdh.
Western blot
Whole proteins from cells were extracted using RIPA solution containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were assessed with the bicinchoninic acid (BCA) Protein Quantification kit. Proteins were then subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked with 5% skim milk for 1 h at room temperature and then probed with the following primary antibodies overnight at 4 °C: RIP1(E8S7U) XP rabbit mAb (1:1000; Cell Signaling Technology), RIP3 (E7A7F) XP rabbit mAb (1:1000; Cell Signaling Technology), Phospho-RIP3 Rabbit mAb (1:1000; Cell Signaling Technology), Anti-MLKL antibody (1:2000; Abcam), phospho-MLKL (Ser345) (D6E3G) rabbit mAb (1:1000; Cell Signaling Technology), Atg16L1 (D6D5) Rabbit mAb (1:1000; Cell Signaling Technology), Phospho-Atg16L1 (Ser278) (E7K6H) Rabbit mAb (1:1000; Cell Signaling Technology), ULK1 (D8H5) Rabbit mAb (1:1000; Cell Signaling Technology), Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb (1:1000; Cell Signaling Technology), and LC3B (D11) Rabbit mAb (1:1000; Cell Signaling Technology). After washing, the membranes were incubated with HRP-conjugated anti-rabbit IgG for 1 h at room temperature. The protein bands were visualized by Luminol ECL reagent (Thermo Fisher Scientific). β-actin, β-tubulin, and GAPDH were used as endogenous controls.
Differentially expressed gene (DEG) analysis
The GSE125637 (GPL1261) dataset for Atp7b-/- mouse, whose hallmarks are similar to Wilson’s disease, is downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). The autophagy-related genes included LC3II, ULK1, ATG16L1, RIPK1, RIPK3, and MLKL.
The Network-Analyst online tool (https://www.networkanalyst.ca/) and limma R package (version 3.44.3) were used to analyze differentially expressed genes (DEGs) between different groups. The adjustive P < 0.05 and |Log2 fold change (logFC)| >1 were set as the parameters to identify DEGs. The Wilcoxon test was employed to compare the statistical differences between the two groups.
Statistical analysis
The results were expressed as mean ± standard error of the mean or median (Min, Max). Group comparisons were performed using Student’s t-test, Mann–Whitney U test, or one-way ANOVA followed by Tukey’s multiple comparison test, as appropriate. Statistics and graphs were generated using Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). P < 0.05 was considered statistically significant.