MicroRNA-155 (miR-155) is a key regulator of numerous cellular processes that affects cell development and homeostasis. Here, we found that miR-155 was highly expressed in early iNKT cells upon thymic selection, and then its expression is gradually downregulated during iNKT cell development. However, the mice with miR-155 germline deletion had normal iNKT cell development. To address if downregulated miR-155 is required for iNKT cell development, we made a CD4Cre.miR-155 knock-in (KI) mouse model with miR-155 conditional overexpression in the T cell lineage. Upregulated miR-155 led to interruption of iNKT cell development, diminished iNKT17 and iNKT1 cells, augmented iNKT2 cells, and these defects were cell intrinsic. Furthermore, defective iNKT cells in miR-155KI mice resulted in the secondary innate-like CD8 T cell development. Mechanistically, miR-155 modulated multiple targets and signaling pathways to fine tune iNKT cell development. MiR-155 modulated Jarid2, a critical component of a histone modification complex, and Tab2, the upstream activation kinase complex component of NF-κB, which function additively in iNKT development and in promoting balanced iNKT1/iNKT2 differentiation. In addition, miR-155 also targeted Rictor, a signature component of mTORC2 that controls iNKT17 differentiation. Taken together, our results indicate that miR-155 serves as a key epigenetic regulator, coordinating multiple signaling pathways and transcriptional programs to precisely regulate iNKT cell development and functional lineage, as well as secondary innate CD8 T cell development.The development of invariant natural killer T ( Unlike conventional T cells, iNKT cells acquire an effector function and differentiate into iNKT1 (PLZFloT-bethi), iNKT2 (PLZFhiRORγt–), and iNKT17 (PLZFintRORγt+) based on transcriptional factors, including T-bet, PLZF, GATA3, and RORγt, before thymic export. Alternatively, iNKT1 cells develop through all stages and finally mature in stage 3, which also highly express cytolytic effectors  and specific chemokines (such as CCL5) and their receptors. However, iNKT2 and iNKT17 cells terminate at stage 2. Stage 2 iNKT cells have a high proliferation capability, and most iNKT cells at this stage emigrate to peripheral organs. Upon antigenic stimulation, iNKT1/2/17cells rapidly secrete a broad range of cytokines such as IFN-γ, IL-4, and IL-17, respectively, which mediates functions that link innate and acquired immunity in a broad spectrum of diseases, including infection, cancer, allergy, and autoimmunity , a class of 21- to 25-nt single-stranded non-coding small RNAs are emerging as key regulators of numerous cellular processes that affect cell development, homeostasis, and disease development . MiRNAs function . Using d fitness ; miR-150ng c-Myb ; miR183-ng Foxo1 ; and Letpression . Global pression  and uprepression . By usinpression . In addifunction . HoweveriNKT cell development and differentiation. To explore the potential role of miR-155 in iNKT cell development and differentiation, two distinct but complementary approaches were used. Germline miR-155 deletion in mice did not significantly interrupt iNKT cell development, which is largely consistent with previous report  by miR-155 may be the cause of the self-limiting expression of miR-155 during iNKT thymic development. Therefore, the dynamic regulation of miR-155 expression is essential for normal iNKT cell thymic development, differentiation, and homeostasis.In the present study, we confirmed the dynamic changes of miR-155 expression during thymic s report . However+miR-155KI, miR-155KI), and the littermate CD4Cre–miR-155KI [wild-type (WT)] mice served as controls. Mice were 6- to 10-week-old, gender-matched, and housed in a specific pathogen-free barrier unit. Handling of mice and experimental procedures were in accordance with requirements of the Institutional Animal Care and Use Committee.Conventional miR-155 knockout (miR-155KO) mice in a C57BL/6 background and wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory . Mice carrying the Rosa26 miR-155 knock-in (KI) allele (Jackson Laboratory) were bred with CD4Cre transgenic mice (Jackson Laboratory) to obtain mice expressing CD4Cre with one or two copies of the miR-155 KI allele. Mice with two copies of miR-155 KI allele, miR-155KI mice (CD4Cre6 per staining well) were stained with antimouse PBS57-loaded CD1d-tetramer and PBS57-unloaded CD1d-tetramer (provided by the NIH Tetramer Core Facility). The following conjugated mAbs were used: TCRβ (H57-597), CD24 (30-F1), CD44 (IM7), CD122 (5H4), CD69 (H1.2F3), NK1.1 (PK136), IL17RB (MUNC33), Slamf1 (TC15-12F12.2), Slamf6 (330-AJ), PLZF (9E12), RORγt (B2D), T-bet (eBio17B7), GATA3 (TWAJ), CD1d (1B1), IL-4 (11B11), IFN-γ (XMG1.2), IL-17a (eBio17B7), CD45.1 (A20), CD45.2 (104), CD4 (GK1.5), CD8 (53-6.7), CXCR3 (CXCR3-173), and Eomes (Dam11mag). All mAbs were purchased from eBioscience, Biolegend, or TOBON. Apoptosis assays were carried out by staining with Annexin V (eBioscience), according to manufacturer’s instructions. Cell surface staining was performed with staining buffer. Intranuclear staining for GZMA, PLZF, T-bet, RORγt, Bcl-2, and Ki-67 was performed with eBioscience Fixation/permeabilization buffer. The flow cytometry assay was performed through BD FACSCelesta, and data were analyzed using FlowJo V10.2 software. Gating strategy: after gating on lymphocyte, doublets were excluded by using forward scatter (FSC) and side scatter (SSC), mouse iNKT cells were then identified as TCRβ+ CD1d-tetramer+  and incubated with Fc block (clone 2.4G2). Cells  supplemented with 10% FBS, 100 units/ml penicillin, 100 units/ml streptomycin, 25 mM HEPES, 1 mM sodium pyruvate, 1 × non-essential amino acids, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol, in the presence of phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (1 μM) for a total of 4 hours at 37°C. Brefeldin A was then added in the last 2.5 h at a final concentration of 1 μM. Anti-IFN-γ, anti-IL-4, and anti-IL-17 were detected by intracellular staining and flow cytometry.Thymocytes and spleen cells total 4 × 10+CD45.2+) were lethally irradiated with 900 rad. Donor bone marrows were harvested from age- and sex-matched SJL (CD45.1+) mice and miR-155KI mice (CD45.2+) by flushing bones with a syringe containing sterile basal tissue culture medium (RPMI 1640 with 10% FBS). After erythrocyte lysis, mature T cells (CD3+) were depleted from bone marrow from each donor mouse by biotin-conjugated antimouse CD3  mAbs and antibiotin magnetic beads (BD Biosciences) using Magni SortTM. Over 90% of mature T cell depletion was confirmed by flow cytometry. CD45.1+ SJL and CD45.2+ miR-155KI or WT control bone marrows were mixed at a 1:1 ratio, and total of 10 × 106 cells per mouse (in a volume of 100 μl) were then injected into the irradiated recipient mice by tail vein injection. The chimeras were analyzed 8 weeks after reconstitution.To generate bone marrow chimeras, 7- to 8-week-old B6.SJL-recipient mice  stained with anti-CD8 biotin Ab  for thymocytes or anti-CD8 biotin Ab and anti-B220 biotin Ab  for spleen cells. CD8+ T cells and B220+ cells were then depleted with antibiotin beads (MagniSortTM) using auto-MACS (Miltenyi Biotec). Negatively selected cells were then stained with anti-TCR-β, anti-CD1d tetramers, anti-NK1.1, and anti-CD44 Abs. Total or different stages of iNKT cells were then sorted by BD FACS Aria II.Total thymocytes and spleen cells were first prepared at a concentration of 1 × 10iNKT cells from WT controls and miR-155KI mice were sorted, and total RNA was extracted by using the EXIQON isolation. RNA was reverse transcribed using the PrimeScriptTM RT reagent kit, and RT-qPCR was performed on an Applied Biosystems 7900 Real-Time PCR system with the following primer pairs: Jarid2 (Forward: 5′-GACACCAAACCCAATCACCAC-3′ and Reverse: 5′-GTTCAACCTGCCACTGACCTT-3′); Rictor (Forward: 5′-TGAGTACCGTTCTTCTCAC-3′ and Reverse: 5′-TGCAATGGAGGG CGCTTTA-3′); Tab2 (Forward: 5′-GGATAGAATAAGCGAAGCCCGGAA-3′ and Reverse: 5′-CTC TTTGAAGCCGTTCCATCCT-3′). Expression levels of miR-155 were normalized with snoRNU202, and expression levels of Jarid2, Rictor, and Tab2 were normalized with L32. Fold change was calculated using the 2CT−ΔΔ method.RNA concentration was determined using the Qubit RNA broad range assay in the Qubit Fluorometer (Invitrogen), and RNA integrity was determined using the Eukaryote Total RNA. Nano Series II ChIP on a 2100 Bioanalyzer (Agilent). Three independent biological replicates were pooled for RNA-seq. RNA-seq libraries were prepared using 4 μg of total RNA with the TruSeq RNA sample prep kit following manufacturer’s protocol (Illumina).1 was used to quantify protein expression levels from digital images of Western blots. IL-4 protein concentration in mouse blood serum was measured using the IL-4 mouse ELISA Ready-Set-Go! Kit (eBioscience) following the manufacturer’s instructions.Cell lysates were prepared by lysis in RRAP buffer containing protease inhibitor and Western blots were performed using standard methods. Image Jt-test with 95% confidence interval). A P-value < 0.05 was considered statistically significant. The processing of RNA-Seq data was performed with R version 3.6.02.GraphPad Prism 8.0 was used for statistical analysis , stage 2 (CD44hiNK1.1–), and stage 3 (CD44hiNK1.1+) iNKT cells. MiR-155 expression was then detected by TaqMan real-time PCR. MiR-155 was detected in all developmental stages of iNKT cells, and expression was dramatically decreased during iNKT cell development, almost to the lowest level at the terminal developmental stage 3  mice. Nevertheless, we did not observe a significant iNKT cell developmental defect in thymus and peripheral lymph organs in mutant mice. The frequencies and absolute numbers of iNKT cells in thymus, spleen, lymph nodes, and liver peripheral organs were comparable between miR-155KO and WT mice  of interested from miR-155 deletion cells in the earliest stages of iNKT cells may not be enough for