In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif  at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCFcyclin F E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G HIV-1 has devised numerous mechanisms to evade the host immune system and establish itself successfully within the host. To accomplish this, HIV-1 exploits a multitude of cellular host factors and even mimics some of their functions. The host system in turn employs numerous competitive strategies to inhibit the invasion of virus for its self-protection. A productive infection, hence, is the outcome of continuous conflicts between the host and the virus where the latter is able to outrun the host defense.Restriction factors constitute an expanding group of cellular proteins that create powerful barriers to the virus, the most well characterized of them being the APOBEC3 family of proteins , 2, TRIMHIV-1 viral infectivity factor (Vif),+ T cells of elite controllers  promoter is reported to be most active in this phase  virus. Infection progression was monitored using a p24 antigen capture ELISA. We observed that the infection peaked at 48 h post-infection (hpi) (A). Cell cycle progression monitored using propidium iodide staining along the same time points showed significant G2/M arrest at 48 hpi (B). On the basis of the above time kinetics, cDNA from cells harvested 48 hpi was used to perform gene expression analysis. Uninfected cDNA from the same time point served as control. From the results obtained, a comparison of gene expression analysis identified several genes as modulated during HIV-1 infection. Genes that showed more than 2-fold modulation are listed in supplemental Table 1. The modulated genes are also listed on the basis of the cell cycle phases in which they are prominently present (+ T cells from two seronegative donors post-infection (C).To carry out this objective, we performed gene expression profiling using a real time-based cell cycle PCR array containing primers of 84 cell cycle pathway-associated genes. CD4on (hpi) A. Cell ct 48 hpi B. On thenfection C.CCNF) displayed the highest down-regulation at the mRNA level among the genes that showed significant modulation. Further expression analysis of cyclin F at mRNA (A) and protein (B) levels in infected CD4+ T cells confirmed its significant down-regulation with infection. We also observed cyclin F down-regulation in infected Jurkat cells (human CD4+ T cell line) at both the mRNA (C) and protein levels (D). Similar down-regulation was also obtained in another CD4+ T cell line, CEM-GFP, the results for which are shown in Fig. 4A. These observations led us to further characterize the potential implication of cyclin F down-regulation in HIV-1 infection.The cyclin F gene . The cell culture supernatants collected and used to compare virus production using p24 antigen capture ELISA also did not show significant differences in the production of virions with cyclin F overexpression . Further, the viral supernatants were normalized and were used to compare viral infectivity using a TZM-bl indicator assay as described under “Experimental Procedures.” Here, surprisingly, we observed a significant decrease in progeny virion infectivity of viral supernatants from cyclin F-overexpressed conditions .The down-regulation of cyclin F expression in HIV-1 infection led us to speculate that cyclin F could have a possible role in viral pathogenesis. As cyclin F undergoes down-regulation during infection, to understand its role in HIV-1 pathogenesis, we overexpressed cyclin F in CEM-GFP T cells. After 24 h of transfection, the cells were infected with 0.5 m.o.i. HIV-1 NL4-3 virus. Cells were harvested 48 hpi, and immunoblotting for p24 gag protein showed no significant difference in the relative cellular expression levels of p24 A, top. Tpression A, middlenditions A, bottomB, top) and production of HIV-1 virions . On comparison of viral infectivity of supernatants from control and cyclin F-silenced cells, we observed a significant enhancement in viral infectivity of virions produced by silencing cyclin F . The above results therefore point to an important functional implication of cyclin F in regulating the infectivity of virions produced during HIV-1 infection.To confirm this observation, endogenous cyclin F was silenced using siRNA pool  against cyclin F in CEM-GFP cells. Non-targeting control siRNA pool served as the control. After 24 h of transfection, cells were infected with 0.5 m.o.i. NL4-3 virus. Cells were harvested 48 hpi, and gene silencing was confirmed by immunoblotting using cyclin F antibody. Further we analyzed the expression of p24 using immunoblotting, and the culture supernatants collected were used to detect virus production as well as perform viral infectivity assays after normalization. In agreement with the overexpression results, we did not observe any significant variations in cellular p24 expression B, top an virions B, middlecyclin F B, bottomA). To further analyze this finding, HEK293T cells were co-transfected with increasing concentrations of pCMV-FLAG-cyclin F along with pNL4-3. Protein expression analysis showed a significant and dose-dependent reduction in Vif expression with cyclin F overexpression (B). To confirm the inverse correlation of cyclin F and Vif expression, we co-transfected Vif expression construct along with increasing doses of cyclin F in HEK293T cells. Here again, we observed a gradual loss of Vif expression with increasing amounts of cyclin F, indicating that cyclin F-mediated Vif down-regulation is direct and is independent of the presence of other viral proteins (C). To confirm the specificity of cyclin F in Vif regulation, HEK293T cells were then co-transfected with increasing doses of cyclin F along with HIV-1 Nef, another viral protein known to be important for viral infectivity (D). The regulatory effect of cyclin F on Vif was further confirmed by cyclin F knockdown in HEK293T cells, where dose-dependent siRNA-mediated silencing of cyclin F led to a simultaneous increase in Vif expression (E). Additionally, we also performed a transient transfection of three different lentiviral shRNA constructs of cyclin F along with Vif in HEK293T cells to analyze the effect on Vif expression. Cells harvested 72 h post-transfection showed silencing of cyclin F by two among the three shRNA constructs (shRNA1 and shRNA3) with simultaneous stabilization of Vif expression as compared with control (F), further strengthening the negative regulation of Vif by cyclin F. The sequences and clone IDs of the shRNA used are given under “Experimental Procedures.” Further, to understand the significance of cyclin F in the context of HIV-1 infection, we performed overexpression and knockdown of cyclin F in TZM-bl as well as CEM-GFP cells followed by 0.5 m.o.i. HIV-1 infection. Vif expression was found to be down-modulated with the overexpression of cyclin F and up-regulated with the silencing of cyclin F during HIV-1 infection in both TZM-bl and CEM-GFP cells (G and H). Interestingly, we also observed that cyclin F expression did not alter Vif mRNA expression . These results thus substantiate that cyclin F negatively regulates the cellular expression of HIV-1 Vif protein during infection.As HIV-1 Vif is known to be intimately associated with viral infectivity, we speculated that cyclin F would have a possible effect on Vif expression. The expression profile of cyclin F and Vif at the protein level was simultaneously analyzed in HIV-infected CEM-GFP cells. Cyclin F, as expected, showed a progressive down-regulation, whereas Vif, a late expressing viral protein, increased and stabilized its expression with the progression of infection A. To furpression B. To conproteins C. To conectivity , 37. Howectivity D. The repression E. Additi control F, furtheFP cells , G and HA, upper panel). Reverse co-immunoprecipitation assay using Vif antibody for pulldown and subsequent immunoblotting with cyclin F antibody substantiated the interaction between the two proteins . The interaction was further confirmed similarly under endogenous conditions by means of co-IP and reverse co-IP using cyclin F and Vif antibodies in HIV-1-infected (0.5 m.o.i.) CEM-GFP cells harvested 72 h post-infection . Co-IP analysis of Skp1 with cyclin F as well as Vif pulldown served as a positive control for the presence of the intact SCFcyclin F complex in the infected lysates . As Skp1 links F-box protein cyclin F to the SCF complex and thereby to the ubiquitin machinery . Immunostaining of cyclin F and Vif performed in HIV-1-infected TZM-bl cells showed co-localization of both the proteins in nuclear as well as cytoplasmic compartments .Cyclin F, although reported to be functionally relevant in the cell nucleus as well as the centrosome , 29, is Fig. 5XL/RXI, on its substrates  with several binding sites in the cyclin domain of cyclin F . The intermolecular protein-protein interactions and surface interface areas of the docked complexes were determined using the PISA server. The selected docked complex of cyclin F-Vif showed an interaction having an interface area of 1745.1 Å2 and solvation free energy (ΔiG) of −10.4 kcal/mol. Docking analysis showed that Arg-167 and Lys-168 of the Vif protein are involved in cyclin F-Vif binding, which involves cyclin F-specific residues Met-309, Ile-312, Asp-315, Trp-316, Glu-319, Thr-322, Met-323, Leu-352, Gln-353, Leu-380, and Thr-381 . The docking study thus suggests a crucial role for Arg-167 and Lys-168 residues of the Vif protein in retaining the interactions with cyclin F protein.Analysis of the docking models showed an