The molecular mechanism by which non-enveloped viruses penetrate biological membranes remains enigmatic. The non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and cause infection. We previously demonstrated that SV40 creates its own membrane penetration structure by mobilizing select transmembrane proteins to distinct puncta in the ER membrane called foci that likely function as the cytosol entry sites. How these ER membrane proteins reorganize into the foci is unknown. B12 is a transmembrane J-protein that mobilizes into the foci to promote cytosol entry of SV40. Here we identify two closely related ER membrane proteins Erlin1 and Erlin2 (Erlin1/2) as B12-interaction partners. Strikingly, SV40 recruits B12 to the foci by inducing release of this J-protein from Erlin1/2. Our data thus reveal how a non-enveloped virus promotes its own membrane translocation by triggering the release and recruitment of a critical transport factor to the membrane penetration site. Polyomavirus (PyV) is a non-enveloped DNA tumor virus that causes debilitating human diseases especially in immunocompromised individuals. At the cellular level, PyVs such as the simian PyV SV40 must enter a host cell and penetrate the ER membrane to reach the cytosol in order to cause infection. Prior to ER membrane transport, SV40 reorganizes select ER membrane proteins including the J-protein B12 to potential membrane penetration sites on the ER membrane called foci where B12 facilitates virus extraction into the cytosol. How B12 reorganizes into the foci is unclear. Here we find that two closely related ER membrane proteins Erlin1 and Erlin2 (Erlin1/2) bind to B12. During infection, SV40 induces release of this J-protein from Erlin1/2 to enable B12 to reorganize into the foci. Our data reveal how a non-enveloped virus mobilizes a specific ER membrane component to a membrane penetration structure to promote its own membrane transport. Intriguingly, recent reports suggest that the non-enveloped polyomavirus (PyV) creates its own membrane transport structure to enter the host cell and cause infection -S, or B12 [WT]-FRB-S construct, and the experiments conducted with or without adding rapalog-1. Only cells expressing the S-tagged construct was scored for the expression of large T antigen. As expected , B12 knoThese results predict that exogenously expressed B12 [WT]-FRB-S, in the presence of the Erlin2-FKBP-FLAG binding partner, would be restricted from mobilizing into the foci upon addition of rapalog-1 during SV40 infection. Indeed, B12 [WT]-FRB-S can largely mobilize to the BAP31-positive foci during SV40 infection in the absence of rapalog-1, similar to B12 [WT]-S  in the ER through a direct interaction with the SREBP-SCAP complex . SREBP iPolyclonal Hsp90 and monoclonal SV40 large T antigen antibodies were purchased from Santa Cruz Biotechnology , a monoclonal PDI antibody from Abcam , a monoclonal BiP antibody from BD , a monoclonal FLAG antibody from Sigma-Aldrich , polyclonal Erlin1, B12, B14, and TMUB1 antibodies from Protein Tech Group , a polyclonal Hsc70 antibody and a monoclonal BAP31 antibody (CC-1) from Thermo Fisher Scientific , and a polyclonal Erlin2 antibody from Cell Signaling Technology . Monoclonal antibodies against SV40 VP1 were generous gift from Dr. W. Scott (University of Miami), and polyclonal antibodies against SV40 VP3 were from Dr. A. Helenius . FLAG M2 antibody-conjugated agarose beads, 3xFLAG peptide, Triton X-100 and Brefeldin A (BFA) were purchased from Sigma-Aldrich, PMSF, digitonin, and Deoxy Big Chap from EMD Millipore Chemicals , Protein G magnetic beads, Lipofectamine 2000, Lipofectamine RNAi max, and Hygromycin B from Thermo Fisher Scientific, Fugene HD from Promega , Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI) from Polysciences Inc. , the rapamycin-1 anologue heterodimerizer from Takara Bio Inc. , and Blasticidin S HCl from Corning . CV-1 and HEK 293T cells  were cultured in DMEM (Thermo Fisher Scientific) containing 10% fetal bovine serum (Corning), 10 U/ml penicillin (Thermo fisher Scientific), and 10 μg/ml streptomycin (Thermo Fisher Scientific).G-GTT-ATG-ATT-TAC-ATC-GAT-AGG 272, and Erlin2: 241 GGT-GTG-ATG-ATT-TAC-TTC-GAT-CGG 265, where the underlines denote the introduced silent mutation. The resulting PCR products were inserted into pCDNA3.1(-) in frame with the FLAG tag sequence by standard cloning methods. FLAG-B12 [WT] and FLAG-B12 [H138Q] were previously described -FRB-S-Erlin2-FKBP-FLAG interaction was analyzed similarly. Briefly, approximately 3x105 CRISPR B12 KO CV-1 cells were plated on a 6-cm plate, incubated for 24 h, and transfected with the indicated DNA constructs using PEI. 24 h after DNA transfection, cells were infected with mock or SV40 (MOI~100) for 6 h and further incubated in the absence or presence of 0.5 μM rapalog-1 heterodimerizer for 10 h, harvested, and analyzed as above.Approximately 1x10The target sequences of the siRNAs and their final concentrations used in this study are as follows: Erlin 1 siRNA: Thermo Fisher Predesigned Silencer siRNA, ID# 1473100 (50 μM), Erlin 2 siRNA: GAACUAUACUGCUGACUAU[dT][dT] (10 μM), PAN-Erlin siRNA: Thermo Fisher Predesigned Stealth RNAi siRNA, ID# HSS116356 (50 μM).Allstar negative control siRNA  was used as a scrambled control siRNA. CV-1 cells were reverse-transfected with each siRNA at the indicated concentration using Lipofectamine RNAi MAX and incubated for 24 h (Erlin1 and PAN-Erlin siRNAs) or 48 h (Erlin 2 siRNA) prior to SV40 infection.Evaluation of XBP1 splicing was described previously in Ravindran et al., 2015 .CV-1 cells transfected with siRNAs were infected with SV40 (MOI~0.3) for 24 h and fixed with 1% paraformaldehyde. Following permeabilization with 0.2% Triton X-100, fixed cells were stained with a mouse monoclonal SV40 large T antigen antibody, followed by Alexa Fluor 488-conjugated secondary antibodies. In each experiment, approximately 1,000 cells were