IRF7) and UNC-93 homolog B1 (UNC93B1). PBMC single-cell RNA-Seq done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and human leukemia monocytic THP1 cells showed that both variants individually suppressed TLR3-driven IRF3 transcriptional activity and the type I IFN response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I IFN response upon HSV-1 challenge. This study reports an infant with recurrent HSV-1 disease complicated by encephalitis associated with deleterious variants in the IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent, severe HSV.Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We evaluated a male infant with neonatal skin/eye/mouth (SEM) HSV-1 disease, who had complete recovery after acyclovir but developed HSV-1 encephalitis at 1 year of age. An immune workup showed an anergic PBMC cytokine response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IFN-regulatory factor 7 (  Neonatal herpes simplex virus (HSV) presents in neonates in the first 28 days of life as 1 of 3 subtypes: skin/eye/mouth (SEM), CNS, or disseminated disease. SEM disease is localized to the skin or mucosal membranes, and it is the most common presentation of neonatal HSV. CNS disease is infection localized to the CNS with or without SEM involvement. Disseminated disease is the most severe form and is defined as the involvement of multiple organ systems, most commonly the lungs and the liver. CNS and SEM involvement can also be present in disseminated disease , UNC-93 homolog B1 (UNC93B1), and IFN-regulatory factor 3 (IRF3), among others, increase susceptibility to HSE  on evaluation, which was initially attributed to testing positive for multiple respiratory viruses including adenovirus, coronavirus, and rhino/enterovirus. The infant was admitted to our pediatric intensive care unit for status epilepticus and was found to have HSE with CSF positive for HSV-1. He was also noted to have 500 copies/mL HSV-1 on plasma quantitative PCR (qPCR). Brain MRI revealed findings consistent with diffuse herpes encephalitis, which was worse in the left temporal lobe, right shift of the septum pellucidum, and left uncal herniation. He was treated with i.v. acyclovir at 15 mg/kg/dose 3 times daily for 21 days, followed by oral lifelong prophylaxis at 20 mg/kg/dose 3 times daily. To control his refractory seizures, he required a midazolam drip and later a 48-hour pentobarbital-induced coma. After adequate seizure control was achieved and respiratory support was weaned, the medical team transferred the patient to the Rehabilitation Service, where he stayed for 2 months prior to his discharge home. The patient had severe loss of motor function, cognition, tone, mobility, and communication.The infant presented to the emergency department on day 7 of life with a worsening vesicular rash, which started on his back and spread to his chest. A full infectious workup was performed including tests for HSV. There was no maternal history of HSV infection. HSV cultures of the vesicular lesions, rectum, and mouth returned positive for HSV-1. HSV PCR of the cerebrospinal fluid (CSF) returned negative. HSV PCR of the blood was positive, with 76,000 copies/mL HSV-1. The patient did not demonstrate any other signs of disseminated HSV such as liver involvement. However, because of the diffuse nature of his vesicular rash, he received 21 days of i.v. acyclovir at 20 mg/kg/dose 3 times daily and was discharged home with a 6-month prescription for oral acyclovir for suppressive therapy at 300 mg/m10 copies. Mitogen and antigen Candida stimulation was normal, indicating normal T cell function. The patient had normal granulocyte oxidative burst and normal NK cell function. His IgG was borderline low, which was likely due to his acute illness, and he received 1 dose of i.v. Ig. His IgM, IgA, and IgE levels were normal. Subsequent IgG levels, NK cell count, and T cell counts were normal when repeated 1 month after the acute phase of the illness. The patient exhibited an antibody response to tetanus in the intermediate range (0.04 IU/L) and in the low range to Haemophilus influenzae (<0.11 mg/L) related to impaired antibody formation or simply to waning immunity. The HIV antibody screen was nonreactive. The child’s PBMC functional response to specific TLR ligands was evaluated by a Clinical Laboratory Improvement Amendments–certified (CLIA-certified)laboratory (ARUP Laboratories). This evaluation showed severely attenuated IL-1β, IL-6, and TNF-α induction with TLR3 [poly(I:C)] stimulation, but normal responsiveness to TLR1/-2, TLR2/-6, TLR4, TLR5, and TLR7/-8 ligands when compared with simultaneously run controls (https://doi.org/10.1172/JCI154016DS1). Clinical exome sequencing with targeted analysis of known HSV susceptibility and immune genes revealed heterozygous missense variants in IRF7  and UNC93B1 .The medical team performed an immunological workup. Flow cytometry initially showed T cell lymphopenia and NK cell lymphopenia with a normal B cell count. Naive Th and naive cytotoxic T cell counts were low. The exact laboratory values are shown in n = 364) and our proband  of PBMCs when our patient was 83 months old and did not have an acute illness. We leveraged our existing Genomic Answers for Kids (GA4K) PBMC scRNA-Seq data set from 364 individuals aged 1 month to 25 years as controls. We identified 4 major immune cell populations  as well as several other subpopulations  followin proband . Since oFOS, FOSB, and JUN and of NF-κB, such as NFKB1, were downregulated. Because AP-1 and NF-κB are transcriptional regulators of cytokine responses to stress and bacterial and viral infections, the proband’s transcriptomic signature suggested a potential for a repressed immune response   . TRAF3 in adults , 21. In IRF7  gene, with a minor allele frequency (MAF) of 0.00007 . The variant was predicted to be deleterious using SIFT, Polyphen II, and MAPP. IRF7 is a transcription factor, and its activation is required for an effective type I IFN response to viruses . We used poly(I:C), a synthetic analog of dsRNA and activator of TLR3 signaling, to stimulate THP1 cells transfected with WT IRF7 or IRF7  alleles (IRF7 enhanced the baseline responsiveness to poly(I:C), implying that IRF7 amplifies TLR3-induced IRF3 transcriptional activity, as has been shown by studies noting that IRF7-IRF3 heterodimers regulate type I IFN responsiveness  variant suppressed even baseline poly(I:C)-induced IRF3 transcriptional activity. We next examined TLR3-stimulated type I IFN responsiveness with the WT and variant IRF7 alleles. Poly(I:C)-stimulated IFNA and IFNB gene expression induced by WT IRF7 was abolished in cells transfected with the IRF7  variant  variant -induced IRF3 transcriptional activity and type I IFN responses.The  of IRF7  and is i alleles . We tran variant . These r variant . These dUNC93B1  gene, which has a mean allele frequency of 0.0006  , the UNC93B1  allele repressed IRF3 transcriptional activity (IFNA and IFNB expression induced by poly(I:C) was enhanced by the WT UNC93B1 allele, but strongly suppressed by the UNC93B1 variant allele -mediated IRF3 transcriptional activity and type I IFN secretion.Our patient was also heterozygous for a missense mutation in the 7021545) . UNC93B17021545) . Luciferactivity . Similart allele . As the  to TLR3 . These dIRF7 and UNC93B1 into THP1 cells, followed by challenge with poly(I:C). Poly(I:C)-mediated IRF3 transcriptional activity  in THP1 cells, mimicking the anergy to poly(I:C) noted in the child’s PBMC responses. Our studies also revealed that UNC93B1 and IRF7 variants repressed RNA expression of NFKB1, IL1B, and TRAF3 both in vivo and in vitro.We next pursued studies to examine the deleterious effect of both variants combined in our cell culture system to mimic the clinical scenario in our proband. We transfected both the WT and variant alleles of activity  and typeactivity  were sevonocytes . Interespression . ExpressP1 cells . These dIRF7 and UNC93B1 variants disrupted the TLR3-dependent response to extracellular stimulation with poly(I:C), we postulated that identified variants would impair the immune response to HSV-1. Human fibroblasts display a TLR3-dependent response to HSV and poly(I:C) and have previously been used to characterize functional defects in innate immune genes  proteins  to stimulate generated fibroblast lines showed similar results, with the UNC93B1 and IRF7 variants resulting in partial and total loss of type I IFN responsiveness, respectively , NFKB1, and IL1B ; poly(I:C), a synthetic analog of dsRNA (TLR3 ligand); LPA ultra-pure Salmonella minnesota LPS (TLR4 ligand); flagellin purified from Salmonella typhimurium (TLR5 ligand); and CLO97 imidazoquinoline compound (TLR7-TLR8 ligand).TLR function testing was performed by functional assay on PBMCs (ARUP Laboratories). TLRs were tested independently by stimulation with TLR-specific ligands. PBMC cytokine production of IL-1β, IL-6, and TNF-α was determined by multianalyte fluorescence detection. The TLR-specific ligands included PAM3CSK4, a synthetic bacterial lipoprotein (TLR2-TLR1 ligand); zymosan cell wall particles from The GA4K is a large pediatric rare disease program at Children’s Mercy Research Institute in Kansas City that collects genomic data and health information for families with a suspected genetic disorder. The goal of GA4K is to expand diagnostic capabilities and catalog rare disease genomes and phenotypes within a health care system. Patients are referred from various specialties for inclusion in GA4K. After a patient is referred for the study, informed written consent is obtained from all participants prior to study inclusion. DNA extracted from the proband’s blood samples was used for whole-exome sequencing (WES) at the Center for Pediatric Genomic Medicine (CPGM) at Children’s Mercy Hospital. Exome libraries were prepared according to the manufacturer’s standard protocols for Illumina TruSeq library preparation and IDT XGen Exome Enrichment (Integrated DNA Technologies). Briefly, 250 ng high-quality genomic DNA (gDNA) was sheared by Covaris sonication to an average size of 450 base pairs. DNA fragments underwent end-repair, a-tailing, adapter ligation, and associated AMPure bead cleanups on a Hamilton NGS Star liquid handler. After adapter ligation, 10 cycles of standard PCR were performed with KAPA HiFi master mix (Roche) and primers specific to the ligated adapters, followed by a bead cleanup. The resulting libraries underwent quality assessment for the appropriate concentration and final library size. Six