interaction between the C-terminal region of Vif  at amino acid residues 167–169 using site-directed mutagenesis. The generation of the point mutant was confirmed by nucleotide sequence analysis. This CY Mut Vif was further used for co-immunoprecipitation assays with cyclin F. Co-transfection of HEK293T cells with the wild-type or CY Mut Vif along with cyclin F was followed by co-immunoprecipitation analysis at 48 h post-transfection. Lysates were pulled down using cyclin F antibody followed by probing with Vif antibody. Interestingly, we observed that cyclin F could not pull down the CY Mut Vif along with it (B). As a positive control, co-IP of cyclin F with Skp1 was also analyzed, which showed an interaction of both in wild-type as well as CY Mut-transfected cells (B). Positive immunoprecipitation for cyclin F is also shown, as detected by anti-FLAG antibody (B). Further, the expression of wild-type and mutant Vif was analyzed in the presence of overexpressed cyclin F. As seen previously, the expression of wild-type Vif decreased as a result of cyclin F overexpression, but the CY Mut Vif showed intact expression with respect to the control (C). These experiments suggest that cyclin F interaction occurs at the CY motif (167Arg-Lys-Leu169) on HIV-1 NL4-3 Vif and that this interaction is essential for cyclin F-mediated regulation of Vif expression.To analyze the bioinformatics-based predictions of the cyclin F-Vif interaction, as well as on the basis of previous reports on cyclin F-interacting amino acid motif, a Vif point mutant (RKL/AAA-CY Mut Vif) was constructed B at amin with it B. As a ped cells B. Positiantibody B. Furthe control C. These cyclin F E3 ligase, which interacts with its specific substrates and directs them for proteasomal degradation, we hypothesized that the same mechanism could be involved in the cyclin F-mediated down-regulation of HIV-1 Vif expression. To scrutinize this possibility, we first analyzed the mRNA level expression changes of HIV-1 Vif with cyclin F overexpression. TZM-bl cells were used here, as they can be efficiently transfected and also can be infected. Cyclin F was overexpressed in these cells followed by 0.5 m.o.i. infection and were analyzed for Vif expression at the mRNA level, which did not show any significant differences between the vector control and cyclin F overexpressed conditions (A), indicating that cyclin F-mediated regulation of Vif expression could be post-translational.As cyclin F is the substrate-binding subunit of the SCFnditions A, indicam) was added to the media 12 h prior to harvesting the infected cells. Complete restoration of Vif expression was observed with the addition of MG132 in the cyclin F overexpressed condition (B), demonstrating that cyclin F mediates proteasomal degradation of Vif during infection. This was further validated by a cycloheximide pulse-chase assay in HEK293T cells, where we found that the turnover kinetics of Vif was accelerated upon overexpression of cyclin F (C).Next we analyzed the effect of proteasomal inhibitor MG132 on Vif expression in infected TZM-bl cells in the presence of cyclin F. MG132 .Cyclin F forms a complex with SCF machinery through Skp1 interaction via its F-box domain . To confFig. 7in vivo ubiquitination assays of Vif in the presence of cyclin F during HIV-1 infection. TZM-bl cells were transfected with either empty vector or cyclin F and infected with 0.5 m.o.i. NL4-3 virus after 24 h of transfection. Cells were treated with MG132 for 12 h prior to harvesting at 48 hpi. The prepared lysates were used for immunoprecipitation using Vif antibody followed by immunoblotting using Lys-48 linkage-specific polyubiquitin antibody. Enhanced ubiquitin linkages were detected in cyclin F-overexpressed lysates (E), thus substantiating that cyclin F regulates HIV-1 Vif expression through polyubiquitination-mediated proteasomal degradation. Additionally, we also analyzed whether HIV-1 Vif possesses any reciprocal regulatory effect on cyclin F, as reported in the case of MDM2 E3 ligase (F), thus indicating that Vif may not be directly responsible for the cyclin F down-regulation observed during infection (A). Thus, overall, the above results delineate that HIV-1 NL4-3 Vif is a substrate of the SCFcyclin F E3 ligase that leads to its ubiquitination and subsequent proteasomal degradation.On confirming the proteasomal degradation of Vif by cyclin F, we performed  lysates E, thus s3 ligase . However3 ligase F, thus infection A. Thus, A). Co-transfection of Vif and A3G showed degradation of A3G, in line with previous reports. However, in cells where all three constructs were co-transfected, A3G expression was found to be restored along with the degradation of Vif . This experiment thus indirectly implicates cyclin F in retaining cellular APOBEC3G levels by the down-regulation of Vif expression.HIV-1 Vif is essential for the production of infectious virions in primary cells and certain non-permissive T cell lines including H9 and CEM , 42, as Fig. 8B, left and upper right panel). Culture supernatants were collected and infectivity assays were performed using TZM-bl infectivity assay. We observed that the effect of cyclin F on viral infectivity was seen only in the presence of A3G in HEK293T cells .Further, to understand the physiologic relevance of the above observations, we co-transfected cyclin F along with pNL4-3 in the presence and absence of A3G in HEK293T cells. Cells were harvested at 48 h post-transfection, and immunoblot analysis of lysates demonstrated that cyclin F-mediated degradation of Vif leads to augmentation in the expression of A3G . The effect of cyclin F on Vif expression was therefore studied using overexpression studies. Surprisingly, here we observed that Vif was down-regulated dose-dependently with cyclin F overexpression, which confirmed that cyclin F and Vif share an inverse correlation. Knockdown studies of endogenous cyclin F further reaffirmed a negative impact of cyclin F on Vif expression.Vif is a late expressing viral protein , 47, andFig. 3Previous reports have shown that Vif undergoes post-translational modification by ubiquitination  and can cyclin F E3 ubiquitin ligase, wherein cyclin F functions as the substrate-binding protein.To resolve our questions regarding cyclin F and Vif, we investigated the association of both these proteins. Co-immunoprecipitation and co-localization assays were performed, which demonstrated that cyclin F interacts physically with HIV-1 Vif. Analysis of the endogenous interaction of cyclin F and Vif in infected T cells demonstrated that although cyclin F is down-regulated during infection, the residual endogenous cyclin F can still interact with Vif under physiological conditions. This implies that HIV-1 Vif could be a potential substrate of the SCFXL/RXI) through a hydrophobic patch in its cyclin domain   containing 10% FBS and penicillin-streptomycin (Invitrogen) at 37 °C in a humidified 5% CO2 incubator. For CEM-GFP cells, the medium was supplemented with 500 μg/ml G418 (Invitrogen).A buffy coat of seronegative blood donors was procured from the Indian Serological Institute Blood Bank, Navi Peth, Pune, India, and PBMCs were isolated using Histopaque 1077 (Sigma) by gradient centrifugation. CD4o. 3655)  and TZM-o. 3655)  were obtCCNF siGENOME SMARTpool siRNA (M-003215-02)  was used for cyclin F silencing. The control siRNA used was non-targeting #1 siGENOME Control Pool (D-001206-13-20) . The cyclin F shRNA lentiviral constructs from Open Biosystems was a kind gift from Dr. Michael R. Green. The sequences and clone IDs of the constructs are: shRNA1, 5′-TATGGATGCTTTGTGAGTC-3′ (clone ID: V2LHS_150290); shRNA2, 5′-AGGTTTATCCGCTTCACCT-3′ (clone ID: V3LHS_322806); shRNA3, 5′-TATTCTTCGCTTTGTAGGA-3′ (clone ID: V3LHS_322803); and non-silencing shRNA, 5′-TCTCGCTTGGGCGAGAGTAAG-3′.pNL4-3 , a molecular clone of HIV-1, was obtained from the National Institutes of Health AIDS repository . pCMV-FLThe antibodies against cyclin F , HIV-1 Vif , HIV-1 Vif , and GAPDH  were procured from Santa Cruz Biotechnology. The Skp1 antibody  was procured from Rockland Immunochemicals. APOBEC3G antiserum  and p24 antiserum  were procured from the National Institutes of Health AIDS repository. Polyclonal anti-sheep Nef antibody was a kind gift from Prof. Mark Harris . Monoclog for 10 min, filtered through a 0.45-μm filter, and concentrated by ultracentrifugation at 28,000 rpm for 2.5 h at 4 °C. The viral pellet was then resuspended in RPMI 1640 containing a final concentration of 50 mm HEPES. Aliquots were made and stored at −80 °C. A p24 antigen capture ELISA (Advanced Bioscience Laboratories) was used to calculate the concentration of virus in the stock.The HIV-1 pNL4-3 molecular clone was transfected in HEK293T cells using a CalPhos mammalian transfection kit (Clontech-Takara Bio) as per the manufacturer's instructions. Cell culture medium was collected 36 h post-medium change, clarified at 1800 × + T cells were infected with 0.5 m.o.i. HIV-1 NL4-3 virus for 4 h at 37 °C in the presence of Polybrene (1 μg/ml) with intermittent mixing. The cells were then washed, suspended in complete medium supplemented with recombinant human IL-2 (Roche Applied Science) at 20 units/ml, and incubated until harvested. Jurkat, CEM-GFP, and TZM-bl cells were infected similarly. Culture supernatants from the infected cells were used to determine virus production by p24 antigen capture ELISA (Advanced Bioscience Laboratories).PHA-activated PBMCs/CD4For the calculation of infectivity of virus generated from overexpression/silencing experiments, culture supernatants collected