counted to assess the extent of large T antigen expression. For the Erlin1/2 rescue experiments, cells were initially transfected with siRNA, incubated for 24 h, and further transfected with FLAG-tagged constructs using Fugene HD. 24 h post DNA transfection, cells were infected with SV40, fixed, and permeabilized as described above. Fixed cells were stained with a mouse monoclonal SV40 large T antigen antibody and a rabbit monoclonal FLAG antibody followed by Alexa Fluor 594-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Large T antigen expression was evaluated in cells expressing the FLAG-tagged protein. At least 100 cells for each sample were counted to assess the extent of large T antigen expression. B12 rescue experiments were similarly performed, except that parental CV-1 or B12 CRISPR KO cells were used without siRNA transfection. As indicated in the result and discussion sections, overexpressed B12 often mislocalized in the nucleus. To avoid artifacts caused by B12 mislocaliztion, only cells expressing FLAG-B12 [WT] or [1–355] that display a clear ER-localization patterns were counted as “FLAG-B12 [WT] or [1–355]-expressing” cells, unless otherwise noted.CV-1 cells transfected with the indicated siRNAs or DNA constructs were infected with SV40 (MOI~30–50) for the indicated time. Cells were fixed and subjected to an immunofluorescence-based method as described in the SV40 infection assay, except that a rat monoclonal BAP31 antibody, the indicated primary antibodies, Alexa Fluor 594-conjugated anti-mouse IgG, and Alexa Fluor 488-conjugated anti-rabbit IgG were used. For triple staining, a rat monoclonal BAP31 antibody, a rabbit polyclonal B12 antibody, and a monoclonal mouse FLAG antibody were used as primary antibodies, while Alexa Fluor 594-conjugated anti-rat IgG, Alexa Fluor 488-conjugated anti-rabbit IgG, and Alexa Fluo-350-conjugated anti-mouse IgG secondary antibodies were used as secondary antibodies.4 cells/well, incubated for 24 h, and transfected with DNA constructs using Fugene HD. 24 h after DNA transfection, cells were infected with SV40 (MOI~0.3) for 6 h and further incubated in the absence or presence of 0.5 μM rapalog-1 heterodimerizer for 18 h. 24 hpi, cells were fixed and subjected to an immunofluorescence method using a mouse monoclonal SV40 large T antigen antibody and a rabbit polyclonal S-tag antibody as described above. Fixed cell samples were imaged using Nikon Eclipse TE2000-E equipped with a 40x objective and a 12-bit CCD camera. To avoid potential overexpression-induced artifacts, cells were counted and examined for the large T antigen expression when expression of B12 [WT]-S or B12 [WT]-FRB-S was localized in the ER without forming nuclear aggregates and that their signal values range from 200 to 1000 under 12-bit resolution. At least 100 cells were counted to assess the extent of large T antigen expression. For SV40-induced foci formation assay, cells were transfected in the same manner, infected with SV40 (MOI~30–50) for 6 h and further incubated in the absence or presence of 0.5 μM rapalog-1 heterodimerizer for 10 h. Following the immunofluorescence procedure using anti S-tag and BAP31 antibodies, cell images were taken as in the infection assay, except that instead of assessing the large T antigen expression, BAP31 images were captured. At least 100 cells were counted to assess the extent of BAP31 and S double-positive foci formation in each experiment.Parental and B12 CRISPR KO CV-1 cells were plated on coverslips in a 24-well plate at a density of approximately 2.5x10Cells transfected with truncated CMV promoter-driven FLAG-B12 [WT] or FLAG-B12 [1–355] were fixed, subjected to the above-mentioned immunofluorescence method using a rabbit monoclonal FLAG antibody and Alexa Fluor 488-conjugated anti-rabbit IgG, and the samples were imaged as described above. To compare the expression level of FLAG-B12 [WT] and FLAG-B12 [1–355], all images were taken using the same exposure time. Due to the varied expression level of FLAG-tagged proteins among cells, cells were counted and assessed for mislocalization of FLAG-tagged B12 constructs only when the signal values of the ER-localized FLAG-tagged B12 constructs ranged from 500 to 1500 under 12-bit resolution and/or the FLAG-tagged B12 constructs formed small nuclear aggregate structures. By contrast, cells were considered to have overexpressed exogenous B12 and removed from analysis when the signal values of ER-localized FLAG-tagged B12 constructs were above 1500 under 12-bit resolution and/or the FLAG-tagged B12 constructs formed massive nuclear aggregated structures. At least 100 cells were counted to assess mislocalization of FLAG-tagged B12 constructs in each experiment.Flp-In 293 T-REx cells (Thermo Fisher Sciences) were co-transfected with pOG44 (Thermo Fisher Sciences) and pcDNA5 FRT/TO encoding 3xFLAG-B12 [WT] using Lipofectamine 2000. 24 h post transfection, cells were split and cultured in DMEM medium containing 100 μg/ml hygromycin and 5 μg/ml blasticidin for 10–15 days. Hygromycin-resistant colonies were cloned.PX330 and PX459 used for generating B12 KO cells were gifts from Dr. F. Zhang (Addgene plasmid # 42230 and 62988) [S1 Fig(TIF)Click here for additional data file.S2 Fig(A) CV-1 cells transfected with Erlin1*-FLAG were infected with SV40 for 16 h, fixed, and subjected to immunofluorescence analyses using antibodies against BAP31 and FLAG. Images were taken by confocal microscopy. Inset shows a 2.5x enlarged image corresponding to the enclosed white square. Bar represents 10 μm. (B) As in A, except that Erlin2*-FLAG was used.(TIF)Click here for additional data file.S3 FigAs in (TIF)Click here for additional data file.S4 FigCells transfected with scrambled siRNA were fixed and subjected to immunofluorescence analyses using antibodies against B12 and BAP31. Images were taken by confocal microscopy. Bar represents 10 μm.(TIF)Click here for additional data file.