libraries were pooled together at 1,000 ng each for exome enrichment. Pooled libraries were blocked to prevent nonspecific binding and lyophilized with a vacuum concentrator. Pools were reconstituted with enrichment buffer (Integrated DNA Technologies [IDT]), standard exome baits, CNV baits, and custom mitochondrial baits. This exome enrichment reaction was subjected to a 4-hour hybridization, followed by incubation with streptavidin beads and serial washes with IDT’s enrichment wash buffers on a PerkinElmer SciClone liquid handler. An additional 10 cycles of standard PCR were performed with KAPA HiFi master mix and primers specific to the adapters, followed by a final bead cleanup. The resulting enriched pools underwent quality assessment for the appropriate concentration and final library size, as well as a TaqMan qPCR assay (Thermo Fisher Scientific) to ensure successful enrichment of target regions before standard Illumina Free-Adapter Blocking was performed. Cleaned, adapter-blocked pools were loaded onto an Illumina NovaSeq 6000 with a run configuration of 151 × 8 × 8 × 151. Resulting data were processed with Illumina’s bcl2fastq software. Sequence alignment and variant detection were performed with the Illumina DRAGEN Bio-IT Platform (versions 3.02–3.6.3) against the GRCh37 reference genome.http://genetics.bwh.harvard.edu/pph2/) and SIFT . RUNES incorporated data from Ensembl’s Variant Effect Predictor software, produced comparisons with dbSNP, ClinVar, the Exome Aggregation Consortium (ExAC), gNOMAD (gnomad.broadinstitute.org), and known disease variants from the Human Gene Mutation Database , 36, 37.and SIFT , 41.6 pooled PBMCs following the 10x Genomics’ Nuclei Isolation for Single Cell Multiome ATAC + GEX Sequencing protocol using a lysis time of 3 minutes. After transposition, bel beads in emulsion were generated by loading 16,100 nuclei into each of 4 lanes of a Chromium Next GEM Chip J Single Cell , and libraries were prepared with the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle  following the manufacturer’s protocol. Completed libraries were sequenced on an Illumina NovaSeq 6000. Gene expression (scGEX/scRNA) libraries were sequenced to a depth of 4.83 × 108 reads per library with a run configuration of 28 × 10 × 10 × 90. Single-cell assay for transposase-accessible chromatin sequencing (scATAC) libraries were sequenced to a depth of 2.66 × 108 reads per library with a run configuration of 52 × 8 × 24 × 52. Only single-nucleus (scRNA-Seq) data were used for analysis in this study.PBMC samples from 32 individuals/pool were thawed and pooled proportionally on ice. Nuclei were isolated from an aliquot of 1 × 10https://www.ncbi.nlm.nih.gov/gap/) under the accession number phs002206.v4.p1. To process results from (RNA + ATAC) multiome sequencing experiments, we ran cellranger-arc (version 2) on all pools sequenced. From the cellranger-arc outputs, we determined the sample identity of each cell barcode with demuxlet using a genotype that had been imputed on the TopMed imputation server. Next, we used Seurat (version 4) and Signac in R to analyze the cellranger-arc/demuxlet results. Then, from the individual ATAC peak regions for all 88 captures, we created a common set of peak regions. We then applied QC filters to the cells  which: (a) kept only cell barcodes whose RNA and ATAC demuxlet identities were both classified as singlets , and (b) whose standard Seurat filters fell within a “normal” range . Once the individual captures were filtered, we split the data set into 2 sets (A and B) by randomly assigning half the captures of each pool to each set . For each of the remaining steps, we did the analysis separately for data sets A and B. Using the common peak set that was previously created, we merged the Seurat objects from each capture into an aggregate. With the aggregate, we then created a new joint multimodal (RNA + ATAC) assay using the weighted nearest neighbor (WNN) method in Seurat. From the WNN graph, we then built a joint uniform manifold approximation and projection (UMAP) visualization. To determine cell-type clusters, we transferred cell-type labels to a previously studied multiomic PBMC data set. Then, we ran differential expression using Seurat to analyze the difference in RNA expression between sample cmh004121-01 and all other samples in the GA4K data set. This was done separately for each cell-type cluster. All data are deposited in dbGaP  reporter gene that is induced upon the addition of bacterial TLR ligands, as well as an IRF3-inducible secreted luciferase upon stimulation with high-molecular-weight (HMW) poly(I:C) (InvivoGen), a TLR3 ligand. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue, a SEAP detection reagent, and QUANTI-Luc, a luciferase detection reagent (InvivoGen). THP1-Dual was used to transfect the WT, mut, or mock (Ø) plasmids from UNC93B1 and IRF7 using Lipofectamine LTX Reagent with PLUS Reagent according to the manufacturer’s protocol (Thermo Fisher Scientific). Cells were used 48 hours after transfection. The HEK293T cells were used to transfect the WT, mut, or Ø plasmids from IRF7 using Lipofectamine 3000 following the manufacturer’s protocol (Thermo Fisher Scientific).A commercial THP1 monocyte cell line  that had 2 inducible reporter constructs integrated, one for measuring NF-κB transcriptional activation and another for IRF3 transcriptional activity, was purchased from InvivoGen . Cells were grown in RPMI 1640 media containing 10% FBS and antibiotics according to the manufacturer’s protocol in a humidified incubator containing 5% COIRF activation with an IFN regulatory factor–inducible Lucia luciferase assay using the QUANTI-Luc detection system (InvivoGen) according to the manufacturer’s instructions. Briefly, cell culture supernatants (in triplicate) obtained from transfected THP1 cells 20 hours after poly(I:C) treatment were used. As quickly as possible, 20 μL supernatant was incubated with 50 μL QUANTI-Luc assay solution, the solution and supernatant were mixed by tapping the plate, and the plate was put into the plate reader. The luciferase was measured by the luminometer with a 0.1 second read time.We detected IRF7 cDNA was amplified from pCMV3-IRF7  with forward primer 5′-catGCTAGCcaccatggccttggctcctgagagggcag-3′ and reverse primer 5′-ctaCTCGAGctaggcgggctgctccagctccataagg-3′ and cloned into pIRES-EGFP-puro (Addgene) between NheI and XhoI. The G299C mutation  was introduced by 2-step PCR with 5′-CTTCCGCTGCGCACTGCCCAGCACGCGTCGCTTCG-3′ and its reversed sequence. Human UNC93B1 cDNA was amplified from pUC-hUNC93B1  with forward primer 5′-catGCTAGCcaccatggaggcggagccgccgccta-3′and reverse primer 5′-ctaGAGCTCtcactgctcctccggcccgtctcc-3′ and cloned into pIRES-EGFP-puro (Addgene) between NheI and SacI. The C1210T mutation was introduced by 2-step PCR with 5′-cacgcccggtgTccctggtggcc-3′ and its reversed sequence. FLAG-tagged WT or mut IRF7 was amplified and subcloned into a lentiviral vector of pHIV-dTomato (Addgene) with 5′-CGTCTAGAACCATGGCCTTGGCTCCTGAGAG-3′ and 5′-CGGGATCCTTACTTATCGTCGTCATCCTTGTAATCGGCGGGCTGCTCCAGCTCCATAA-3′. For the construction of a lentiviral vector expressing both WT and mut IRF7 and WT and mut UNC93B1, cDNA of UNC93B1 WT or mut (without the stop codon) were inserted in front of FLAG-IRF7 interspaced with a P2A peptide (GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT). DNA sequencing was done to confirm our constructs (Eurofins Genomics). Expression of IRF7 and UNC93B1 was confirmed by Western blotting of cell lysates from HEK293T cells transfected with lentiviral vectors.Human 2 at 37°C. HEK293T cells were used to create a stable cell line for the WT and mut IRF7 plasmids. Briefly, the lentiviral vector and packing vectors psPAX2 and pMD2.G were transfected into HEK293T cells using calcium phosphate transfection to produce lentiviral particles , IRF7 WT:UNC91B1 WT, IRF7:UNC93B1 mut, IRF7 mut:UNC93B1 WT, and UNC93B1 mut:IRF7 WT.Newborn human foreskin fibroblasts were purchased from Coriell Institute for Medical Research (GM21808) and grown according to the manufacturer’s protocol in a humidified incubator containing 5% COIRF-Lucia luciferase assay that is unique to the THP1-Dual monocytes was run following the manufacturer’s protocol (Thermo Fisher Scientific) and read on a luminometer plate reader. The assay is designed to monitor the activity of the IRF pathway by assessing the activity of the Lucia luciferase.The IRF7 reporter luciferase assay examines the ability of WT and mut IRF alleles to induce IRF7 transcriptional activity. We performed this assay using both transient transfection of IRF7 alleles into HEK293T cells and into the 293T/Ø-dTomato, 293T/IRF7-FLAG-dTomato and 293T/IRF7mut-FLAG-dTomato cell lines we generated. PGL4-4X IRF7-minP-Luc was generated by inserting 4 copies of IRF7 consensus–binding sites (ACGAAAGCGAAAGTACGAAAGCGAAAATACGAAAGCGAAAGTACGAAAGCGAAAAT) upstream of the minimal promoter-driven luciferase reporter of pGL4.29 (Promega) , CFP-TLR3 (Addgene), and pIRES-Puro-EGFP-IRF7 (or mut) plasmids were cotransfected into HEK293T cells. The luciferase assay was applied using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions after 2 days of transfection. In HEK293T stable cell lines expressing WT or mut IRF7 alleles, we transfected pGL4-4X IRF7-minP-Luc, Renilla luciferase vector (pRL-TK), and CFP-TLR3. Two days after transfection, the luciferase reporter assay was performed in cell lysates.The Promega) . pGL4-4XHSV-1 was purchased commercially from American Type Culture Collection (ATCC) . The virus was propagated in Vero cells  and titrated as previously described .5/well) and infected with HSV-1 at a MOI of 10 for 1 hour, after which the media were exchanged for fresh culture media. Sixteen hours after infection, the fibroblasts were lysed with TRIzol (Thermo Fisher Scientific), and gDNA was isolated according to the manufacturer’s protocol (QIAGEN). The HSV-1 copy number and cell population were quantified by qPCR with the following HSV-1–specific primers and fibroblast primers: HSV-1 TK (forward), ATGGCTTCGTACCCCTGCCAT; HSV-1 TK (reverse), GGTATCGCGCGGCCGGGTA; fibroblast (forward), CCCGTGTTTAGCCTTGTTAAAG; fibroblast (reverse), CTAGGAATCCCGGACAGTTTG . RNA was made into cDNA using iScript, following the manufacturer’s protocol (Bio-Rad). PCR for IFN-α and IFN-β was performed on a Bio-Rad CFX machine using primers manufactured by MilliporeSigma with SYBR (EVA) green from Biotium according to the manufacturer’s protocol.RNA was harvested from THP1-Dual monocytes transfected with WT or mut