from these experiments were quantified using a p24 ELISA, and equal amounts of viral p24 units were used subsequently to infect TZM-bl reporter cells at a confluency of 50–60%. Infectivity was calculated and compared using β-gal staining after fixing the cells with 0.25% glutaraldehyde (48 hpi). The infectivity of the virus stock was also calculated using the same β-gal staining method.For overexpression and knockdown studies, HEK293T/TZM-bl cells were co-transfected with the indicated expression vectors or siRNA using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol followed by subsequent transfection/infection wherever indicated. Transfection in CEM-GFP cells was performed by nucleofection with Amaxa Cell Line Nucleofector kit V (Lonza) using program X-001. In all of the experiments, the cells were harvested 48 h post-transfection/infection for further analysis. All transfection experiments were normalized using empty vector control.m Tris-HCl, pH 7.4, 5 mm EDTA, 0.12 m NaCl, 0.5% Nonidet P-40, 0.5 mm NaF, 1 mm DTT, and 0.5 mm PMSF) supplemented with protease inhibitor mixture (Roche Applied Science). Nuclear and cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic reagents (Thermo Scientific). Equal protein concentration was resolved on a 10–12% SDS-PAGE, and the protein was then transferred to a PVDF membrane , blocked using 5% nonfat dry milk, and probed with the indicated antibodies. All densitometric analyses of the immunoblots were performed by normalization to respective GAPDH levels. Co-immunoprecipitation assays were performed using the clarified lysates incubated with the indicated antibodies. The antigen-antibody complex was pulled down using an equal mixture of protein A- and G-agarose beads (Invitrogen) and resolved on 10–12% SDS-PAGE. Proteins transferred to a PVDF membrane were probed with the indicated antibodies. The blots were developed using the ECL Prime system . Immunofluorescence analysis was performed in infected TZM-bl cells by fixing the cells using paraformaldehyde. Cells were permeabilized using 0.1% Triton-X-100, blocked using 5% FCS, and incubated overnight with the indicated antibodies. α-Rabbit-Cy3 and α-mouse-Cy2 secondary antibodies (Chemicon) were used against the cyclin F and Vif antibodies, respectively. Cells counterstained using DAPI were acquired on an Olympus Fluoview image analyzer.For immunoblotting, cells were lysed in lysis buffer . The -fold change values were calculated as: -Fold difference = 2T−ΔΔC, where ΔCT = CT (target) − CT (GAPDH) and ΔΔCT = ΔCT (treated) − ΔCT (control).Expression of genes was analyzed by quantitative real-time RT-PCR in a reaction mixture containing SYBR Green IQ Supermix (Bio-Rad) and a 10-pmol concentration of hGAPDH or gene-specific oligonucleotide primer pairs and amplified on a Realplex2 Profiler PCR array (PAHS 020A)  was used according to the manufacturer's protocol. Details of the primers used for gene expression analysis and PCR array data validation are given in For the cell cycle gene expression analysis, the Human Cell Cycle RTCCNF) from human was retrieved from the NCBI-RefSeq , having 33% identity, was chosen to build the homology model. The template sequence of cyclin A protein and that of the target cyclin F sequence were then aligned using ClustalW (4N9F), and chain B of Vif protein (176 residues long) was used for the docking study. Docking studies were performed between the known Vif structure and the modeled structure of cyclin F protein with PatchDock server  for 12 h prior to harvesting. The prepared lysates were immunoprecipitated using HIV-1 Vif antibody (sc-69732) followed by immunoblotting using Lys-48 linkage-specific polyubiquitin antibody  .Endogenous ubiquitination assays were performed by treating the cells with proteasomal inhibitor MG132  ≥ 0.05; *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001.Statistical analysis of the experimental data was performed using Student's T. A., D. M., M. K. S., and P. C. contributed to the concept of the paper. T. A., M. K. S., and D. M. designed the experiments. T. A., K. G., S. I., and P. G. performed the experiments. T. A., P. C., P. G., and D. M. wrote the paper.


Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases. Coronary artery disease (CAD), the most common type of heart disease and the single most common leading cause of death in developed countries, starts with a progressive impairment of endothelial integrity and function , 2. Emerin vitro and neovascularization in vivo [Since the first identification of EPCs in 1997 , EPCs ha in vivo .In addition to regulating protein-coding genes in EPCs, emerging evidence shows that the expression of various microRNAs (miRNAs) is also important for the function of EPCs and consequently in the pathogenesis of CAD. In our previous report, we detected and compared the levels of miRNA in peripheral blood and umbilical cord blood ECFCs, we found that the ECFCs show high and low vasculogenic ability, respectively. We also showed that miR-31 was more abundant in umbilical cord blood-derived ECFCs by small RNA sequencing (smRNA-seq) . TransfeExpression of many miRNAs is associated with the severity of CAD , 32. RecThis study was approved by the institutional Ethics Committee Review Boards of Tri-Service General Hospital and the Taipei Veteran General Hospital (IRB#: 1-103-05-061). Written informed consents were obtained from all participants. The protocols of this study were consistent with the ethical guidelines of 1975 Helsinki Declaration.2, ECFCs with typical phenotype of cobblestone-shaped and monolayer growth pattern were obtained. All the ECFCs used for functional analysis and RNA extraction were cultured within six passages.ECFCs were isolated from peripheral blood samples of HLTY individuals and CAD patients and characterized by flow cytometry (FACS) analysis using hematopoietic stem cell and endothelial lineage surface markers as previously described . CAD pat® Green Master Mix kitsUniversal cDNA Synthesis and SYBR . Real-time PCR reactions were performed with miRCURY LNATM Universal RT microRNA PCR LNATM PCR primer set (EXIQON) using CFX connectTM real-time PCR detection system  in Bio-Rad CFX96 Real-Time PCR Detection System. The expression of miRNAs was normalized to U6 small nuclear RNA. For mRNA detection, 2 μg of total RNA was used for RT with Oligo(dT)12-18 using SuperScriptIII RT (Invitrogen). The expression of each gene was quantified by iQTM SYBR Green Supermix (BioRad) using CFX96 Real-Time PCR Detection System and normalized to GAPDH.Total RNA was extracted from ECFCs using TRIzol reagent  and plasma using TRIzol LS reagent (Invitrogen) following the manufacturer’s protocol. For miRNA detection, 150 ng of total RNA was used for RT using www.targetscan.org) was used to predict target genes and binding sites of miR-146a-5p and miR-146b-5p.Total RNAs purified from plasma and ECFCs of HLTY individuals and CAD patients were sequenced using Illumina MiSeq  following the standard manufacturer’s procedure. Sequencing results were analyzed using an in-house bioinformatics pipeline . Express5’-ATAGGATCCATCATGCATGGCTCATTTTT-3’; reverse: 5’-ATAGGATCCAGCTACTTGGAACCCTGCTT-3’; and miR-146b-5p primer pairs: forward: 5’-ATAGGATCCAGACCCTCCCTGGAATAGGA-3’; reverse: 5’-ATAGGATCCGAGCACTGAGGAAGGACCAG-3’. miRZipTM lentivector-based anti-miRNAs expresses anti-miRNA-146a-5p (5’-GTGAGAACGGAATTCCATGGATTCTTCCTGTCAGAACCCATGGAATTCAGTTCTCATTTTT-3’) and anti-miR-146b-5p (5’-GGATCCGTGAGAACCGAATTCCATCGGATCTTCCTGTCAGAGCCTATGGAATTCAGTTCTCATTTTTGAATTC-3’) was purchased from System Biosciences . Lentiviruses expressing miRNAs and anti-miRNAs were produced in 293T cells and used for transduction.Genomic DNA fragments containing precursor sequence of miR-146a-5p and miR-146b-5p was cloned into pL4-V5-CPO-B, a lentiviral-based expression vector using miR-146a-5p primer pairs: forward: 4 ECFCs in 100 μl of EGM2 medium were seeded on the upper chamber with 8 μm pore size of Costar Transwell Polycarbonate Permeable Supports . Six hours after incubation at 37°C with 5% CO2, suspension cells were removed and the membranes were fixed with 4% paraformaldehyde for 15 minutes at room temperature. The migrated cells were then stained with Hoechst 33342 reagents (Sigma) for 30 minutes and counted under fluorescent microscope in five representative fields. The images were quantified using MetaMorph .For migration assay, 600 μl of EGM2 medium supplemented with 10% FBS were added to the lower chamber, while 5x10in vitro tube formation assay, 96-well plate were coated with Basement Membrane Extract  (50 μl per well) at 37°C for an hour as described previously [4 ECFCs in 100μl medium were placed onto the matrix and incubated at 37°C for 3 hours. The tube structures were visualized under an inverted light microscope (100 X). Five microscopic fields were captured in each group and total tube length was calculated using MetaMorph (Molecular Devices). All data were obtained from three independent experiments with triplication.For 5’-ATTACTAGTGCAACTCGCTTTAGGTCAGC-3’; reverse: 5’-ATTAAGCTTGGCCATGTGTCACAGCATAA-3’ and RHOJ, forward: 5’-ATTGAGCTCTTTTCCATGTATTGCCACGA-3’; forward: 5’-ATTAAGCTTTTACATCATTGGGCACAGGA-3’. The luciferase reporter plasmids containing mutations on miR-146a-5p and miR-146b-5p binding sites in the 3’UTR of RHOJ was created using QuikChange Site-Directed Mutagenesis Kit . Reporter assays were performed by transiently co-transfecting 293T cells with reporter plasmids and vector overexpressing miR-146a-5p or miR-146b-5p using Lipofectamine 2000 reagent (Life Technologies). Renilla luciferase