an obvious interruption of iNKT cell development.To assess the role of miR-155 in  WT mice . iNKT ceal stage , which wal stage , suggestiNKT cell differentiation , and WT littermate control  were used in experiments without specific indication  cells.To assess the role of miR-155 overexpression in 5KI mice . These diNKT cell developmental defects in miR-155KI mice, we examined the rate of Ki-67 expression to measure iNKT cell proliferation capability. Compared with WT controls, miR-155KI iNKT cells exhibited a higher proliferating status . Normally, T-bet is initially expressed in stage 1/2 and is maximally expressed in stage 3, while PLZF is gradually downregulated along this developmental process. Here, we found that miR-155KI iNKT cells failed to upregulate T-bet at stage 2, resulting in less PLZFhi/int T-bet+iNKT cells from stage 2 progressing to stage 3 , CD69 (Cd69), and NKG2D (Klrk1) in miR-155KI thymic iNKT cells more specifically, in the NK1.1– counterpart  and GZMA (Gzma) were significantly reduced in the miR-155KI iNKT NK1.1– counterpart, suggesting the downregulated cytotoxic capability of iNKT cells from miR-155KI mice  are typically located in stages 1/2 iNKT cell pool. Compared with WT, thymic iNKT cells from miR-155KI mice exhibited a significant increased PLZF level  and IL17RB (Il17rb)  iNKT cell pool   were alsell pool . Given t of IL-4 , we then5KI mice . In addiKT cells . Even th WT mice . The iNK5KI mice . TherefoiNKT17 is a distinct subset of iNKT cells characterized by the expression of transcription factor RORγt (Rorc)  . These c stage 0 . Here, w stage 1 . We thenph nodes . In supp with WT . These diNKT cells and precursors but also in conventional T cells. To evaluate the role of miR-155 overexpression in conventional T cell development, we checked on the gross conventional T cell phenotypes with flow cytometry. Interestingly, miR-155KI mice showed increased frequencies and numbers of both CD8 single positive (CD8 SP) and CD4 SP T cells but decreased frequencies and numbers of DP T cells  BM with miR-155KI or WT littermate BM (CD45.2+) was transferred to lethally irradiated SJL/B6-recipient mice (CD45.1+CD45.2+)  chimera experiment. A 1:1 mixture of SJL (CD45.1CD45.2+) . Eight wntiation . These rin vitro stimulation  in the NK1.1–iNKT cell pool, and 26 down- and 52 upregulated genes  in the NK1.1+iNKT cell pool  was identified for genes within the miR-155 target gene set and confirmed a transcriptome-wide reduction of gene expression in miR-155KI NK1.1– and NK1.1+iNKT cell pools. With this strategy, we identified 21 predicted miR-155 targets that were significantly repressed in miR-155KI NK1.1–iNKT cells (+iNKT cell pool (data not shown). It is important to point out that neither Itk nor Ets1 were in the downregulated gene list for both NK1.1– and NK1.1+iNKT cells in miR-155KI mice, even though both were previously reported as potential targets of miR-155 during iNKT cell development  list of genes was prepared and used as input for a preranked GSEA analysis  against KT cells . Howeverelopment . TherefoSeq data . In suppJarid2 is a member of the JmjC domain-containing protein family, a novel component of polycomb repressive complex 2 (PRC2). A previous study confirmed that Jarid2 is a negative regulator of Zbtb16 (encode PLZF) mediated by H3K9me3 demethylation  complex 2 mTORC2 . These data strongly support the idea that miR-155 inhibits iNKT17 lineage differentiation and controls iNKT survival through targeting Rictor. Thus, downregulation of miR-155 to derepress Rictor during iNKT thymic development may be necessary for overall iNKT development and iNKT17 lineage differentiation.These data suggested that miR-155 inhibits KT cells . Rictor 2 mTORC2 . Here, wia q-PCR . The delpression . Alternaelopment . InteresKT cells . Strikinr/mTORC2 , was fouTab2 (TGF-β activated kinase 1-binding protein 2), one of the predicted targets of miR-155, was also repressed in miR-155 KI iNKT cells (Tab2 is required for transforming growth factor-β-activated kinase 1 (TAK1)-dependent signaling pathways, including the transforming growth factor-β (TGF-β) signaling pathway and the NF-κB signaling pathway (iNKT cell development at multiple levels (iNKT cells (iNKT1/2 cell differentiation and secondary innate CD8 T cell development by targeting Jarid2 and controls iNKT17 differentiation and iNKT homeostasis by targeting Rictor. Additionally, miR-155 modulates the NF-κB signaling pathway and TGF-β signaling pathway during iNKT cell maturation and cell function (Furthermore, we found that KT cells . Tab2 is pathway , both ofe levels . In suppKT cells . Taken tfunction .iNKT cells. Overexpression of miR-155 perturbed iNKT1 but promoted iNKT2 differentiation by targeting Jarid2, the component of a negative epigenetic regulator of PLZF, while miR-155 restrained iNKT17 differentiation by targeting Rictor, an obligatory component of mTORC2. Our discovery adds further complexities of miR-155-mediated immune regulation and emphasizes the cell type, developmental, and differentiation stage-specific regulation characteristics and molecular mechanisms of miR-155-mediated immune regulation.MiR-155 was one of the first discovered mammalian miRNAs and is expressed in a variety of immune cells. Extensive studies have revealed the complex and versatile functions of miR-155 in different immune cells. In B cells, miR-155 overexpression leads to the transformation and development of lymphoma , which iiNKT cell lineage. PLZF directs the iNKT cell effector program, lineage differentiation, and function and affects cell migratory properties (iNKT cells (iNKT development, the dynamic upregulation of let-7 miRNA is involved in the gradual downregulation of PLZF and iNKT1 differentiation and maturation (iNKT cell lineage differentiation through targeting Jarid2, which epigenetically restricts the expression of PLZF, further expanding the complexity of the molecular networks in controlling PLZF and iNKT cell differentiation and maturation. Previous studies have indicated that either Egr2 or Jarid2 and miR-155 are upregulated by TCR activation (iNKT cells. Nevertheless, the magnitude of the exponential surge of Egr2 expression far outweighs the slight upregulation of Jarid2 after TCR activation, which indicates the dominant regulatory role of Egr2 in the initial PLZF transcriptional boost and in the early stages of iNKT cell development and effector programs (Jarid2 elevation resulting from miR-155 downregulation and IL-15-promoted Let-7 expression are critically involved in the gradual downregulation of PLZF, which is essential for iNKT1 differentiation and maturation (iNKT cells, in which miR-155 deletion-mediated Jarid2 elevation may not be enough to perturb the early PLZF boost dominated by the Egr2 surge.PLZF is a signature transcription factor of the operties . PreviouKT cells . During turation . Our curtivation , and thaprograms . Howeverturation . These miNKT developmental stage may be mediated through the NF-κB signaling pathway. Nevertheless, iNKT cells with miR-155 overexpression showed downregulated NF-κB signaling pathway, possibly through directly targeting Tab2, the upstream activation kinase complex component of NF-κB (Rictor. The NF-κB signaling pathway is known to be critical for iNKT cell differentiation. NF-κB-deficient mice, including NF-κB1, c-Rel, and RelA KO mice, have shown perturbed iNKT maturation: especially in RelA KO mice (iNKT1 phenotypes in miR-155KI mice, particularly with regard to interrupted stage 3. Thus, the defective NF-κB signaling pathway in iNKT cells with miR-155 overexpression may cooperate additively with the perturbed Jarid2 and derepression of PLZF in the overall defective iNKT1 phenotype of miR-155KI mice. On the other hand, the cell-intrinsic NF-κB activity was shown to be essential for the survival of iNKT cells (iNKT cells with miR-155 overexpression. Furthermore, the interaction between miR-155 and the NF-κB signaling pathway forms a negative feedback loop, which could be the reason for the transient or self-limiting manner of miR-155 expression induced by TCR activation, such as the gradual downregulation of miR-155 during iNKT thymic development and differentiation identified in the current study.MiR-155 transcription was reported to be positively regulated through NF-κB signaling pathway. Therefore, the upregulation of miR-155 after TCR activation during the early of NF-κB . Neverthof NF-κB . Furtherof NF-κB . Hence,  KO mice , which cKT cells , which ciNKT17 cells and in iNKT homeostasis (iNKT17 lineage differentiation and controls iNKT survival through targeting Rictor, the required component of mTORC2. On the other hand, iNKT cells with miR-155 overexpression showed a downregulated TGF-β signaling pathway, while TGF-β signaling, particularly its SMAD4-dependent pathway, is required for both the survival of RORγt+iNKT cells during their development and IL-17 production (iNKT17 differentiation and iNKT homeostasis in iNKT cells with miR-155 overexpression. Nonetheless, studies by Jarid2. These studies, combined with our data, emphasize the spatial, temporal, and delicately balanced characteristics of miR-155-mediated regulation. Our data also bring miRNAs and chromatin modification together by showing how miR-155 targets the chromatin protein Jarid2, which in turn regulates the key transcription factor for iNKT development and effector program, PLZF.mTOR signaling integrates immune signals and metabolic cues in orchestrating T cell responses . mTOR sieostasis . Our resoduction . TherefoiNKT and Treg cell interaction. The number of Tregs cells in thymus was found increased in miR-155KI mice (iNKT cells and that Tregs in thymus may suppress the proliferation, cytokine production, and cytotoxic activity of iNKT cells in local environment (iNKT cell development and function, which need to be further investigated.We do realize there are some limitations in our current study, including the dissection of role of miR-155 in 5KI mice . A previironment . Our curiNKT cells is essential for the development, functional lineage differentiation, and homeostasis of iNKT cells. Even though the early upregulation of miR-155 after iNKT positive selection is not a dominant factor for the initial PLZF transcription boost, the potential miR-155-NF-κB-negative feedback loop controlling miR-155 downregulation is essential for downstream iNKT differentiation and homeostasis. Downregulated miR-155 relief the suppression to Jarid2 and the NF-κB signaling pathway, which further controls PLZF expression and balances iNKT1/iNKT2 differentiation cooperatively. On the other hand, downregulated miR-155 relieves the suppression of Rictor and the TGF-β signaling pathway, additively cooperating for the differentiation of the iNKT17 lineage and promotion of overall iNKT homeostasis. Consequently, the dynamic regulation of miR-155 expression during thymic iNKT cell development is essential, and miR-155 functions as a moderator connecting and coordinating multiple signaling pathways and transcriptional programs for a consolidated regulation of iNKT development, functional lineage differentiation, and secondary innate CD8 T cell development (Overall, the dynamic expression of miR-155 and its regulation in thymic elopment .