UNC93B1, variant UNC93B1, or Ø plasmids. Cells were lysed with Pierce IP lysis buffer (Thermo Fisher Scientific) combined with protease and phosphatase inhibitors (MilliporeSigma). The clarified cell lysates were immunoprecipitated using protein G Surebeads  following the manufacturer’s protocol, with a small aliquot taken out prior to IP for Western blot analysis. Western blots were run for protein analysis following standard protocols. IP was completed using mouse anti-TLR3 . The antibodies used for Western blot analysis were: rabbit anti-TLR3 , anti-UNC93B1 , mouse anti–IRF-7 , and mouse anti–β-actin . Densitometry was performed using ImageJ Software (NIH), and changes were normalized to β-actin or TLR3 (IP).THP1-Dual monocytes were transfected with WT P value of less than 0.05 as assessed using an unpaired samples t test. The IPA system  was used for subsequent bioinformatics analysis, which included canonical pathways, upstream analysis, diseases and functions, and regulator effects. For analyses, the –log >1.3 (P < 0.05) was set as the threshold, and a z score of ± 2 was defined as the threshold for significant activation and repression.Gene expression data from scRNA-Seq were obtained from differentially expressed genes and defined as a P value of less than 0.05 was considered significant. For cell culture experiments, data from a minimum of 3 independent experiments with adequate technical replicates were used for quantification. RNA quantification and PCR were done with 2–3 technical replicates. Analysis was initially performed to determine whether the distribution of the data was Gaussian using the D’Agostino-Pearson omnibus normality test. If data were normally distributed, then ANOVA with a post hoc Tukey test was used for analysis. If data did not meet Gaussian assumptions, a 2-tailed Mann-Whitney U test was used for analysis. For most analyses, fold changes were calculated for the expression/changes in the Ø controls. Statistical analysis was done using Graphpad Prism 9.0 (GraphPad Software). For scRNA-Seq data, a Wilcoxon rank-sum test with Bonferroni’s correction was applied for multiple comparisons.Data are presented as the mean ± SD or the median with the IQR, and a Written informed consent was obtained from the parent of the proband prior to performing the studies. The proband’s immunological workup and exome sequencing were done as part of the clinical workup. In vitro functional studies were conducted under the approval of the Institutional Biosafety Committee of Children’s Mercy Hospital (protocol no. 00025). The patient was later referred and consented to the GA4K study for further genomic analysis. This study was approved by the Children’s Mercy IRB (study no. 11120514).MHT analyzed data, drafted the initial manuscript, and reviewed and revised the manuscript. WY planned and conducted experiments, acquired and analyzed data, wrote certain parts of the manuscript, and reviewed and revised the manuscript. The order of the co–first authors’ names was determined alphabetically. HM conducted experiments, acquired and analyzed data, drafted figures and figure legends, and reviewed and revised the manuscript. SX conducted experiments, acquired and analyzed data, and reviewed and revised the manuscript. CFS acquired and analyzed the single-cell data and reviewed and revised the manuscript. DL and MG were involved in performing the scRNA-Seq experiments and reviewed and revised the manuscript. TP conceived the scRNA analysis, supervised the scRNA-Seq experiments, and reviewed and revised the manuscript. NR analyzed the data and reviewed and revised the manuscript. VS conceptualized and designed the research studies, analyzed the data, drafted the initial manuscript, and reviewed and revised the manuscript.


Although rotavirus A (RVA) is the primary cause of acute viral gastroenteritis in children and young animals, mechanisms of its replication and pathogenesis remain poorly understood. We previously demonstrated that the neuraminidase-mediated removal of terminal sialic acids (SAs) significantly enhanced RVA-G9P[13] replication, while inhibiting RVA-G5P[7] replication. In this study, we compared the transcriptome responses of porcine ileal enteroids (PIEs) to G5P[7] vs. G9P[13] infections, with emphasis on the genes associated with immune response, cholesterol metabolism, and host cell attachment. The analysis demonstrated that G9P[13] infection led to a robust modulation of gene expression (4093 significantly modulated genes vs. 488 genes modulated by G5P[7]) and a significant modulation of glycosyltransferase-encoding genes. The two strains differentially affected signaling pathways related to immune response, with G9P[13] mostly upregulating and G5P[7] inhibiting them. Both RVAs modulated the expression of genes encoding for cholesterol transporters. G9P[13], but not G5P[7], significantly affected the ceramide synthesis pathway known to affect both cholesterol and glycan metabolism. Thus, our results highlight the unique mechanisms regulating cellular response to infection caused by emerging/re-emerging and historical RVA strains relevant to RVA-receptor interactions, metabolic pathways, and immune signaling pathways that are critical in the design of effective control strategies. Rotaviruses (RVs) are a leading cause, globally, of acute viral gastroenteritis in both children and production animals . The gloSedoreoviridae family, Rotavirus genus, this nonenveloped virus possesses 11 segments of double-stranded RNA (dsRNA) in a triple-layer viral capsid. These 11 segments encode dsRNA for structural proteins  and non-structural proteins (NSP and NSP1-5/6)  (G9P[13] strain) and RVA G5P[7] were used in the study. Intestinal contents were diluted at a 1:10 ratio in sterile minimal essential media . Contents were then centrifuged at 2095× 5 fluorescent foci units/mL) were activated prior to infection using 10 µg/mL trypsin  for 30 min at 37 °C and diluted in complete medium without growth factors (CMGF-) to achieve the desired MOI (1.0). PIEs were incubated with RVAs for 1 h at 37 °C and then washed twice with CMGF- and placed in 96-well plates in triplicate and harvested at 24 hours post-infection. Mock control PIE cells were treated identically, but inoculated with CMGF- with 10 ug/mL trypsin without RVA. Plates were kept at −80 °C until RNA was extracted from homogenized cells using a MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit .The rotavirus infection protocol was described by Guo et al. . BrieflySus scrofa 11.1 genome release 97  using the Rsubread aligner  vs. G5P[7] replication on the host (PIE) gene expression profile, we used transcriptome analysis. We demonstrated that G9P[13] replication in PIEs resulted in significant modulation of the expression of 4093 genes , with 19F11R . Infection with both RVAs only marginally modulated the expression of gene encoding integrin αvβ3 (data not shown). However, G9P[13] infection of PIEs led to a significant upregulation of tight junction protein JAM-A—F11R , while tynthesis ) after iRIG-1) by G5P[7] ; molecules associated with different functions including immune regulation [“role of double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in interferon induction and antiviral response” and “N-formyl-Met-Leu-Phe (fMLP) signaling in neutrophils”] . Furthery G5P[7] B. This fAdditionally, IPA identified the top significantly affected molecules and G9P[13] infection had a staggering >200 fold-change increase of ANPEP and TMIGD1, suggestive of an increased granulocyte and cell recovery response . Of intep-value—1.944)—while G5P[7] infection was not associated with significant changes in cholesterol-related pathways. Ceramides are important bioactive sphingolipids involved in a variety of cellular processes such as cell growth and differentiation, inflammation, and apoptosis, including the biosynthesis of oligosaccharides and, therefore, gangliosides  resulted in a significant upregulation of only one pathway associated with lipid metabolism—the ceramide pathway , the dominant negative inhibitor of basic helix-loop-helix proteins (ID1) , the groGlobally, we face a prevalence of emerging RVA strains with seemingly increased interspecies transmission potential, such as G9P[13] ,53,54,55In sharp contrast with previous observations suggestive of more efficient G5P[7] vs. G9P[13] replication in vivo and in vitro , ourThe significant modulation of the expression of the genes encoding sialyltransferases and fucosyltrasferases and some attachment factors following G9P[13] infection suggests that this RVA could have evolved a mechanism regulating the availability of cell attachment factors, including unmasking additional receptors that can be covered by glycoconjugates. Importantly, sialyltransferases are enzymes responsible for SA transfer to nascent carbohydrates and they can regulate the availability of RVA attachment sites. Infection with the sialidase-dependent RVA strain, G9P[13], significantly modulated (mostly downregulated) the expression of the genes encoding sialyltransferases of the four major families, ST3Gal, ST6Gal, ST8Sia, and ST6GalNAc , which mABO with both cholesterol  induced a significant upregulation of the IL-6 and IL-8 signaling pathways that have been previously shown to be involved in the pathogenesis of acute RVA-associated gastroenteritis . HoweverApart from the potential link to T1 diabetes, RVAs have not been demonstrated to have long-term health effects, to the best of our knowledge . HoweverTaken together, this study demonstrated strain/genotype-specific modulation of the host cellular response to RVA infection, further emphasizing the unique interactions between SAs and other host factors and G9P[13] and G5P[7]. Additionally, our results suggest that long-term circulation and adaptation to swine might have increased the within-host viral fitness of G5P[7] RVA, resulting in higher replication rates and a reduced transcriptome/immune response. In contrast, G9P[13] replication was associated with a robust modulation of the host transcriptome and a significant downregulation of sialyltransferase expression. Importantly, the latter can lead to decreased sialylation and the unmasking of additional attachment factors, including gangliosides. We plan to further investigate these results, using reverse genetics and CRISPR-Cas9 gene editing to mechanistically analyze G9P[13]-specific interactions with SAs to increase its replication, and to determine how to manipulate this mechanism to reduce its growing global prevalence.


PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle–specific deletion of Cdk4’s target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber–specification that is of relevance to metabolic and muscular diseases.Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of   The variable temporal advancement of type 2 diabetes (T2D) characterized by progressive dysfunction in multiple targeted organs renders ineffective most therapeutic modalities –3. IneffSkeletal muscle’s ability to perform diverse functions, including movement, thermogenesis, and metabolism, is largely attributed to the plasticity of muscle fibers that inherently differ in their contractile ability, mitochondrial content, and metabolic properties . SkeletaInk4a  that is also linked to muscle fiber–type specification and metabolic disease  mice and constitutively active Cdk4R24C/R24C (Cdk4R/R) mice (Cdk4R/R mice harbor the arginine to cysteine point mutation at the 24th amino acid position on the Cdk4 protein (hence the term Cdk4R24C). This mutation renders the Cdk4R24C kinase insensitive to inhibition by p16Ink4a — a product of the CDKN2A locus (Cdk4R/R mice were longer and heavier (https://doi.org/10.1172/JCI162479DS1), with a significant increase in skeletal muscle mass. When fed regular chow diet (RD), the mice exhibited lower fasting glucose levels (Cdk4R/R mice exhibited improved glucose tolerance (We used Cdk4-deficient (/R) mice . Cdk4R/R2A locus , 40. Cdke levels , with noe levels , D and Ee levels . In respolerance , enhanceolerance , reducedolerance , I–K.Cdk4R/R mice maintained lower body weight and reduced fat mass  fat mass . Food in fed HFD . HFD-fed feeding . These dean mass , reducedean mass , and decean mass . Moreoveean mass  and imprean mass . In addiean mass , suggest markers . Reduced markers , A and B markers , C and E markers , F–I. TaCdk4R/R mice exhibited significantly higher whole-body glucose uptake, specifically in skeletal muscle  and mitochondrial genes  were elevated in muscles, but not liver, WAT and BAT from Cdk4R/R mice  and IIA (MyhcIIa) fibers; in contrast, muscles like the extensor digitorum longus (EDL) and quadriceps contain more of the faster type IIB (MyhcIIb) fibers that have lower mitochondrial numbers , with no change in fast/glycolytic specific markers   , A–D.Cdk4R/R muscle exhibited higher cell proliferation (Cdk4R/R and Cdk4WT mice (MyhcI and MyhcIIa) were significantly increased at 7 and 14 days after CTX injection in Cdk4R/R mice, without any change in fast/glycolytic marker gene expression  muscle injury model . CTX wasferation . Consistferation , A and B4WT mice , L and Mpression . In agrenjection . These oCdk4KO mice exhibited severe reductions in MyhcIIa-positive oxidative myofibers and markers of type 1 fibers  pyrrolo  (IDCX-treated) C2C12 cells during muscle differentiation  mice. E2F3fl/fl mice (E2F3mKO mice were born at the expected Mendelian ratio without significant changes in their body weight and lean mass (E2F3mKO mice displayed significantly increased fat mass (E2F3mKO muscle exhibited decreased expression of slow/oxidative muscle fiber–specific genes (MyhcI and MyhcIIa), Myf5, Mef2c, MyoD1, Pgc-1α, and muscle/mitochondrial markers  differentially affect myoblast proliferation and its regeneration potential (based on the CTX injury model) and, (b) upon differentiation, these signals selectively regulate the expression of specific muscle fiber–type markers and muscle mitochondrial genes.To obtain further molecular insight into how p16yoblasts , A and B markers . Differec fibers . In contc fibers . CollectLepob/ob) mouse and the HFD-induced obese/diabetic mouse models. Significantly reduced expression of muscle-specific, energy sensing, fatty acid oxidation, and mitochondrial genes along with reduced levels of Cdk4-E2F3-PGC-1α transcripts were seen in skeletal muscles of Lepob/ob mice  and obese  individuals compared with individuals with healthy BMI (less than 25 kg/m2) (PGC-1α expression (P = 0.062) but that the correlation was significantly driven by BMI (P < 0.001) and sex (P = 0.008), with no effect of age (P = 0.479). We observed statistically significant negative associations of E2F3 and PGC-1α levels with BMI, percentage body fat, total body fat mass, total lipid content of the vastus lateralis, and intramyocellular lipid content of the vastus lateralis muscle prior to biopsy (E2F3 and PGC-1α levels with insulin sensitivity index from frequently sampled intravenous glucose tolerance test (FSIVGTT), suggesting a correlation of both proteins with improved insulin sensitivity  . Express5 kg/m2)  and posipression . Multiplo biopsy . We furtsitivity .CDKN2A gene, which codes for the Cdk4 inhibitor p16Ink4a, was prominently associated with T2D, raising the possibility that Cdk4 — the primary substrate for p16Ink4a — may be involved in glucose disposal and insulin sensitivity. Here, we report that Cdk4 regulates skeletal muscle fiber–type proportions and muscle mitochondrial function. Specifically, we showed that Cdk4-E2F3-PGC-1α signals preferentially increase oxidative myofibers in skeletal muscle and stimulate mitochondrial biogenesis and bioenergetics, enabling greater exercise capacity, improved glucose tolerance, and protection from diabetes and obesity. It has long been considered that skeletal muscle fiber composition is genetically determined  mice, E2F3fl/fl mice (n = 5–8 of each genotype) at 6–8 weeks of age.The generation of eviously . Mice we/fl mice , 51 were/fl mice . In vivon = 5–8 of each genotype at 6–8 weeks of age). These analyses were done as described previously  using man = 9–11 of each genotype) were used at 8–10 weeks of age.Grip strength was measured using the inverted screen test as described previously . ExercisQuadriceps, TA, and EDL muscle types were used. 10 μm–thick sections were stained with antibodies according to standard protocols. Details of the antibodies and primers used in the characterization studies are provide in n = 6 of each genotype) were anesthetized with 1%–3% isoflurane/O2 and injected with 50 μL of 20 μM CTX (Sigma-Aldrich) solution into TA muscle to induce muscle injury. Simultaneously, mice were provided water supplemented with IdU/CldU (0.1% solution) ad libitum and euthanized 7 or 14 days after CTX injection for analysis.Muscle injury was induced using a protocol described elsewhere . Eight- MEFs and C2C12 cells were grown in DMEM media supplemented with 10% FBS and 1% antibiotics. Primary myoblasts isolated from mouse hind limb muscles were grown in DMEM/F10 media supplemented with 20% FBS, 1% antibiotics, and 10 ng/mL bFGF. Cdk4, E2F3, or p16 knockdown in C2C12 cells or primary myoblasts was accomplished using shRNA in pLKO.1-puro lentiviral vectors (Sigma-Aldrich). pCMV-E2F3 plasmid or adenovirus Ad-human CDK4 (Vector Biolabs) was used for ectopic overexpression in C2C12 cells. Myogenic differentiation of C2C12 or primary myoblasts was initiated by switching the cells to medium containing 2% or 5% horse serum and continued for 4–6 days, until myotubes were visible. Cdk4 inhibitor , was included in myocyte differentiation media during the entire differentiation period.C2C12 cells were grown in normal DMEM medium containing 10% FBS and 1% antibiotics to achieve 70% confluency. Cells were trypsinized and incubated with Cdk4 inhibitor  and monitored for oxygen consumption using BD oxygen plate.14C] oleic acid) oxidation was measured using a method similar to that described previously , and placed into a sealed chamber connected to an air pump (Thomas Scientific # 7893B05) and a 50 mL tube containing 3 M NaOH solution to trap expired 14CO2. One milliliter of aliquots was taken 30 and 240 minutes after injection of [1-14C] oleic acid to calculate the oxidation of [1-14C] oleic acid to 14CO2.In vivo fatty-acid ([1-eviously . Mice we5 cells/well were transfected with 1 μg/well of PGC-1α reporter and E2F plasmids together with 0.1 μg/well of Renilla-luc plasmid using Fugene6 transfection reagent (Promega). Luciferase activity was measured using the Promega dual luciferase assay kit.2 × 10ChIP assay was performed using the chip-IT kit (Active Motif), following the manufacturer’s instructions and as described elsewhere . E2F3 bin = 3 each group). mRNAs were purified from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), and RNA integrity and quantitation were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and sequenced on the Illumina NovaSeq6000 with 150 bp paired-end reads. For bioinformatics analysis, paired-end clean reads were aligned to Mus musculus mm10 reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software. FeatureCount was used to count the read numbers mapped of each gene, and then reads per kilobase of exon model per million mapped reads (RPKM) of each gene was calculated for each gene as a measure of expression level. Differential expression analysis was performed using DESeq2 R package. Genes with an adjusted P value of less than 0.05 found by were DESeq2 assigned as differentially expressed. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted with the clusterProfile R package to identify differentially expressed genes at the biologically functional level.Quadriceps and soleus muscles were used  institutional review board–approved study for extensive metabolic phenotyping  that included a dual energy x-ray absorptiometry scan, a FSIVGTT, a maximal exercise stress test, magnetic resonance spectroscopy (MRS) of the vastus lateralis muscle, and a biopsy of the vastus lateralis muscle , 86. All2; mean ± SD) were admitted to the Clinical Research Unit, NIDDK, to participate in a longitudinal study to determine risk factors of diabetes and obesity . Before participation, volunteers were fully informed of the nature and purpose of the study, and written informed consent was obtained. Volunteers were between 18 and 55 years and were determined to be healthy by physical examination, medical history, and laboratory tests. Upon admission, volunteers were placed on a daily weight-maintaining balanced diet  for at least 3 days prior to any metabolic assessment. Glucose tolerance was assessed by a 3-hour 75 g OGTT, and all volunteers were free from diabetes according to the ADA diagnostic criteria. After at least 3 days on the weight-maintaining diet and following an overnight fast, individuals underwent a needle biopsy of the vastus lateralis muscle under local anesthesia with 1% lidocaine. Total RNA was extracted, and the cDNA was analyzed using the Human Exon 1.0 ST Array microarray chips (Affymetrix) as previously described in detail . RNA-Seq data were deposited in GEO (GSE185154).YJB and HY conducted most of the experiments, analyzed results, and prepared figures. OG and DAS performed muscle metabolism and exercise-related experiments in mice. IP helped with confocal imaging for the CTX experiment. PMZ performed electron microscopy. BSA and MCS provided human-related data. ACM provided expertise on muscle phenotyping studies and helped with drafting the manuscript. PP provided human muscle biopsies data, correlations, and figures. SGR conceived the idea for the project and wrote the paper with input from YJB, HY, BSA, PP, OG, and ACM.


Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer. Liver cancer is the third most fatal cancer, accounting for 8.3% of total deaths in 2020  . HepatocEmerging evidence suggests that targeting cancer metabolism could be a promising avenue for therapeutic interventions . Given tThe roles of numerous amino acid transporters in cancer cells have been examined by several studies . ParticuOur previous study has identified a significant loss of BCAA catabolism and a corresponding accumulation of intracellular BCAAs in HCC tumors. Moreover, decreasing the amount of intracellular BCAAs by either reducing or removing dietary BCAA intake or increasing BCAA catabolism significantly suppressed HCC cell proliferation in vitro and improve overall survival in vivo.  BCAAs, p0 type of solute carrier (SLC) transporters are responsible for transporting BCAAs into hepatocytes. Thus, we first investigated the L-type and B0-type SLC expression in individual samples from the Cancer Genome Atlas (TCGA) data. We found that L-type amino acid transporters (LATs) are upregulated in most tumor samples compared to adjacent non-tumor samples. LAT1 (encoded by slc7a5 gene) was most highly upregulated in tumor samples among the four LATs  and LAT1 wildtype control (LAT1WT) were selected for this study. PCR analysis, sanger sequencing around the sgRNA targeted site (KO demonstrated a 40% reduction in BCAA levels compared to LAT1WT (WT (KO or LAT1WT cells were injected into the flank of immunodeficient mice (n = 6), and tumor growth was continually monitored with calliper measurement. Consistent with in vitro studies, lack of LAT1 led to significant suppression of tumor growth compared with wildtype control in xenograft model . To clarify the in vivo tumor growth suppression mechanism presented in LAT1KO injected mice, we conducted immunohistochemistry with Ki67 cell proliferation marker. Histology of the LAT1KO and LAT1WT xenografts was similar, but we observed a reduction of Ki67 positive cells in the LAT1KO injected group  or corresponding empty vector (EV) were transduced into LAT1KO or LAT1WT cells. When LAT1 stably re-expressed into LAT1KO by lentivirus transduction, the LAT1 protein level became similar to that in LAT1WT transduced by EV  analysis for both LAT1WT and LAT1KO samples. We characterize the relationship between groups by a Principal Component Analysis (PCA). The processed PCA data revealed distinct clustering patterns within each sample group  on genes with a False Discovery Rate (FDR) < 0.05. Notably, we found that genes in the Mammalian target of rapamycin complex 1 (mTORC1) signaling, 70-kDa ribosomal protein S6 kinase (p70S6K) signaling, and eukaryotic initiation factor 2 (eIF2) and eukaryotic initiation factor 4 (eIF4) signaling were significantly over-represented in LAT1KO . sgRNAs that have BbsI restriction endonuclease site overhangs were synthesized, annealed, and sub-cloned into pSpCas9-2A-GFP plasmids  that is digested with BbsI  using T4 DNA ligase . The resultant constructs were subjected to Sanger sequencing to verify the correct sgRNA sequences.The gene sequences of human 2.Huh7, Hep3B, and HepG2 cells were maintained in high glucose Dulbecco’s Modified Eagle’s Medium  with 10% Fetal Bovine Serum  and 0.1 mg/mL Penicillin ad Streptomycin . All cells were cultured at 37 °C in humidified atmosphere with 5% CO2) plates using JetPRIME  transfection reagent according to the manufacturer’s instructions. Briefly, 3 μg of plasmid was diluted into 200 μL of jetPRIME buffer and mixed well before adding 7.5 μL of jetPRIME reagent. This mixture was incubated in room temperature for 10 min and thereafter added dropwise to the cells in serum containing DMEM medium. The medium was changed after 4 h of transfection. 48 h after transfection, cells were subjected to FACS isolation of cells with high levels of GPF using BD FACS Aris III instrument . The cells were detached using TrypLE, then single cell suspension was collected by passing through a 50 μm mesh and sorted for a desired GFP fluorescence levelled cells. Non-transfected cells were used for background fluorescence level. Isolated cells were plated in clonal conditions  and grown for further detection of indels. Each clone was picked and analysed for LAT1 expression by DNA sequencing and confirmed by immunoblotting. Finally, one clone in each cell lines for LAT1 KO were selected for this study.Huh7, Hep3B, and HepG2 cells were transfected with plasmids encoded with sgRNA. Plasmids were transfected into 80% confluent cells in 6-well plate , Sulforhodamine B (SRB) assay, and Bromodeoxyuridin (BrdU) incorporation assay. For live cell proliferation, cells were plated in the E-Plates and monitored proliferation every 30 min interval from the time of plating until the end of the experiment. The SRB assay relies on the stoichiometric binding of SRB dye to proteins under mild acidic conditions and its subsequent extraction under basic conditions. The amount of dye extracted is a proxy for cell number in a sample and this assay allows its use for analysis of cell proliferation. For SRB assay, cultured cells were fixed with 10% TCA after an incubation period on plates and stained with SRB for 30 min then excess dye was removed by washing with 1% acetic acid and dried. The bounded SRB is solubilized in 10 mM Tris base solution for optical density measurement at 510 nm using a microplate reader. For BruU assay , cells were plated in 96 well plate and incubated for 12 h, then 10 μM of BrdU was added into each cell. After 4 h of further incubation, plasma membranes were permeabilized followed by DNase I digestion. BrdU was detected by anti-BrdU antibody followed by HRP-conjugated secondary antibody incubation. The incorporated BrdU is measured at 450 nm using a microplate reader. In each proliferation assay, cells were plated in quadruplicate per cell line.Cell proliferation of LAT1Cellular BCAA content was measured with Branched Chain Amino Acid Assay Kit  and followed the manufacturer’s protocol. Briefly, Cells were plated into six-well plates in triplicate per cell line. 24 h after seeding, cells were washed twice with pre-warmed HBSS and then pre-incubated with pre-warm HBSS for 2 min. Cells were then incubated with pre-warmed DMEM for 2 h then homogenized and centrifuged to remove cell debris and insoluble materials. Each sample with assay buffer was incubated for 30 min then measured optical density at 450 nm in a microplate reader. BCAA concentrations of test samples were calculated by standard curve (from 0 to 10 nmol) and corrected background by subtracting the value derived from the zero BCAA standards. These measurements were normalized to cellular protein content measured by BCA assay with the remaining lysate of each sample.For Western blot analysis, cells were washed with ice-cold PBS twice and then lysed with NP-40 buffer (50 mM Tris-HCl (pH = 7.2), 150 mM NaCl, 1% NP-40, 5 mM EDTA) freshly contained protease inhibitor  and phosphatase inhibitor . Cell lysates were cleared by centrifugation at 13,000 rpm for 15 min in cold conditions and protein concentration was determined using the BCA protein assay kit . Thirty micrograms of cell lysates were separated by SDS-PAGE at the appropriate percentages and transferred onto nitrocellulose membranes. Western blot analysis was conducted following the manufacturer’s instructions and using specific antibodies against LAT1 , LAT2 , LAT3 , LAT4 , 4EBP1 , p-4EBP1 , ULK1 , p-ULK1 , mTOR , p-mTOR , p70S6K , p-p70S6K , Raptor , p-Raptor , TSC1 , TSC2 , S6RP , p-S6RP , eIF2a , p-eIF2a , eIF4b , p-eIF4b , GAPDH . Western blot analysis for mouse liver tissues were conducted using specific antibodies against LAT1 , LAT3 , LAT4 .slc7a5 gene was amplified by polymerase chain reaction (PCR) with primers designed additional Not1  and EcoR1 . PCR product was separated, cut, purified then sub-cloned into pLenti-EF1a-PGK-Puro that is digested with Not1 and EcoR1  using T4 DNA ligase . The resultant constructs were subjected to Sanger sequencing to verify the correct slc7a5 sequence. The constructed plasmid was co-transfected with pMDLg/pRRE , pRSV-rev , and pMD2.G  into HEK293 cells and the amplified recombinant viruses were isolated via ultracentrifuge. The virus titers were measured with qRT-PCR in AML12 cells. Cells were infected with ten multiplicity of infection of lentivirus and infected cells were selected with puromycin.To construct a lentivirus-mediated overexpression for LAT1, the KO clone or LAT1WT control of Huh7 cells were suspended in PBS and mixed with Matrigel  in 1:1 volume ratio to give a final concentration of 5 × 107 cells/mL. The cell suspension was subcutaneously injected into the lower flank of 8-week-old male CB-17 SCID mice . Tumor growth was monitored every three days and the size of tumor was measured by callipers to calculate volumes.All animal procedures were performed by approved protocol by the Institutional Animal Care and Use Committee (IACUC #191480) of the Agency for Science, Technology, and Research (A’STAR) of Singapore. And this study was carried out in strict accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Biological Resource Center IACUC guidelines for cancer research in mice. Cultured LAT1Immunohistochemistry was performed on 4-μm-thick sections of paraffin-embedded specimens using anti-Ki67  at 4 degrees in a moist chamber overnight. Then treated with the biotin-labelled secondary antibody, a VECTASTAIN Elite ABC peroxidase kit , followed by colour development with DAB. 40× images were acquired with Ni-E brightfield microscope slc7a5 , slc7a8 , slc43a1 , slc43a2 , gapdh .Total RNA from DEN-induced mouse liver tumors and non-tumor liver tissues were extracted using RNeasy mini kit  according to manufacturer’s protocol. 1.2 ug of total RNA was used for reverse transcribed into cDNA by Superscript III reverse transcriptase . Real-time PCR reactions were performed with the Fast Real-Time PCR System , using 1 μg of cDNA product in a reaction volume 10 μL. The primers were as follows: slc7a5 gene were subcloned into pRRL lentiviral vector. To generate lentiviral knockdown constructs, the following oligonucleotides were used for vector construction: slc7a5-1: TGCTGTTGACAGTGAGCGAAAGGGTGATGTGTCCAATCTATAGTGAAGCCACAGATGTATAGATTGGACACATCACCCTTCTGCCTACTGCCTCGGA; slc7a5-2: TGCTGTTGACAGTGAGCGATGGAAAGTAGCTGCTAGTGAATAGTGAAGCCACAGATGTATTCACTAGCAGCTACTTTCCACTGCCTACTGCCTCGGA; slc7a5-3: TGCTGTTGACAGTGAGCGCTCCCTCCTTTGTTTACTTATATAGTGAAGCCACAGATGTATATAAGTAAACAAAGGAGGGAATGCCTACTGCCTCGGA. The sequence of non-targeting control (NTC) is as follow: CCTAAGGTTAAGTCGCCCTCG.Short hairpin oligonucleotides targeting human depmap from the Cancer Dependency Map (DepMap) databases [Transcriptomics and proteomics data of HCC patients were acquired from the Cancer Genome Atlas (TCGA) and Clinical Proteome Tumor Analysis Consortium (CPTAC), respectively. Dependency scores of CRISPR KO screening were downloaded by R package atabases . All thep-values < 0.05 were defined as differentially expressed genes (DEGs). Transcripts per kilobase million (TPM) of DEGs were used for hierarchical clustering and heat map visualization. Pathway analysis were performed by Ingenuity Pathway Analysis (IPA) [Total RNA was extracted from Huh7 cell lines with TRIzol Reagent according to the manufacturer’s instructions. RNA samples were treated with DNase I  and cleaned with the Ribo-Zero Magnetic Core Kit . A total of 500 ng RNA was used for library preparation with TruSeq Stranded Total RNA with Ribo-Zero Gold kit . Cutadapt and RSeQC were executed for adaptor trimming and quality control of paired-end RNA sequencing data then aligned to the UCSC hg19 human reference genome with TopHat2.0 . Read-pais (IPA) .survival.Survival analysis were performed by ToPP  based on


Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR–Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing. Cas12g is an RNA-guided ribonuclease (RNase) that displays both collateral RNase and single-stranded DNase activities distinguishing it from other Cas12 proteins discovered so far that targeting DNA. RNA-targeting CRISPR-Cas effectors have been developed into various tools for manipulating specific RNA molecules. Currently, RNA-targeting applications primarily rely on the RNA-specific type VI systems, particularly Cas13a-13d, which possess HEPN domains that are exclusively involved in RNA cleavage. Although both Cas12g and Cas13 effector proteins possess the ability to cleave RNA, they exhibit significantly different characteristics in terms of structure and molecular mechanisms. To harness Cas12g and apply it as an effective RNA editing tool, it is important to understand the molecular basis for sgRNA recognition as well as target RNA recognition and cleavage by Cas12g. Our findings not only improve understanding of Cas12g at molecular level but also provide useful information for rational engineering of the CRISPR-Cas12g system and its application in RNA editing and other fields. CRISPR–Cas is part of the defense systems of the adaptive immunity of bacteria and archaea against bacteriophages and mobile genetic elements. It uses Cas effector proteins to target and cleave exogenous nucleic acid sequences that match the guide RNA –4. Classin vitro with or without tracrRNA, suggesting that additional endogenous factors are required for in vivo crRNA biogenesis [Cas12g, which is a thermostable protein identified from a hot spring metagenome locus, functions as an RNA-guided ribonuclease (RNase) [Recently, the cryo-EM structures of Cas12g bound to sgRNA and sgRNA-target RNA have been published . AlthougTo address these questions, we present the structures of two different states: apo-Cas12g and the Cas12g-sgRNA binary complex. The structural analysis of these two states provides insight into the recognition and interaction of Cas12g with sgRNA. Upon binding to sgRNA, we found that Cas12g undergoes conformational rearrangements, creating a positively charged central channel that accommodates sgRNA. Notably, the conformational helix-to-loop transition of the lid motif in RuvC induced by sgRNA binding leads to partial exposure of the RuvC active site, preparing it for substrate binding and cleavage. Additionally, in the apo-Cas12g structure, we identified two adjacent zinc finger motifs within the Helical 1 subdomain II aa 201–382). Overall, this study confirms the significant role of these zinc finger motifs in the substrate cleavage process. By integrating our findings with previous structural research [1–382. OvThe crystal structure of apo-Cas12g was determined at a resolution of 2.24 Å. We observed a single Cas12g in the asymmetric unit of the crystal structure. Apo-Cas12g exhibits a compact domain arrangement that differs from the extended conformations observed in apo-Cas12a [The WED domain is divided into WED-I and WED-II subdomains, which can fold into two antiparallel β-sheets (β1-β2-β6-β7 and β3-β4-β5) and act as a hinge connecting the REC and NUC lobes . The RuvThe Cas12g-sgRNA binary complex was formed by incubating the catalytically inactive Cas12g (D513A) mutant with a 139-nucleotide (nt) sgRNA, which was determined by cryo-EM at a resolution of 3.1 Å and showed a clear density map . CompareThe sgRNA used in the complex was generated by fusing a 45-nt crRNA with a 94-nt tracrRNA . The crRIn the Cas12g-sgRNA complex, the sgRNA is bound to the outer concave surface formed by the Helical 1, Helical 2 and WED domains, which establishes extensive charged interactions. The 3’-end nucleotides A (-3)-A (+1) of crRNA are guided from this outer concave surface into the positively charged central channel through an opening between the WED and Helical 2 domains . The chaAlanine substitutions of key residues 375-FRY-378 decreased the cleavage activity of Cas12g. However, substitutions of residue K221 or R368, which form hydrogen bonds with nucleotides U(-71) and U(-70) of the U-shaped turn of the stem 1 region, had little effect on the cleavage activity of Cas12g. The short R:AR duplex 2 is located at the entrance of the central channel and forms extensive hydrogen bonds with residues from the Helical 2 and RuvC domains . Mutatin2+ ion and is referred to as Zn2+A (HCCC) and Zn2+B (CCHC) hereafter, respectively. Interestingly, only two α-helices of the Helical 1 subdomain II are visible, showing discontinuity in the Cas12g-sgRNA binary complex due to the limited resolution. Although the remaining electron density of the Helical 1 subdomain II region (aa 232–355) that contains these two zinc finger motifs cannot be traced in the binary complex, the orientation can be unambiguously discerned using unsharpened density maps  and the CCHC-type zinc finger (Cys228-Cys240-His335-Cys339) . Each moaps Figs  and S2C.aps Figs . Deletinaps Figs .2+B ion abolish the RNA cleavage activity of Cas12g, similar to the effect observed with the deletion of the aa 232–355 region. Moreover, mutations in the HCCC zinc finger coordinating Zn2+A significantly reduced nuclease activity  and tested the target RNA cleavage abilities of these Cas12g mutant variants. As expected, the CCHC zinc finger motif mutations coordinating the Znactivity . Altoget . In the Importantly, the conformational change of the lid motif contributes to the formation of the central channel and plays a key role in regulating substrate cleavage. An activated catalysis pocket with the three Asp-Glu-Asp conservative nuclease sites is observed in Cas12 systems. In Cas12g, three conserved residues, namely, Asp513, Glu655 and Asp745, located in the RuvC domain have been identified as the nuclease active site via alanine substitution of Asp and Glu residues in the RuvC domain . AnalysiIn the inactive state of apo-Cas12g, the lid motif is positioned above the catalytic active site, obstructing the catalytic core region and preventing substrate binding and cleavage . RecogniIn apo-Cas12g, the lid motif interacts with the Helical 1 subdomain II and Helical 2 domains and is held in place above the active site through hydrogen bonding between residues R661-R224, E666-R226 and K672-V632 . This coCas12g is a newly discovered RNA-guided RNase identified through the analysis of the prokaryotic metagenome database . Unlike (232–355) plays an essential regulatory role in the activation of Cas12g, the structural characteristics remain unclear due to limited resolution [2+, were identified. Structural analyses of apo-Cas12g, its binary complex and the previously reported ternary complexes revealed that the Helical 1 subdomain II undergoes distinct movement and that the Helical 1 subdomain II region (aa 232–355), which is located away from the protein core in apo-Cas12g, can move toward the exit of the central channel in the Cas12g binary complex, eventually attaching to the guide-target RNA heteroduplex in the Cas12g ternary complex [Although a previous study suggested that the C-terminal region of Helical 1 solution . Fortuna complex  S6 Fig)(232–355)Zinc finger motifs exhibit notable diversity, and some have been shown to interact with various molecules other than DNA, including RNA, proteins and lipids . ClassicPrevious studies on type V Cas effectors, including Cas12a , Cas12b 4-H motifs in HEPN domains [Although Cas12g exhibits RNA-guided RNase activity, its architecture and functional domains differ from those of Cas effectors of type VI systems  –43, whic domains ,45. In cE. coli Rosetta (DE3) competent cells. The bacterial cultures were grown in LB medium at 37°C until OD600 reached ~0.8–1.0, after which the overexpression of the recombinant protein was induced with 0.2 mM IPTG at 16°C for 12 h. Afterward, the cells were pelleted by centrifugation and resuspended in ice-cold lysis buffer (50 mM Tris-HCl (pH 8.5), 1 M NaCl, 5 mM β-ME, 1 mM PMSF). The bacteria were then sonicated and centrifugated to separate the supernatant from cell debris. The clarified supernatant was loaded onto a Ni-NTA affinity column and allowed to flow through the column at a flow rate of 0.8 mL/min. After extensive washing, the target protein was eluted with elution buffer (50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 200 mM imidazole, 1 mM PMSF). The target protein was then purified with a HiTrap Heparin column  using a salt gradient between buffer A (50 mM Tris-HCl (pH 8.5), 500 mM NaCl, 1 mM MgCl2, 1 mM DTT) and buffer B (50 mM Tris-HCl (pH 8.5), 1.5 M NaCl, 1 mM MgCl2, 1 mM DTT), followed by pooling and concentrating the fractions containing target protein in buffer C (50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgCl2, 1 mM DTT). The concentrated target protein was further purified with Mono S 10/100 column  using salt gradient between buffer D (50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgCl2, 1 mM DTT) and buffer E (50 mM Tris-HCl (pH 8.5), 1.5 M NaCl, 1 mM MgCl2, 1 mM DTT). Fractions containing purified target protein were pooled, concentrated, and purified by size-exclusion chromatography  in buffer D (50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgCl2, 1 mM DTT). Peak fractions were concentrated and stored at -80°C until further use.The codon-optimized sequence of the Cas12g full-length gene  was