was used to normalize transfection efficiency. Forty-eight hours after transfection, total cell lysates were measured for the luciferase activities by the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's instruction.Luciferase reporter plasmids were constructed by cloning the predicted miRNA-binding site on 3’-UTR of CAV1 and RHOJ into pGL3-Basic plasmid  using specific primers for CAV1, forward: Student’s-t test and indicated as n.s: not significant; *p<0.05; **p<0.01; ***p<0.001.Statistical analysis of gene and miRNA expression measured by qPCR was performed using Cobblestone-like ECFCs obtained from the peripheral blood (PB) of HLTY donor and CAD patients were first characterized by fluorescence-activated cell sorter (FACS) as described . Both EC480 to avoid background miRNA signals from red blood cells. A pilot study of exosome miRNAs sequencing confirmed similar miRNA signature of exosome and plasma miRNAs. To simplify the processing step, we aimed to purify the miRNA from plasma. Total RNA from plasma of 5 individuals were pooled for preparation of a library for smRNA-seq. About 4 to 5 million of raw reads were obtained using Illumina MiSeq. Total reads were first trimmed for adapter sequence and filtered with read length. Reads with more than 90% of the bases that reached a score of ≤Q30 (Phred score) were preserved for further analysis. Unique read counts and length distribution peaks were summarized (Circulating miRNAs have been found in exosomes . In addimmarized . Reads wmmarized . The resmmarized .10), the 12 miRNAs that were up-regulated in both CAD plasma and ECFC were ranked among the top 50% of highly unregulated plasma miRNAs, as highlighted on top of To identify miRNA candidates that are dysregulated in CAD ECFC, we compared the miRNA expression profile of CAD plasma and ECFC to that of HLTY subjects. We found 82 and 83 miRNAs were up-regulated in CAD plasma and ECFC versus HLTY control, respectively . Heat maWe hypothesized that miRNAs up-regulated in CAD ECFCs might be secreted in plasma and uptake by HLTY ECFCs, thereby dysregulating mRNAs expression and functionality of HLTY ECFCs and making them more CAD ECFCs-like. Therefore, we performed cDNA microarray analysis to identify genes that were up- or down-regulated in CAD ECFCs. By using RNA prepared from ECFCs isolated from two CAD patients and one HLTY individual, we identified 410 and 411 genes that were at least 2-fold up- and down-regulated in CAD ECFCs, respectively . Followiin silico data was validated by detecting the levels of the five miRNAs in plasma and ECFCs from patients with CAD and HLTY control individuals. Among the five miRNAs examined, levels of circulating miR-26a-5p, miR-146a-5p and miR-146b-5p in plasma from CAD patients showed significant up-regulation compared to HLTY control , one binding site for miR-146b-5p (TSS 1736) on the 3’-untranslated region (3’-UTR) of CAV1  but not Endothelial damage is considered as the first step of the cascade in arterial pathogenesis and therefore has long been believed to play a pivotal role in the development of CAD. Repair of defective endothelium at adult stage is not solely a result of proliferation of local endothelial cell, but also depends largely on differentiation of blood mononuclear cells derived ECFCs into endothelial cells . ReducedThe miRNAs expression profiles of HLTY and CAD ECFCs have been extensively studied. There is emerging evidence showing many anti-angiogenic miRNAs, which target angiogenic genes such as VEGF, are up-regulated in CAD ECFCs and consequently suppresses ECFC activities –35. It iin vitro [in vivo [miRNAs play a key regulatory role in gene expression at the posttranscriptional level. It is well recognized that miRNA represses gene expression at both the level of mRNA stability by inducing mRNA degradation and the level of translation inhibition by binding to 3′UTR of the target mRNAs . Here, win vitro , 61 and [in vivo . HoweverIn summary, our study has revealed miR-146a-5p and miR-146b-5p as new potential angiogenesis regulatory miRNAs that are up-regulated and potentially secreted by CAD ECFCs. Here, we provide indirect evidence showing that circulating miR-146a-5p and miR-146b-5p, including free and exosome-associated forms, may be taken up by HLTY ECFCs and resulting in amplification of diseased ECFCs . Our resS1 FigExpression of indicated molecules in HLTY and CAD ECFCs were stained and analyzed by flow cytometer.(TIF)Click here for additional data file.S2 Fig(A) Heat map of 82 miRNAs significantly higher expressed in CAD plasma (RPM > 5 and > 2-fold change). (B) Heat map of 83 miRNAs significantly highly expressed in in CAD ECFCs (RPM > 5 and > 3-fold change).(TIF)Click here for additional data file.


Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. For HIV, cellular invasion requires merging viral and cellular membranes, an event achieved through the activity of the viral fusion protein Env. Env consists of three gp120 and three gp41 subunits symmetrically arranged on the viral surface. The gp120 subunits bind cellular receptors, which, in turn, coordinate gp41 conformational changes that promote membrane fusion. Understanding these structural rearrangements illuminates the mechanism of viral membrane fusion, and also spurs development of targeted inhibitors of viral entry and vaccine candidates that elicit antiviral immune responses. In this study, we employed a novel strategy to investigate individual subunits in the context of functioning Env complexes. The strategy links distinct gp120-receptor interactions to conformational changes that expose specific gp41 subunits. We found that, despite the initial symmetric arrangement of its subunits, Env conformational changes most often proceed quite asymmetrically, leading to exposure of only one-third of the gp41 trimer for much of the fusion event. This finding might explain why attempts to elicit potent anti-HIV antibodies to a fully exposed gp41 trimer have been largely unsuccessful. The study gives us a glimpse of the early structural transitions leading to Env-mediated membrane fusion and provides a framework for interrogating the fusion proteins of other membrane-encapsulated viruses. Entry of human immunodeficiency virus type 1 (HIV-1) into target cells involves fusion of viral and cellular membranes, a process mediated by the viral surface protein Env gp160) –5. In hi0 –5. In In mediating HIV-1 entry, Env undergoes a series of coordinated structural transformations initiated when gp120 binds cellular CD4 –15. ThisThe molecular mechanisms that guide global structural changes of Env are largely unknown. For instance, it remains unclear how the conformational transitions of the gp41 subunits are allosterically coupled to CD4 and coreceptor binding to gp120. As a first step in unraveling these mechanisms, this study focuses on the nature of the native-to-PHI transition that exposes the N-HR and C-HR regions of the gp41 ectodomain. In the current model, the transition is envisioned as a concerted process, with the three gp120 subunits simultaneously disengaging from the gp41 trimer leading to full (symmetric) exposure of the N-HR coiled coil and three C-HR regions , 33. In Probing initial receptor-induced conformational transitions of Env is complicated by the homotrimeric oligomerization of the glycoprotein. The three identical CD4- and coreceptor-binding sites, the three-fold symmetry of the N-HR coiled coil, and the three identical C-HR regions make it difficult to assess the structural transformations of individual gp120 and gp41 subunits. Here, we circumvent this obstacle by studying two types of asymmetric Env trimers. The first type is a heterotrimer of functionally complemented Env protomers, one deficient in CD4 binding and the other deficient in coreceptor binding , 35. TheFusion inhibitors bind the gp41 N-HR and C-HR regions exposed in a kinetic window between receptor activation of Env and formation of the gp41 TOH  39–54].–54.39–54The kinetic properties of fusion inhibition make these inhibitors particularly useful in monitoring late conformational changes in gp41, the duration of the PHI, and the steric environment around the N-HR and C-HR regions during membrane fusion , 55–60. 