Understanding the current status of influenza A viruses of swine (IAVs-S) and their evolution at the farm level is important for controlling these pathogens. Efforts to monitor IAVs-S during 2015 to 2019 yielded H1N1, H1N2, and H3N2 viruses. H1 genes in Japanese swine formed a unique clade in the classical swine H1 lineage of 1A.1, and H3 genes originating from 1999–2000 human seasonal influenza viruses appear to have become established among Japanese swine. A(H1N1)pdm09-derived H1 genes became introduced repeatedly and reassorted with endemic IAVs-S, resulting in various combinations of surface and internal genes among pig populations in Japan. At the farm level, multiple introductions of IAVs-S with phylogenetically distinct HA sequences occurred, or IAVs-S derived from a single introduction have persisted for at least 3 years with only a single mutation at the antigenic site of the HA protein. Continued monitoring of IAVs-S is necessary to update and maximize control strategies. To assess the current status of influenza A viruses of swine (IAVs-S) throughout Japan and to investigate how these viruses persisted and evolve on pig farms, we genetically characterized IAVs-S isolated during 2015 to 2019. Nasal swab samples collected through active surveillance and lung tissue samples collected for diagnosis yielded 424 IAVs-S, comprising 78 H1N1, 331 H1N2, and 15 H3N2 viruses, from farms in 21 sampled prefectures in Japan. Phylogenetic analyses of surface genes revealed that the 1A.1 classical swine H1 lineage has evolved uniquely since the late 1970s among pig populations in Japan. During 2015 to 2019, A(H1N1)pdm09 viruses repeatedly became introduced into farms and reassorted with endemic H1N2 and H3N2 IAVs-S. H3N2 IAVs-S isolated during 2015 to 2019 formed a clade that originated from 1999–2000 human seasonal influenza viruses; this situation differs from previous reports, in which H3N2 IAVs-S derived from human seasonal influenza viruses were transmitted sporadically from humans to swine but then disappeared without becoming established within the pig population. At farms where IAVs-S were frequently isolated for at least 3 years, multiple introductions of IAVs-S with phylogenetically distinct hemagglutinin (HA) genes occurred. In addition, at one farm, IAVs-S derived from a single introduction persisted for at least 3 years and carried no mutations at the deduced antigenic sites of the hemagglutinin protein, except for one at the antigenic site (Sa). Our results extend our understanding regarding the status of IAVs-S currently circulating in Japan and how they genetically evolve at the farm level.IMPORTANCE Understanding the current status of influenza A viruses of swine (IAVs-S) and their evolution at the farm level is important for controlling these pathogens. Efforts to monitor IAVs-S during 2015 to 2019 yielded H1N1, H1N2, and H3N2 viruses. H1 genes in Japanese swine formed a unique clade in the classical swine H1 lineage of 1A.1, and H3 genes originating from 1999–2000 human seasonal influenza viruses appear to have become established among Japanese swine. A(H1N1)pdm09-derived H1 genes became introduced repeatedly and reassorted with endemic IAVs-S, resulting in various combinations of surface and internal genes among pig populations in Japan. At the farm level, multiple introductions of IAVs-S with phylogenetically distinct HA sequences occurred, or IAVs-S derived from a single introduction have persisted for at least 3 years with only a single mutation at the antigenic site of the HA protein. Continued monitoring of IAVs-S is necessary to update and maximize control strategies. Influenza A virus of swine (IAV-S) is a major respiratory pathogen and causes broadly prevalent disease in pigs worldwide. IAV-S typically causes mild symptoms, such as fever, loss of appetite, dullness, and low mortality rates , but coiWorldwide circulation of IAV-S changed dramatically after the pandemic caused by A(H1N1)2009 [A(H1N1)pdm09] viruses. Before 2009, three IAV-S subtypes, H1N1, H1N2, and H3N2, predominated in swine throughout the world . Classic11–17–25–The pig industry in Japan has risen to intensive production recently; fewer but bigger pig farms are distributed around Japan, raising a total of about 9 million pigs and producing about 900,000 tons of pork in Japan in 2019. To evade the introduction of pathogens from foreign countries, imported live pigs are quarantined for 15 days at an animal quarantine station. Classical swine H1N1 IAVs-S are thought to have been introduced into the swine population in Japan during the late 1970s , 39. Cas42–To understand the status of IAVs-S among pig populations in Japan, from 2015 through 2019, we collected nasal swab samples from pigs on farms in 21 prefectures in Japan for virus isolation and phylogenetic analyses. Simultaneously, we monitored several pig farms for at least 3 years to examine how IAVs-S circulated within these populations. The findings from our study will improve our understanding of IAVs-S currently circulating in Japan and how they evolve at the farm level.The swabs collected through the active surveillance efforts in this study yielded 370 IAVs-S, giving an overall IAV-S isolation rate of 5.2% . In addition, 54 isolates were obtained from the specimens submitted for diagnosis during 2015 to 2019. Overall, 78 of the 424 total IAVs-S were subtyped as H1N1, 331 as H1N2, and 15 as H3N2. In addition, the lineages of the HA and NA genes were determined through phylogenetic analysis. Specifically, the H1 HA genes of 37 H1N1 and 319 H1N2 viruses belong to the 1A.1 classical swine lineage, whereas those of 41 H1N1 and 12 H1N2 viruses belong to the 1A.3.3.2 lineage [A(H1N1)pdm09] pdm09 virus, except for a single strain isolated in Tochigi prefecture: A/swine/Tochigi/38-7119/2019 (H1N2) inherited all of its genes from Japanese pig isolates that circulated during the 1990s and 2000s  were circulating from October 2015 to September 2016 . PhylogeIn the case of farm 007, two phylogenetically distinct 1A.1 classical swine H1 IAVs-S were introduced  and B. OAt two farms, 006 and 012, H1N2 IAVs-S with 1A.1 classical swine H1 and human seasonal N2 genes derived from a single introduction had circulated for more than 3 years  and 9. OThe aims of this study were to illustrate the current situation of IAVs-S in pig populations in Japan and to show how IAVs-S genetically evolved at farms where they were isolated over a long period of time. Our phylogenetic analyses of HA genes revealed that 3 types of IAVs-S have been isolated in Japan. As reported previously –44, the –25–IAVs-S with an H3 HA gene of human seasonal strains have been reported sporadically in Japan , 40, 41,Vaccination is one of the most frequently implemented strategies to control IAV-S on farms. However, the vaccine will be ineffective when the vaccine strain and field strains differ in antigenicity; when mutations occur at antigenic sites of the HA gene in a field strain of IAV-S that is of the same lineage as a vaccine strain; or when a virus possessing HA of a different lineage from the vaccine strain circulates in the field . The vacMultiple introductions of IAVs-S with the same subtype but different lineages of the HA genes might have contributed to the long-term isolation of IAVs-S on farms 001 and 007, as we previously suggested . The levIn summary, our phylogenetic analysis reveals the current situation of IAVs-S in Japan and the genetic evolution of IAVs-S on various farms. Whereas the 1A.1 classical swine H1 IAVs-S and human seasonal H3 IAVs-S have become established among pig populations in Japan, 1A.3.3.2 A(H1N1)pdm09 IAVs-S have become introduced repeatedly from the human population. Therefore, IAVs-S surveillance should be continued to understand the status of IAVs-S among the pig population in Japan and to support vaccine strategies compatible with the current situation in Japan.Through active surveillance efforts during 2015 through 2019, we collected 7,133 nasal swabs from pigs in various age groups at farms in Japan. During the same period, nasal swab samples and lung tissue samples collected from pigs suspected to have swine influenza were transferred from prefectural animal hygiene centers to the National Institute of Animal Health, Japan, for diagnosis. Through active and passive surveillance, we collected samples from 21 of all 47 prefectures. Nasal swabs collected through active surveillance were put promptly into minimum essential medium containing penicillin , streptomycin , amphotericin B (25 μg/ml), HEPES (0.01 M), and 0.5% bovine serum albumin and were kept on ice packs until transport to the National Institute of Animal Health, Japan. There, the swabs were removed and the medium aliquoted and stored at –80°C until virus isolation. From each sample, 200 μl was used for RNA extraction and virus detection.RNA was extracted from swab samples by using RNeasy minikits , as described previously , 56. TheAll gene segments of isolated viruses were sequenced by using next-generation sequencing; consensus sequences were generated and the HA and NA genes of each isolate were subtyped by using FluGAS software as described previously . BrieflyTo calculate molecular estimates of divergence times from ancestral IAVs for selected clusters containing HA and NA genes of IAVs-S, maximum clade credibility trees were constructed as previously reported .Sequences for the polymerase basic protein 1 (PB1) genes of IAVs isolated from humans  and swine  were downloaded from the GISAID database, and nonredundant sets of human and swine sequences were produced by using CD-HIT software  at an idSee Table S4 for a complete list of the EpiFlu accession numbers of the IAVs-S in the present study.