The A2B receptor (A2BR) can mediate adenosine-induced tumor proliferation, immunosuppression and angiogenesis. Targeting the A2BR has proved to be therapeutically effective in some murine tumor models, but the mechanisms of these effects are still incompletely understood. Here, we report that pharmacologic inhibition of A2BR with PSB1115, which inhibits tumor growth, decreased the number of fibroblast activation protein (FAP)-expressing cells in tumors in a mouse model of melanoma. This effect was associated with reduced expression of fibroblast growth factor (FGF)-2. Treatment of melanoma-associated fibroblasts with the A2BR agonist Bay60-6583 enhanced CXCL12 and FGF2 expression. This effect was abrogated by PSB1115. The A2AR agonist CGS21680 did not induce CXCL12 or FGF2 expression in tumor associated fibroblasts. Similar results were obtained under hypoxic conditions in skin-derived fibroblasts, which responded to Bay60-6583 in an A2BR-dependent manner, by stimulating pERK1/2. FGF2 produced by Bay60-6583-treated fibroblasts directly enhanced the proliferation of melanoma cells. This effect could be reversed by PSB1115 or an anti-FGF2 antibody. Interestingly, melanoma growth in mice receiving Bay60-6583 was attenuated by inhibition of the CXCL12/CXCR4 pathway with AMD3100. CXCL12 and its receptor CXCR4 are involved in angiogenesis and immune-suppression. Treatment of mice with AMD3100 reduced the number of CD31+ cells induced by Bay60-6583. Conversely, CXCR4 blockade did not affect the accumulation of tumor-infiltrating MDSCs or Tregs. Together, our data reveal an important role for A2BR in stimulating FGF2 and CXCL12 expression in melanoma-associated fibroblasts. These factors contribute to create a tumor-promoting microenvironment. Our findings support the therapeutic potential of PSB1115 for melanoma. In the tumor microenvironment stromal cells, including endothelial cells, fibroblasts and several types of immune and inflammatory cells, communicate with neoplastic cells through multiple soluble factors. Stroma-derived growth factors, cytokines and chemokines can directly induce the proliferation and migration of neoplastic cells and support angiogenesis. At the same time, they create a chronic inflammatory milieu that attracts lymphoid and myeloid cells with immunosuppressive features, allowing tumor cells to escape the immune response. Furthermore, tumor stromal cells such as cancer-associated fibroblasts can also induce resistance to chemotherapy and/or targeted therapy, in a soluble factor- and cell adhesion-dependent manner . To. To17]. Previous reports show that the A2BR is expressed in fibroblasts –26. TherBased on the results shown in Figure Tumor-associated fibroblasts are critical component of tumor stroma that produce and secrete various tumor-promoting factors, including CXCL12 or stromal-derived factor 1 α (SDF1α) –30. Thusin vitro as described in the Methods section. A representative image of spindle-shaped, vimentin-positive melanoma-associated fibroblasts is shown in Figure To support a critical role of the A2BR in promoting stromal cells activation in tumors, we evaluated the response to Bay60-6583 in fibroblasts isolated from melanoma tissues of C57Bl/6 mice. Melanoma-associated fibroblasts were isolated and cultured Normal mouse fibroblasts isolated from skin were also used to assess the response to Bay60-6583. When these cells were exposed for 24 hours to a hypoxia-inducing treatment (100 μM CoCl2) as a tumor-relevant stressor  and thenFGF2 is a growth factor strongly implicated in tumor cell growth . To inveTaken together, these results indicate that FGF2 released from fibroblasts upon A2BR stimulation induces melanoma cells proliferation, as reported by other authors , 52–54.in vivo. We previously described a role for A2BR in inducing tumor accumulation of MDSC, which suppress anti-tumor T-cell responses [Next, we investigated whether A2BR-induced CXCL12 expression could contribute to the tumor promoting effects of Bay60-6583 esponses , and tumesponses . CXCL12 esponses , 35 and esponses . We usedesponses , 35 . B16.F10 murine melanoma cells were purchased from American Type Culture Collection . Animal studies were approved by Italian Health Ministry (protocol n° 12827) according to institutional animal care guidelines, Italian Law 26/2014 based on the European Community Law for Animal Care 2010/63/UE.5 B16-F10 cells were subcutaneously (s.c.) injected in the right flank of anesthetized mice [2×10zed mice , 17. At zed mice –21 or wized mice   and then placed in a Petri dish with RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin  in a tissue culture incubator, allowing fibroblasts to migrate from the tissue within 5-7 days. Fibroblasts were also isolated from skin of C57Bl6 mice as reported by Seleuanov et al., . Brieflyin vitro experiments as it achieved optimal effects in preliminary studies. Some experiments were also performed in the presence of 100 nM PSB1115. In additional experiments, fibroblasts isolated from skin tissue were exposed to 100 μM CoCl2 used as hypoxia inducer [Fibroblasts isolated from either melanoma or skin tissues, grown on polylysine-coated surfaces, were treated with Bay60-6583  for further analyses. 10 nM Bay60-6583 was selected to perform  inducer  at diffeConfocal microscopy immunofluorescence analysis was performed on frozen sections of melanoma tissue or fibroblasts isolated from either melanoma or skin tissues. Frozen sections of melanoma tissue, fixed in 4% paraformaldehyde (PFA), were incubated with 20% goat serum containing 0.5% TritonX-100 for 30 minutes at room temperature to block nonspecific binding sites. Fibroblasts plated on poly-lysine-coated slides were washed twice in PBS, fixed in 4% PFA, and nonspecific binding sites were blocked using 20% goat serum or 5% bovine serum albumin (BSA) for 30 minutes at room temperature, as appropriate. Slides were stained overnight with the following primary antibodies: anti-FAPα (H-56) or anti-FGF-2 (147)  were detected with Alexa Fluor® 488 or 555 Goat Anti-Rabbit IgG (H+L) (1:5000) secondary antibodies ; anti-CXCL12  (clone P-159X) was detected with the secondary antibody Alexa Fluor® 488 Goat Anti-Mouse IgG (H+L) (1:5000); anti-vimentin (E-5)  or anti-fibronectin (A17)  were detected with the secondary antibody Alexa Fluor® 555 Goat Anti-Mouse IgG (H+L) (1:5000); anti-CXCR4 (clone L276F12)  was detected with Alexa Fluor® 555 Goat Anti-Rat IgG (H+L) (1:5000); FITC-conjugated anti-CD31 (MEC 13.3)  was also used. Primary antibody for A2B receptor: A2B-R (N-19)  was detected with the secondary antibodies DyLight™ 549-conjugated AffinePure Donkey anti-goat IgG (H+L) . DAPI was used to counterstain nuclei. In all staining experiments, isotype-matched IgG and