3 or B3 homotrimers were poorly infectious on CD4+CXCR4+CCR5- target cells  variants, denoted in this paper A and B. Env A was competent in binding CD4 but deficient in binding CXCR4, while Env B was deficient in binding CD4 but competent in binding CXCR4 , top. InHXB2 trimers with 2 CD4/1 CXCR4 binding sites (A2B) and EnvHXB2 trimers with 1 CD4/2 CXCR4 binding sites (AB2) have similar fusogenic activities.To characterize the fusogenic activities of these Env heterotrimers, we adjusted the relative protomer expression while keeping total Env level in virus producing cells constant . HIV-1 i2B and AB2 heterotrimers on Mm and Ww viruses mimicked mutant and wild type Env homotrimers in terms of symmetry in their gp41 ectodomains. However, when gp41 mutations were incorporated into either Env A or Env B, the viral populations  contained functional Env heterotrimers with asymmetric gp41 trimers , while the same trimers on Mw viruses appear to preferentially expose a low affinity site and have significantly poorer T20 sensitivity. Hence, A2B trimers (2 CD4 / 1 CXCR4 binding site) appear to uncover N-HR regions from CD4-binding Env A protomers before the entire N-HR coiled coil is uncovered. Curiously, AB2 trimers (1 CD4 / 2 CXCR4 binding sites) from Wm and Mw viruses had similar T20 sensitivities, as expected if all three T20 binding sites were equally exposed. The results suggested that single CD4 binding to Env leads to symmetric gp41 N-HR exposure  binds the C-HR region with extremely high affinity and displays kinetically restricted inhibitory potency. A mutant version (denoted 5HLAVA) binds less tightly and displays affinity-dependent inhibitory potency. We employed resistance mutation N656D in the C-HR region that reduces 5-Helix binding affinity by more than three orders of magnitude  and 37 residues from the C-HR sequence (C37) and solved its structure (1), three-fold crystallographic symmetry did not run through the center of the N-HR coiled coil as it does in almost all previous gp41 TOH structures. Instead, the structure was markedly asymmetric in that only two of the three N40 segments were folded into α-helices throughout their entire length. The N-terminal region (residues 545–554) of the third N40 segment assumed a distorted, largely extended conformation (Propagating HIV-1 (NL4-3 strain) in the presence of fusion inhibitor C37-KYI led to the selection of the escape mutation Q552R. With its additional bulk and positive charge, we suspected that the Argtructure . Althougormation , S4A Figormation .552 residues occupied distinctly different chemical environments. One Arg552 side chain extended from an intact N-HR helix into the coiled-coil interior, where it formed a hydrogen bond with the backbone of the other intact N-HR helix  radially away from the center of the TOH. We speculated that the spatial arrangement of the three Arg552 side chains minimized their electrostatic repulsion, which would be particularly destabilizing if all three were confined within the interior of the N-HR coiled coil.As a consequence of the asymmetric distortion of the N-HR coiled coil, the three ArgHR helix  Figs. ThUnlike the N40 coiled coil, all three C37 peptides in the Q552R-mutant TOH structure folded into their normal helical structures, even in the asymmetric zone of the N40 segment . The bace-position of the N-HR hydrophobic heptad repeat, see 656, Glu657 and Leu660 and the backbone of the C-HR helix  ):f represents the fraction of inhibition events that titrate with IC50.1. The data fit best with f = 0.31, IC50.1 = 8 nM, and IC50.2 = 180 nM  binding guide the structural transformation of HIV-1 Env from a constrained, metastable native state through an extended PHI configuration and ultimately into a compact, low energy TOH conformation , 28. StrLAVA, potency was largely unaffected by which protomer contained the mutation. The results suggested that the C-peptide binding sites on the gp41 N-HR coiled coil were exposed in piecemeal fashion, while the gp41 C-HR regions were exposed symmetrically. Our interpretations were based on experiments complicated by multiple levels of Env manipulation . Such alterations, and perhaps even fusion inhibitor binding itself, could have shifted the conformational landscape of the trimer and played a more active role in promoting what appeared to be an asymmetric transition. It was also possible that our observations were a consequence of the fact that CD4 and CoR could not bind the same protomer in the Env heterotrimer. To partially address these concerns, we analyzed the fusion inhibitor sensitivity of a naturally asymmetric Env homotrimer (EnvQ552R/N637K) that has three intact CD4 and CXCR4 binding sites. Inhibitor titrations revealed that C37-KYI blocked viral entry with multiple potencies, as if the C-peptide bound to sites of different affinities and these sites were asymmetrically exposed. These measurements of viral entry inhibition remain an indirect reporter of Env structural changes. More direct methods, such as cryo-EM structural determination combined with inhibitor binding/precipitation assays, will be required to confirm our results.To interrogate these early conformational transitions, we employed functionally complemented Env heterotrimers consisting of protomers deficient in either CD4- or CXCR4-binding. Fusion inhibitor-escape mutations were then selectively introduced into either protomer, and the impact on inhibitor potency was measured. For N-HR-targeted T20, potency depended on whether the escape mutation was incorporated into the CD4- or CXCR4-binding protomer. By contrast, for C-HR-targeted 5HHXB2  is sufficient to promote both N-HR coiled-coil formation and rapid shedding of gp120 . In. In70]. In developing an HIV-1 vaccine, the gp41 N-HR coiled coil has always been imagined as a viable target for neutralizing antibodies based on how well C-peptides inhibit viral entry . Toward SF162) from Drs. L. Stamatatos and C. Cheng-Mayer  by ultracentrifugation . Pellets were resuspended in PBS with 2% BSA, and viral titers were quantified by p24 antigen ELISA. To assess Env incorporation into virus, samples containing equal p24 content were separated by SDS-PAGE and analyzed by Western Blot as described above. The Env modifications used in this study did not alter the incorporation of Env into the HIV-1 particles  using a Vydac C-18 column and a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. The identity of T20 was confirmed using MALDI-TOF mass spectrometry.HXB2 sequence), a trypsin-cleavable linker (GGRGG), a 37-residue C-HR segment (625–661) and a C-terminal hexahistidine tag (GHHHHHH). Briefly, the protein was expressed in E. coli RP3098 and purified from lysates using metal chelate chromatography . The trimeric protein was subject to trypsin digestion , and C37 was purified to homogeneity using rp-HPLC as described above. C37-KYI contains the affinity enhancing mutations N637K/T639I. Di-C37 is a dimerized form of C37 connected via an engineered disulfide bond at the C-terminus of each monomer.C-peptides C37, C37-KYI and di-C37 were prepared from a recombinant gp41 TOH construct NC1 as previously described , 55. NC1E. coli RP3098 as previously described [LAVA contains the affinity-disrupting L556A/V570A mutations in all three N-HR segments.5-Helix proteins were recombinantly expressed and purified from escribed , 50. 5-H50 values.The concentrations of T20, PIE12, C37 constructs and 5-Helix proteins were determined by absorbance at 280 nm by the method of Edelhoch . The intNL4-3 propagated in the presence of C37 and C37-KYI. The Q552R and V549E mutations disrupt C-peptide binding affinity approximately 5000-fold and confer resistance to C37 (150-fold) and the higher affinity C37-KYI (6-fold) [Crystal structures were obtained using NC1 proteins (see above) containing either the double mutation Q552R/L555M or single mutation V549E. These mutations were detected in HIV-1Q552R/L555M using 20% w/v PEG 8000, 0.3 M Calcium Acetate, 0.1 M Na Cacodylate, (pH 6.5) and 6.35mg/mL of protein. NC1V549E crystallized into a hexagonal prism of 50 μm in 3 M Sodium Chloride, 0.1 M Sodium Acetate (pH 4.5) and 6 mg/mL of protein. Crystals were frozen in mother liquor supplemented with 27% v/v ethylene glycol and stored in liquid nitrogen until exposed.Crystallization conditions were screened  by sitting-drop vapor diffusion method using robotically dispensed 100 nL sample volumes . Following optimization, diffraction quality crystals with hexagonal prism shape of 150 μm were obtained for NC1Diffraction data sets were collected on beamline's X6A at the National Synchrotron Light Source  and 23ID-B at the Advanced Photon Source, Argonne National Laboratory. The images were indexed and integrated using DIALS  and scalS1 FigHXB2 trimers (WT or L544S) or Env A/Env B trimers . Env A and Env B were expressed equally in viral producing cells. Each bar represents the mean ± SEM of three or more independent experiments. A Western blot depicting the expression of gp41 in viral progenitor cell lysates is shown below the graph. (B) Fusion inhibitor titrations of HIV-1 pseudotyped with wild-type EnvHXB2 (black squares) or the L544S mutant variant (red circles). Titrations were performed with di-C37  and T20 . (C-G) Fusion inhibitor titrations of HIV-1 generated from cells expressing EnvA and Env B at ratios of 1:1 (C), 1:9 (D-E) or 9:1 (F-G). Viral populations Ww (black), Wm (red), Mw (green) and Mm (blue) were inhibited using di-C37  or T20 . Data points represents the mean ± SEM of at least three independent experiments and have been fit to a simple Langmuir equation (solid lines) to extract IC50 values. All infections were performed using U87.CD4.CXCR4 target cells.(A) Infectivity of HIV-1 pseudotyped with Env(PDF)Click here for additional data file.S2 FigHXB2 trimers (WT or V549A) or Env A/Env B trimers . Env A and Env B were expressed equally in viral producing cells. Each bar represents the mean ± SEM of three or more independent experiments. A Western blot depicting the expression of gp41 in viral progenitor cell lysates is shown below the graph. (B) Fusion inhibitor titrations of HIV-1 pseudotyped with wild-type EnvHXB2 (black squares) or the V549A mutant variant (red circles). Titrations were performed with C37-KYI  and T20 . (C-G) Fusion inhibitor titrations of HIV-1 generated from cells expressing EnvA and Env B at ratios of 1:1 (C), 1:9 (D-E) or 9:1 (F-G). Viral populations Ww (black), Wm (red), Mw (green) and Mm (blue) were inhibited using C37-KYI  or T20 . Data points represents the mean ± SEM of at least three independent experiments and have been fit to a simple Langmuir equation (solid lines) to extract IC50 values. All infections were performed using U87.CD4.CXCR4 target cells.