In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis  The complement system is a vital, evolutionarily conserved arm of the innate immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear pathogens from an organism. Activation of complement on pathogen surfaces initiates a cascade of proteolytic events that result in the formation of complement component split products and complexes that act as proinflammatory signals, mediate phagocytosis by opsonization, or induce cell lysis –3.The complement cascade can be initiated through three different pathways , but all may result in eventual cleavage of C5 by C5 convertase. C5 convertase is a protein complex containing the C3 cleavage product, C3b, and other complement components that are specified by the initiating pathway ,3. Cleavex vivo inhibition of complement-mediated lysis of red blood cells is a clinically validated biomarker for C5-targeted therapeutics carbodiimide hydrochloride)/N-hydroxysuccinimide coupling chemistry. Anti-C5 monoclonal antibodies were captured through their Fc regions by an anti-human Fc-specific antibody immobilized on the sensor chips and were tested for binding to the human C5  and recombinant monkey , recombinant cynomolgus monkey C5 (MfC5.mmH),); as well as C5b/6 isolated from serum. Varying concentrations of C5 and C5 split product proteins were injected over sensor surface-captured REGN3918 at 25°C and pH 7.4. Binding signal changes were recorded, and kinetic binding parameters were determined using a 1:1 binding model with mass transport limitation.The mouse C5 locus was humanized using a vector that contained genomic human C5 sequence including exons 2–41, partial intron 1, the 3’UTR, and approximately 1.5 kb downstream sequence of the human C5 gene, followed by a neo cassette flanked by 2 loxP sites to replace the corresponding region of mouse C5 (the mouse sequence replaced downstream of the 3’UTR was roughly 900bp). The neo cassette was subsequently removed. The humanized locus encodes the secreted human C5 protein and a portion of the murine N-terminal signal sequence.P control antibody. Then, free anti-hFc was blocked with non-binding hIgG. Biosensor tips with surface-captured mAbs were immersed in wells containing soluble C5. These pre-complexed mAb-C5 biosensor tips were then immersed in solutions of each mAb as the second antibody binding step. Binding responses (nm) of the second antibodies to the surface-captured mAb-C5 was recorded for each combination. All samples were prepared in HBS-ET containing 0.1 mg/mL BSA. Data were processed and analyzed using Scrubber software version 2.0c.The ability of in-house eculizumab to compete with REGN3918 for binding to C5 was examined using real-time, label-free bio-layer interferometry on an Octet instrument at pH 7.4 and 25°C. Anti-hFc biosensor tips were immersed in a well containing either in-house eculizumab, REGN3918 or an IgG4All mice were generated in-house at Regeneron and monkeys were provided by and housed at Covance. All mouse and non-human primate studies were conducted under the protocols approved by the appropriate Institutional Animal Care and Use Committees (IACUCs)  and the animal ethics committee approved all the animal work performed for this study in its entirety. The studies were conducted in compliance with all applicable sections of the Final Rules of the Animal Welfare Act regulations , the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the Office of Laboratory Animal Welfare, and the Guide for the Care and Use of Laboratory Animals from the National Research Council. IACUC protocol # 169 was used for mouse studies. If a mouse was determined to be in overt pain/distress, or appeared moribund and beyond a point where recovery appears reasonable, the animal was euthanized. Mice were monitored for the following signs of illness, i.e. matted fur, changes in body weight from insufficient food/water intake, little/no movement, dull eyes and body condition scoring. Any and all observations are documented. For euthanasia of mice, overdose of anesthetic  was administered.All non-human primate studies were conducted at contract research organization (CRO) laboratories fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC), in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare under the Regeneron stuy number: REGN2913-PK-15063 (CRO study number 8324374). The study was conducted according to a protocol and procedures approved by an Institutional Animal Care and Use Committee (IACUC). Veterinary care was available throughout the study by an attending Laboratory Animal Veterinarian. In accordance with applicable animal welfare guidelines, any medical treatment necessary to prevent unacceptable pain and suffering, including euthanasia, is the sole responsibility of the attending Veterinarian. Animals were housed in stainless steel cages, individually housed and co-mingled according to laboratory standard operating procedures (SOP). Animal rooms were temperature controlled with 12-hour light/12-hour dark cycles. Animals were provided feed and water (ad libitum), and other appropriate treats including fruits, as well as in-cage enrichment devices in accordance with applicable laboratory SOPs. Animals were monitored on a daily basis via cage side observations for general health and appearance. Upon completion of the in-life portion of the study animals were returned to the laboratory stock colony.P control antibody on day 0. Mice were serially bled at 6 hours, 1, 2, 3, 8 and 14 days via tail bleeds. All blood was processed into serum and stored at -80°C until analysis.Female WT C57BL/6J mice, 10 weeks old (n = 4), received a single SC injection of 15 mg/kg of anti-C5 mAbs or IgG4C5hu/hu mice, 20–30 weeks old (n = 3–5), were stratified based on initial C5 concentrations obtained via tail bleed. Mice received a single SC injection of 15 mg/kg of anti-C5 mAbs or IgG4P control antibody on day 0. Mice in a staggered sacrifice study had terminal blood samples taken via cardiac puncture at 0 (predose) 1, 10, 30 and 35 days. Mice in other PK studies were serially bled at 6 hours, 1, 2, 3, 7, 10, 13, 21, 30, 45 or 59 days or 2, 4, 7, 10, 14 or 18 days via tail bleeds with terminal bleeds taken via cardiac puncture. All blood was processed into serum and stored at -80°C until analysis.Male and female C5hu/hu mice, 16 weeks old (≥14 mice/group), were stratified based on initial C5 concentrations obtained via tail bleed. All mice received SC injections of 15 mg/kg of REGN3918 or in-house eculizumab at 0, 15 and 29 days. Mice for PK analysis were serially bled via tail vein at 0 (predose), 1, 2, 7, 15, 16, 17, 18, 22, 29, 30, 31, 32, 35 and 43 days (n = 2–13/timepoint). Bleeds on dosing days were taken prior to dosing. Mice for PD analysis were terminally bled via cardiac puncture on 0, 15, 29 and 43 days. All blood was processed into serum and stored at -80°C until analysis.Male and female PK parameters for mouse studies were determined by non-compartmental analysis using Phoenix WinNonlin software (version 6.3) and an extravascular dosing model. All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Regeneron Pharmaceuticals.P control antibody were determined by an immunoassay specific for detecting human IgG using either the GyroLab xPlore™ (Gyros) or a sandwich enzyme-linked immunosorbent assay (ELISA). For Gyros, briefly, a biotinylated goat anti-hFcγ polyclonal antibody (Jackson ImmunoResearch Laboratory) was captured onto streptavidin coated beads in order to capture the human IgG present in the sera. After affinity column capture, bound human IgG antibody in samples was detected with Alexa-647 labeled goat anti-hFcγ polyclonal antibody (Jackson ImmunoResearch Laboratory). Fluorescent signal on the column allowed for the detection of bound IgG and response units (RU) were read by the instrument. Sample concentrations were determined by interpolation from a standard curve that was fit using a 5-parameter logistic curve fit using the Gyrolab Evaluator Software. The lower limit of quantitation (LLOQ) for this assay is 0.02 μg/mL. For ELISA, briefly, a goat anti- hFcγ polyclonal antibody (Jackson ImmunoResearch Laboratory) was passively adsorbed to a microtiter plate overnight at 4°C followed by a nonspecific binding block. Serum samples were allowed to bind to plates for one hour at room temperature. After washing, plate captured human IgG antibodies were detected using a horseradish peroxidase (HRP)-conjugated goat anti-hFcγ polyclonal antibody (Jackson ImmunoResearch) followed by addition of the chromogenic HRP-substrate, 3,3',5,5'-tetramethylbenzidine . TMB was used to detect HRP activity and the resultant OD450 was read on a Perkin Elmer Victor X4 Multimode Plate Reader. Sample concentrations were determined by interpolation from a standard curve that was fit using a 4-parameter logistic curve fit using Prism GraphPad (version 6). The LLOQ for this assay is 0.035 μg/mL.Total antibody concentrations of anti-C5 mAbs or IgG4kappa light chain in-house generated mAb. Fluorescent signal on the column allowed for the detection of bound IgG and response units (RU) were read by the instrument. Sample concentrations were determined by interpolation from a