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Experiments using animal were approved by the Institutional Animal Care and Use Committee of Henry Ford Health System.LZ and Q-SM conceived and designed the study. JW, KL, XZ, GL, TL, and XW performed the experiments. JW, KL, and LZ analyzed the data. SB performed mouse irradiation for BM transfer. LZ, JW, and Q-SM wrote the manuscript, which was commented on by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Withania somnifera-derived triterpenoid, is an anticancer natural product. The anticancer effect of nonionizing radiation such as ultraviolet-C (UVC) as well as the combined treatment of UVC and WFA is rarely investigated. Low dose UVC and/or WFA treatments (12 J/m2 and/or 1 μM) were chosen to evaluate antioral cancer cell line effects by examining cytotoxicity, cell cycle disruption, apoptosis induction, and DNA damage. For two cancer cell lines (Ca9-22 and HSC-3), single treatment (UVC or WFA) showed about 80% viability, while a combined treatment of UVC/WFA showed about 40% viability. In contrast, there was noncytotoxicity to normal oral cell lines (HGF-1). Compared to single treatment and control, low dose UVC/WFA shows high inductions of apoptosis in terms of flow cytometric detections for subG1, annexin V, pancaspase changes as well as Western blotting for detecting cleaved poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) and luciferase assay for detecting Cas 3/7 activity. Low dose UVC/WFA also showed high inductions of oxidative stress and DNA damage in terms of flow cytometric detections of reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX) generation, and membrane potential (MitoMP) destruction, γH2AX and 8-oxo-2’deoxyguanosine (8-oxodG) types of DNA damages. For comparison, low dose UVC/WFA show rare inductions of annexin V, Cas 3/7 activity, ROS, MitoSOX, and MitoMP changes to normal oral HGF-1 cells. Therefore, low dose UVC/WFA provides a novel selectively killing mechanism to oral cancer cells, suggesting that WFA is a UVC sensitizer to inhibit the proliferation of oral cancer cells.Withaferin A (WFA), a  Radiotherapy and chemotherapy are commonly used for curing oral cancer . HoweverSimilarly, high dose radiation is commonly associated with radioresistance to cancer such as prostate cancer cells . Low dosIn addition to X-ray radiotherapy for oral cancer, several types of nonionizing radiation were developed for dental curing, including light-emitting diodes (LED), quartz-tungsten-halogen (QTH), argon lasers, and plasma arc curing (PAC) . Except Withania somnifera, shows antioxidant properties [Withaferin A (WFA), a triterpenoid isolated from operties ,12. In aoperties ,14 and ooperties ,16,17,18operties . HoweverWe hypothesized that low dose WFA had an UVC radiosensitizing effect to oral cancer cells. To address this hypothesis, a low dose combined treatment (UVC/WFA) and individual treatments of oral cancer cells were compared for their changes of cytotoxicity, cell cycle, apoptosis, oxidative stress, and DNA damage.2O2  were used as a positive control treatment.Two human oral cancer cell lines (Ca9-22 and HSC-3) and normal oral cell lines (HGF-1) were commercially obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank  and the American Type Culture Collection . Ca9-22 cells were derived from gingival squamous carcinoma. HSC-3 cells were derived from tongue squamous carcinoma with high metastatic potential. HGF-1 cells were derived from gingival fibroblasts. The relative proliferation rate for Ca9-22, HSC-3, and HGF-1 cells was 1.9:1.6:1.0, respectively . Their rCytotoxicity was evaluated by tetrazolium-based 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) kit  as described previously . The cel2/sec [2. Subsequently, medium containing WFA was added for post-treatment. The control contained DMSO only without UVC and WFA treatments. All experiments including DMSO only, UVC and/or WFA treatments had the same concentration of 0.01% DMSO.After removing medium, cells were treated with a germicidal UVC lamp 254 nm) in a laminar flow chamber at a rate of 2 J/m4 nm in aDNA was stained with 7-Aminoactinmycin D (7AAD)   for a cell cycle assay . Flow cy® 3/7 Assay  based on luminescent detection as described previously [A flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) was used to measure apoptosis in terms of annexin V /7AAD  and a paeviously . Reactive species-detecting probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA)  was interacting with ROS to generate fluorescence, which was determined by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) .Reactive species-detecting probe MitoSOX Red   was interacting with MitoSOX to generate fluorescence, which was determined by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) [2(3)   was used to determine MitoMP by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) [MitoMP-sensitive probe DiOCckinson) . ®488  (1:10000 dilution), and 7AAD (5 μg/mL) were chosen for flow cytometry reaction [After cell harvesting and fixation, γH2AX primary antibody  (1:500 dilution), secondary antibody conjugated Alexa Fluorreaction  and analAfter cell harvesting and fixation, fluorescein isothiocyanate (FITC) conjugated 8-oxodG antibody  (Santa Cruz Biotechnology) were chosen for flow cytometry reaction at 4 °C for 1 h and analyzed by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson).The significance for multiple comparisons were tested by One-way ANOVA with the Tukey Honestly Significant Difference (HSD) post hoc test using JMP12 software . Groups showing no overlapping letters indicate significant differences.2 and 1 μM) around 80% viability were used to evaluate the cytotoxic effect of a combined treatment (UVC/WFA) of oral cancer Ca9-22 and HSC-3 cells and normal oral HGF-1 cells , or UVC/WFA. For Ca9-22 cells  staining, the MitoMP depletion was detected as oxidative stress by flow cytometry. According to a MitoMP assay for oral cancer Ca9-22 and HSC-3 cells  jointed to induce antiproliferation of colon cancer SW480 and DLD-1 cells [Cryptocarya concinna  and UVC  improve antiproliferation against oral cancer cells compared to single treatment [Recently, the radiosensitizing studies using low dose clinical drugs and UVC were reported. For example, low dose cisplatin (10 μM) and UVC  values of cisplatin to oral cancer Ca9-22 and HSC-3 cells in a 24-h MTS assay are 7.9 and 9.6 μM, respectively. At a low dose, UVC (12 J/m2) and WFA (1 µM) were used in the present study to demonstrate a higher antiproliferation ability to oral cancer Ca9-22 and HSC-3 cells  than cisplatin alone. It is noted that the oral cancer Ca9-22 cell line is recently described for being problematic in terms of showing contamination with the cell line MSK922  radical scavenging ability in vitro . WFA alsa in rat ,43. In ca in rat  and lunga in rat  cancer ca in rat  through a in rat ,45.2) [2 [2 (ROS modulating is one of the strategies for antiproliferation of cancer cells . Both hi2)  and WFA 2) ,18 trigg2) [2  and 12 J2) [2 [2 ; ~80% vi2) [2 [2  for ~80%2) [2 [2 . These rCellular oxidative stress enhances DNA damages . High doHigh dose UVC ,51 or WFFurthermore, the DNA repair system may be inhibited for drug radiosensitization. For example, withanolide D is helpful in radiosensitization of ovarian cancer SKOV3 cells by suppressing DNA repair such as non-homologous end joining (NHEJ). This warrants a detailed examination of the role of DNA repair in UVC/WFA synergistic effects to oral cancer cells in the future.UVC/WFA accumulated a larger subG1 population than single treatment in both oral cancer Ca9-22 and HSC-3 cells. However, UVC/WFA induced more G2/M arrest in Ca9-22 cells than upon single treatment but no G2/M arrest appeared in HSC-3 cells. Because Ca9-22 and HSC-3 cells are gingiva and tongue squamous cell carcinoma cell lines , it is pThe present study examined the combined effect of low dose UVC/WFA on regulating cell proliferation of oral cancer cells. Single treatment at low dose UVC or WFA showed low cytotoxicity to oral cancer Ca9-22 and HSC-3 cells. Combined treatment of low dose UVC/WFA highly induces oxidative stress and resulted in apoptosis and DNA damage of oral cancer cells. Moreover, low dose UVC/WFA showed highly cytotoxic, oxidative stress, and apoptosis induction in oral cancer cells without effects on normal oral cells. Therefore, low dose UVC/WFA shows a selective killing potential and effectively inhibits oral cancer cell proliferation with no cytotoxic side effects to normal oral cells. In the future, detailed investigations in vivo, such as those using mouse models, should be conducted to further validate the promising effects of a low dose UVC/WFA combined treatment in oral cancer therapy.