(A) Infectivity of HIV-1 pseudotyped with Env(PDF)Click here for additional data file.S3 FigHXB2 or the N656D variant. Each bar represents the mean ± SEM of three or more independent experiments. The Western blot shows gp41 from lysates of viral progenitor cells expressing either EnvHXB2 trimers (WT or N656D) or Env A/Env B trimers . Env A and Env B were expressed at a 1:1 ratio. (B) Fusion inhibitor titrations of HIV-1 pseudotyped with wild-type EnvHXB2 (black squares) or the N656D mutant variant (red circles). Titrations were performed with 5HWT  and 5HLAVA . (C-G) Fusion inhibitor titrations of HIV-1 generated from cells expressing EnvA and Env B at ratios of 1:1 (C), 1:9 (D-E) or 9:1 (F-G). Viral populations Ww (black), Wm (red), Mw (green) and Mm (blue) were inhibited using 5HWT  or 5HLAVA . Data points represents the mean ± SEM of at least three independent experiments and have been fit to a simple Langmuir equation (solid lines) to extract IC50 values. Infections of HIV-1 pseudotyped with Env homotrimers were performed using HOS-CXCR4+ cells, while infections of HIV-1 pseudotyped with Env A/Env B mixtures were performed using U87.CD4.CXCR4 target cells.(A) Infectivity of HIV-1 pseudotyped with wild type Env(PDF)Click here for additional data file.S4 Fig552, Met555, and Ile559 (stick representations) reflecting consecutive a-, d- and a-positions of the canonical 3,4-hydrophobic heptad repeat (see (A) Lateral view of the six-helix bundle formed by a 40 amino-acid N-HR segment  and 37 amino-acid C-HR segment . The N-terminus of N40 and C-terminus of C37 are found at the top of the structure. The unwound region of the distorted N40 segment is colored orange. Arrows designate the levels of Argpeat see . The ori(PDF)Click here for additional data file.S5 Fig(Top) Ribbon diagrams depicting structures of the wild type  and mut(PDF)Click here for additional data file.S6 FigRibbon diagrams depicting the wild type structure are shown in gray  and blue . (A) The Q552R variant (red) aligns to the wild type structure with a backbone Cα RMSD of 0.69 Å. (B) The V549E variant (green) aligns to the wild type structure with a backbone Cα RMSD of 0.94 Å. The diagrams are oriented with the N-termini of the N-HR segments and C-termini of the C-HR segments at the top of the structures.(PDF)Click here for additional data file.S7 Fig(A and C) Ribbon diagrams depicting the structures the wild type trimer-of-hairpins  and the(PDF)Click here for additional data file.S8 Fig2B heterotrimers from Mw viruses. The N-HR helices from the two Env A protomers (gray) contain the L544S (purple asterisk) or V549A (orange asterisk) substitution. The N-HR helix from the Env B protomer (olive) has a wild type sequence. The resulting N-HR coiled coil contains 1 high affinity and 2 low affinity T20 binding sites. A steric barrier (green) blocks C-peptide access to the N-HR coiled coil until the second CD4-Env interaction. If only the site formed by the two Env A protomers is initially exposed, then T20 binds with low affinity regardless of whether the heterotrimer contains the L544S or V549A substitution. However, if two C-peptide binding sites are exposed, then T20 will have access to at least one high affinity site on either the L544S or V549A mutant heterotrimer. As arbitrarily drawn here, barriers covering the g-positions of the Env A N-HR helices are removed, allowing T20 access to two low affinity sites for L544S mutant heterotrimers. However, removing the same steric barriers now uncovers one high affinity site on V549A mutant heterotrimers (thick arrow). Under these circumstances, T20 should poorly inhibit L544S mutant heterotrimers but potently inhibit V549A mutant heterotrimers.(Top) Helical wheel diagram of the gp41 TOH showing the relative positions of mutations L544S and V549A on the N-HR coiled coil. For each Env protomer, these residues point into different C-HR binding sites. (Bottom) Modeled exposure of the N-HR coiled coil of A(PDF)Click here for additional data file.S9 Fig(A) Influenza virus hemagglutinin . (B) Eb(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Epidermal growth factor receptor (EGFR) is commonly overexpressed in breast cancer and is associated with poor clinical outcomes; however, an increasing number of patients have shown a poor effective response to EGFR tyrosine kinase inhibitors (EGFR-TKI). Here, we found that AXL expression was positively correlated with poor progression in breast cancer patients. Suppression of AXL by an anti-tumor protein, E1A, enhanced EGFR-TKI  sensitization, resulting in significant inhibition of tumor growth in breast cancer cells. Additionally, AXL overexpression dramatically impaired E1A-mediated EGFR-TKI sensitization. These findings show that downregulation of AXL expression by E1A contributes to sensitization to EGFR-TKI in breast cancer, suggesting that combinatorial therapy of AXL inhibitors or E1A gene therapy with EGFR-TKI may be a potential therapeutic strategy for treatment of breast cancer patients. EGFR is one of four structurally related receptor tyrosine kinases (RTKs) of the ErbB family . Recent Adenovirus-5 early region 1A (E1A) exerts its anti-tumor activity through various mechanisms, such as increasing drug sensitivity through the downregulation of HER2/neu , 13; enhAXL, a receptor tyrosine kinase is a member of the TAM (Tyro-Axl-Mer) family, that is overexpressed and activated in various human cancers  and is cIn this study, we found that inhibition of AXL (E1A gene therapy or R428) increases the sensitization of breast cancer cells to EGFR-TKI. Notably, we also found that higher expression of AXL is correlated with poor overall survival, advanced tumor stage, and lymph node status of breast cancer patients. Taken together, our findings suggest that a combination of E1A gene therapy or an AXL inhibitor and EGFR-TKI will improve treatment of breast cancer. These results suggest that combinational therapeutic strategies for treatment of breast cancer patients should be developed.AXL expression (n = 36) were significantly correlated with poor survival outcomes compared to those with low levels of AXL (n = 37)  Figure . The Oncs Figure  and lymps Figure  of breasTo confirm whether AXL inhibition enhances the sensitization to EGFR-TKI, we knocked down AXL by specific short hairpin RNA (shRNA) in MDA-MB-231 and HBL100 cells and determined the cell viability after treatment with EGFR-TKI Figure . Suppreshttp://www.oncomine.org) and found that the expression of EGFR is positively correlated with AXL in breast cancer patients  analysis. We found that the stability of AXL mRNA was not significantly changed in MDA-MB-231/vector and MDA-MB-231/E1A cells  of the AXL gene and performed luciferase reporter assays. As shown in Figure AXL has been reported to be involved in resistance to EGFR-TKI and was correlated with poor prognosis , 33, ands Figure . Becauses Figure . MDA-MB-s Figure . To furtin vivo, we performed an orthotopic breast tumor growth assay in an animal model. MDA-MB-231/E1A/Ctrl and MDA-MB-231/E1A/AXL cells were orthotopically injected into the mammary fat pads of SCID mice, and tumor growth was measured. Four weeks after implantation of the cells, mice were treated with gefitinib (100 mg/kg/day) by oral gavage, and the tumor volumes were measured. After gefitinib treatment, the tumor volumes of MDA-MB-231/E1A/AXL-injected mice were higher than those of MDA-MB-231/E1A/Ctrl-injected mice, suggesting that downregulation of AXL is essential for E1A-mediated sensitization of EGFR-TKI , the results will be valuable for developing an efficient combination of E1A gene therapy with other agents in the future. Moreover, the toxicity was tolerable and the maximum tolerated dose (MTD) was determined. However, given the value of its application in cancer treatment, more studies and examination of its therapeutic effects should be carried out in the future.In the past few years, E1A has been shown to be a tumor suppressor that enhances the anti-neoplastic effect of several chemotherapeutic drugs through regulation of apoptotic molecules. Although a clinical trial of E1A gene therapy using tgDCC-E1A (E1A-lipid complex) combined with paclitaxel treatment for patients with platinum-resistant ovarian cancer was terminated at phase I/II of the study due to low accrual , which analyzed 16 STR loci. All cells were frozen within 1–2 weeks after authentication. MDA-MB-231, HS578t, and MDA-MB-453 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12. MCF-7 and T47D cells were grown in RPMI-1640 medium. MDA-MB-468 and HBL100 cells are grown in DMEM. Mediums were supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/ml streptomycin. MDA-MB-231 and its E1A/vector-stable transformants have been described previously ), mice were randomly allocated into eight groups to receive gefitinib (100 mg/kg/day) or vehicle by oral gavage [Four-week-old female severe combined immunodeficient (SCID) mice were supplied by the National Laboratory Animal Center  and were used for the orthotopic xenograft studies. According to the 3Rs , there were six mice per group. For the tumor growth assay, 5 × 10l gavage , and tumTotal RNA was extracted from cells with TRIzol reagent  and reverse transcribed into cDNA with MMLV reverse transcriptase (Invitrogen). The reactions were incubated at 37°C for 50 min, 72°C for 10 min, and 4°C for 5 min. qRT- PCR was performed using a Roche LightCycler 480 qRT-PCR system. All reactions were carried out in triplicate.AXL promoter  was amplified by PCR. In the PCR protocol, samples were first denatured at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 1 min and 72°C for 3 min, with a final extension at 72°C for 10 min. The primer pairs for constructs (p1627-p480) of the AXL promoter are shown in Renilla luciferase construct  were cotransfected (1 μg each) into each well. After transfection for 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).The 1,726 bp n = 73).The breast cancer tissues were from the Tissue Bank Core Facility for Genomic Medicine of the National Taiwan University Hospital  and were obtained with Institutional Review Board approval. Patients had not received neoadjuvant chemotherapy or radiation therapy. Patients who died within 30 days of surgery were excluded from the survival analysis. Survival was calculated from patients whose survival information was available for analysis . Data were approximately normally distributed and are presented as the mean ± s.e.m. from at least three independent experiments. Statistical analysis among the experimental groups was conducted using a two-tailed Student's