standard curve that was fit using a 4-parameter logistic curve fit using the Gyrolab Evaluator Software. The LLOQ for this assay is 0.2 μg/mL.Total antibody concentrations of REGN3918 or in-house Eculizumab were determined by an immunoassay specific for detecting human IgG using the Gyros. Briefly, a biotinylated mouse anti-human IgG1/IgG4 in-house generated monoclonal antibody (mAb) was captured onto streptavidin coated beads in order to capture the human IgG present in the sera. After affinity column capture, bound human IgG antibody in samples was detected with Alexa-647 labeled mouse anti-human P control was determined by a sandwich ELISA. Briefly, a mouse anti- human C5 in-house generated mAb was passively adsorbed to a microtiter plate overnight at 4°C followed by a nonspecific binding block. Serum samples were allowed to bind to plates for one hour at room temperature. After washing, plate captured C5 was detected using a mouse anti-C5 in-house generated mAb followed by detection with HRP-conjugated goat anti- mouse Fcγ polyclonal antibody (Jackson ImmunoResearch). The chromogenic HRP-substrate, TMB (BD Pharmingen) was used to detect HRP activity and the resultant OD450 was read on a Perkin Elmer Victor X4 Multimode Plate Reader. Sample concentrations were determined by interpolation from a standard curve that was fit using a 4-parameter logistic curve fit using Prism GraphPad (version 6). The LLOQ for this assay is 0.02 μg/mL.In the PK/PD studies and the switch study, concentrations of total human C5 in mice prior to dosing with anti-C5 antibodies or IgG427,28. Therefore, the measured amounts are likely to include only negligible amount, if any, of C5b fragment.In the cynomolgus monkey PK/PD study and the mouse PK/PD and mouse switch studies, concentrations of total human C5 in serum were determined by measuring the concentration of a unique tryptic peptide LQGTLPVEAR (LQG) in the C5 protein as a surrogate for C5. Theoretically, this method could also detect C5b, the cleaved product of C5. However, due to the instability of free C5b, the concentration of C5b in serum are generally low with the majority of C5b being bound to cell surfaces as MAC complexes 13C615N4). Calibration standards were prepared at concentrations ranging from 3.9 to 250 μg/mL in blank monkey serum . Calibration standards, blanks, and study samples (10 μL each) were dried in Speed-Vac and then denatured in 100 μL of 8M urea/20mM Tris(2-carboxyethyl) phosphine (TCEP) buffer at 37°C for 1 hour. After 10 μL of 25 nM internal standard was added, samples were then alkylated with 10 mM of 2-iodoacetamide at room temperature for 30 minutes before diluted using 50 mM ammonium bicarbonate to a final volume of 500 μL. The samples were then digested by trypsin (1:20 w/w) overnight at 37°C. LQG derived from C5 was detected and quantified by LC-MRM-MS (m/z 542.3→571.3) using a Waters Xevo TQ-S coupled with ACQUITY UPLC system. 10 μL of digestion mixture was directly injected onto a Waters BEH Peptide C18 Column  maintained at 40°C. The flow rate was 0.5 mL/min using 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phase A (MA) and B (MB), respectively. Initial gradient was set as 93% MA from 0 to 1.0 min; from 1.0 to 13 min, changed from 93% MA to 65% MA; at 13.1 min to 5% MA and maintained at 5% MA until 14 min; then changed to 93% MA at 14.2 min through the end of the run. Data was processed by Masslynx Analyst 4.2 . Calibration curve was fitted using peak area ratio of LQG/SIL-LQG with the linear regression and 1/x weight factor. The lowest concentration of C5 reference standard (3.9 μg/mL) was within the dynamic range of the assay and was defined as the assay’s LLOQ.For analysis of total C5 in monkey serum, C5  was used as the reference standard for the calibration; and a peptide bearing a C-terminal stable isotope-labeled (SIL) arginine residue was used as the internal standard (LQGTLPVEAR--/-). Serum from C5-/- mice was also used as a negative control (blank). The tryptic peptide LQGTLPVEAR derived from C5 was detected and quantified by LC-MRM-MS using a Waters Xevo TQ-S with ACQUITY UPLC system at the flow rate of 0.6 mL/min. The rest of procedure is as described for monkey serum samples.For analysis of total C5 in mouse serum, standards were prepared in serum from in house-generated C5 knock-out mice, in which the mouse C5 gene was deleted  were stratified based on initial C5 concentrations. Monkeys received a single IV bolus injection of 15 mg/kg of anti-C5 antibody REGN3918, H4H12161P or H4H12170P on day 0. Blood samples were collected from each animal at 0 (predose), 5 minutes, 4 and 8 hours, 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, and 49 days post dose. All blood was processed into serum. Monkey study was performed at Covance Laboratories, Inc.Pre-study concentrations of C5 in cynomolgus monkey serum were measured using a commercial enzyme- linked immunosorbent assay kit  according to manufacturer’s recommendations.Total antibody concentrations of anti-C5 antibodies in monkey serum were measured using a direct enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated with an in-house generated mouse anti-human IgG1/IgG4 Fc mAb. REGN3918, H4H12161P and H4H12170P were then captured on the plate and detected using an in-house generated biotinylated mouse anti-human IgG4 Fc mAb, followed by NeutrAvidin conjugated with horseradish peroxidase (NeutrAvidin HRP). A luminol based substrate specific for peroxidase was then added to achieve a signal intensity that is proportional to the concentration of total REGN3918, total H4H12161P and total H4H12170P. The lower limit of quantitation (LLOQ) is 0.078 μg/mL in neat monkey serum.hu/hu mice or cynomolgus monkey following SC injection of REGN3918, in-house eculizumab or IgG4P control antibody was evaluated ex vivo using a hemolysis assay. Hemolysin-sensitized sheep red blood cells (SRBCs) were used for the classical complement pathway initiated hemolysis assay. Briefly, SRBCs were washed and resuspended at 1x109 cells/mL in gelatin veronal buffer with CaCl2 and MgCl2 (GVB++). To sensitize the SRBCs, cells were incubated with 0.75 mg/mL hemolysin at 37°C for 20 minutes. Sensitized SRBCs were further diluted in GVB++ to 2x108 cells/mL. Sera from C5hu/hu mice were used at a 1:10 final dilution and were ex vivo supplemented with hC3 at final concentrations of 90 μg/mL. Monkey sera were diluted at 1:20 final dilution. A total of 1x108 sensitized SRBCs in a volume of 100 μL were plated into round bottom 96-well plates followed by addition of 100 μL of diluted test serum. Sensitized SRBCs without addition of test serum (GVB++ only) were included to determine background hemolysis, or were lysed by addition of distilled water to determine maximal hemolysis signal. Cells were gently mixed and incubated at 37°C for 10 or 60 minutes. Cells were pelleted by centrifugation at 1,250xg at 4°C for 7 minutes. A total of 100 μL of the supernatant was transferred to a fresh 96-well flat bottom plate and the OD412 was read on a Spectramax microplate reader. The hemolytic activity was calculated as follows:For hemolysis assays, sheep and rabbit red blood cells were obtained from Colorado serum . The hemolytic activity in serum samples from C58 cells/mL in gelatin veronal buffer with 40mM MgCl2 and 40mM EGTA (GVB-Mg2+/EGTA). A total of 2x107 RbRBCs in a volume of 100 μL were plated into each well of 96 well round bottom plates followed by addition of 100 μL of test serum . RbRBCs without addition of test serum (GVB Mg2+/EGTA only) were included to determine background hemolysis. The maximal hemolysis signal was determined by addition of distilled water to lyse the cells. Cells were gently mixed and incubated at 37°C for 60 minutes. Subsequently, hemolysis was quantified as described above.Rabbit RBCs (RbRBC) were used for the alternative pathway initiated hemolysis assay. In brief, RbRBCs were washed and resuspended at 2x10P control antibody. C5a protein levels were measured using the MicroVue C5a ELISA kit according to the manufacturer’s instructions. Briefly, samples were diluted and incubated on plates pre-coated with capture antibody (mouse anti-C5a specific for a neo-epitope on human C5a). Human C5a protein provided by the manufacturer was used as a standard for calibration. C5a in the supernatants was detected by horseradish peroxidase (HRP)-conjugated detection antibody . The chromogenic HRP-substrate, 3,3',5,5'-tetramethylbenzidine, was added to detect HRP activity. A solution of 1N hydrochloric acid was used to stop the reaction, and the optical density at 450nm (OD450) was measured on a SpectraMax plate reader. Data were analyzed using nonlinear regression (4-parameter logistics) in GraphPad Prism. C5a concentration was expressed as ng/mL of supernatant.C5a protein levels were determined in supernatants from the CP hemolysis assay using complement-preserved normal human serum (NHS) pre-incubated with REGN3918 or IgG4++, followed by incubation for 60 minutes at 37°C with 2-fold serially diluted test sera (1:10 to 1:640) to initiate hemolysis via the CP. Complement-preserved sera from at least 5 WT or C5hu/hu mice each were pooled for this analysis. Serum from C5hu/hu mice was supplemented