omission of the primary Ab were used as controls. Slides were observed using a Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH). Samples were vertically scanned from the bottom of the coverslip with a 63 × or 40 x (1.40 NA) Plan-Apochromat oil-immersion objective. Images were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH).Fibroblasts were collected and suspended in RIPA buffer (Radio-Immuno Precipitation Assay Buffer). Forty micrograms of total protein were analyzed by 10% denaturing polyacrylamide gels and then transferred electrophoretically to PVDF membranes . Anti-A2BR (H-40) (Santa Cruz Biotechnology), anti HIF1α (A300-286A)  or anti CD73   or phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) or p44/42 MAPK (Erk1/2) (137F5)  primary antibodies were used. Immunoreactive protein bands were visualized by enhanced chemiluminescence reagents  and analyzed to Las4000 .Tumor-derived fibroblasts, B16-F10 cells or co-cultures of tumor-derived fibroblasts and B16-F10 cells (1:1 ratio) were seeded onto 96 well plates and treated with 10 nM Bay60-6583, 100 nM PSB1115 or both, and cell proliferation assays were performed at 24-48-72 hours. Some experiments with fibroblasts co-cultured with melanoma cells were performed in presence of an anti-FGF2 antibody (100 ng/ml) alone or in presence of 10 nM Bay60-6583. Recombinant FGF-β  was used at the concentrations of 30-100 ng/ml.Mouse CXCL12/SDF-1 alpha ELISA kit  was used to measure the levels of CXCL12 in the supernatant of isolated fibroblasts stimulated with 1, 10 or 100 nM Bay60-6583 for 24 h with or without 100 nM PSB1115.Melanoma tissues were digested with 1 U/ml collagenase , passed through 70-μm cell strainers and red blood cells (RBC) were lysed to prepare single cell suspensions. Cell samples were blocked with anti-mouse CD16/CD32 . Antibodies against CD11c-FITC, CD11b-PeCy5.5, Gr1-PE or Gr1-allophycocyanin, CD3-PeCy5.5; CD8-allophycocyanin or CD8-PE; CD4-allophycocyanin; NK1.1-PE were obtained from eBioscience and BioLegend. Expression of FAP in melanoma tissue was also analyzed by flow cytometry by using an anti-FAP-FITC and expressed as percentage of positive cells. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences).TL . Results are expressed as mean ± SEM. Data were analyzed with GraphPad Prism 6 (GraphPad Software). Two-tailed Student's t test or ANOVA were used as appropriate. P values < 0.05 were considered significant and reported in the figure legends.Results of confocal microscopy immunofluorescence are coming from sections derived from tumors obtained from at least 5-6 different mice per condition, as reported in the figure legends. Two sections were stained for each tumor and positive cells were counted in four-to-five randomly selected fields per tumor section. The results of immunofluorescence in fibroblasts are expressed as mean of 4 separate fibroblast preparations, each isolated from C57Bl6 mice (±S.E.M.). The optical density of the protein bands detected by Western blotting was performed with ImageQuant


Vibrio campbellii, isolated from Penaeus vannamei cultured in a Latin American country. The Tn3-like transposon and pirAB genes were encoded on the plasmid pLA16-2. These data support the geographical variations in the virulence plasmid found among acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio isolates from Latin America and Asia.We report here the complete genome sequence of  Vibrio spp. carrying a virulence plasmid encoding the pirAB gene, has caused significant economic losses in the shrimp-farming industry , which is caused by industry . Initialreported –5. Receneported –. There ide novo assembled by FALCON version 0.2.1, and the Illumina paired-end reads  were mapped to the assembled contigs to improve the accuracy of the genome. Annotation was carried out using the NCBI’s Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/books/NBK174280).Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen). Sequencing was performed by Macrogen, Inc., using a hybrid approach with the PacBio RS II (Pacific Biosciences) (20-kb SMRTbell template library) and Illumina HiSeq 2000 (paired-end short-read data) platforms. The PacBio long-read data  were n = 133), rRNA (n = 37), and noncoding RNAs (n = 4) were encoded on Chr I, except 15 tRNAs and 3 rRNAs on Chr II and one tRNA on pLA16-1. Overall genome similarities among LA16-V1 and other completely sequenced V. campbellii strains were assessed using the orthologous average nucleotide identity algorithm  and Chr II , and a total of four plasmids, designated pLA16-1 , pLA16-2 , pLA16-3 , and pLA16-4 . The two chromosomes showed similar G+C contents (45.6% and 45.4%) and percentages of coding regions (87.1% and 86.8%). Moreover, most of the predicted tRNAs  to those from the AHPND-related plasmids pVPE61a , CP021146 (Chr II), CP021147 (pLA16-1), CP021148 (pLA16-2), CP021149 (pLA16-3), and CP021150 (pLA16-4).The genome of


Here, we reveal that miR-155 levels are downregulated in serum from type 2 diabetes (T2D) patients, suggesting that miR-155 might be involved in blood glucose control and diabetes. Gain-of-function and loss-of-function studies in mice demonstrate that miR-155 has no effects on the pancreatic β-cell proliferation and function. Global transgenic overexpression of miR-155 in mice leads to hypoglycaemia, improved glucose tolerance and insulin sensitivity. Conversely, miR-155 deficiency in mice causes hyperglycemia, impaired glucose tolerance and insulin resistance. In addition, consistent with a positive regulatory role of miR-155 in glucose metabolism, miR-155 positively modulates glucose uptake in all cell types examined, while mice overexpressing miR-155 transgene show enhanced glycolysis, and insulin-stimulated AKT and IRS-1 phosphorylation in liver, adipose tissue or skeletal muscle. Furthermore, we reveal these aforementioned phenomena occur, at least partially, through miR-155-mediated repression of important negative regulators  of insulin signaling. Taken together, these findings demonstrate, for the first time, that miR-155 is a positive regulator of insulin sensitivity with potential applications for diabetes treatment. In the present study, we provide evidence for the first time showing that miR-155 is a positive regulator of insulin sensitivity in mice. Here, we determine that miR-155 levels are downregulated in serum from type 2 diabetes (T2D) patients, and shows a negative correlation with HOMA-IR, suggesting that miR-155 might be involved in glucose homeostasis and insulin action. Global transgenic overexpression of miR-155 in mice leads to hypoglycaemia, improved glucose tolerance and insulin sensitivity. Conversely, miR-155 deficiency in mice causes hyperglycemia, impaired glucose tolerance