The therapeutic efficacy of antibodies can be successfully improved through targeted delivery of potent cytotoxic drugs in the form of antibody-drug conjugates. However, conventional conjugation strategies lead to heterogeneous conjugates with undefined stoichiometry and sites, even with considerable batch-to-batch variability. In this study, we have developed a chemo-enzymatic strategy by equipping the C-terminus of anti-CD20 ofatumumab with a click handle using Sortase A, followed by ligation of the payload based on a strain-promoted azide-alkyne cycloaddition to produce homogeneous conjugates. The resulting antibody-drug conjugates fully retained their antigen binding capability and proved to be internalized and trafficked to the lysosome, which released the payload with a favorable efficacy in vitro and in vivo. Thus, this reported method is a versatile tool with maximum flexibility for development of antibody-drug conjugates and protein modification. Recent successes in the development of ADCs for targeted cancer therapy, such as Brentuximab vedotin . Furthermore, different DAR of ADCs will have different efficacy, pharmacokinetic properties, stability and safety profiles , which rSrtA could recognize substrates containing a highly conserved LPXTG -sorting sequence motif, cleave the amide bond between the threonine (T) and the glycine (G), then, followed by the nucleophilic attack of oligoglycine substrates, produce covalently linked products . Owing t3, GPN, 3) and sortase enzyme are readily available in larger quantities from chemical synthesis and E. coli production respectively, it is conceivable that this design is suitable for scale-up production. Compared with the direct enzymatic attachment of toxic payloads to the antibody , is an emerging tool for protein modification. The reaction of azide with strained cyclooctyne is highly specific, efficient, and proceeds under mild conditions . Herein,antibody B, the ch4S)n, often used to link the antibody formats [nLPETG(GHHHHHH)m sequence , and successfully expressed in HEK293F cells, which still remained the binding affinity to CD20 of cell surface. In order to seek the optimized antibody, six OFA variants were tested for the relative conjugation efficiency using chemo-enzymatic approach, and MMAE covalently coupled to the light and heavy chains was visualized with 3,3’-diaminobenzidine (DAB) color development after western blot of the conjugated antibody (4S)n length after the attachment of His-tag which will be lost in the enzymatic reaction. Therefore, we chose the (G4S)2LPETGGHHHHHH tagged OFA to conjugate toxins using chemo-enzymatic method to generate ADCs in this study.We previously have shown that the direct enzymatic conjugation using a 100-fold excess GGG-vc-PAB-MMAE , 3 yield formats , was insantibody . In acco3, 2) suitable for the SrtA-catalyzed transpeptidation was used to transfer an additional azide functionality onto the antibody at a rate of 100:1 in order to promote enzymatic reaction, while the antibody formats (scFv or Fab) required less oligoglycine-modified substrate for their easy access [We developed a chemo-enzymatic approach by SrtA-mediated ligation and click chemistry to produce ADC . In the y access . In the y access . Moreovey access . Recent y access  and strey access , combine3  and DBCO-PEG-vc-PAB-MMAE , approximately consistent with the expected mass shift of 1049 Da. As expected, we also confirmed the conjugation site on LPETG motif  A. The maTG motif . FuthermTG motif B and hydTG motif C. The chTo investigate the effects of the conjugation processes of toxins on targeted antigen-binding, the qualitative cell-binding experiments were performed with CD20-positive Daudi cells using flow cytometry. The ADCs were found to retain the antigen binding capability, which demonstrated that the modification of MMAE had minimal effect on the binding affinity to cell surface CD20 when compared with the unconjugated antibody . This reNext, we examined the internalization of OFA and the corresponding ADCs, which is a predominant factor in the function of ADC after antigen binding. Daudi cells were incubated for 30 min at 4 °C with serial dilutions of either the unconjugated OFA or the corresponding ADCs, stained with FITC-labeled secondary antibody, and examined by flow cytometry. Moreover, the subcellular trafficking and localization of OFA-GPN-vcMMAE in target cells were further determined using confocal microscope as shown in To study the cellular toxicity, the degree of apoptosis in Ramos cells was determined by Annexin V-FITC/PI staining after incubated with OFA or ADCs at a concentration of 5 μg/mL for 72 h, as shown in 50 > 6.667 × 103 nM)  was slightly less cytotoxic with corresponding IC50 of 5.336 nM and 30.51 nM. The potency of these ADCs correlated well with the DAR value. These IC50 values are comparable with published values about anti-CD20-based ADCs generated by reduced interchain disulfides [50 than unconjugated OFA against two CD20-positive cells, clearly demonstrating that the enhanced potency of the ADCs in comparison to the unmodified antibody. Moreover, both OFA-vcMMAE and OFA-GPN-vcMMAE showed far less potency on CD20-negative K562 cells compared to their effects on the CD20-positive cells, indicating the cytotoxicity is specific for CD20-positive cells.Next, we further evaluated the cytotoxicity in vitro and selectivity of the ADC, two CD20-positive B-lineage lymphoma cell lines  and one CD20-negative cell line (K562) were included. The results showed that unconjugated OFA possessed very low toxicity on two CD20-positive cells (IC 103 nM) . In contsulfides ,23. Sign3 (3–9 fold of the regular starting tumor volume of 50–150 mm3), therapy was initiated once every four days for four times (q4d × 4), and the tumor growth was monitored . During the first week, OFA-GPN-vcMMAE and OFA-vcMMAE groups showed efficient tumor shrinkage. After one week, tumor sizes gradually increased for mice treated with OFA-vcMMAE but not for OFA-GPN-vcMMAE group, indicating OFA-vcMMAE showed lower potency in vivo compared with OFA-GPN-vcMMAE. Remarkably, the tumor disappeared completely in three of five mice in OFA-GPN-vcMMAE treated group, and no tumor recurrence was observed among them for the whole length of the study, leaving two slowly progressing.The in vivo efficacy of chemo-enzymatic ADC was explored using a Ramos B-lymphoma xenograft model in nude mice. Saline solution was used as negative control, and direct enzymatic ADC was included as a comparator. When mean tumor size in each group reached approximately 450 mmonitored A. TumorsAt the same time, mice were weighed to evaluate the in vivo toxicity of ADC B. SlightFor acute toxicity evaluation, mice were sacrificed 24 h after the last administration, and histological sections of the major organs  of the mice were examined after H&E staining. No obvious histomorphologic alterations were observed in any sections of organs except that hepatic steatosis was found in OFA-vcMMAE group C. To furIn this study, we have developed a convenient and simple two-step procedure consisting of SrtA-mediated conjugation followed by a strain-promoted click reaction for the production of the site-specific ADC, which can be efficiently internalized when targeting the noninternalizing CD20 antigen and exhibited potent antitumor activity. Our results demonstrated that the in vitro and in vivo efficacy of such chemo-enzymatic conjugates is superior to those prepared using a direct enzymatic coupling method based on the higher DAR, and revealed the two-step approach is versatile with the potential to be readily scaled up on account of cost reduction and less toxic waste. In addition, OFA-GPN-vcMMAE showed an excellent therapeutic effect on the B-lymphoma mice model for a better cure rate and significantly reduced side effects . Further3 (1), DBCO-PEG-vc-PAB-MMAE (2), and GGG-vc-PAB-MMAE (3) were synthesized by Concortis  and the purity was above 95% according to RP-HPLC analysis. Mouse polyclonal antibody against MMAE was produced and validated in the immunized mice from our laboratory. All reagents were purchased from Sigma-Aldrich  unless otherwise noted.GGG-PEG-N2 atmosphere.Human CD20-positive  and CD20-negative (K562) cell lines were purchased from American Type Culture Collection , and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  at 37 °C in 5% COS. aureus SrtA (∆N59) was synthesized, inserted into pET28a (+) vector, and expressed in E. coli Rosetta (DE3) as a fusion protein with an C-terminal His-tag, followed by the purification as described previously [A DNA sequence encoding residues 60–206 of the eviously . The amiQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK.n sequence was inserted before the LPETG sequence using standard molecular biology