MDC1, XLF, or both MDC1 and XLF in murine vAbl pro-B cell lines and, using chromosomally integrated substrates, demonstrated that MDC1 stimulates the V(D)J recombination in cells lacking XLF. Moreover, combined inactivation of MDC1 and XLF in mice resulted in synthetic lethality. Together, these findings suggest that MDC1 and XLF are functionally redundant during the mouse development, in general, and the V(D)J recombination, in particular.DNA double-strand breaks (DSBs) trigger the Ataxia telangiectasia mutated (ATM)-dependent DNA damage response (DDR), which consists of histone H2AX, MDC1, RNF168, 53BP1, PTIP, RIF1, Rev7, and Shieldin. Early stages of B and T lymphocyte development are dependent on recombination activating gene (RAG)-induced DSBs that form the basis for further V(D)J recombination. Non-homologous end joining (NHEJ) pathway factors recognize, process, and ligate DSBs. Based on numerous loss-of-function studies, DDR factors were thought to be dispensable for the V(D)J recombination. In particular, mice lacking Mediator of DNA Damage Checkpoint Protein 1 (MDC1) possessed nearly wild-type levels of mature B and T lymphocytes in the spleen, thymus, and bone marrow. NHEJ factor XRCC4-like factor (XLF)/Cernunnos is functionally redundant with ATM, histone H2AX, and p53-binding protein 1 (53BP1) during the lymphocyte development in mice. Here, we genetically inactivated  In mammalian cells, DNA double-strand breaks (DSBs) activate the DNA damage response signaling (DDR). During DDR, Ataxia telangiectasia mutated (ATM) protein kinase phosphorylates multiple substrates, including histone H2AX and the scaffold proteins, mediator of DNA damage checkpoint protein 1 (MDC1) and p53-binding protein 1 (53BP1)  and acetV, D, and J gene segments of immunoglobulin and T cell receptor genes. NHEJ is the only known process to recognize and efficiently repair RAG-induced DSBs [XRCC4 or Lig4 resulted in embryonic lethality in mice, while conditional inactivation or knocking down of XRCC4 or Lig4 in lymphocytes blocked the V(D)J recombination and NHEJ [XLF [PAXX [Mri [During the B and T lymphocyte development, both DDR and NHEJ pathways function in response to the recombination activating gene (RAG)-induced DSBs in the process known as the variable (V), diversity (D) and joining (J) gene segments recombination (V(D)J recombination). RAG is the nuclease that generates DSBs adjacent to the ced DSBs ,14. V(D)ced DSBs  and Ku80ced DSBs . Inactivand NHEJ ,18. Acceand NHEJ ,21,22,23HEJ [XLF ,25, PAXXLF [PAXX ,27,28,29AXX [Mri ,13 had nAXX [Mri ,31,32 anAXX [Mri ,28,29,33AXX [Mri ,35, Mri AXX [Mri ,13, and AXX [Mri .ATM [H2AX [MDC1 [53BP1 [XLF and ATM [XLF and 53BP1 [XLF-deficient background [DDR factors were thought to be dispensable for the V(D)J recombination, because germline inactivation of ATM , H2AX [3TM [H2AX ,39, MDC1AX [MDC1 , or 53BP1 [53BP1  resulted and ATM , or XLF nd 53BP1 ,44, resuckground ,43,44.XLF gene in humans result in combined immunodeficiency [XLF gene in mice results in a modest reduction of B and T lymphocytes count [Trp53-deficient mice [XLF is the NHEJ factor. Mutations in the ficiency ,45, and es count ,25. XLF es count . XLF hases count . Moreoveent mice . Togetheent mice . Like XLent mice . Moreoveent mice ,48.−/−XLF−/−MDC1 double-knockout cell lines and demonstrated that MDC1 is stimulating the V(D)J recombination in cells lacking XLF. Moreover, we demonstrated that combined inactivation of MDC1 and XLF resulted in synthetic lethality in mice.Here, we generated +Eμ-Bcl2 and −/−Eμ-Bcl2+XLF vAbl cells were published earlier [−/−Eμ-Bcl2+MDC1 were generated using two three-week-old mice following the procedure described previously [XLF gene was inactivated in +Eμ-Bcl2 vAbl cells to obtain −/−Eμ-Bcl2+XLF cell lines, and in −/−Eμ-Bcl2+MDC1 to generate −/−XLF−/−Eμ-Bcl2+MDC1 vAbl lines, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing approach as described earlier [exon 3 of the XLF gene: sgRNA1_FWD: 5′-CTTAGCATACACCAACTTC-3′; sgRNA1_REV: 5′-GAAGTTGGTGTATGCTAAG-3′; sgRNA2_FWD: 5′-CCACCAACAGGTACTCATA-3′; sgRNA2_REV: 5′-TATGAGTACCTGTTGGTGG-3′. Parental vAbl cells were transduced with lentiviral vectors containing corresponding sgRNA sequences, and up to 200 clones were screened by western blot. The cells lacking the XLF signal were used to validate the deletion of the exon 3 by DNA sequencing (available upon request). Two −/−XLF clones and four −/−XLF−/−MDC1 clones were used for experiments. Mock-treated and parental vAbl cells were used as DNA repair-proficient controls. earlier ,42,43. Feviously ,50. Addi earlier . Briefly earlier . The folThe following antibodies were used for western blot: rabbit polyclonal anti-XLF , swine polyclonal anti-rabbit immunoglobulin-horseradish peroxidase-conjugated , mouse monoclonal anti-β-actin , rabbit polyclonal anti-mouse Ig-HRP , and goat polyclonal anti-mouse Ig-HRP .pMX inversion (pMX-INV) and pMX deletion (pMX-DEL) substrates, as previously described [pMX-INV cassette, the green fluorescent protein (GFP) gene is placed in the reversed orientation and the GFP protein is not expressed. Upon the RAG-induced V(D)J recombination, the GFP gene is placed in the sense orientation leading to the GFP protein expression. The GFP protein is then detected by flow cytometry to estimate the V(D)J recombination efficiency in indicated vAbl cells [pMX-DEL cassettes. During the V(D)J recombination, the CJpMX-DEL cassette results in an intermediate product with hairpin-sealed coding ends that require Artemis nuclease activity to open the hairpins prior DNA ligase 4-dependent DNA ligation, leading to coding joints (CJ). On the contrary, the SJpMX-DEL cassette results in the RAG-dependent generation of blunt signal ends (SE) that can be directly ligated by DNA ligase 4 and do not require Artemis nuclease activity, leading to signal joints (SJ) [V(D)J recombination assays were performed using chromosomally-integrated escribed ,43,49,50bl cells ,49,50. Fnts (SJ) ,43,49,50+/−MDC1 [+/ΔXLF [+Eμ-Bcl2 [+Eμ-Bcl2 transgenic mice were used to generate vAbl pre-B cells and increase cell survival during the experimental procedures [All experiments involving mice were performed according to the protocols approved by the Norges teknisk-naturvitenskapelige universitet (NTNU), FOTS#8319. MDC1+/− , XLF+/Δ  [XLF+/Δ , and Eμ-Eμ-Bcl2+  mice werocedures .2, according to the manufacturer’s instructions. ∆MDC1 HAP1 cells are nearly haploid human cells that were custom-generated by request and provided by Horizon Discovery . The HAP1 cells are human, nearly haploid cell lines derived from the chronic myelogenous leukemia (CML) cell line (KMB-7). The HAP1 model has been recently used to develop knockout human cells  medium in triplicates into 6-well plates. Similarly, fifty thousand human haploid 1 (HAP1) cells were plated in Iscove’s Modified Dulbecco’s Medium  and supplemented with 10% fetal bovine serum, FBS , and 1% penicillin-streptomycin  at 37 °C with 5% COferences ,33,51,54t-test.Both vAbl and HAP1 cells were counted every 24 h using a Countess™ Automated Cell Counter  with Trypan blue staining  and bright-field detection. Statistical analyses were performed using GraphPad Prism 8 , one-way analysis of variance (ANOVA), and MDC1 and Artemis suggests that MDC1 protects or stabilizes RAG-induced DSBs before ligation. In particular, the vAbl cells lacking MDC1 and Artemis possess ATM-dependent degradation of free DNA ends during the attempted V(D)J recombination [+/−Eμ-Bcl2+MDC1 mice and isolated the cells from the bone marrow of three-week-old −/−Eμ-Bcl2+MDC1 animals. We then established Abelson murine leukemia virus kinase-transformed pro-B cells (vAbl) and chromosomally-integrated either pMX-INV or pMX-DEL V(D)J recombination cassettes, as described previously [Mice lacking MDC1 possess nearly wild-type levels of B and T lymphocytes . Combinebination . To furteviously ,42,43,49−/−XLF−/−MDC1 double knockout mice. Individual inactivation of MDC1 or XLF results in live-born mice that possess modest levels of DNA repair defects [+/−XLF−/−MDC1 mice, starting with available heterozygous +/−MDC1 [+/−XLF [+/−XLF−/−MDC1 mice, we obtained and genotyped 104 pups, including 34 +/+XLF−/−MDC1 and 70 +/−XLF−/−MDC1  J recombination, we first attempted to generate the  defects ,42,43,44 MDC1+/−  and XLF+ [XLF+/−  animals./−XLF−/− . Strikin (1:2:0) . We conc−/−XLF−/−MDC1 and control −/−XLF vAbl cells, we inactivated the XLF gene in −/−MDC1 and WT vAbl cells using the CRISPR/Cas9 gene-editing approach . Briefly, we targeted exon 3 of the XLF gene  at 48 and 72 h of the experiment  [−/−XLF cell lines  . Double knockout −/−XLF−/−DNA-PKcs vAbl cells were used as a negative control to establish background levels of the experiments (0% of GFP-positive cells) [To determine the impact of MDC1 on V(D)J recombination, we chromosomally-integrated the cassette-carrying pMX-INV) ,50  ,49,50. Tctively) B–D. StriRAG and most of the known NHEJ factor genes in mice leads to immunodeficiency [XLF, PAXX, or Mri genes results in mice with the nearly normal immune system, due to the overlapping functions between XLF and PAXX [XLF and ATM [DNA-PKcs and ATM [ATM and XLF, or 53BP1 and XLF, resulted in immunodeficient mice of smaller sizes than single knockouts or wild-type controls, with abrogated NHEJ, resembling −/−Ku70 or −/−Ku80 knockouts [DNA-PKcs and XLF [H2AX and XLF [MDC1 and XLF J recombination in developing B and T lymphocytes ,42,43,44The proliferation rate of vAbl cells lacking both XLF and MDC1 was reduced when compared to single-deficient and WT controls  at 72 h.Multiple DDR factors are involved in the V(D)J recombination. Due to the functional redundancy between the DDR and NHEJ pathways, complex genetic in vivo and in vitro models will be appropriate to uncover specific functions of DDR factors in B and T lymphocyte development and further elucidate mechanisms underlying their roles.