clonE. coli Rosetta (DE3) cells cultured in M9 minimal medium supplemented with SeMet, Lys, Phe, Thr, Val, Leu, and Ile. The SeMet-labeled Cas12g D513A mutant was purified following the procedure described above.Selenomethionine (SeMet)-labeled Cas12g (D513A) mutant was expressed in E. coli DH5α cells (Novagen). Amplified plasmids were extracted, linearized by HindIII, and then purified using phenol-chloroform extraction and ethanol precipitation. The linearized DNA templates were used to carry out in vitro RNA transcription with homemade T7 RNA polymerase. Transcribed RNA products were resolved in denaturing polyacrylamide gel containing 8 M urea. Desired RNA bands were excised from the gel and eluted using the Elutrap system . The eluted RNA was desalted, concentrated by ethanol precipitation, and dissolved in diethylpyrocarbonate (DEPC)-treated H2O. All RNA samples were stored at -80°C.The DNA fragments serving as templates for sgRNA and target RNA sequences  were syn2, 1 mM DTT) at a molar ratio of 1:1.2 and incubated at 16°C for 1 hour. The mixture was first purified with the Q column to remove excess sgRNA, followed by further purification using the Superdex 200 Increase 10/300 GL column. Fractions containing purified and homogenous Cas12g-sgRNA binary complex were pooled and concentrated.Before assembling the binary complex, sgRNA samples stored at -80°C were thawed at 4°C, denatured at 75°C for 5 min, and then allowed to be cooled at 4°C. Afterward, Cas12g and sgRNA were mixed in buffer D (50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgClPurified wild-type and mutant Cas12g proteins were detected using Chirascan (Applied Photophysics) at room temperature, with a wavelength range of 190–240 nm in the far-ultraviolet region. The concentration of the samples was diluted to 0.2 mg/ml. Quartz cuvettes with a path length of 0.1 cm were used for the measurements. The baseline was established using the buffer spectrum, and the spectra were obtained by subtracting the baseline. Each sample was measured three times for repeatability.Melting curves of effector proteins were determined through differential scanning fluorimetry (DSF). It was determined by monitoring the changes in tryptophan fluorescence intensity or the tryptophan fluorescence ratio  in the target protein. The wild-type Cas12g and its zinc-finger mutants proteins are diluted to a concentration of 1 mg/ml respectively. Aspirate the prepared sample without introducing any bubbles using a pipette and transfer it to the sample holder. Perform fluorescence monitoring at a rate of 1°C/min, within a temperature range of 20°C to 95°C. The first derivative of the raw fluorescence data was taken in order to determine the Tm of the effector. Each sample was measured three times for repeatability.http://www.ebi.ac.uk/Tools/msa/clustalo/).Download the amino acid sequences of Cas12g from the NCBI database and perform multiple sequence alignment using the Clustal Omega software  performed primary screening of crystallization conditions. Diffraction-quality crystals were grown in 0.1 M BIS-TRIS (pH 6.5) and 25% polyethylene glycol 3,350 (w/v) at the protein concentration of A280 nm ≈ 10. Harvested crystals were cryoprotected in 20% (v/v) glycerol and flash-frozen in liquid nitrogen.X-ray diffraction data were collected using a CCD detector at the beamline BL-17U1 of the Shanghai Synchrotron Radiation Facility (SSRF). Diffraction datasets were processed using XIA2 , followe5 μL of the complex at 3 mg/ml concentration was loaded onto a freshly glow discharged (45 s at 15 mA) holey carbon grid (Quantifoil Au R1.2/1.3) before sample application. Then, the excess liquid is removed by Whatman #1 filter paper at a blot force of 4 s at a blot force of -3 at 4°C, and 100% humidity using a Vitrobot Mark IV plunger (ThermoFisher Scientific) and plunge-frozen in liquid ethane. The prepared frozen-hydrated specimen was loaded on an FEI Titan Krios transmission electron microscope equipped with a Gatan K2 Summit direct detector and Gatan BioQuantum energy filter used for the Date collection. 5,730 movies were collected using SerialEM software that operated the 300 kV transmission electron microscope at a nominal magnification of 165,000× with a pixel size of 0.84 Å, and the movies were recorded with a defocus range of -1.2 to -1.8 μm in super-resolution mode.(232–355) region to improve the local map quality.The 32 frames of each movie in a super-resolution model were aligned, dose-weighted, and summed using MotionCor2 , resultiStructural models of individual domains in apo-Cas12g were docked into the EM density of Cas12g-sgRNA binary complex in UCSF Chimera , and the2), and the sample was incubated at 37°C for 30 min. Afterward, target RNA was added to the prepared binary complex using the same molar ratio as sgRNA, and the cleavage reaction was carried out at 37°C for 30 min. Reactions were terminated by adding EDTA  and Proteinase K  at 37°C for 30 min. Reaction samples were run on 20% PAGE TBE-urea denaturing gels stained with GelRed Nucleic Acid Gel Stain and the results were visualized using Alpha Innotech (Fluouchem TM). Cleavage assays were conducted in triplicate.First, the binary complex was assembled by mixing purified Cas12g with sgRNA at a molar ratio of 4:1 in assay buffer (25 mM Tris-HCl (pH 8.5), 150 mM NaCl 150mM, 10 mM MgClS1 Fig(A) Representative electron micrograph of Cas12g-sgRNA complex embedded in vitreous ice on a Quantifoil grid. The image shows a homogeneous distribution of protein. (B) Representative views of 2D class averages. The 2D averages shows clearly elements of secondary structure, revealing the high resolution of the data. (C) Flowchart of Cryo-EM data processing and 3D reconstruction of the Cas12g-sgRNA binary complex. (D) FSC curves at 0.143 of the final reconstruction of Cas12g-sgRNA binary complex. (E) Local resolution map calculated using Relion for Cas12g-sgRNA binary complex.(TIF)Click here for additional data file.S2 Fig(A) Cryo-EM map and model of the Cas12g-sgRNA complex with each domain of Cas12g color coded as in Fig 1(A). (B) Fitting of nucleic acids to the corresponding cryo-EM map. The atomic models are shown in stick with crRNA and tracrRNA. The crRNA strand and tracrRNA colored in orange and sky blue, respectively. (C) Fitting of the Helical 1 domain. Despite the unresolvable structure Helical 1 subdomain II (aa 232–355) region, its position in the Cas12g-sgRNA complex can be determined based on the density. (D-G) Fitting of the Helical 2 (D), WED (E), RuvC (F) and, Nuc domain (G).(TIF)Click here for additional data file.S3 Fig(A) Overall structure of apo-Cas12g. (B) Overall structure of the Cas12g binary complex. (C) The structure of Helical 1 domain (right). The SAD map around Helical 1 subdomain II discussed in article shown in left. (D-G) Structures of Helical 2, Nuc, WED and RuvC domains of Cas12g. Domains are colored according to Fig 1(A).(TIF)Click here for additional data file.S4 Fig(A) The alignment of the amino acid sequences of Cas12g from different bacterial strains. The amino acids related to the zinc finger motifs are marked with black boxes. (B) Circular dichroism (CD) spectra of wild-type and mutants of zinc finger motifs in Cas12g. (C) Collateral cleavage of unrelated ssDNA (left) and ssRNA (right) by Cas12g. The results shown are representative of three experiments.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(A) The cryo-EM map of the Cas12g-sgRNA-target RNA complex (PDB code: 6XMG). Helical 1 subdomain II (aa 232–355) region is marked with a red box. (B) Structural representation of Helical 1 subdomain II (aa 232–355) region in Cas12g-sgRNA-target RNA complex (PDB code: 6XMG).(TIF)Click here for additional data file.S7 Fig(A).Each domain of Cas12g color coded as in Fig 1(TIF)Click here for additional data file.S8 Fig(A).Domains and residues are colored according to Fig 1(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.Estrogen receptor positive (ER Of all breast cancers, 80% have positive ER status [+ BCa cells are activated by the ER, which is a ligand-dependent transcription factor that is believed to regulate cancer growth by inducing the expression of downstream target genes involved in cell growth, survival, and proliferation [Hormone receptor positive , selective ER degraders , and aromatase inhibitors  [The ER pathway is the primary driver of ERtrozole) . While mtrozole) . Althougtrozole) ,8. This Semaphorins are a group of signaling proteins with an emerging role in cancer. Recent research indicates that Semaphorin 3C (SEMA3C), a member of the semaphorin family, significantly contributes to cancer progression by promoting tumor growth, invasion, and metastasis in a variety of tumors including prostate, breast, lung, colorectal, ovarian, pancreatic and glioblastoma cancers ,10,11. Wp < 0.001) [+ breast cancer cells.In breast cancer, SEMA3C silencing has been shown to inhibit the cell invasion of MCF7 and MDA-MB-231 cells . Further< 0.001) . However+ breast cancer patients and cell lines, and its expression is modulated by the estrogen receptor. Importantly, we demonstrate that silencing SEMA3C inhibits the MAPK and AKT signaling pathways, promotes apoptosis, and suppresses the growth of both tamoxifen-sensitive and resistant ER+ breast cancer cells. We further demonstrate the druggable potential of SEMA3C by using the B1SP Fc fusion protein, a SEMA3C pathway inhibitor that has displayed inhibitory effects on the growth and signaling of ER+ breast cancer cells comparable with those observed during SEMA3C silencing. These findings underscore the emerging role of SEMA3C in promoting ER+ breast cancer growth and survival and suggest that targeting SEMA3C may represent a potential therapeutic strategy for ER+ breast cancer patients. Here, we show that SEMA3C is significantly upregulated in ERMCF10A and T47D were purchased from the American Type Culture Collection . MCF7 , TamR3 and TamC3  according to the manufacturer’s protocol) were kindly provided by Dr A. Cherkasov (Vancouver Prostate Centre). MCF10A were maintained in growth medium as recommended by the American Type Culture Collection . MCF7 and T47D cell lines were maintained as recommended in . TamC3 wAntibodies to the following were used at 1:1000 dilution unless specified: p-EGFR (Y1068), p42/44 pMAPK(T202/Y204), p42/44 MAPK, Akt (9272), p-Akt , cleaved PARP (Asp214), PARP (9542), Estrogen Receptor alpha (D8H8) cleaved caspase-3 , caspase-3 (9662), cleaved caspase-8 , caspase-8   SEMA3C ; Plexin B1 , t-EGFR  , Actin , Vinculin (1:5000) , LICOR secondary