with human C3 at 40, 80, and 120 μg/mL in the 1:10 dilution followed by 1:2 serial dilution .SRBCs were sensitized with hemolysin in GVB™ 3+ A4F Separation System (Wyatt Technology) coupled to an Agilent 1200 Series HPLC system equipped with a ultraviolet (UV) diode array detector, Wyatt Technology Dawn HELEOS® II laser light scattering instrument (LS), and an Optilab® T-rEX differential refractometer (RI) detector.In-house eculizumab was mixed with C5 in 1X DPBS, pH 7.4 and allowed to incubate at room temperature for 1 hour. Then REGN3918 was added to the pre-formed complexes to yield final concentrations of 0.7 μM in-house eculizumab, 0.8 μM C5, and 4.3 μM REGN3918. The final solution was allowed to incubate for an addition 1 hour prior to analysis on an A4F-MALLS system composed of an Eclipse™ short channel fitted with a W350 spacer foil  and using a 10 kDa MWCO Nadir regenerated cellulose membrane. The channel was pre-equilibrated with the mobile phase buffer , prior to the injection of each sample. The fractionation method consisted of four steps: injection, focusing, elution, and a channel “wash-out” step. Each sample  was injected at a flow rate of 0.2 mL/min for 1 minute and subsequently focused for 5 minutes with a focus flow rate of 1.0 mL/min. The sample was eluted with a channel flow rate of 1.0 mL/min with the linear gradient cross flow from 2.0 mL/min to 0 mL/min over 45 minutes. Finally, the cross flow was held at 0 mL/min for an additional 5 minutes to wash out the channel. Bovine serum albumin  was fractionated using the same parameter settings as a system suitability control. Data was collected using ASTRA software (Version 6.1.2 Wyatt Technology) and data analysis was performed using ASTRA software (Version 7.1.0 Wyatt Technology) with an experimentally determined dn/dc value and extinction coefficient at 215 nm (corrected for glycosylation) derived from the protein conjugate analysis provided in the software.All the samples were maintained unfiltered at 4°C prior to injection into an Eclipse2O labeling solution (10 mM PBS in D2O at pD 7.0) for various time-points. The deuteration was quenched by transferring 50 μL sample to 50 μL pre-chilled quench buffer  and then was incubated at 1.0°C for two minutes. The resulting sample was injected into a Waters HDX Manager for online pepsin/protease XIII digestion, trapped onto VanGuard pre-column at 0°C and eluted to ACQUITY BEH C18 1.0 × 50 mm column  for a 9-minute gradient separation by 5%-40% MB . The mass spectrometer was set at cone voltage of 37 V. LC- MSE data were processed and searched against the database including human C5, pepsin, and their randomized sequence via Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported to DynamX software, which, based on preset criteria, automatically determined deuterium uptake of each peptide across multiple time points based on retention time and mass accuracy (<10ppm).HDX-MS was performed on an integrated Waters HDX/MS platform, consisting of a LEAP Tech HDX PAL system , a Waters Acquity M-Class  and Synapt G2-Si mass spectrometer. For deuterium labeling, 3.8 μL of C5 (EMD Millipore) or C5 premixed with the REGN3918 in 1:1 molar ratio was incubated with 56.2 μL DS1 Checklist(PDF)Click here for additional data file.S1 Figin vitro. The percent block in CP- and AP-mediated hemolysis assays using either human  and cynomolgus monkey  serum was determined over a range of concentrations (0.4 to 400nM) for each antibody to generate dose-response curves; REGN3918 is shown in blue. All hemolysis assays are representative experiments that were performed in duplicate at each mAb concentration; each experiment was repeated with multiple sera from different donors (n > 3). Data is plotted as mean ±SD.(A-D) Sera from human and monkey were used to examine the efficacy of anti-C5 mAbs to block hemolysis mediated by the classical and alternative complement pathways (TIFF)Click here for additional data file.S2 FigC5hu/hu mice by ELISA, which revealed a difference between male  and female  mice. (C) Serum concentrations of antibodies were measured by ELISA at 6 hours, 1, 2, 3, 8 and 14 days post dosing in WT mice administered a single 15 mg/kg subcutaneous dose of anti-C5 mAbs or IgG4P control antibody. REGN3918 is shown in blue. (D) Antibody serum concentrations were measured by ELISA at 2, 4, 7, 10, 14 and 18 days post dosing in C5hu/hu mice administered a single 15 mg/kg subcutaneous dose of anti-REGN3918 (blue) or H4H12170P (grey). (E-F) RbRBCs in GVB++-Mg2+/EGTA were incubated for 10 minutes with 5% normal human serum (E) or monkey serum (F), resulting in CP-mediated hemolysis. Pre-incubation of serum with increasing concentrations of REGN3918 or in-house eculizumab blocked hemolysis in human serum a dose-dependent manner. REGN3918 also blocked hemolysis in monkey serum in a dose-dependent manner, but in-house eculizumab failed to block hemolysis in monkey serum. (G) Serum collected from terminally bled C5hu/hu mice administered with REGN3918 alone (blue), in-house eculizumab alone (red), or in-house eculizumab/REGN3918 switched (green) were supplemented with hC3 and the percent of AP-mediated hemolysis using ex vivo assays was assessed. Hemolysis figures are representative experiments that were performed in duplicate at each mAb concentration; each experiment was repeated with multiple sera from different donors (n > 3). Data is plotted as mean ±SD.(A) Schematic of the strategy used for humanizing the mouse C5 gene (top). A vector containing human genomic C5 sequence, including exons 2–41, partial intron 1, the 3’UTR, and approximately 1.5 kb downstream sequence of the human C5 gene, followed by a neomycin (neo) cassette flanked by loxP sites was used to replace the corresponding region of mouse C5 (middle). The neo cassette was subsequently removed (bottom). (B) Serum C5 levels were determined in (TIFF)Click here for additional data file.S3 Fig(A) The ability of in-house eculizumab to compete with REGN3918 for binding to C5 was examined using real-time, label-free bio-layer interferometry. Binding responses indicating noncompetitive binding of in-house eculizumab and REGN3918 are indicated in bold, whereas competitive binding is highlighted in grey shaded fields. (B) In-house eculizumab:C5 complexes (orange) were analyzed by asymmetric flow field-flow fractionation coupled to multi-angle laser light scattering (A4F-MALLS). Fractograms from individual samples of in-house eculizumab (red), C5 (green), and REGN3918 (blue) are also overlaid. Relative UV absorbance at 215 nm as a function of retention time is shown for each sample and the measured molar masses of resolved peaks are indicated.(TIFF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Urothelial carcinoma (UC), including upper tract urothelial carcinoma (UTUC) and urinary bladder urothelial carcinoma (UBUC), is a common malignant disease in developed countries. Oncogenic metabolic lesions have been associated with UC development.Methods: Using data mining, a series of studies were performed to study the involvement of SLC14A1 in UC specimens, animal models and UC-derived cell lines.Results: In two cohorts of UTUC (n = 340) and UBUC (n = 295), the SLC14A1 protein level was an independent prognostic factor. Epigenetic silencing contributed to SLC14A1 downregulation in UCs. Total and membranous SLC14A1 played tumor suppressive roles through the inhibition of cell proliferation and metastasis in distinct UC-derived cells and animal models. Functional SLC14A1 prevented the accumulation of arginine and urea, enhanced mitochondrial fusion and aerobic respiration, inhibited glycolysis by altering the expression levels of several related proteins and sensitized arginine-deprivation treatment in ASS1-deficient UC-derived cells. In vitro and in vivo, SLC14A1 inhibited the mTOR signaling pathway and subsequently tumorigenesis, supported by reduced arginine concentrations in vitro. Nuclear SLC14A1 transrepressed HK2 and DEGS1 genes via recruitment of HDAC1 and/or SIN3A to maintain metabolic homeostasis and thereafter impeded tumorigenesis.Conclusion: Clinical associations, animal models and in vitro indications provide solid evidence that the SLC14A1 gene is a novel tumor suppressor in UCs. Total and membranous SLC14A1 prevents urea and arginine accumulation via the mTOR signaling pathway. Nuclear SLC14A1 recruits HDAC1 to transrepress oncometabolite genes. Urothelial carcinoma (UC), also known as transitional cell carcinoma, is one of the most common malignancies worldwide SLC14A1 gene encoding the type B urea transporter protein was mapped to chromosome 18q12.3, adjacent to another urea transporter, SLC14A2, locus SLC14A1 knockout mice, high urea concentrations caused DNA damage and apoptosis in bladder urothelium SLC14A1-rs10775480, a variant at intron 6, is highly associated with susceptibility in bladder cancer SLC14A1 may play a causal or regulatory role.The human SLC14A1 mRNA was correlated with low-stage lung adenocarcinoma SLC14A1 mRNA and its corresponding protein levels High SCL14A1 transcript level, 36 UBUCs and 42 UTUCs with normal, pTa-pT1 and pT2-pT4 specimens were evaluated. For immunohistochemistry, another 340 primary UTUCs and 295 UBUCs