Thus, our data strongly suggest that the interplay between receptors promotes or restricts transmission and further indicate that Syndecan 4 is the molecular switch controlling HCV susceptibility after sexual contact.Sexually transmitted Hepatitis C virus (HCV) infections and high reinfections are a major concern amongst men who have sex with men (MSM) living with HIV-1 and HIV-negative MSM. Immune activation and/or HIV-1 coinfection enhance HCV susceptibility via sexual contact, suggesting that changes in immune cells or external factors are involved in increased susceptibility. Activation of anal mucosal Langerhans cells (LCs) has been implicated in increased HCV susceptibility as activated but not immature LCs efficiently retain and transmit HCV to other cells. However, the underlying molecular mechanism of transmission remains unclear. Here we identified the Heparan Sulfate Proteoglycan Syndecan 4 as the molecular switch, controlling HCV transmission by LCs. Syndecan 4 was highly upregulated upon activation of LCs and interference with Heparan Sulfate Proteoglycans or silencing of Syndecan 4 abrogated HCV transmission. These data strongly suggest that Syndecan 4 mediates HCV transmission by activated LCs. Notably, our data also identified the C-type lectin receptor langerin as a restriction factor for HCV infection and transmission. Langerin expression abrogated HCV infection in HCV permissive cells, whereas langerin expression on the Syndecan 4 expressing cell line strongly decreased HCV transmission to a target hepatoma cell line. These data suggest that the balanced interplay between langerin restriction and Syndecan 4 transmission determines HCV dissemination. Silencing of langerin enhanced HCV transmission whereas silencing Syndecan 4 on activated LCs decreased transmission. Blocking Heparan Sulfate Proteoglycans abrogated HCV transmission by LCs  Viral hepatitis is responsible for an estimated 1.3 million deaths from acute infection, hepatitis-related liver cancer and cirrhosis in 2015 –9. AlthoLCs reside in mucosal tissues and are therefore among the first immune cells to encounter viruses, they are thought to be involved in sexual transmission of HIV-1 –22 InterPrimary LCs are also present at mucosal sites of the foreskin and within the anal tissue , 23, thetrans-receptors to infect hepatocytes. This transmission mechanism implicates that the balanced interplay between langerin and Syndecan 4 on activated LCs dictates HCV susceptibility after sexual contact. Therapeutical interventions targeting Syndecan 4 on mucosal LCs could represent a novel strategy to counteract LC-mediated dissemination of HCV.Here our data show that upon activation LCs acquire the ability to retain and transmit HCV using HSPGs. Further analyses showed that the major HSPG involved is Syndecan 4. Strikingly, Syndecan 4 induction upon activation of LCs counteracts langerin restriction of HCV transmission. Ectopic expression of langerin on the Syndecan 4 expressing cell line enhanced HCV capture but decreased HCV transmission to a target hepatoma cell line Huh 7.5. Moreover, silencing langerin on Mutz-LCs enhanced HCV transmission which strongly support a restrictive role for langerin in mucosal HCV transmission. Together our data strongly suggest that activated LCs upregulate Syndecan 4 to capture HCV via their heparan sulfate chains and exploit them as in The following antibodies were used : Heparan Sulfate (clone F58-10E4) (Amsbio), digested Heparan (clone F69-3G10) (Amsbio), Syndecan 1 (DL-101) (Santa Cruz), Syndecan 2-FITC (H-7) (Santa Cruz), Syndecan 3 (M-300) (Santa Cruz), Syndecan 4 (clone F94-8G3), CD207-PE (langerin) mouse IgG1 (#IM3577) , CD1a-APC mouse IgG1  CD1a-PE (clone SK9) mouse IgG2b (BD Bioscience), HLA-B27-FITC (clone HLA-ABC-m3), mouse IgG2a (Abcam), HLA-DR-FITC (clone G46-6), mouse IgG2b (BD Bioscience), CD80-PE, mouse IgG1 (BD Pharmingen), CD83-PE, mouse IgG1 (eBioscience), CD86-FITC, mouse IgG1 (BD Pharmingen), FITC-conjugated goat-anti-mouse IgM (#31992) (Invitrogen), AF488-conjugated goat-anti-mouse IgG1 (#A21121) (Invitrogen), AF488-conjugated donkey-anti-mouse IgG2b (Invitrogen).Flavobacterium heparium, EC 4.2.2.8, Batch 010 (Amsbio). 123Count eBeads, REF# 01-1234-42, LOT# E133305, 1.011.000 eBeads/ml (eBioscience).The following reagents were used: Unfractionated (UF) heparin, 5.000 I.E./ml (LEO). Low Molecular Weight (LMW) heparins: dalteparin, 10.000 IE anti-Xa/ml (Pfizer), tinzaparin, 10.000 IE anti-X1/0.5ml (LEO), enoxaparin, 100 mg/ml (Sanofi). 4-Nitrophenyl β-D-xylopyranoside  (SigmaAldrich). Heparinase III from nef position (1.35 ug) and genotype 1a pHCV_H77_E1_E2(AF009606) (0.6 ug). Transfection was performed in 293T/17 cells using genejuice  transfection kit according to manufacturer's protocol. At day 3 or day 4, pseudotyped HCV virus particles were harvested and filtered over 0.45 um nitrocellulose membrane . Replicative JFH1-AM120-Rluc in vitro transcribed RNA, containing a luciferase reporter gene, was generated according to manufacturer's instructions  and electroporated into Huh 7.5 cells as previously described (3 to 4 × 103.The following plasmids were provided by Dr. Takaji Wakita at Tokyo Metropolitan Institute of Neuroscience: Genotype 2a HCV genomic RNA clone pJFH1 (APP1025) . pNL4.3.escribed . Virus p+ human AML cell line Mutz3 progenitors in the presence of GM-CSF , TGF-β  and TNF-α (2.5 ng/ml), R&Dsystems) and cultured as described before  and Namalwa cells stably expressing human Syndecan 1, Syndecan 2, Syndecan 3, Syndecan 4  were mad before . Cell suLangerin expression plasmid pcDNA3.1 were obtained from Life Technologies and subcloned into lentiviral construct pWPXLd (Addgene). HIV-1-based lentiviruses were produced by co-transfection of 293T cells with the lentiviral vector construct, the packaging construct  and vesicular stomatitis virus glycoprotein envelope  as described previously . Huh 7.52 and cultured in Iscoves Modified Dulbeccos's Medium  supplemented with 10% FCS, gentamicin , penicillin/streptomycin . Activated LCs were generated as described before  or Namalwa Syndecan 1-4 cell line  or immature LCs  or activated LCs  were exposed to replicative HCV genotype 2a strain containing a luciferase reporter gene (JFH1-AM120-Rluc) or pseudotyped HCV (HIV-1 NL4.3Δenv pseudotyped with HCV env glycoproteins E1 and E2) pre-incubated with 250 U or 500 U UF heparin or LMW heparins either for 4 or 24 h, harvested, extensively washed to remove unbound virus and co-cultured with Huh 7.5 for 3 or 5 days at 37°C and analyzed for luciferase reporter activity. Luciferase activity (relative light units (R.L.U.)) was measured using the Luciferase assay system  or Reporter gene assay system  according to manufacturer's instructions. For the ex vivo transmission model epidermal sheets were culture for 24 h and exposed to medium or UF heparin pre-incubated replicative HCV or pseudotyped HCV for another 24 h. After 48 h cells were harvested, extensively washed to remove unbound virus and co-cultured with Huh 7.5 for 5 days at 37°C, transmission was determined by luciferase reporter activity. Luciferase activity (relative light units (R.L.U.)) was measured using the Luciferase assay system  or Reporter gene assay system  according to manufacturer's instructions.Namalwa cell line (1.0 × 10MUTZ-LCs and primary LCs were silenced by electroporation with Neon Transfection System (ThermoFischer Scientific). The siRNA were specific for langerin , siRNA Syndecan 4  or non-targeting siRNA  as control. Silencing of the targets was verified by real-time PCR, flow cytometry. Cells were used for experiment 72 h after silencing. Silencing of the targets was verified by real-time PCR or flow cytometry.∧Ct(GAPDH) – Ct(target). For relative mRNA expression, control siRNA sample was set at 1 for each donor.mRNA was isolated with an mRNA Capture kit (Roche) and cDNA was synthesized with a reverse-transcriptase kit (Promega) and PCR amplification was performed in the presence of SYBR green in a 7500 Fast Realtime PCR System (ABI). Specific primers were designed with Primer Express 2.0 (Applied Biosystems). Primer sequences used for mRNA expression were for gene product: GAPDH, forward primer (CCATGTTCGTCATGGGTGTG), revers primer (GGTGCTAA GCAGTTGGTGGTG). For gene product: langerin, forward primer (CACAGTGGCATTCTGGAGTCC), reverse primer (CCACCCCTCCCACTTTAACC). For gene product: Syndecan 4, forward primer (AGGTGTCAATGTCCAGCACTGTG) reverse primer (AGCAGTAGGATCAGGAAGACGGC). The normalized amount of target mRNA was calculated from the Ct values obtained for both target and household mRNA with the equation Nt = 26 cells/ml were treated in D-PBS/0.25% BSA with 140 miliunits heparinase III (Amsbio) for 2 h at 25°C, washed and used in subsequent experiments. Enzymatic digestion was verified by flow cytometry using antibodies directed against heparan sulfates and digested heparan sulfates.Namalwa Syndecan 4 cell line were cultured in the presence of 1.0 mM or 2.5 mM PNP-Xyl for 72 h to inhibit HSPG biosynthesis and used in subsequent experiments. The expression of cell surface heparan sulfates was assessed by flow cytometry using antibodies directed against heparan sulfates. 