and insulin resistance. In addition, consistent with a positive regulatory role of miR-155 in glucose metabolism, miR-155 positively modulates glucose uptake in all cell types examined, while mice overexpressing miR-155 transgene show enhanced glycolysis, and insulin-stimulated AKT and IRS-1 phosphorylation in liver, adipose tissue or skeletal muscle. More importantly, we reveal that these aforementioned phenomena occur, at least in part, through the miR-155-mediated coordinated repression of multiple negative regulators  of insulin signaling. Diabetes is recognized as one of the most important health threats of our time–4. HowevAs a multifunctional miRNA, miR-155 plays crucial roles in various physiological and pathological processes, such as haematopoietic lineage differentiation, cardiovascular diseases and cancer. Our repIn the present study, we investigate the roles of miR-155 in blood glucose homeostasis, as well as the underlying mechanisms. Our findings show, for the first time, that miR-155 enhances insulin sensitivity through coordinated regulation of multiple genes in mice, including important negative regulators  of insulin signaling.P = 0.0069, P = 0.1548, We evaluated the levels of miRNAs in serum of T2D patients. miR-146a levels were decreased in serum and periAs described in the Materials and Methods section, to examine the roles of miR-155 by gain of function, Rm155LG transgenic mice  for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were generated by us.To achieve global overexpression of miR-155, we crossed Rm155LG mice with EIIa-Cre mice. Procedure for producing RL-m155 transgenic mice  which can globally overexpress miR-155 transgene and express mRFP and luciferase (Luc) reporter transgenes in multiple organs and tissues is detailedly illustrated in As mentioned in “Introduction section”, miR-155 might be physiologically required for normal glucose homeostasis. This feature prompted us to firstly explore the influences of miR-155 on pancreatic β-cell proliferation, β-cell mass and β-cell function by using both RL-m155 mice and miR-155 knockout (KO) mice. We founOur above-mentioned results exhibited that the gain and loss of miR-155 function in mice didn’t have influences on pancreas morphology, islet architecture, β-cell proliferation and mass, and insulin and glucagon immunoreactivity , leadingWe observed that fasting and fed-state blood glucose levels were lower in RL-m155 male  and femaTo evaluate insulin sensitivity of peripheral tissues, we performed insulin tolerance tests (ITTs) in RL-m155 mice. We noticed that RL-m155 mice were more sensitive to insulin than controls . To demoAdditionally, the glucose metabolic performance in RL-155 mice maintained on an HFD (high-fat diet) was explored in this study. When mice were maintained on the chow diet, the RL-155 mice exhibited lower fasting serum glucose levels than control mice . The lev-/- mice to perform the following loss-of-function experiments. miR-155-/- mice exhibited increased blood glucose levels  uptake assays] using human hepatocellular carcinoma 7402 cells, and murine hepa1-6 and C2C12 cell lines. Here we designed a method for in vitro evaluation of cellular 18F-FDG uptake by microPET/CT Inveon scanner, as described previously.To quantitate miRNA and mRNA expression, total RNA was extracted from cells or from various tissues of transgenic mice and gene knockout mice with the use of TRIzol reagent (TaKaRa). The isolation of microRNA from the serum of healthy subjects and T2D patients was performed using the microRNA RNeasy Mini Kit (Exiqon) following the manufacturer’s recommendations. qRT-PCR was performed with standard methods on Stratagene Mx3005P qRT-PCR System, which are described in detail previously. The primRFP expression in the postnatal organs and tissues of RL-m155 transgenic mice was assayed under stereo fluorescent microscope , as described previously, 58, 59.Bioluminescence imaging for multiple organs and tissues obtained from RL-m155 transgenic and control mice was measured using the IVIS LuminaIIimaging system , as described previously, 58.For glucose tolerance test (GTT), mice were fasted overnight for 12h, and then injected intraperitoneally with glucose (2g/kg body weight). For insulin tolerance test (ITT), mice were fasted overnight for 6h, and then injected intraperitoneally with human insulin (Novo Nordisk) at a dose of 0.75IU/kg body weight. Blood glucose levels were measured from mouse tail vein with an automatic glucometer  before glucose and insulin injection and at the indicated time after injection. Serum insulin levels were measured by enzyme-linked immunosorbent assay according to vendor’s instructions .immunohistochemistry (IHC). The primary antibodies are anti-insulin , anti-glucagon , anti-Ki67  and anti-BrdU .For immunohistology, mouse tissues were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin as described previously–62, thenWestern blot was performed with standard methods, which are described in detail previously–64. For The mouse or human miR-155 mimics, mimics control, miR-155 inhibitor and inhibitor control were purchased from RiboBio Co., Ltd . miRNAs were transiently transfected into cells  at a working concentration of 100nM using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer’s procedure. All RNA oligonucleotides treatments proceeded for 48-72h. The effect of in vitro overexpression of miR-155 by using miRNA mimics and the repression of endogenous miR-155 expression by a miR-155 inhibitor on insulin-stimulated AKT phopshorylation was examined in hepa1-6 cells. After stimulation with 50IU/L human insulin (Novo Nordisk) for 15 min at 37°C , the medMouse hepatoma (Hepa1-6) cells were obtained from the Cell Bank of Chinese Academy of Sciences Shanghai Institute of Cell Biology. Hepa1-6 cells were seeded in triplicate in 96-well plates and cultured overnight. The dual luciferase reporter gene plasmids [pLuc-C/EBPβ-3’-UTR-wt or pLuc-HDAC4-3’-UTR-wt ] were cotransfected into hepa1-6 cells with the miR-155 mimics, mimics control or miR-155 mimics plus miR-155 inhibitor using Lipofectamine 2000 Reagent (Invitrogen), respectively. Luciferase and Renilla activities were assayed 48 hours after transfection using the Dual Luciferase Reporter Assay Kit (Promega) following the manufacturer’s instructions.An siRNA targeting mouse PDK4 (siG140711153320), an siRNA targeting mouse C/EBPβ (siG09723100032), an siRNA targeting mouse HDAC4 (siB1271190307) and a control siRNA were purchased from RiboBio Co., Ltd . Western-blot and qRT-PCR analysis showed successful reduction of C/EBPβ, HDAC4 and PDK4 expression in cells transfected with siC/EBPβ, siHDAC4 and siPDK4, respectively . Hepa1-65/well) for microPET/CT scan. After 24 h, culture medium was