techniques. Thus, the sequence variants appended to the C-terminus of the light and heavy chain were (GGGGS)nLPETG(GHHHHHH)m . Finally, sequences were confirmed by DNA sequencing . Primers used above are detailed in The full-length human light and heavy chain sequences of OFA  were tagFor the expression of the tagged antibodies, relevant expression plasmids were transiently transfected into human embryonic kidney cells (HEK293F) in suspension culture with polyethylenimine as described previously . The rec3  in the presence of SrtA (50 μM) in reaction buffer  for 12 h at 37 °C. The His-tag on the OFA was removed during this reaction. The azide-functionalized antibodies (OFA-GPN) were purified by protein A chromatography. The buffer of eluted antibodies was exchanged to 50 mM Tris-HCl (pH 7.5), 150 mM NaCl by ultrafiltration, and sterile filtered and stored at −80 °C.OFA with C-terminal LPETG sequence (2 μM) was conjugated to GGG-PEG-NFor the strain-promoted azide-alkyne cycloaddition (SPAAC), OFA-GPN was incubated with DBCO-PEG-vc-PAB-MMAE , adjusted to pH 7.4 in PBS at RT overnight. After the incubation, the samples were washed repeatedly with PBS by ultrafiltration to remove excess of DBCO-PEG-vc-PAB-MMAE (2), and sterile filtered and stored at −80 °C.Equivalent proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes, followed by the incubation with blocking solution containing phosphate-buffered solution (PBS) (pH 7.4) with 5% skimmed milk and 0.1% Tween-20 for 1 h at 37 °C, and then with an anti-MMAE primary antibody (1:1000 dilution) for 2 h at 37 °C. Blots were then washed three times with 0.1% Tween-20/PBS (TPBS), incubated with a 1:1000 dilution of horse radish peroxidase (HRP)-labeled secondary antibody  for 2 h at 37 °C and washed three times with TPBS again. Protein bands were visualized with DAB Horseradish Peroxidase Color Development Kit .m/z), 2000–8000.Analysis was done on a Waters Xevo-G2S Q-TOF mass spectrometer coupled to a Waters ACQUITY UPLC. Samples were diluted to a final concentration of 2 mg/mL, and then the disulfide bonds were reduced in the presence of 12 mM DTT at 37 °C for 40 min. Desalting and chromatographic separation were performed using a 12 min linear gradient between 10% B and 90% B. . Mass spectrometry parameters were as followed: capillary (kV), 2.5; sampling cone (V), 60; source temperature (°C), 120; desolvation temperature (°C), 500; desolvation gas flow (L/h), 800; mass range . As elution buffer A, 0.1% trifluoroacetic acid in water was used. Elution buffer B was composed of acetonitrile, exclusively. Elution conditions were as followed: 0–3 min 25% buffer B; 3–25 min 25%~50% buffer B; 25–27 min 50%~95% buffer B; 27–28 min 95%~25% buffer B; 28–30 min 25% buffer B at 0.6 mL/min. Samples were previously reduced by incubation with DTT at 37 °C for 30 min before performing RP-HPLC.4)2SO4, 25 mM Na3PO4, pH 7.0 and elution buffer B-75% 25 mM Na3PO4, pH = 7.0, 25% isopropanol (IPA).HIC was performed on a TOSOH Butyl-NPR column  at 0.8 mL/min with a 15 min linear gradient between elution buffer A-1.5 M (NH6 cells were incubated with 5 μg/mL antibody or ADCs in staining medium containing PBS (pH 7.4) with 1% (w/v) bovine serum albumin (BSA) on ice for 30 min, and then washed twice with ice-cold PBS to remove unbound antibody or ADCs. Cells were stained with FITC-labeled secondary antibody  at a dilution of 1:250 in ice-cold staining medium, incubated 30 min on ice, and washed as above. Labeled cells were examined by flow cytometry gated to exclude nonviable cells. Data was analyzed using CXP Analysis 2.2  and the background-corrected mean fluorescence intensity was determined.Binding of antibody or ADCs to cell-surface CD20 was assessed by flow cytometry  on CD20-positive Daudi cells. 1 × 10Briefly, Daudi cells were incubated with serial dilutions of antibody or ADCs on ice for 30 min and then washed twice. Next, cells were incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibody for 30 min on ice, followed by the analysis of mean fluorescence intensity (MFI) after wash. The MFI-concentration curve was produced, and the linear range was selected as the standard curve, indicating that the percentage of internalization could be calculated by MFI within this range. Simultaneously, cells in triplicate were incubated with 2 μg/mL antibody or ADCs on ice for 30 min. After washing, cells were incubated at 37 °C for 2 h to drive internalization, and then cell supernatants were collected after centrifugation. Antibody or ADCs may be shed from the cell surface to the media, and Daudi cells were incubated with the centrifuged supernatants containing antibody or ADCs on ice for 30 min. Then cells surface-bound antibody or ADCs were stained with FITC-labeled secondary antibody and analyzed by flow cytometry after wash. The concentration was calculated according to the linear MFI-concentration standard curve after subtracting the background value of MFI derived from the untreated control. The volume of incubation above was fixed to 200 μL. The percentage of internalization of antibody or ADCs was calculated based on the following formula: Internalization (%) = /4 °C total surface-bound amount ×100%.5 cells/mL and treated with 5 μg/mL ADC at 37 °C with 5% CO2. After a 6-h incubation, the cells were gently washed twice with PBS (pH 7.4) to remove unbound ADC, and then seeded on slides by centrifugation. Cells were fixed with 4% paraformaldehyde solution for 15 min. After PBS washing, cells were permeabilized for 10 min with 0.1% Triton X-100, 0.2% bovine serum albumin (BSA)-PBS, followed by a blocking step with 2% BSA-PBS for 30 min. After the blocking, cells were stained by rabbit anti-lysosome-associated membrane protein-1 (LAMP-1) antibody  at a dilution of 1:500 in 1% BSA-PBS (pH 7.4) for 45 min. After gentle washing, cells were stained by FITC-labeled goat anti-human IgG (H + L) polyclonal antibody  and Cy5-labeled goat anti-rabbit IgG (H + L) polyclonal antibody  for 45 min, and then gently washed. Nuclei were further stained with DAPI for 3 min, and excessive dye was washed away. After staining, cells were covered with coverslip. Fluorescence images of the optical section were acquired with a Zeiss LSM 800 confocal laser scanning microscopy  and processed with ZEN 2 (blue edition) image analysis software .Daudi cells were plated on 12-well plates  at a density of 5 × 104 cells/mL in a 6-well plate , and then exposure to 5 μg/mL antibody or ADCs for 72 h. At 72 h post-exposure, cells were stained with Annexin V-FITC and PI for apoptosis analysis. The percentages of apoptotic cells (AnnexinV+/PI−) and dead cells (AnnexinV+/PI+) were determined by flow cytometric analysis of each population.Apoptosis and cell death were assessed with the Annexin V-FITC Apoptosis Detection Kit . Ramos cells were seeded at a density of 5 × 102 atmosphere. After 24 h, a serial dilution of antibody or ADCs in growth medium were added, resulting in final concentrations ranging from 10−3 ng/mL to 106 ng/mL. The PBS (pH 7.4) was used as negative control. After exposure to antibody or ADCs for 96 h, cells were incubated with 10% CCK-8 agent for 4 h, and then the absorbance at 450 nm was measured by the BioRad Model 680 Microplate Reader. GraphPad Prism software was used for data analysis, including IC50 calculations.In vitro potency was assessed using Cell Counting Kit-8  to determine cell viability on two CD20-positive tumor cell lines  and one CD20-negative tumor cell line (K562). Cells were cultured into log-phase growth, and then plated on 96-well plates  in 100 μL growth medium (RPMI-1640 with 10% FBS) at a density of 10,000 cells/well and incubated at 37 °C in a humidified incubator in a 5% CO6 Ramos cells were implanted subcutaneously into the right flank of female Balb/c nude mice . When the tumors reached an average volume of 450 mm3, the animals were divided into six per group treated intravenously with saline, OFA-vcMMAE (5 mg/kg) and OFA-GPN-vcMMAE (5 mg/kg) respectively once every four days for four times (q4d × 4). The body weights of mice in each group were continuously monitored until the end of the experiment, and tumor volumes (mm3) were calculated according to the formula: V = (L × W2)/2, where L and W refer to the tumor length and width (mm), respectively.To establish a localized xenograft model of human B-lymphoma, 8 × 10Possible heart, liver or kidney toxicity was examined by morphological examination of relevant tissue cells after H&E staining. Briefly, tissues for H&E staining were obtained from experimental groups randomly (one mouse per group), 24 h after the last administration of ADCs. The heart, liver and kidney tissues obtained were fixed in 10% neutral buffered formalin, paraffin-embedded and stained by H&E. The sections were examined using light microscopes at 40× magnification. The histology was evaluated by a trained pathologist. To further determine the maximum tolerated dose (MTD), we randomly selected a tumor-bearing nude mouse to treat intravenously 20 mg/kg OFA-GPN-vcMMAE (q4d × 4) and were examined by histopathology as described above. All procedures related to animal handling, care, and the treatment in this study were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Zhejiang University, China (SCXK 2007-0029).p < 0.05, ** p < 0.01 and *** p < 0.001) was accepted as a significant difference.Differences between the experimental groups were tested for significances on the basis of one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test using GraphPad Prism software . A probability value of less than 0.001, 0.01 and 0.05 (*