with available tissue blocks were used  samples  and QuantiGene™ Plex Assay Kit  based on the user guides , UMUC3 (ATCC) and BFTC905  and one UTUC-derived cell line, RTCC1  SLC14A1 gene. Purified amplicons from the library were ligated to adapters with barcodes and loaded into a MiSeq System  for exon 3-11 sequencing. The bioinformatics analysis workflow was performed . Cells were treated with paraformaldehyde to crosslink proteins and DNA, followed by glycine treatment to terminate the reaction. Chromatin was digested into 150- to 900-bp DNA/protein complexes by micrococcal nuclease. Sonication was further used to breakdown the nuclear membrane. Fragmented crosslinked chromatins were collected and incubated with the complex of protein G and the antibody against H3K27me3 , H3K9me2/3  or HDAC1 . Purified ChIP DNA was subjected to quantitative PCR .SLC14A1_v1 plasmid served as the template to generate a double-mutant pLVX-puro-6HIS-SLC14A1-C25S/C30S_v1 using the QuikChange Lightning Site-Directed Mutagenesis Kit  (Supplementary). The SLC14A1 gene was ligated to a DNA fragment corresponding to the SV40 nuclear localization signal (NLS: PKKKRKV) at the 3' end to generate the pLVX-pur-pHIS-SLC14A1-NLS_v1 plasmid. Cell viability, proliferation, migration, invasion and tube formation with HUVECs were performed according to the methods used in our previous study All chemicals were purchased from Sigma‐Aldrich (USA) unless stated otherwise. The pLVX-puro-6HIS-A plasmid containing a mitochondrial targeting sequence fused to a red fluorescence tag (pLV-MitoDsRed) was used to generate replication-incompetent lentivirus. Transduction of the lentiviral particles containing MitoDsRed showed the morphology and localization of each mitochondrion within a single cell, and 4',6-diamidino-2-phenylindole (DAPI) staining was used to identify nuclei. Mitochondrial fusion and fission were measured as in our previous study The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFp Analyser (Agilent) according to the manufacturer's instructions (Supplementary). Glucose uptake  was performed according to the manual. NOD/SCID mice  were randomly grouped and subjected to subcutaneous and tail vein injections with UMUC3 cells . All aniCoimmunoprecipitation was performed using the Pierce™ Co-Immunoprecipitation Kit (ThermoFisher). Protein lysates were incubated with anti-human SLC14A1 antibody and subsequently, magnetic beads. The protein complexes were eluted from antibody/beads after washing and subjected to immunoblot analysis by probing anti-SIN3A , -HDAC1 , -ARID4B (Proteintech) or -SUDS3  antibody.mock- and SLC14A1-NLS-overexpressing J82 cells in a 96-well white plate by using PolyJet™ In Vitro DNA Transfection Reagent . After transfection for 24 h, the medium was replaced and incubated for another 24 h. The luminescence intensities were measured using the Dual-Glo® Luciferase Reporter Assay System (Promega) (Supplementary).pKM2L-phHKII  and pGL4.54[luc2/TK] Vector  were cotransfected into the t-test was used to examine significant differences in the relative mRNA levels, different phases of the cell cycle, percentages of cell viability, ability of cell migration, invasion, HUVEC tube formation and mouse xenografts (tumor volume). The comparison of SLC14A1 transcription levels between different groups were determined using the Mann-Whitney U test.Statistical analysis was performed using the SPSS software . The protein expression levels of SLC14A1 and those involved in glycolysis and mTOR activation as determined by IHC were dichotomized into high (≥ median H-score) and low (< median H-score). The associations of these proteins were evaluated by Chi-square test. The correlations between the SLC14A1 protein level and specific clinicopathological parameters were evaluated using the Chi-square test. The disease-specific survival (DSS) and metastasis-free survival (MeFS) were estimated by the log-rank test and plotted using Kaplan-Meier curves. A multivariate Cox proportional hazards model was used to evaluate independent prognostic impacts for selected parameters. A two-tailed SLC14A1 transcripts was found to be significantly correlated with a high pT status in UBUC patients  and UBUC (n = 36) subsets, SLC14A1 mRNA levels were downregulated in tumor specimens compared to their normal counterparts , negative nodal metastasis (P < 0.001), low histological grade (P < 0.001), negative vascular invasion (P < 0.001), negative perineural invasion (P < 0.05) and low mitotic rate (P < 0.01) in two large cohorts of UTUC (n = 340) and UBUC (n = 295) patients  datasets, GSE32894 and GSE31684, downregulation of the s Figure . In the SLC14A1 mRNA and its corresponding protein levels were highly expressed in RTCC1 compared to other UC-derived cell lines. SLC14A1 protein was highly expressed in low-grade Ta specimens compared to high-grade ones  database , indicating that SLC14A1 mRNA levels and methylation status are negatively correlated  in J82 and UMUC3 cells compared to the control   genes was verified by mRNA upregulation (Supplementary SLC14A1(C25S/C30S) gene did not upregulate membranous (ATP1A1: membrane marker) or nuclear SLC14A1 protein (H2B: nuclear marker) level compared to those in SLC14A1-overexpressing J82 and UMUC3 cells (Supplementary SLC14A1(C25S/C30S) gene was not able to induce G1 cell cycle arrest, reduce HUVEC tube formation (Supplementary SLC14A1 gene in RTCC1 and BFTC905 cells downregulated SLC14A1 mRNA (P < 0.05) and the corresponding protein levels, while it promoted cell cycle progression to S and G2 phases  (Supplementary P < 0.05), increased cell proliferation (P < 0.05) from 24 to 72 h after seeding  and cell invasion (P < 0.05)  in the ) Figure D compareSLC14A1-overexpressing UMUC3 cells than in the mock (P < 0.05), while it was restored to the similar size as the mock in xenografts from SLC14A1(C25S/C30S)-overexpressing cells -overexpressing UMUC3 cells compared to the mock -overexpressing UMUC3 cells metastasized  and SLC14A1(C25S/C30S) compared to SLC14A1-overexpressing UMUC3 cells  gene in J82 and UMUC3 cells showed the opposite pattern. Knockdown of the SLC14A1 gene in RTCC1 and BFTC905 cells displayed similar results to those from overexpression of the nonmembranous SLC14A1(C25S/C30S) gene, suggesting that SLC14A1 regularizes the urea cycle by continually releasing urea and subsequently maintains the catabolism of arginine and prevents excessive biogenesis of polyamines. Particularly, arginine was upregulated in SLC14A1-knockdown and ASS1-deficient RTCC1 cells (P < 0.05), indicating that SLC14A1-suppressed arginine synthesis is independent of ASS1. Manipulation of the SLC14A1 level was not able to consistently alter the mRNA abundance of several metabolic enzymes including OTC, ASS1, ASL and ARG in 4 UC-derived cell lines  gene in J82 and UMUC3 cells promoted mitochondrial fission  and UBUCs (n = 148)  counteracted these effects in UMUC3 cells  and ASS1-deficient RTCC1 (shLacZ) cells decreased cell viability . These observations indicated that regarding cell viability, arginine upregulation due to SLC14A1- knockdown in ASS1-deficient cells enhances the resistance to arginine removal by ADI-PEG 20. Accordingly, functional SLC14A1 promotes mitochondrial fusion/respiration; nevertheless, it inhibits aerobic glycolysis along with altering the expression levels of several related proteins in UC specimens and UC-derived cells. Furthermore, upregulation of SLC14A1 sensitizes arginine-deprivation treatment in ASS1-deficiency cells.Interestingly, under arginine-deprivation, knockdown of the SLC14A1 (n = 19) were sensitive, while low SLC14A1 (n = 7) cell lines were resistant to a PI3K/mTOR dual inhibitor, BEZ235, treatment  SLC14A1-overexpressing cells but restored in SLC14A1(C25S/C30S)-overexpressing J82 and UMUC3 cells as well as in SLC14A1-knockdown RTCC1 and BFTC905 cells. However, the level of pAKT1(S473) or pEIF4EBP1(S65) protein in the abovementioned cells was not consistently correlated to the SLC14A1 status  and UBUCs (n = 148) , pRPS6(S235) and Ki-67 were notably downregulated, and these downregulated proteins were restored in xenografts from SCL14A1(C25S/C30S)-overexpressing UMUC3 cells compared to the mock , pMTOR(S2448), pRPS6S235), pEIF2EP1(S65) and Ki-67 proteins were highly expressed in high-grade UCs  and the corresponding protein levels . Three potential SLC14A1-interacted proteins, namely, ARID4B, SIN3A and SUDS3 were identified in UMUC3 and J82 cells  imaging technology for clinical tumor detection. A meta-analysis showed that high HK2 levels conferred a poor prognosis in solid tumors of the digestive system Hk2 was required for tumor initiation and maintenance in Kras-driven lung cancer and Erbb2-driven breast cancer. Without any adverse effects, mice with lung tumors could be controlled by systematic Hk2 deletion HK2 transcription and subsequent translation.Although SLC14A1 was originally identified as a transmembrane urea transporter, we found that SLC14A1 protein was also expressed in the nucleus and cytosol in UC specimens. Importantly, the fact that nuclear SLC14A1 protein gradually faded away in the invasive front of UC specimens promoted us to investigate the nuclear roles of SLC14A1. We