replaced, and cells were incubated in fresh medium containing 18F-FDG (about 20μCi/well) for 1h. Then, cells were washed with phosphate-buffered saline five times, and image was obtained by microPET/CT Inveon scanner (Siemens).Cells were seeded on a 24-well plate  Schematic strategy for generating RL-m155 transgenic mice. Procedure for producing transgenic mice which can globally overexpress mouse miR-155 transgene in multiple organs and tissues of mice was detailedly illustrated in S1A Fig. Briefly, at 6–8 wk of age, the heterozygous Rm155LG transgenic mice[#, 4# or 6#)  were intercrossed to produce F2 , including Luc- and mRFP-positive F2 animals with white fur (6#). Finally, both Luc- and mRFP-positive F2 animals with white fur and without Cre gene (determined by PCR-based genotyping) were intercrossed to produce RL-m155 transgenic mice   which can globally and constantly express mRFP ((B) Whole-body fluorescence (b) and bioluminescence (c) imaging for newborn offspring derived from mating heterozygous Rm155LG transgenic mice with homozygous EIIa-Cre mice. (C) In vivo mRFP (b) and luc (c) imaging for newborn offspring derived from intercrossing of both Luc- and mRFP-positive F1 animals  . (D) Whole-body fluorescence (b) and bioluminescence (c) imaging for newborn RL-m155 transgenic mice. Both Luc- and mRFP-positive mice   are RL-m155 transgenic mice. (E) Whole-body fluorescence (b) and bioluminescence (c) imaging for adult RL-m155 transgenic mice. (F) PCR-based genotyping for Cre, mRFP and Luc transgenes in RL-m155 transgenic mice. These RL-m155 transgenic mice  were individually analyzed by PCR for the genomic integration of Cre, mRFP and Luc transgenes with tail biopsy-derived DNA. PCR products were amplified by the primer pair P1/P2 (specific for mRFP), by the primer pair P3/P4 (specific for Luc) and by the primer pair P5/P6 (specific for Cre), respectively. P1: 5'-GGGAGCGCGTGATGAAC-3', P2: 5'-CGTTGTGGGAGGTGATGTC-3';P3: 5'-AGATACGCCCT GGTTCCTGG-3',P4: 5'-ACGAACACCACGGTAGGCTG-3';P5: 5'-GAACCTGATGGACATGTTC AGG-3', P6:5'-AGTGCGTTCGAACGCTAGAGCCTGT-3'. lane 1: Cre positive control DNA from EIIa-Cre mouse as template; lane 2: positive control (pRm155LG as template); lane 9: negative control using genomic DNA from WT mouse as template. Data are representative of three independent PCR experiments that yield similar results.enic mice to generess mRFP , Luc (S2ess mRFP  and miR-ess mRFP  transgen(TIF)Click here for additional data file.S2 Fig(A) mRFP and Luc expression in multiple organs and tissues of RL-m155 transgenic mice. The left organ samples in each figure were obtained from one control littermate, while the right organ samples in each figure were isolated from one RL-m155 transgenic mouse. mRFP expression in the postnatal organs and tissues of RL-m155 transgenic mice was assayed under stereo fluorescent microscope , while bioluminescence imaging for multiple organs and tissues obtained from RL-m155 transgenic mouse and littermate controls was measured noninvasively using the IVIS LuminaIIimaging system . Muscle and pancreas from RL-m155R transgenic mice (the right samples in each figure) can be distinguished from their wildtype littermates according to their deep red color under daylight ((B) qRT-PCR analysis of the expression of miR-155 transgene in multiple organs and tissues of RL-m155 transgenic mice. BAT: brown adipose tissue; WAT: white adipose tissue. (C) RL-m155 (right) and control (left) male littermates at 20 weeks of age. (D) RL-m155 (right) and control (left) female littermates at age 12 weeks. (E-F) Body weight of control mice vs. RL-m155 transgenic mice at different ages. Values are mean ± SD; n = 5–10 mice per time point. *, P < 0.05 compared with control mice;**, P<0.01 compared with control mice;#, P<0.001 compared with control mice.daylight . (B) qRT(TIF)Click here for additional data file.S3 Fig(A-C) Blood glucose concentrations in fed-state (A), 12-hour–fasted (B) and 24-hour–fasted (C) mice at indicated ages. (D) Serum insulin concentrations in fed-state and 12-hour–fasted mice (5m). (E-F) GTT in 12-hour–fasted mice (E) and AUC (F) for this GTT (E). (G) Serum insulin measurements performed in 12-hour–fasted mice (3m) during a GTT (E). (H) ITT performed on 12-hour–fasted 3-month-old control and RL-m155 mice. (I) AUC calculated from mice in (H). (J) ITT of 12-hour–fasted control and RL-m155 mice. (K) AUC analysis for this ITT (J). Values are statistically significant at *P<0.05; **P<0.01 and #P<0.001.(TIF)Click here for additional data file.S4 Fig(A) Blood glucose concentrations in 24-hour-fasted control (Con) and RL-155 mice (7m) maintained on chow or an HFD (high-fat diet) (60% fat). (B) GTT in 12-hour–fasted mice (maintained on chow or an HFD). (C) ITT performed on 12-hour–fasted 7-month-old control and RL-m155 mice maintained on chow or an HFD. 28-week-old mice were maintained on chow or were fed an HFD beginning at age 12 wk. For RL-155 mice vs. control mice fed chow: *P <0.05, **P<0.01, ***P<0.001; for RL-155 mice vs. control mice fed a high-fat diet: #P<0.05, ##P<0.01, ###P<0.001.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigHepa1-6 cells transfected with miR-155 mimics or mimics control were challenged with human insulin at 50 IU/L or 100 IU/L for 0, 15 and 30min, respectively. Protein levels of total and phosphorylated AKT were detected by Western blotting. The results were obtained from three independent experiments and a representative immunoblot is shown.(TIF)Click here for additional data file.S7 FigA) Sequence alignment of 3’-UTR of human (Hsa), mouse (Mmu), rhesus (Mml) and rat (Rno) SOCS1 highlighting miR-155 binding site. (B) Sequence alignment of 3’-UTR of human and mouse SOCS1 with respect to miR-155 mature sequence, showing possible interaction through a conserved binding site. (C) Immunoblot analysis of SOCS1 expression in liver of RL-m155 transgenic mice and KO mice.((TIF)Click here for additional data file.S8 FigP < 0.05 compared with siSCR.SCR: scrambled siRNA. *, (TIF)Click here for additional data file.S9 FigP < 0.05; **P < 0.01; NS, not significant.Data are presented as fold changes in the miR-155-expressing cells compared to the control cells. UC: untransfected cells. *(TIF)Click here for additional data file.S10 FigP < 0.05; **P < 0.01; NS, not significant.Data are presented as fold changes in siRNA-transfected cells compared to control cells. UC: untransfected cells. *(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S1 Data(XLS)Click here for additional data file.S2 Data(XLS)Click here for additional data file.S3 Data(XLS)Click here for additional data file.S4 Data(XLS)Click here for additional data file.S5 Data(XLS)Click here for additional data file.S6 Data(XLS)Click here for additional data file.


BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression.The anti-apoptotic protein B cell lymphoma/leukaemia 2 ( L), BCL-2-like protein 2 (BCL-W) and myeloid cell leukaemia sequence 1 (MCL-1)] and pro-apoptotic members  [Numerous cancer types have been associated with aberrations in genes encoding B cell lymphoma/leukaemia 2 (BCL-2) family proteins . The BCLt (BID)] –6. Incret (BID)] –13.BCL-2 [BCL-2 inhibition using RNA interference caused an apoptotic response in cell lines with moderate-to-high BCL-2 levels. These findings could be confirmed with the small-molecule BCL-2 family inhibitor ABT263, which inhibits the anti-apoptotic activity of BCL-2, BCL-XL, BCL-W and MCL-1 with inhibition constant (Ki) values of 0.044, 0.055, 7 and 224 nmol/L, respectively [BCL-2 levels responded better to ABT263 treatment than low BCL-2-expressing cell lines. ABT263 furthermore delayed the onset of tumor formation in mice injected with high BCL-2-expressing neuroblastoma cells [BCL-2-expressing neuroblastoma tumors. Unfortunately, the administration of ABT263 in phase I/II clinical studies for adult cancers was associated with dose-limiting thrombocytopenia due to concomitant inhibition of anti-apoptotic BCL-XL, a key survival factor for circulating platelets [Neuroblastoma is the most commonly diagnosed extracranial solid cancer in children, accounting for approximately 15% of all pediatric cancer deaths . A largeBCL-2 . Previouectively . Neuroblma cells . These olatelets –22. Therlatelets , 23, 24.L (Ki of 48 nmol/L), BCL-W (Ki of 245 nmol/L) and MCL-1 (Ki of >444 nmol/L), while maintaining its activity against BCL-2 (Ki of <0.01 nmol/L) [Compared with ABT263, ABT199 displays less activity against BCL-X nmol/L) , 25. ABT-W Ki of 5 nmol/L  nmol/L) . In the 50 and LC50 values of ABT199 were established for 21 classical neuroblastoma cell lines and 3 tumor-initiating cell lines (TIC)  versus micromolar IC50 and LC50 values  for the other cell lines tested. Evaluation of the BCL-2, MCL-1, BCL-XL, BCL-W and BIM protein levels . As ABT199 acts by displacing pro-apoptotic BCL-2 family members including BIM from BCL-2 [MYCN status. MYCN-amplified neuroblastoma cell lines responded more potently to ABT199 than MYCN single copy cell lines, with IC50 values ranging from 10 nmol/L-20 μmol/L versus 10-18 μmol/L, respectively , 13% (KCNR) and 5% (SJNB12) for the high BCL-2-expressing cell lines versus 0% (SKNAS) and 1% (SHEP2) for the low BCL-2-expressing cell lines. Effects on sub-G1 were dose-dependent, with maximum increases observed after treatment with 10 μmol/L ABT199 . Next, in vitro effects of ABT199 on the activation of the intrinsic apoptotic pathway were studied. As ABT199 inhibits the activity of BCL-2 by displacement of pro-apoptotic proteins, we first studied the effects of ABT199 on BIM displacement from BCL-2. Treatment of the high BCL-2-expressing neuroblastoma cell lines with 62.5 nmol/L ABT199 was already sufficient for almost complete displacement of BIM from BCL-2  shows that the effects of ABT199 obtained in CHP126, KCNR and SJNB12 are indeed caused by BLC-2 inhibition rather than off-target effects.In addition, overexpression of BCL-2 in the low BCL-2-expressing neuroblastoma cell line SY5Y resulted in a strong increase in sensitivity to SY5Y . The remin vivo in mice with high BCL-2-expressing KCNR neuroblastoma xenografts. Once-daily oral treatment with 100 mg/kg ABT199 for three consecutive weeks resulted in significant tumor growth inhibition relative to the vehicle treated controls . In vitro efficacy studies furthermore showed that BCL-2 protein and the BIM/BCL-2 complex levels are better predictive biomarkers for ABT199 sensitivity than BCL-2 mRNA levels. Although less strong, MYCN amplification status was also observed to be a predictive biomarker for sensitivity to ABT199.Based on previously performed preclinical studies , the BCLin vivo comparison of ABT199 with ABT263 for solid cancer treatment. Although ABT199 induced a strong apoptotic response, comparison of the efficacy of equal doses of ABT199 and ABT263 in mice with KCNR neuroblastoma xenografts expressing high levels of BCL-2 showed superior antitumor activity for ABT263. As maximum BIM displacement from BCL-2 was observed after ABT199 treatment, the most plausible explanation for the superior efficacy of ABT263 is its additional activity against anti-apoptotic proteins BCL-XL and BCL-W. Results indicate that the simultaneous inhibition of multiple anti-apoptotic proteins is more effective for treatment of BCL-2-dependent neuroblastoma than inhibition of BCL-2 alone.The current manuscript describes the first side-by-side in vitro and in vivo treatment with ABT199 resulted in upregulation of the anti-apoptotic protein MCL-1 and sequestration of released BIM by MCL-1. Upregulation of non-targeted anti-apoptotic BCL-2 family proteins taking over the function of BCL-2 has earlier been described as one of the key mechanisms of tumor resistance to BCL-2 inhibitors including ABT199 [L that sequestered BIM [L were observed after ABT199 treatment, indicating that MCL-1 plays a more important role in neuroblastoma resistance to ABT199 than BCL-XL.The importance of inhibiting multiple anti-apoptotic proteins is strengthened by the observations that g ABT199 –32, 34. ered BIM . No chanin vitro and in vivo treatment with ABT199, which has not been reported for BCL-2 inhibitors before. Since complexation of BIM with MCL-1 stabilizes MCL-1 independent of Noxa [MCL-1 upregulation after ABT199 treatment could be explained by Noxa downregulation and MCL-1 stabilization by BIM. Binding of Noxa to MCL-1 triggers Mule-mediated ubiquitination and proteasomal degradation of the anti-apoptotic protein, resulting in decreased MCL-1 levels , 35–40.  