1.0 × 10t-test for paired observations (differences within the same donor) or unpaired observation (differences between different donors) was performed. For unpaired, non-parametric observations a Mann-Whitney test was performed. Statistical analyses were performed using GraphPad Prism 7 software and significance was set at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.A two-tailed, parametric Student's ex vivo transmission model   created The datasets generated for this study are available on request to the corresponding author.Human skin tissue was obtained from healthy donors undergoing corrective abdominal surgery in accordance with our institutional guidelines. This study was reviewed and approved by the Medical Ethics Review Committee of the Amsterdam University Medical Centers, location Academic Medical Center (AMC), Amsterdam, the Netherlands, reference number: W15_089 # 15.0103. All samples were handled anonymously. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.BN performed and designed the research study, analyzed the data, and wrote the paper. JE performed the research study and analyzed the data. CL performed the research study and analyzed the data. TK performed the research study. SM contributed essential material. PZ contributed essential material. CR designed the research study and contributed essential material. TG was involved in all aspects of the study. All authors contributed to the manuscript, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Additionally, the CATT5/C and CATT7/G haplotypes were not detected in any groups.Expression level of mRNA in ectopic tissue of endometriosis patients with CATT6,7/CC haplotype, were significantlyhigher compared to other haplotypes including CATT5,5/GG , CATT5,5/GC  andCATT6,6/GG . Homozygote of CATT6/C and CATT7/C haplotypes in endometriosis patients. Increased MIF expression is affected bygenetic variants in the MIF promoter in ectopic endometrial tissues. This promoter haplotype might play an importantrole in the development and establishment of endometriosis.We report, for the first time, a strong linkage between the decreased repetition of CATT and G allelein control and CATT Endometriosis is an inflammatory, estrogen-dependentdisease that is characterized by presence of ectopicendometrial-like tissue outside of the uterine cavity ,2. EndoMacrophage migration inhibitory factor (MIF) isa pleiotropic pro-inflammatory cytokine , 9 thatMIF gene is located in the chromosome22q11.2 region and consists of three exons (MIF promoter (5–8 (rs5844572), were shown byseveral meta-analyses to increase susceptibility someimmune and inflammatory diseases and their severity and each participantsigned an informed consent form. The stage of endometriosislesions was categorized based on the revised classificationof the American Fertility Society (rAFS). In the currentstudy, 106 patients with diagnosed endometriosis, whohad undergone laparoscopy from 2015 to 2017 and hadendometrioma cysts confirmed by histological tests, wereenrolled. The endometrioma tissues were collected duringlaparoscopic surgery in the secretory phases of menstrualcycle (days 16-19). The 110 controls were recruited fromsubjects who were not diagnosed with endometriosis,underwent diagnostic laparoscopy or fertile women with nosign of endometriosis in Doppler ultrasonography. None ofthe participants had endometrial hyperplasia, neoplasia, orinflammatory and autoimmune disorders, and none of themwere receiving anti-inflammatory or hormonal medicationfor at least 3 months before laparoscopy. The subjects’ agewas between 20 and 40 years old and the control individualswere matched with the endometriosis patients in terms ofbody mass index (BMI).DNA was extracted from whole blood anticoagulatedwith ethylenediamine tetraacetic acid (EDTA)-2Na.Patients’ genomic DNA was extracted by using theGene All® kit (Korea), according to the manufacturer’sinstructions. Salting-out method was used to obtain thecontrols’ genomic DNA.The polymerase chain reaction (PCR) was used toamplify the studied fragments of MIF gene. The PCRincluded a hot start at 95˚C for 5 minutes, followed by 35PCR cycles, each including denaturation for 30 secondsat 94˚C, primer annealing (depending on the primer pairs)for 30 seconds, and extension for 60 seconds at 72˚C. Afinal extension step was conducted at 72˚C for 10 minutes.Restriction fragment length polymorphism (RFLP)was performed to detect -173G/C SNP. PCR was used toamplify a 303 bp fragment.Sense primer was:5´- CCT-CCT-GGC-GAC-TAA-CAT-CGG-TGA-CT-3´and the anti-sense primer was: 5´- TAC-GTG-CCT-GAC-TTC-TCG-GAC-ACC-ACT -3´The annealing temperature was set at 63˚C. Theresulting fragment was digested using AluI restrictionendonuclease  for15 minutes at 37˚C, and the digested fragments wereresolved using 1.7% agarose gel stained with ethidiumbromide, and visualized using Molecular Imager® GelDoc™ XR+  under ultraviolet(UV) light. The GG genotype revealed a single band (303bp) because no cutting site for this enzyme, while twosmall PCR fragments containing 98 and 205 bp representCC genotype. The RFLP pattern for heterozygous GCwas characterized using the following 3 bands: 303, 205and 98bp. More than 10% of the samples with differentgenotypes, were randomly selected to be sequenced to confirm thegenotypes obtained by PCR- RFLP method.Oligonucleotide primers (sense primer:5´-TAT- GGA-TTG-CAC-CTA-TCA-GAG-ACC-3´ and anti-sense primer: 5´-TCT-CAT-AGA-GCC-CTT-GGT-GAAT-3´),5-8 promoter region. The annealing temperature of PCR cyclewas 58˚C. Purified PCR products of -794(CATT)5-8 andORF region were sequenced using an ABI automated DNAsequencer . were designed to amplify a 250 bp segment of the -794(CATT)MIF promoter. The RNA was isolated using TRIzol basedon the manufacturer’s instructions. cDNA was amplifiedby One-Step Reverse transcriptase (RT)-PCR using atranscriptase kit  in the presence of random hexamers. The reactionwas incubated at 25˚C for 5 minutes, 42˚C for 60 minutes,and 70˚C for 5 minutes. The RT-PCR products were runon agarose gel. Quantitative real-time PCR was performedin an ABI 7000 Thermal Cycler . Each standard PCR reactioncontained 2 μl cDNA templates, 1 μl of each primer , and 10 μl SYBR Green PCRMaster Mix containing Taq DNA polymerase buffer, deoxynucleotidetriphosphate mix, SYBR green I, MgCl2, and Taq DNApolymerase. After denaturation (for 4 minutes at 95˚C),amplification and quantification were repeated 40 times. The primer pairs (in the 5’-3’direction) used forhuman MIF:Endometriotic tissues were collected from 17endometrioma lesions in women who were genotyped forthe [(sense:5´-AGA-ACC-GCT-CCT-ACA-GCA-AG-3´antisense:]5´-GAG-TTG-TTC-CAG-CCC-ACA-TT-3´and amplicon size: 121bp) and(sense:5´-CAA-GAT-CAT-TGC-TCC-TCC-TG-3´and antisense:5´-ATC-CAC-ATC-TGC-TGG-AAG-G-3´ and amplicon size: 90bp)β-Actin were described in our previous study   and 95% confidence interval (CI). Differences were considered statisticallysignificant at P<0.05. All statistical analyses wereperformed using Statistical Package for the Social Sciences software.Comparison of Hardy-Weinberg equilibriumtest results was made and allelic and genotypedistributions were compared between endometriosispatients and controls using the Pearson’s Chi-squareanalysis by SHEsis (io-x.cn) .The homval (CI). For hapAll endometriosis participants had severe endometriosis(stage III and IV). The two groups matched on age witha mean distribution of 31 ± 3.74 and 31.75 ± 3.52 yearsin endometriosis patients and controls, respectively. BMIhas not significant difference between both groups . All participants in the current studyhave a regular menstruation cycle.5-8 polymorphisms (5-8 and -173G/C), we continued to evaluate the frequencies of -173G/C polymorphism in 53 endometriosis patients and 53controls with homozygous genotypes of -794(CATT)5-8polymorphism . Additionally, theCATT5/C and CATT7/G haplotypes were not detectedin any group . As shownin Table 1, the CATT8 allele was not detected neitherin endometriosis patients nor in controls. Homozygoteof CATT7 was observed only in endometriosis whilstCATT6 and CATT7 alleles were more prevalent inendometriosis patients and CATT5 allele was moreprevalent in controls but these differences were notsignificant (P>0.05). Therefore, at the first step, westudied the -794 (CATT)morphism . The datmorphism .Finallyquencies . Since tny group . Therefo5-8 and function with MIF mRNA expression,we assessed the levels of mRNA using fluorescentquantitative polymerase chain reaction (FQ-PCR) in 17patients with endometrioma who were also genotypedfor promoter polymorphisms. The data were normalizedagainst the mRNA level of the CATT5/G samples. Wefound an interaction between the -173C and more copiesof repetitions of CATT in ectopic endometriotic tissues.Promoter activity and subsequent expression of mRNAin ectopic tissue of patients with CATT6,7/CC haplotypewere significantly higher compared to other haplotypesincluding CATT5,5/GG , CATT5,5/GC  and CATT6,6/GG . However, the higher transcriptional activity inindividuals carrying CATT6,6/GC  andCATT6,6/CC  was not significantlydifferent from those of subjects carrying CATT6,7/CChaplotype aplotype .MIF gene in promotingthe inflammatory processes and establishment of ectopictissues, we investigated possible associations betweengenetic variants of MIF promoter