found that nuclear SLC14A1 is able to recruit HDAC1/SIN3A complex to transrepress the DEGS1, and its protein product catalyzes the final step of de novo biosynthesis from dihydroceramide to ceramides in the sphingolipid signaling pathway. Knockdown of the DEGS1 gene using small inhibitor RNA led to the accumulation of endogenous dihydroceramide, reduced cell growth and induced G1 cell cycle arrest in SMS-KCNR neuroblastoma cells DEGS1-knockout embryonic fibroblasts DEGS1 in preadipocytes prevented adipogenesis, resulting in an increase in oxidative stress, cellular death and cell cycle arrest due to dihydroceramide upregulation DEGS1 transcription and translation, SLC14A1 may also inhibit tumor growth/cell proliferation in UC-derived cells through hindering lipotoxicity.Another SLC14A1-transrepressed gene is 2. Of these, PDHA1 contains the E1 active site and plays a pivotal role in the function of the PDH complex. Furthermore, downregulation of protein levels involving the glycolytic pathway suggests that SLC14A1 disfavors glycolysis. Thus, earlier studies strongly fortified our findings, consistent with the development of novel glycolytic inhibitors as a new class of anticancer agents.Prominently, we found that SLC14A1 overexpression promotes mitochondrial fusion and inhibits glycolysis, which was supported by mitochondrial elongation, an increased OCR, decreased ECAR and glucose uptake, downregulation of several glycolysis-related enzymes including SLC2A1, HK2, PKM, enzymes for the final step of anaerobic glycolysis, and LDHA/C and upregulation of MFN2 and PDHA1 protein levels in vitro and/or in an animal model. These aspects were also supported by significant correlations between SLC14A1 and related protein levels in UC specimens. Indeed, mitochondria are dynamic organelles and constantly change owing to coordinated fission, fusion or movement with the microtubular structure de novo DNA synthesis and repair, providing another indication to support our conclusions.We verified that functional SLC14A1 maintains a normal urea cycle by the prevention of arginine and urea accumulation and downregulation of downstream polyamines in vitro. Increasing evidence substantiates that tumor cells reprogram metabolism to expand nitrogen and carbon usage for cell proliferation. In normal cells, enzymes involved in the urea cycle are dynamically expressed to adapt to cellular requirements. However, in cancer cells, expression levels of these enzymes are adjusted to exploit nitrogen for DNA and/or protein biosynthesis ASS1-deficient UC-derived cell line. Arginine is a nonessential amino acid in humans, and ASS1 catalyzes the penultimate step of the arginine biosynthetic pathway in the urea cycle. Arginine auxotrophy due to ASS1 loss/mutation is one recurrence characteristic in human malignancies ASS1 negativity was detected in ~40% of bladder cancer, and a multivariate analysis indicated worse disease-specific and metastasis-free survival rates ASS1-deficient RTCC1 cells, strengthening the finding that SLC14A1 also prevents arginine accumulation in addition to urea. Thus, SLC14A1 is critical to maintain a normal urea cycle and metabolic homeostasis in the urothelial system.Moreover, we identified that functional SLC14A1 sensitizes arginine deprivation treatment in an RPS6 encodes the 70 kDa serine/threonine kinase and regulates the translation of a subset of mRNA with an oligopyrimidine fragment at the 5' untranslated region. These mRNAs represent ~20% of entire cellular mRNA and are central components for translation apparatus HK2 and inhibits arginine/MTOR-mediated HK2 translation. Instead, DEGS1 ablation activates the Akt/mTOR signaling pathway in mouse embryonic fibroblasts In vitro and in the animal model, we identified that SLC14A1 downregulated pMTORS2448), which is an active component of mTORC1 8, which SLC14A1 gene and both hypermethylation and histone/lysine methylation in CpG island of the SLC14A1 promoter region cause low transcriptional and subsequent translational activities. Truly, epigenetics catalyzes diverse biochemical modifications to either the DNA or the histone proteins to adjust chromatin conformation, which further regulates gene expression temporally and spatially. Akin to most human malignancies, urological cancers are characterized by extensive epigenetic changes, especially histone methylation in euchromatin, resulting in the silencing of tumor suppressor genes and genomic instability SLC14A1 gene in this study. The mRNA and protein levels of the EZH2 gene, which encodes a polycomb-group protein catalyzing H3K27me2/3, were upregulated in muscle-invasive urothelial carcinoma EZH2 and EHMT2 genes. Although the SLC14A1 protein was frequently lowly expressed in several and advanced cancers, this is the first study to identify that epigenetic silencing contributes to its low transcription and subsequent low translation.In clinical specimens and UC-derived cell lines, we unearthed that negative mutations in the coding DNA sequence in the SLC14A1 promoter resulted in low SLC14A1 transcription and subsequent translation. In vitro, xenograft and/or tail vein injection experiments in mouse models provided evidence that total and membranous SLC14A1 inhibited cell viability, proliferation, migration, invasion, ECAR, tumor growth and metastasis, induced mitochondrial fusion, increased the OCR along with upregulation of proteins related to mitochondrial respiration and downregulated proteins associated with aerobic glycolysis, which may be mediated by downregulation of arginine and phosphorylated/active MTOR and RPS6. Functional SLC14A1 further sensitized arginine deprivation therapy in ASS1-deficient cells. Nuclear SLC14A1 recruited HDAC1 to transrepress HK2 and DEGS1 genes to maintain metabolic homeostasis.Taken together, we identified that low SLC14A1 is a poor prognostic factor for disease-specific and metastasis-free survival in UTUC and UBUC patients. Epigenetic modifications including DNA hypermethylation and H3K27 and H3K9 histone methylations on the All data generated or analyzed during this study are included in this published article and its supplementary documentation file.Supplementary materials and methods, figures, tables.Click here for additional data file.


V1, RCC1LV2 and RCC1LV3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1LV1 and RCC1LV3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1LV2. Overexpression and silencing of RCC1LV1 or RCC1LV3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, , and pseudouridylation proteins, . All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly.Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1L Mitochondria are essential organelles responsible for the production of most of the energy required by the eukaryotic cells and hence their proper function is essential for cell survival. About 1,500 proteins are estimated to be present in human mitochondria but new proteins are still being reported as mitochondrial. While most proteins present in mitochondria are synthesized in the cytosol and then imported into mitochondria, 13 proteins encoded in the mitochondrial DNA are synthesized inside mitochondria by a dedicated set of proteins. RCC1L (WBSCR16) has recently been described as a protein involved in mitochondrial protein synthesis and more precisely in mitochondrial ribosome biogenesis. Three different RCC1L transcripts resulting in three different isoforms have been described; however, their role in mitochondrial translation has not been fully studied. Here we show that all three isoforms are present in mitochondria and that at least two of them play a role in mitochondrial ribosome biogenesis through the interaction with specific proteins that play a role in the assembly of either the large or the small ribosome subunit. This protein is an example of how biological processes may be more complex than anticipated as different isoforms of the same gene can have different roles in the cell. Val or mt-tRNAPhe have also been found as constituents of the mtLSU subunit, replacing bacterial and eukaryotic 5S rRNA . The. TheVal Ribosome assembly is a complex series of events in which the processing and modification of rRNAs needs to be coordinated with the incorporation of MRPs. This process requires the action of many different factors such as nucleases, rRNA modifying enzymes, RNA helicases, GTPases, chaperones and maturation factors , 7. GTPaIn human mitochondria, five GTPases involved in ribosome biogenesis have been described to date: MTG1 (GTPBP7) couples mtLSU assembly with bridge formation between mtLSU and mtSSU  10], MT, MT10], V1-3, all of them sharing the same N-terminus and, therefore, all three predicted to be mitochondrial , in , in V3 iof mtSSU . C4ORF14of mtSSU . While aV1 and RCC1LV3, are essential for correct biogenesis of mitochondrial ribosome subunits. RCC1L isoforms interact with different GTPases to promote mitoribosome subunit assembly but also their expression has to be coordinated in order to balance the biogenesis of each subunit and subsequently the formation of the monosome. The presence of a third isoform, RCC1LV2, showing DNA binding properties but little effect on mitoribosome biogenesis is very intriguing