MARC1 A165T substitution leads to reduction in MARC1 protein expression in HepG2 cells.

(A) Plasmids with empty vector (Vector), wild-type and common variants of human MARC1 (CMV promoter, C terminal 1x Flag tag) were expressed in HepG2 cells. 48 hours after transfection, cells were split into two parts. First part of cells were solubilized in RIPA buffer, proteins separated on SDS-PAGE (4–20%) gels and blotted with corresponding antibodies, (B) immunoblot quantitation was performed. (C) Second part of cells was used to measure MARC1 mRNA levels using Real-Time PCR. Mean ± s.e.m. are shown in all graphs, ****p (TIF)</p

Feeding Marc1 KO male mice with different NASH/NAFLD-causing diets revealed minimal difference in liver lipids and plasma metabolites compared to Marc1 WT littermates.

Mice fed with chow diet were sacrificed at age 13 weeks. For other diets—8–10 weeks old WT and Marc1−/− male mice were fed with HFHFD for 35 weeks or CDAA-HFD for 18 weeks or high fat diet (HFD) for 11 weeks or high sucrose diet (HSD) for 9 weeks, respectively. At the end of experiments mice were sacrificed, liver and serum collected and indicated measurements done as described in Methods. Each group contains 7–12 mice. All mice were sacrificed after ad lib feeding state (non-fasted). Mean ± s.e.m. are shown for all measurements. In bold–all measurement with *p (TIF)</p

Generation and validation of <i>Marc1</i> knockout mice.

(A) Homologous recombination was used to replace the second and third exons of mouse Marc1 gene. The antibiotic selection marker (Neo) located between two LoxP sites was deleted with Cre recombinase under self-deleting protamine promoter, leaving β-galactosidase gene (LacZ) and one LoxP site in the flanking intron. (B) Total DNA was extracted from the wild type, heterozygous and homozygous Marc1 knockout male mice livers using commercial kit. Genotyping was performed by PCR with dedicated primers (green arrows) to amplify a 4 kB fragment from genomic DNA flanking Marc1 gene second and third exons. Amplified PCR DNA fragments were digested by EcoRV restriction enzyme (present in LacZ, but not in mouse genome form Marc1 Exon 2 to 3) and size separated on agarose gel by electrophoresis. (C) mRNA (qRT-PCR) and (D) immunoblotting analysis of hepatic Marc1, Marc2 and cofactor proteins in 16-week-old male WT, Marc1 +/- and Marc1 -/- mice. Liver lysates were prepared for Western Blot as described under “Methods”. Each sample (45 μg) was size-fractionated by SDS-PAGE (4–15%), and immunoblotting was performed using a rabbit anti-mouse Marc1 polyclonal antibody (DB0161 (1ug/ml); antibody targeting C-terminus of mouse Marc1 protein). Calnexin served as protein loading control for the experiment. PCR–polymerase chain reaction. SA—splice site acceptor.</p

Marc2 is the main benzamidoxime (BAO) reducing enzyme in mouse hepatocytes.

12 weeks old male Cas9 transgenic mice were injected in tail vein with 3x1010 genome copies (GC) of AAV8 containing Cas9 gRNA against Marc1, Marc2, Marc1/2 (edit both genes), and control (red fluorescent protein (RGF)). (A) 3 weeks after virus injection mice were sacrificed and primary hepatocytes isolated, counted and plated on poly-D-lysine pre-coated plates, then cells were left in incubator to attach to plate for 4 hours. After attachment, cells were washed with PBS once and incubated in serum free media with 0.5 mM benzamidoxime for 2 hours, then cell media were collected and protein from all primary cell plates was extracted, size fractioned on 4–20% SDS-PAGE gel and blotted with corresponding antibodies. Densitometry was used to evaluate protein reduction after Marc1 or Marc2 gene editing and normalized against house-keeping gene (calnexin). (B) BAO and its metabolite benzamidine (BA) were extracted from the collected cell media and measured by UV-HPLC. Controls: no cells–no primary hepatocytes plated for incubation of 0.5 mM BAO in serum free media for 2 hours (control for spontaneous conversion from BAO to BA); no BAO–no benzamidoxime added to RFP gRNA treated cells with serum free media (control for BAO/BA in media or cells). Experiment repeated once with comparable results. Red arrows–% reduction of BAO conversion.</p

MARC1 pLOF variants show association with reduced liver fat and protection from various etiology liver diseases.

AAF—alternate allele frequency, PDFF-MRI—proton density fat fraction by magnetic resonance imaging. (TIF)</p

MARC2 pLOF coding variants have no effect on plasma ALT levels.

MARC2 pLOF coding variants have no effect on plasma ALT levels.</p

<i>MARC1</i> A165T substitution leads to reduction in MARC1 protein expression.

(A) Plasmids with empty vector (Vector), wild-type and common variants of human MARC1 (pTK promoter, C terminal 3x Flag tag) were expressed in HuH-7 cells. 48 hours after transfection, cells were split into two parts. First part of cells were solubilized in RIPA buffer, proteins separated on SDS-PAGE (4–20%) gels and blotted with corresponding antibodies, (B) immunoblot quantitation was performed. (C) Second part of cells was used to measure MARC1 mRNA levels using Real-Time PCR. (D) Human liver samples DNA were extracted, and human MARC1 gene third exon that contains MARC1 variants p.165A/T and p.187M/K was amplified by PCR and Sanger sequenced, and samples with homozygous MARC1 p.187M variant selected. Liver samples proteins were extracted, size fractioned and blotted as described in Methods. (E) Western Blot signals were measured with ECL imager build-in software, (F) RNA was extracted from the same livers, and MARC1, MARC2 and house-keeping gene (36B4) expression levels were detected by qRT-PCR. For human MARC1 primary antibody control MARC1 deficient HuH-7 cell line (CAS9/Crispr) was used as negative control. For MARC2 primary antibody control MARC1 deficient HuH-7 cell line (expressing no MARC1 and 2) was used as negative control and the same cell line with transient overexpression of recombinant human MARC2 without tags, as positive control. Red arrows–reduction (%) of MARC1 protein. Mean ± s.e.m. are shown in all graphs, **p<0.01.</p

Human MARC1 p.165T variant causes mislocalization of MARC1 protein in hepatic cells.

HuH-7 cells were transfected with plasmids containing wild type or 2 common genetic variants (p.A165T and p.M187K) of human MARC1 (with C terminal Flag tag and gene expression driven under CVM promoter). 48 hours after transfection cells were incubated with Mitotracker Orange CMTMRos staining (Thermo Fisher) solution in serum free media for 20 minutes, cells were washed, fixed, permeabilized and blocked as described in Methods. After additional washing, cells were incubated with Flag antibody (Sigma) for 1 hour, washed and incubated with Alexa Fluor 647 conjugated secondary antibody. After final wash, cells were incubated in mounting medium with DAPI (Ibidi) and imaged with Leica confocal microscope. The experiment was repeated once with the equivalent results. (TIF)</p

MARC1 pLOF variants are associated with lower plasma ALT levels.

Rare variant adjusted by common variants. ALT (U/L); AAF—alternate allele frequency. (TIF)</p

Marc1 gene deletion does not affected Mendelian patterns of allele inheritance in mice.

Genotypes of pups from Marc1 heterozygous knockout mice breeding (F2-F4) were detected, results grouped by sex and genotype. (TIF)</p