and susceptibility toendometriosis. The results revealed that genetic variantsof MIF, including the 7 repetitions of the CATT STRin homozygote, were only detected in subjects withendometriosis. Exceptionally, the CATT8 allele, which isa rare allele, was not detected in this study.Considering the importance of the 6/C and CATT7/C haplotypes can be consideredas moderate risk factors and CATT5/G has an almostprotective effect against endometriosis. Additionally, theCATT5/C and CATT7/G haplotypes were not detected inany of the groups.Based on our results, the -173C allele is more common inpatients carrying -794(CATT)5-8 homozygotes suggestingit as a risk factor for endometriosis. This finding wasconfirmed by haplotype analysis which revealed thatCATT5 were less susceptible to autoimmuneinflammation , juvenile rheumatoid arthritis (JRA),inflammatory bowel disease (IBD) and cancers  andthe susceptibility towards endometriosis, because thishaplotype causes the simultaneous presence of AP-4,Pit-1 and ICBP90 transcription factors and consequentlyenhances MIF promoter activity. The results showedan increased expression of MIF mRNA in individualswith 6C compared to those with 5G and 7C haplotypes.Thus, observed simultaneous attendance of longer CATTrepeats at -794 and the -173C allele in the gene wasassociated with elevated MIF production and correlatedwith increased risk of endometriosis. This was confirmedin haplotype analysis which revealed that CATT5/G hasstrongly protective effect against endometriosis. Takentogether, our findings indicate that increment of MIFexpression is associated with genetic variants of MIFpromoter in ectopic endometriotic tissues.At the promoter region, the CATT repetition containsseveral identification regions pituitary-specific factor1 (Pit-1) binding sites. Pit-1 is a transcription factor inneuroendocrine and mononuclear cells , 34. Alsroblasts. Pit-1 apetition. This maIncreased MIF activates a cascade of events and stronglystimulates cyclooxygenase 2 (COX2) and prostaglandinE2 (PGE2) expression. This finally leads to increased localsynthesis of estrogen in ectopic tissues, which is involvedin maintenance and progression of endometriosis .Thus, i5/G have a protective effecton endometriosis. As well, increased repetitions ofCATT and C allele in MIF promoter were associatedwith increased susceptibility to endometriosis in ourpopulation and this was related to transcriptional activityof MIF. These findings provide the first insight that MIFpromoter polymorphisms may have a significant effecton susceptibility towards endometriosis; however, furtherstudies are required to determine contribution of MIF todevelopment of endometriosis.We believe that the CATT


Ehrlichia chaffeensis (E. chaffeensis) exploits evolutionarily conserved Notch and Wnt host cell signaling pathways to downregulate innate immune host defenses and promote infection. The multifunctional E. chaffeensis TRP120 effector which has HECT E3 ubiquitin ligase activity, interacts with the host nuclear tumor suppressor F-BOX and WD domain repeating-containing 7 (FBW7). FBW7 is the substrate recognition subunit of the Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) known to negatively regulate a network of oncoproteins . In this study, we demonstrate that TRP120 and FBW7 colocalize strongly in the nucleus by confocal immunofluorescent microscopy and interactions between TRP120 and FBW7 FBOX and WD40 domains were demonstrated by ectopic expression and co-immunoprecipitation. Although FBW7 gene expression increased during E. chaffeensis infection, FBW7 levels significantly decreased (>70%) by 72 h post infection. Moreover, an iRNA knockdown of FBW7 coincided with increased E. chaffeensis infection and levels of Notch intracellular domain (NICD), phosphorylated c-Jun, MCL-1 and cMYC, which are negatively regulated by FBW7. An increase in FBW7 K48 ubiquitination was detected during infection by co-IP, and FBW7 degradation was inhibited in infected cells treated with the proteasomal inhibitor bortezomib. Direct TRP120 ubiquitination of native and recombinant FBW7 was demonstrated in vitro and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. This study identifies the tumor suppressor, FBW7, as a TRP120 HECT E3 Ub ligase substrate, and demonstrates that TRP120 ligase activity promotes ehrlichial infection by degrading FBW7 to maintain stability of Notch and other oncoproteins involved in cell survival and apoptosis. E. chaffeensis is an obligately intracellular bacterium that replicates in mononuclear phagocytes by secreting effectors that manipulate host cell processes and exploit evolutionarily conserved pathways. This investigation reveals the complex and expanding role of the E. chaffeensis TRP120 moonlighting effector as a ubiquitin (Ub) ligase targeting host nuclear proteins. Herein, we demonstrate that E. chaffeensis TRP120 HECT Ub ligase targets the nuclear tumor suppressor Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex substrate recognition subunit, F-BOX and WD domain repeating-containing 7 (FBW7) for degradation. FBW7 is a central regulator of broadly acting host cell oncoproteins involved in cell proliferation and survival. The reduction in FBW7 through TRP120-mediated ubiquitination increases cellular oncoprotein levels and promotes E. chaffeensis infection. This study illuminates novel bacterial effector-host interactions, the importance and interplay of both host and bacterial Ub ligases and the Ub-proteasome system for infection, and mechanisms whereby evolutionarily conserved signaling pathways are hijacked by obligately intracellular pathogens. Ehrlichia chaffeensis (E. chaffeensis) is an obligately intracellular, gram-negative bacterium that exhibits tropism for mononuclear phagocytes and resides in microcolonies within membrane-bound cytoplasmic vacuoles known as morulae [E. chaffeensis survival in the mononuclear phagocyte is dependent in part on pathogen-host interactions involving tandem repeat protein (TRP) effectors that are secreted via the type-1 secretion system and interact with a diverse array of host targets [ morulae ,2. E. ch targets –5. TRPs  targets .E. chaffeensis effectors is TRP120, a moonlighting effector that has several defined functions. Early studies demonstrated that surface expressed TRP120 plays a role in host cell entry, but once ehrlichiae are internalized, TRP120 rapidly (<3 h) translocates to the host cell nucleus where it functions as a nucleomodulin, interacts with chromatin-associated proteins and directly binds genes associated with transcriptional regulation, signal transduction and apoptosis [One of the most studied poptosis –9. TRP12poptosis . TRP120 poptosis ,11.E. chaffeensis infection [There is a large group of functionally diverse host proteins that interact with TRP120, including FBOX and WD repeat domain-containing 7 (FBW7), the substrate recognition subunit of the eukaryotic Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) . FBW7 coE. chaffeensis TRP120 effector is able to activate Notch signaling in human monocytes, which appears to be critical for establishment and maintenance of intracellular infection [Recently, our laboratory reported that the nfection . Howevernfection ,19. Estanfection . Lesser nfection ,25,26. HE. chaffeensis TRP120 effector and the tumor suppressor FBW7. The interaction ultimately enables the E. chaffeensis TRP120 HECT Ub ligase to engage FBW7 as a substrate to initiate a degradation process that involves K48 ubiquitination and proteasomal degradation. The degradation of FBW7 during ehrlichial infection increases Notch intracellular domain (NICD) levels and stabilizes other prominent FBW7-regulated oncoproteins resulting in ehrlichial infection enhancement. This study provides further insight into how evolutionarily conserved signaling pathways are hijacked by obligately intracellular pathogens.In this investigation, we have characterized a novel nuclear interaction between the E. chaffeensis-infected THP-1 cells  where the intensities of green and red fluorophores  were calculated. The brightest points of colocalization were also quantified using Mander’s coefficients  with a 58% of total analyzed regions of interest with >0.8, indicating a very strong colocalization correlation between TRP120 and FBW7. We also determined the nuclear colocalization of TRP120 with FBW7 is specific with confocal microscopy demonstrating that FBW7 does not colocalize with E. chaffeensis Dsb (disulfide bond formation), a protein that is observed in the periplasm of E. chaffeensis  where both domains were shown to be bound to TRP120  compared to uninfected cells, while TRP120 nuclear level was also reduced (~40%) with significantly increased TRP120 presence on the morulae. The fluorescent intensities of FBW7 and TRP120 from the confocal microscopic images were quantified using image J  and MCL1 were also significantly elevated  in uninfected control cells, which is consistent with maintenance of FBW7 levels by K48 autoubiquitination. In infected cells, K48-ubiquitinated FBW7 levels increased in bortezomib-treated cells. Moreover, decreased FBW7 was observed at 24, 48 and 72 hpi in untreated cells, but was stable throughout infection in bortezomib-treated  cells  cells . These rin vitro