and its characterization may contribute to a better understanding of the relationship between mitochondrial nucleoids, the RNA granules and the translation machinery.Collectively, our results show that the RCC1L gene produces three mitochondrial isoforms and that two of them, RCC1L2.Human embryonic kidney cells HEK293T  and epitheloid cervix carcinoma HeLa cell lines were maintained in high-glucose medium (Gibco) supplemented with 10% tetracycline-free FBS (Merck Biochrom), 1% penicillin-streptomycin and at 37°C in a humidified atmosphere of 5% COV1, RCC1LV2 and RCC1LV3, were cloned into pcDNA5/FRT/TO plasmid (Invitrogen) with a carboxy-terminal STREP2 followed by a FLAG tag or just HA tag. Inducible transgenic HEK and HeLa cell lines were established by transfection with the STREP2-FLAG constructs using Lipofectamine 2000 (Invitrogen). Control and transgenic cell lines were cultured in the presence of 15 μg/ml of blasticidin and 100 μg/ml of Zeocin or 100 μg/ ml of hygromycin and 15 mg/ml of blasticidin, respectively. Transgene expression was induced with 10 ng/ml doxycycline for 3–6 days and cells were collected or mitochondria purified for further analysis -methionine was added to the medium after treatment with emetine dihydrochloride and labelling was performed for 1 h, as previously described -ATP using T4 polynucleotide kinase (New England Biolabs) and purified with G-50 columns . RCC1LV1-3 isoforms were partly purified from sucrose gradient-purified mitochondria using Srep-Tactin Sepharose beads (iba), as described above. Elution fractions were dialysed overnight at 4°C in 20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 2 mM DTT, 1 mM PMSF, 50% glycerol. HEK parental sucrose gradient-purified mitochondria followed the same procedure and it was used as negative control. Increasing concentrations of dialysed protein  were incubated with 10 nM [γ32P]-end-labelled oligonucleotide for 30 min at 37°C in binding buffer . The mixtures were loaded onto native 6% polyacrylamide gels with 10 mM Tris-HCl pH 9.4, 75 mM glycine, 0.1 mM EDTA and run at 80V for 1.5 h. Gels were then dried and exposed to Typhoon phosphorscreens, with products visualized and quantified with ImageQuant software .Two homopolymer RNA oligonucleotides, rUAll experiments were done in duplicate or at least triplicate, when quantifications were required. X-ray films were digitalised and analysed with ImageJ software and data analyses were performed in Microsoft Excel. The data are presented as mean ± standard deviation (SD) or percentages of control. Student’s two-tailed unpaired t-test were used for comparisons.S1 Fighttp://www.ensembl.org/index.html). Representative mammalian species (common names on the left), representing nine different orders (on the right) and one outgroup species (chicken) were taken into consideration. Distinction between prosimian primates and anthropoid primates is marked in the figure. Red and white boxes indicate the presence or absence, respectively, of the isoform in each species based on Ensemble data. Pink boxes mean that the isoform is not reported in Ensemble for that species but it is in other species that share a common ancestor with the former ones. Therefore, they may have not been described yet in the database or less likely, they have been lost during evolution in those species.The presence of RCC1L isoforms was carried out in in Ensemble ((TIF)Click here for additional data file.S2 Figin silico predictions with different programs: Mitoprot (https://ihg.gsf.de/ihg/mitoprot.html), TargetP (http://www.cbs.dtu.dk/services/TargetP/), MitoFates (http://mitf.cbrc.jp/MitoFates/cgi-bin/top.cgi), TPred (https://tppred2.biocomp.unibo.it/tppred2) and Predotar (https://urgi.versailles.inra.fr/predotar/). The position of the predicted amino acid position cleavage for the mitochondrial targeting sequence (MTS) is also reported for each program.Probability scores of mitochondrial localisation based on (TIF)Click here for additional data file.S3 FigV1 and RCC1LV1 lacking 37 amino acids of the predicted N-terminal mitochondrial targeting signal (MTS) were stained with DAPI for the nucleus, MitoTracker Red for mitochondria and anti-FLAG antibody followed by Alexa 488 conjugated secondary antibody for the overexpressed RCC1L proteins. Co-localisation of MitoTracker and RCC1L-specific green signal appears yellow to orange, depending on the abundance, in the merged images. Panels from HeLa and RCC1LV1 are the same as those shown in Intra-cellular localisation of RCC1L isoforms by immunofluorescence. Parental and transfected HeLa cells expressing a STREP2-FLAG-tagged version of RCC1L(TIF)Click here for additional data file.S4 FigV1 and RCC1LV2 isoforms and the levels of the 37 kDa RCC1L containing RCC1LV3 are presented along with the FLAG signal from the overexpressed protein . In the case of RCC1LV3 overexpression, RCC1L antibody allowed the quantification of endogenous and overexpressed isoform, RCC1LV3 and RCC1LV3-FLAG, respectively, on the same blot. Transparent blue, orange and yellow colours mark the fractions where the 28S mtSSU (fractions 8–9), 39S mtLSU (fractions 6–7) and 55S monosome (fractions 4–5) peak, respectively whereas non-assembled subunit peaks are left unmarked (fractions 10–14). See Mitochondrial endogenous and overexpressed RCC1L profiles from isokinetic sucrose gradients from cells induced with 10ng/ml doxycycline for 3 days presented in (TIF)Click here for additional data file.S5 FigV1 and RCC1LV2, while the 37 kDa band (red arrowhead) corresponds to isoform RCC1LV3. The STREP2-FLAG-tagged RCC1L proteins  are also marked (grey arrowheads) in each case. Antibodies against structural components  and assembly factors (DDX28) of the mtLSU were used for immunodetection of proteins of interest. In the case of mtSSU analysis, immunoblot analysis was performed using antibodies against structural components (bS6 and mS35). Transparent blue, orange and yellow colours mark the fractions where the 28S mtSSU (fractions 8–9), 39S mtLSU (fractions 6–7) and 55S monosome (fractions 4–5) peak, respectively whereas non-assembled subunit peaks are left unmarked (fractions 10–14). Equal volume of each fraction was loaded for all cell lines.Mitochondrial ribosome profile in parental and RCC1L overexpressing cells after induction with 10ng/ml doxycycline for 3 days. Equal amounts of mitochondrial lysates from each of the four cell lines were separated on a 10–30% (v:v) isokinetic sucrose gradient and fractions were analysed by immunoblotting. In the immunoblots of endogenous RCC1L, the 50 kDa band (black arrowhead) contains isoforms RCC1L(TIF)Click here for additional data file.S6 FigP < 0.05, **P < 0.01. See Quantification of overall protein level of all the markers used in the isokinetic sucrose gradients presented in (TIF)Click here for additional data file.S7 FigA) Co-immunoprecipitation of STREP2-FLAG-tagged RCC1L isoforms from purified mitochondria after induction of HEK cells with 3–10 ng/ml doxycycline for 3–4 days. In the immunoblots of endogenous RCC1L, the endogenous isoforms (37 or 50 kDa band) are marked (empty arrowhead) in the elution fractions. Immunoblots for mitochondrial ribosomal proteins and biogenesis factors are presented. ACO2 was used as mitochondrial negative control for unspecific binding. Additional immunoblots not shown in B) Longer exposure of the panel shown in (A). (C) Quantification of mitochondrial DNA and RNA by quantitative PCR (qPCR) in the elution fractions after DNAse I, RNAse A and Benzonase treatment. Data represent the average of MT-CO1, MT-CYB and 12S rRNA from two independent experiments and relative to the untreated samples. See ((TIF)Click here for additional data file.S8 FigV1  and RCC1LV3  isoforms and combined silencing of RCC1LV1 and RCC1LV3  and all three isoforms  were performed in HEK cells. Mock (M) and AllStars dsRNA (S) transfected HEK cells were used as negative controls. Data represent mean ± SD from four independent experiments. t-test: *P < 0.05, **P < 0.01, ***P < 0.001. See Quantification of isoform-specific mRNA levels by quantitative PCR (qPCR) in silencing experiments shown in (TIF)Click here for additional data file.S9 FigV1  and RCC1LV3  isoforms and combined silencing of RCC1LV1 and RCC1LV3  and all three isoforms  were performed in HEK cells. Mock (M) and AllStars dsRNA (S) transfected HEK cells were used as negative controls. Data represent mean ± SD from two independent experiments. t-test: *P < 0.05, **P < 0.01, ***P < 0.001. See Quantification of protein levels in silencing experiments shown in (TIF)Click here for additional data file.S10 FigV1 (RNAi 1–3) and RCC1LV3 (RNAi 4–6) isoforms and combined silencing of RCC1LV1 and RCC1LV3 (RNAi 7–8) and all three isoforms  in HEK cells. Mock (M) and AllStars dsRNA (S) transfected cells were used as negative controls. Data represent the average of MT-CO1, MT-CYB and 12S rRNA normalised to APP from three independent experiments. See Quantitative PCR (qPCR) analysis of mitochondrial DNA copy number in silenced HEK cells. Silencing of RCC1L(TIF)Click here for additional data file.S1 TableSequence of the Stealth dsRNAi used for RCC1L silencing.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.