antibodies- anti Rabbit IR680 and IR800, anti mouse IR680 and IR800  and anti Goat680 . Antibodies were used according to manufacturer’s protocols. The following chemicals were used: Tamoxifen , Fulvestrant , Estradiol , Charcoal Strip Serum , RNase A , Propidium Iodide  and Insulin-transferrin-selenium .We utilized three publicly available databases : (1) CbiTotal RNA was extracted from cultured cells using the PureLink RNA kit (Invitrogen). Total RNA (1.5–2ug) was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Real-time monitoring of PCR amplification of cDNA was performed using SEMA3C (Hs00170762_mL) and GAPDH (Hs03(9)29097_gl (Applied Biosystems) on a ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) with SYBR gene expression Master Mix . Target gene expression was normalized to GAPDH levels in respective samples as an internal standard.Cells were lysed with 100 uL lysis buffer  containing Complete and PhosStop phosphatase inhibitors . Whole cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. The protein concentration in cleared whole cell lysates was identified using BCA kit . Protein lysates (30–50 ug) were analyzed by SDS-PAGE (6–10%) and transferred by Western Blot to nitrocellulose membranes . Membranes were blocked in PBS containing 5% BSA or TBS Odyssey buffer  if probing with phospho-antibodies. The primary antibody dilution was conducted as per the manufacturer’s protocol. Membranes were washed 3x with TBST followed by appropriate IRdye-conjugated secondary antibodies for 1h. Blots were washed again 3X for 5 min with TBST. Detection was via image analysis using a LICOR infrared imager and Image Studio Lite version 3.1 software. 5 cells/well in a six-well plate in RPMI medium containing 10% FBS for two days. After two days, the medium was replaced with RPMI phenol-red-free and serum starved for 24h. MCF10A cells were treated similarly under their respective recommended media, and for serum starvation we utilized MEBM without phenol red for 5 h. The medium was changed again just prior to stimulation to remove endogenous-secreted SEMA3C. For SEMA3C stimulations: MCF7, T47D and MCF10A were treated with SEMA3C for 10 min at the indicated concentrations followed by a wash with PBS. For B1SP, MCF7 and T47D, cells were treated with B1SP for 90 min, and following B1SP treatment cells were treated with SEMA3C for 10 min without removing media followed by a wash with PBS. To study the effect of tamoxifen on SEMA3C expression in tamoxifen-resistant cells, TamC3 and TamR3 were seeded at 5 × 105 cells/mL in a 10 cm dish in their growth media conditions except with 0.1% ethanol or 1 uM tamoxifen, respectively, for three days in order to see the effect. After three days, these cells were seeded into a 6-well plate at 1 × 105 cells/well and were treated with either Ethanol (95%) or tamoxifen (0.1 uM) for 48 hrs. All of these treatments were carried out at 37 °C in 5% CO2.MCF7 and T47D were seeded at 1 × 10siRNA-targeting Plexin-B1 and SEMA3C were validated: siPlexin-B1-1 - CCGGGUGGAAUUUAUCCUUGAUU, siSEMA3C-1 - CACCAUCCUUUAGACUACATT. MCF7 and T47D cells were reverse transfected with either scramble siRNA (siScr) (AUCAAACUGUUGUCAGCGCUGUU), siPlexin-B1-1 (25 nM) or siSEMA3C-1 (25 nM) using RNAiMax  as described by the manufacturer. For immunoblotting, transfected cells were incubated for 72 h then washed with PBS and then lysed. Plexin-B1 (45 ug) and SEMA3C (40 ug) levels were analyzed with SDS-PAGE. For cell growth assays, cells were reverse transfected in a 96-well flat bottom plate and proliferation was monitored over 4 days (siPlexin-B1) and 5 days (siSEMA3C) with readings taken at every alternate day starting from Day 0.50, cell growth inhibition was by B1SP and during synergy assay we allowed cells to attach to the wells of the 96-well plate overnight and then administered B1SP, Fulvestrant or tamoxifen the next day at the indicated concentrations in respective experiments (see results section).The cell growths of MCF10A (2000 cells/well), MCF7 (1500 cells/well) and T47D (1500 cells/well), TamC3 (1500 cells/well) and TamR3 (2200 cells/well) were assessed in 96-well plates using Presto Blue Viability Reagent  according to the manufacturer’s protocol. Fluorescence was measured at excitation 535 nm and emission 612 nm using a Tecan F500 plate reader . To detect cell growth for (1) siRNA silencing, we conducted reverse-transfection within a 96-well plate and analyzed data at different time points. (2) For showcasing the dependence of cells on SEMA3C, we used Plexin-B1 complex for growth, and exogenous SEMA3C (1 uM) was added to wells in presence of charcoal strip serum. (3) For ICThe flow cytometry analysis of propidium iodide-stained nuclei was performed as shown previously . MCF7 an8), which were seeded and cultured in a 5 L Wave Bag  using 75% Freestyle CHO expression medium/25% Optimem supplemented with 8 mM glutamine and penicillin/streptomycin (5.0 ml/L)  at 37 °C and 8% CO2. CM were harvested three times over a period of 4 weeks and B1SP was purified using the specific purification process described previously [For SEMA3C: FL, cells (40 million) were seeded in Cellstack (2-stack) culture chambers  in a volume of 200 mL DMEM, supplemented with 5% FBS , heparin (25 μg/mL) and blasticidin (5 μg/mL). Conditioned media (CM) were collected after 48 h and then replenished with the same medium containing 2% FBS. The CM were collected every 48 h and replenished. Harvested CM were then centrifuged at 2000 RPM for 5 min and then filtered (0.22 μm). Filtrates were purified over HisTrap excel . Recombinant SEMA3C-FL was then buffer exchanged and concentrated on an Amicon Ultra-15 centrifugal filter device , to 2.0 mg/mL . The protein sample was then desalted on a PD midiTrap G-25 column, as directed by the manufacturer , and then filter sterilized using a 0.22 μm filter. B1SP was produced from CHO-S B1SP Cells (2 × 10eviously .t-test and ANOVA. Data were expressed as the mean ± standard error of the mean (SEM). Comparisons between two means were performed using a Student’s t-test. Comparisons among multiple means were performed with a one-way ANOVA followed by Fisher’ protected least significant difference test . GraphPad Prism software (Version No. 8.4.3) was used to calculate the statistical significance and generate graphs. ChIP-Atlas was used as a source for CHIP-Seq and DNA-seq studies, and the IGV (Integrative Genomics Viewer) browser (Version 2.14.0) was used to analyze the files. Densitometry analysis of SEMA3C expression and phosphorylation of MAPK and Akt under B1SP treatment were performed using ImageJ 1.53 e  based on two independent experiments. The Synergy Finder Online tool was used to calculate synergy scores. The thresholds of statistical significance were set at * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.The clinical data and in vitro data were assessed using Student’s + breast cancer, we analyzed publicly available clinical datasets to investigate the association between SEMA3C expression and patient characteristics, as well as the correlation of SEMA3C with known breast-cancer-associated genes. SEMA3C is a protein that has been implicated in various aspects of cancer development and progression and is overexpressed in several types of cancer, including breast, prostate, and lung cancer. From the analysis of normalized RNA expression data from the TCGA datasets, we found that breast tumors exhibited the highest levels of SEMA3C expression (proteinatlas.org) compared to other cancer types, and by comparing its expression in breast normal vs. breast tumors using GEPIA2, we confirmed higher mRNA expression in breast tumors than matched normal TCGA and GTex samples (p < 0.05) [To assess the clinical relevance of SEMA3C in ER < 0.05) ,27   ,21. We f 0.0001) A. Furthe 0.0001) B. p < 0.0001)  between SEMA3C and ESR1 mRNA expression  with 1100 samples . We perf 0.0001) C. Interepression C. Taken p < 0.0001) [To investigate the potential functional or regulatory relationship between SEMA3C and ESR1, we performed a data mining analysis of GEO datasets  generated by Carroll et al. , studyin 0.0001)   and T47D  from the ChIP-Atlas database ,30. Base+ breast cancer cells and examine whether SEMA3C can activate RTK signaling pathways that are commonly observed in breast cancer. Our previous findings indicate that SEMA3C functions as a secreted soluble autocrine growth factor that drives cancer growth by transactivating multiple RTKs such as EGFR, HER2 and MET via Plexin B1 [To investigate SEMA3C’s potential role as a driver of breast cancer, we needed to induce SEMA3C signaling via the stimulation of ERlexin B1 . To dete+ breast cancer patients, the Akt and MAPK signaling pathways are known to be constitutively active due to downstream effects of ER [+ breast cancer cells, we treated MCF7 and T47D cells with recombinant SEMA3C in a dose-dependent manner. Our results indicated that SEMA3C treatment activated MAPK (Thr202/Tyr204) and Akt (Ser473) proteins in a dose-dependent manner  of Plexin-B1 attenuated cell signaling and growth, even in the presence of recombinant SEMA3C  on MCF7 and T47D cells, and 72 h post-treatment we analyzed cell signaling pathways known to be upregulated in ER+ breast cancer via immunoblotting. We observed a decrease in phospho-Akt and phospho-MAPK signals  in a six-well plate and collecting conditioned media, along with whole cell protein lysate, we stained the cells with propidium iodide and observed a higher percentage of cells in the sub G0/G1 fraction, which was indicative of apoptotic cell death D. Throug+ breast cancer, as it drives the activation of RTK signaling pathways, and SEMA3C knockdown significantly impacts cell growth. To assess its drugability potential within ER+ breast cancer, we conducted proof-of-concept testing using B1SP, a novel SEMA3C pathway inhibitor, to lay the groundwork for the development of SEMA3C-targeted therapies in ER+ breast cancer.Our study indicates that SEMA3C is a potential therapeutic target in ER+ breast cancer, we tested the effect on normal mammary epithelial cells, MCF10A, and ER+ breast cancer cells MCF7 and T47D (p < 0.01) growth inhibitory effects on MCF7 and T47D cells within 72 h , the next step was to understand the relevance of SEMA3C-dependent signaling in endocrine therapy resistant cells. To being with, we analyzed the expression levels of SEMA3C protein expression in tamoxifen-resistant (TamR) cells generated via two different conditions, representing distinct endocrine therapy strategies vs. tamoxifen sensitive (MCF7) ERh MCF10A . We obseh MCF10A  .