ubiquitination assays were performed to demonstrate TRP120 directly ubiquitinates FBW7. The In vitro ubiquitination experiment revealed that in the presence of rTRP120, Ub-FBW7 (~200 kD) was detected at higher levels compared controls. Similarly, increased K48-Ub was detected on FBW7 in the presence of rTRP120 (TRP120 has HECT E3 ligase activity and one host cell substrate (PCGF5) has been identified . Thus, w rTRP120 , which iE. chaffeensis infection using siRNA knockdown of FBW7 in THP-1 cells. E. chaffeensis infection was significantly and progressively increased by 50% by 48 hpi in FBW7-KD cells compared to control transfected with scrambled siRNA  TRP120 is secreted via type-1 secretion system, and translocates to the host cell nucleus via an unknown mechanism; 3) TRP120 ubiquitinates FBW7 in the nucleus; 4) K48-Ub-FBW7 is then degraded by the 26S proteasome most likely in the nucleus; 5) degradation of FBW7 results in increased levels of downstream pro-survival markers  resulting in the maintenance of Notch signaling activation and anti-apoptotic programming; 6) finally, the pro-survival markers activate downstream gene expression to suppress host innate immunity and delay monocyte apoptosis, thus creating a favorable environment for E. chaffeensis replication.Through this investigation, we have gained an understanding of the importance of TRP120 interaction with FBW7 during ed model  that canE. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described [Human monocytic leukemia cells (THP-1) were grown and maintained in RPMI medium 1640 with L-glutamine and 25 mM HEPES buffer , supplemented with 10% fetal bovine serum . Henrietta Lack’s cervical epithelial adenocarcinoma (HeLa) cells were propagated in MEM medium with Earle’s Salts and L-glutamine , supplemented with 10% fetal bovine serum (HyClone). escribed .E. chaffeensis-infected THP-1 cells were harvested at 24, 48 and 72 h post infection (hpi) and whole-cell lysates (uninfected THP-1 cells were used as controls) were extracted three times in complete-RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail tablet  and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were centrifuged at 13,000 x g for 30 s to pellet insoluble material and cleared by centrifugation at 13,000 x g for 20 min at 4°C. The protein concentration was determined using Pierce BCA Protein Assay (Thermo Fisher Scientific). In addition, equal mass amount of N-ethylmaleimide  was added to the whole cell lysates to preserve native ubiquitination of proteins in co-immunoprecipitation experiment performed for E. chaffeensis-infected and uninfected THP-1 cells was isolated with the RNeasy Mini Kit  using on-column DNA digestion with RNase-free DNase reagent (Qiagen). cDNA synthesis was performed with total RNA (1 μg) using qScript cDNA Synthesis Kit . Quantitative real-time PCR was performed with iQ SYBR Green SuperMix  with gene-specific primers. Forward FBW7: 5’-CCACTGGGCTTGTACCATGTT-3’; reverse FBW7: 5’-CAGATGTAATTCGGCGTCGTT-3’. Forward GAPDH: 5’-GCTCTCTGCTCCTCCTGTTC-3’; reverse GAPDH: 5’-TTCCCGTTCTAGCCTTGAC-3’.Total RNA from plus non-targeting siRNA (Dharmacon) designed to have fewer off-targets than standard unmodified negative control siRNAs. Quantification of E. chaffeensis by qPCR after RNA interference has been previously described [Specific FBW7 siRNAs were siGENOME SMARTpool siRNA , which are endoribonuclease-prepared siRNA pools containing heterogenous mixture of four different siRNAs targeting the same human FBW7 mRNA sequence. The control siRNA was ON-TARGETescribed . Fold chE. chaffeensis at a multiplicity of infection (MOI) of 100 for 24, 48 and 72 h. Cells were cytocentrifuged onto glass slides and fixed in ice-cold 4% paraformaldehyde in PBS for 20 min then permeabilized and blocked in 1% Triton-X 100 in PBS with 5% bovine serum albumin for 1 h. The cells were incubated with TRP120 rabbit peptide antisera (1:1000) and anti-FBW7 mouse monoclonal antibody  for 1 h, washed three times with PBS and incubated with anti-rabbit Alexa Fluor 488-IgG (H+L) and anti-mouse Alexa Fluor 594-IgG (H+L) secondary antibodies for 1 h. Slides were washed three times with PBS and mounted with ProLong Gold Antifade reagent with 4’,6-diamidino-2-phenylindole . Images were obtained using a Zeiss Laser Scanning Microscopy 880 with Airyscan and processed using Zeiss ZEN Microscopy Software  and FIJI (FIJI Is Just ImageJ).THP-1 cells were plated in 6-well plates and infected with cell-free E. coli cultures and purified using QIAprep Spin Maxiprep kit (Qiagen). Full-length FBW7 plasmid was purchased as pcDNA3.1+/C-(K) DYK-FBW7 . The same FBW7 domain constructs were also used for purification from HeLa cells through immunoprecipitation to obtain recombinant proteins used in co-IP experiment  were created using PCR amplification from pGEM-FBW7 plasmid  and cloning into the pcDNA3.1/His mammalian expression vector (Thermo Fisher Scientific). Plasmid DNA of FBW7-N, FBW7-FBOX and FBW7-WD40 clones was obtained from transformed TOP10 periment . Full leescribed . RecombiThe plasmid DNA was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Cells ectopically expressing TRP120 or FBW7-His were cytocentrifuged onto glass slides and fixed in ice-cold 4% paraformaldehyde in PBS for 20 min then permeabilized and blocked in 1% Triton-X 100 in PBS with 5% bovine serum albumin for 1 h. The cells were incubated with rabbit anti-TRP120 (1:1000) and mouse anti-His antibodies (1:100) at room temperature for 1 h, then washed and incubated with anti-rabbit Alexa Fluor 488-IgG (H+L) and anti-mouse Alexa Fluor 594-IgG (H+L) secondary antibodies for 1 h. Images were obtained using an Olympus BX61 epifluorescence microscope and analyzed using SlideBook 6 Reader software  and FIJI.Quantification of immunofluorescence microscopy and calculation of Mander’s coefficient were carried out using WCIF-ImageJ , which contains plugin for intensity correlation analysis (ICA) and corrected total cell fluorescence (CTCF). ICA was used to calculate Mander’s overlap coefficient (MOC) by using the selection tool to mark the regions of interest. Similarly, CTCF values were collected using the measurement tool after the selection of the regions of interest in a micrograph.Recombinant TRP120-GST was incubated at 4°C overnight with recombinant FBW7-His or recombinant WD40-His in native equilibration buffer . All proteins were also incubated alone as control. Samples were placed onto cOmplete His-Tag Purification Resin (Sigma-Aldrich) to immobilize the His-tagged bait from the samples overnight at 4°C. Unbound protein was washed away with equilibration buffer containing 5mM, 10mM, or 20mM Imidazole. Protein-protein interaction complexes were eluted using equilibration buffer containing 500mM Imidazole. Western blot analysis was performed on eluted samples. Detection of FBW7 and WD40 was performed with anti-His antibody and TRP120 was detected using both anti-TRP120 or anti-GST antibody.Co-Immunoprecipitation (co-IP) was performed with 3-day post-infection (95–100%) THP-1 cells using Pierce Protein A/G Agarose kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Protein samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and blocked for 1 h at room temperature in Tris-buffered saline with 5% nonfat dry milk and 1% Triton-X 100. Primary antibodies included mouse anti-FBW7 , rabbit anti-TRP120 peptide antisera , rabbit anti-GAPDH , rabbit anti-NICD , rabbit anti-p-c-Jun , rabbit anti-MCL1 , rabbit anti-K48 linkage-HRP conjugated , mouse anti-FK2 , and mouse anti-cMYC (9E10) . Secondary antibodies included horseradish peroxidase-labeled goat anti-rabbit IgG and anti-mouse IgG . Densitometry was performed using ImageJ software.in vitro ubiquitination assays were performed with a Ubiquitinylation kit . FBW7 ubiquitination was performed using purified recombinant TRP120 and native or recombinant FBW7. FBW7 (100 nM) was added to a ubiquitination reaction containing Ub, ATP, inorganic pyrophosphatase and Mg2+ (buffer) in presence of E1, UbcH5b E2 and TRP120 (10 nM) as the E3. Negative control reaction did not contain ATP. The assay was performed according to manufacturer’s protocol. Ubiquitination reaction was performed at 37°C for 4 h, and the reaction was stopped with the addition of Laemmli buffer. The samples were boiled for 5 min and resolved by SDS-PAGE for Western blot analysis using anti-FBW7, anti-TRP120, and anti-K48 antibodies.All t-test with p-values of ≤0.05 considered statistically significant.The results were evaluated using two-tailed Student S1 FigIn vitro pull-down was performed to demonstrate direct interaction between recombinant E. chaffeensis TRP120 tandem-repeat (TR) domain protein with recombinant FBW7 FBOX and WD40 domains. cOmplete His-Tag purification resin was used to pull down FBOX-His and WD40-His proteins, and bound TRP120-TR-GST was detected with anti-GST and anti-TRP120 antibodies.(TIF)Click here for additional data file.S2 FigHis-tagged FBW7 domain constructs were transfected into HeLa cells and whole cell lysates were obtained at 48 hours post-transfection (hpt). Western immunoblots were performed to demonstrate stability of ectopically expressed FBW7 domain constructs detected by anti-His antibody.(TIF)Click here for additional data file.