Moreover, TBC1D25 deficiency increases the phosphorylation levels of TAK1 and its downstream molecular (JNK and p38), whereas overexpressed TBC1D25 inhibits phosphorylation of TAK1, JNK and p38. And TAK1 is the key molecule in this process. Furthermore, we demonstrated that TBC1D25 could directly interacts with TAK1 by immunoprecipitation assay and GST pull-down assay, and the interaction needs the amino acids from at least 138 to 226 in the C-terminal region of TBC1D25 and from 1 to 300 in the C-terminal region of TAK1. We conclude that TBC1D25 suppresses pathological cardiac remodeling via regulating TAK1-JNK/p38 signaling pathway, which suggests that TBC1D25 will likely become a promising therapeutic target for heart failure.Cardiac remodeling is a major early event of heart failure, which is regulated by multiple signaling pathways. Here, we demonstrate that TBC1D25 is upregulated during pathological cardiac remodeling. The aim of this study is to determine the role of TBC1D25 in cardiac remodeling and to illustrate the underlying molecular mechanism. Specifically, cardiac remodeling was induced in TBC1D25-KO mice and their wild-type control mice through partial transverse aortic constriction (TAC) of aortic arch. Knockout TBC1D25 exacerbated cardiac hypertrophy, fibrosis and dysfunction. Meanwhile, TBC1D25 overexpression in both H9C2 cells and NRCMs alleviate Angiotensin II-induced cardiomyocyte hypertrophy  Heart failure (HF), a leading cause of morbidity and mortality in patients over 65 years of age, is a growing public health problem worldwide. Multiple pathological stimuli, such as hypertension, myocardial infarction or ischemia, cardiomyopathy, structural heart diseases, and other cardiovascular diseases are main risk factors in the progression of heart failurein vitro, TBC1D25-overexpressing and control cardiomyocytes were treated with Angiotensin II. The results indicated that TBC1D25 could negative regulate myocardial fibrosis, cardiac dysfunction and hypertrophy. Furthermore, we provided evidence that TBC1D25 could directly inhibit TAK1 phosphorylation, and protect against cardiac remodeling through TAK1- JNK/p38 signaling.Recently, the great progress in molecular biology techniques has made it possible to better understand molecular mechanism and identify new therapeutic targets involved in HF. TBC1Domain Family Member 25 (TBC1D25), also known as OATL1, is a protein contains a TBC (Tre-2/Bub2/Cdc16)/RAB-GTPase-activating protein (GAP) domain. It is identified as a protein participated in autophagy in previous studies in vitro study, TBC1D25 and ANP protein levels were significantly increased in H9C2 cells or neonatal rat cardiomyocytes (NRCMs) stimulated by Ang II  control mice, the loss of TBC1D25 protein in TBC1D25-KO mice was confirmed by Western blotting  or picrosirius red (PSR). After 4 weeks of TAC, TBC1D25-KO mice exhibited a significantly larger heart and cardiomyocytes cross-sectional area Figure A and morin vitro. However, TBC1D25 overexpression dramatically decreased the hypertrophic phenotypes induced by Ang II, compared with control cells  into AdGFP or AdTBC1D25 infected NRCMs, respectively Figure A. AdcaTATo further investigate the molecular mechanism of TBC1D25-regulated cardiac remodeling. We examined whether TBC1D25 could directly regulates TAK1 activation. The result showed that TBC1D25 and TAK1 were completely co-localized in the cytoplasm of 293T cells by immunofluorescent staining Figure A. And thHeart failure is one of the most common cardiovascular diseases with extremely high morbidity and mortality. Mainstream researches suggest that cardiac remodeling, the major early event of heart failure, is the key target in HF therapyin vitro. These findings indicated a new function of TBC1D25 in cardiac remodeling, which is undiscovered in previous study. Furthermore, our results indicate that TAK1 is the key molecule in this process. TBC1D25 could directly interact with TAK1 and significantly inhibit TAK1 phosphorylation in the development of cardiac hypertrophy. JNK and p38, the downstream molecular cascades of TAK1Here, we demonstrate the important function of TBC1D25 in pathological cardiac remodeling, which is unrecognized previously. TBC1D25 , mainly localized outside autophagosomes, is a protein contains a TBC (Tre-2/Bub2/Cdc16) /RAB-GTPase-activating protein (GAP) domain. TBC1D25 is identified as a LC3-banding protein, which could localize at LC3-positive isolation membranes and autophagosomes. Previous researches have shown that TBC1D25 plays an inhibitory role in the maturation step of autophagosomes through binding to LC3 in vivo and in vitro studiesin vivo and in vitro. This means that TBC1D25, even TAK1 did not affect the activation of ERK in this study, which had not been understood and needed further research. Additionally, the molecular mechanism of cardiac remodeling is extremely complicated, future studies are needed to elucidate the upstream of TBC1D25 in this process and clarify whether TBC1D25 participate in the development of other cardiovascular diseases.Transforming growth factor-β-activated kinase 1 (TAK1), also referred to as mitogen-activated protein kinase kinase kinase 7 (MAP3K7), controls a variety of cell functions including transcription regulation, inflammation, and apoptosisin vivo and in vitro experiments demonstrated that TBC1D25 protects against pathological cardiac remodeling. Knockout of TBC1D25 aggravates interstitial fibrosis, myocardial dysfunction and cardiac hypertrophy. Conversely, overexpression of TBC1D25 mitigates cardiac remodeling. The cardioprotective effect of TBC1D25 involves the suppression of TAK1-JNK/p38 signaling pathway. In brief, TBC1D25 will probably become a new therapeutic or research target in cardiac remodeling.In summary, our Experimental procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health . All animal usage protocols were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Zhengzhou University. The related procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Constitutive TBC1D25 knock-out (TBC1D25-KO) mice were purchased from the Texas A&M Institute for Genomic Medicine.Cardiac hypertrophy was induced in mice through partial transverse aortic constriction (TAC) of aortic arch, as previously described with some adaptationsMice were anesthetized with isoflurane (1.5-2%), and echocardiography was performed using a MyLab 30CV ultrasound system (Biosound Esaote Inc.) using a 15-MHz transducer. The size of the left ventricular (LV) cavity and LV wall thickness were acquired from at least 3 consecutive cardiac cycles. The end-systole and end-diastole were defined as the phases in which the smallest or largest LV area was obtained, respectively. The LV end-diastolic dimension (LVEDd) and LV end-systolic dimension (LVESd) were measured from the LV M-mode tracing, with a sweep of 50 mm/s at the mid-papillary muscle level. Fractional shortening (FS%) was calculated using the formula: FS%= (LVEDd-LVESd)/LVEDd×100%.Hearts were obtained from experimental animals 4 weeks after TAC surgery that had been perfused with a 10% potassium chloride solution to induce cardiac arrest at the end of diastole and then harvested and fixed with a 10% formalin solution. After being embedded in paraffin, the hearts were cut into 5-μm transverse sections. Sections were stained with hematoxylin and eosin (H&E) to measure the myocyte cross-sectional area, and the abundance of collagen was assessed after Picrosirius red (PSR) staining. Fibrosis was expressed as a percentage of the average positively stained area relative to the total area. More than 40 fields per group were examined. A quantitative digital image analysis system (Image-Pro Plus 6.0) was used for the image measurements.TBC1 Domain Family Member 25  and TAK1 (NM_003188.3) gene were amplified by nested PCR and then cloned into pHAGE-3×flag vector, or pcDNA5-flag and pcDNA5- HA vector, or GST-HA vector. Primer sequences used for cloning the coding sequences of TBC1D25 and TAK1 were as following: TBC1D25 forward, '-TCGGGTTTAAACGGATCCGGGATGGCAACAGCCTCC-3'; TBC1D25 reverse, 5'-GGGCCCTCTAGACTCGAGTCAAGATGCGGCTGTGGCCT-3'. TAK1 forward, 5'-TCGGGTTTAAACGGATCCATGTCTACAGCCTCTGCCGCCT-3'; TAK1 reverse, 5'-GGGCCCTCTAGACTCGAGTCATGAAGTGCCTTGTCGTTTCT-3'.5 cells/well. Cultured them in DMEM/F12 containing 20 % FCS, BrdU , and penicillin/streptomycin at 37℃ for 48 h. Then, replaced the culture medium with serum-free DMEM/F12 for 12 hours. For further experiments, AdGFP (used as the non-targeting control) or AdTBC1D25 adenoviruses were generated and infected into NRCMs at a multiplicity of infection (MOI) of 10 for 24h. Similarity, AdcaTAK1 (adenovirus expressing constitutively active TAK1) were co-infected into AdGFP or AdTBC1D25 infected NRCMs respectively.Primary neonatal rat cardiomyocytes (NRCMs) were prepared as described previouslyH9C2 cardiomyocytes and HEK-293T cell lines were maintained in DMEM medium containing 10% fetal bovine serum and 1% pencillin/streptomycin. For lentiviral infection, recombinant constructs were firstly co-transfected into HEK-293T cells with lentiviral helper plasmids (pMD2.G and psPAX2) and PEI transfection reagent. After 6 to 16 hours, fresh media were replaced and lentiviral particles were harvested 48 hours later and then used for target cell infection. Briefly, viruses containing TBC1D25 and control particles were added to H9C2 cardiomyocytes in the presence of polybrene (8μg/mL) respectively. Infected cells were selected by puromycin (2μg/mL) for 48 hours and then used for indicated assays.2 and fixed with 3.7% formaldehyde. After permeabilization with 0.1% Triton X-100 in PBS and blocking with a 10% BSA solution at room temperature, cells were immunostained with an α-actinin antibody , followed by staining with a fluorescent secondary antibody . Image-Pro Plus 6.0 software was used to measure the surface area of the cell.Immunofluorescence staining was performed to determine the surface area of the cell. H9C2s and NRCMs were stimulated with PBS or Ang II (1 μmol/L) for 48 h under a condition of 37.0 °C, 5% COFor the RT-PCR assay, total mRNA was extracted from ventricular tissues or cells with TRIzol reagent . Then, cDNAs were reverse transcribed from RNAs using the Transcriptor First Strand cDNA Synthesis Kit . Quantitative real-time PCR was used to detect the expression of selected genes with SYBR Green PCR Master Mix . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The primer pairs used in this study are listed in For the Western blot analyses, total proteins were extracted from ventricular tissues or cell samples using RIPA lysis buffer , and the protein concentration was determined with a BCA Protein Assay Kit . After fractionation by using SDS-PAGE, proteins were transferred to PVDF membranes and blocked with 5% non-fat milk at room temperature for 1h. After incubated with multiple antibodies overnight at 4 °C, the secondary antibodies were added next day, and bands were visualized using a Bio-Rad ChemiDoc XRS+ system (Bio-Rad). The levels of specific proteins were normalized to the levels of GAPDH on the same PVDF membrane. The antibodies used in this study are listed in IP was performed as previously describedEscherichia coli, and then induced them with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an optical density of 600nm (OD600) of 0.6. Escherichia coli extracts were dealt with PBS containing protease inhibitor cocktail tablets . Subsequently, incubated them with glutathione-sepharose 4B beads  for 1h at 4 ℃. The proteins binding to the beads were washed five times with 1ml of PBS, and then incubated them for an additional 4h at 4℃ with immunopurified Flag- TBC1D25 or Flag-TAK1. Then washed the glutathione-sepharose 4B beads with 1ml of the NaCl wash buffer for three times. And the eluted proteins were analyzed by Western blotting using indicated antibodies. In the same conditions, the GST tag was used as the negative control.In glutathione S-transferase (GST) pull-down assayImmunofluorence and confocal microscopy assays were detected as previously described t-test. Differences among more than 2 groups were assessed using one-way analysis of variance (ANOVA), followed by the Bonferroni test  or Tamhane's T2  test. A value of P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS  software, version 21.0.Data are presented as means ± S.D. Comparisons between two groups were performed using a two-tailed Student's