of Noxa , BIM disMCL-1 knockdown with ABT199 treatment resulted in synergistic cell growth inhibition. Potentiating effects of MCL-1 inhibition were confirmed with the small molecule MCL-1 inhibitor A-1210477, but combined effects of ABT199 and A-1210477 were additive rather than synergistic. The less potentiating effects obtained with A-1210477 might be due to the incomplete displacement of BIM from MCL-1, which has been described before. In addition, MCL-1 might have other functions independent of its role in apoptosis such as maintenance of mitochondrial integrity and deregulation of mitochondrial integrity by MCL-1 knockdown in combination with BCL-2 inhibitors might have a more lethal effect on neuroblastoma cells than inhibition of the pro-apoptotic function of MCL-1 alone. Of interest is the observation that several compounds in use or development for cancer treatment also cause MCL-1 inhibition  400/60% phosal 50 propylene glycol (PG) (v/v/v) in final concentrations of 10 mg/mL.ABT199, ABT263 and A-1210477 were purchased from Selleck Chemicals while QVD-OPH was purchased from Sigmaaldrich. For Classical human neuroblastoma cell lines and neuroblastoma tumor-initiating cell (TIC) lines were cultured as previously described , 42. Cel50) and half lethal concentration (LC50) values were derived from dose-response curves. IC50 values at 72 hours were calculated by determining the ABT199 concentrations needed to achieve a 50% reduction in cell viability observed for DMSO-treated cells at 72 hours (set at 100%). LC50 values at 72 hours were calculated by establishing the ABT199 concentrations needed to attain a 50% reduction in the cell viability compared to time point 0.Neuroblastoma cell lines were seeded in triplicate in 96-well  or 48-well (TIC lines) plates using the most optimal confluency for each cell line . Cells w1 fraction. See Cells were treated with 0.1% DMSO (control) or ABT199 using concentration ranges of 7.8 nmol/L to 10 μmol/L. After 72-hour treatment, floating and adherent cells were harvested for FACS analysis to determine the cell-cycle distribution and the apoptotic sub-GCHP126, KCNR, SJNB12, SKNAS and SHEP2 were 24-hour treated with 0.04% DMSO (control) or 62.5 nmol/L to 10 μmol/L ABT199. Floating and adherent cells were harvested to determine cytochrome c in the cytosolic and organelle fractions. See L (clone 54H6) monoclonal antibody ; rabbit anti-human MCL-1 monoclonal antibody ; rabbit anti-human BCL-W (Clone 31H4) monoclonal antibody ; rabbit anti-human Noxa (clone EPR9735B) monoclonal antibody ; rabbit anti-human BIM ; mouse anti-human cytochrome c (clone 7H8.2C12) monoclonal antibody ; rabbit anti-human PARP (Clone 9542S) monoclonal antibody ; rabbit anti-human cleaved caspase 3 (Clone 5AE17) monoclonal antibody ; mouse anti-human α-tubulin (clone DM1A) monoclonal antibody  and horseradish peroxidase (HRP)-conjugated goat anti-rabbit (clone NA9340V) and goat anti-mouse (clone NXA931) secondary antibodies . See The following antibodies were used: rabbit anti-human BCL-2 (clone D55G8) monoclonal antibody ; rabbit anti-human BCL-XL, cells were treated with 0.25% DMSO (control) or 62.5 nmol/L or 1.25 μmol/L ABT199 at 24 hours after seeding and harvested after 24-hour treatment. Co-immunoprecipitation studies have been performed as described in detail in the Cell lines were seeded onto 14-cm culture dishes. For the detection of basal BIM/BCL-2 complex levels, untreated cells were harvested after 72 hours incubation at normal culture conditions. For the detection of BIM displacement from BCL-2 and BIM complexation with MCL-1 and BCL-X5 cells in 4 mL culture medium) and incubated overnight. Next, cells were transfected with non-targeting shRNA  or MCL-1 shRNA (TRCN0000199070 and TRCN0000005518) for the BCL-2 high-expressing cell lines using the PLenti VI system according to the manufacturers protocol (Sigma Aldrich), while SY5Y was transfected with either a luciferase 2 control or a pLenti 6/V5-DEST vector constitutively overexpressing the BCL-2 protein. After 72 hours, cells were transferred into 96-well plates for in vitro MTT assays and synergy studies (see below).CHP126, KCNR, SJNB12 and SY5Y were seeded in 6-cm culture dishes . Effects on cell viability were studied after 72-hour treatment with ABT199, using the MTT cell proliferation assay.CHP126, KCNR and SJNB12 transiently transfected with non-targeting shRNA or For synergy studies between ABT199 and the MCL-1 inhibitor A-1210477, non-transfected cells in 96-well plates were co-treated with ten-fold serial dilutions of ABT199 (0-10 μmol/L) and five-fold serial dilution of A-1210477 (0-50 μmol/L).nu−/nu− mice  were obtained from Harlan) and experiments were performed with permission from and according to the standards of the Dutch animal ethics committee . NMRI nu−/nu− mice with KCNR neuroblastoma xenografts of approximately 268 mm3 were orally treated with 100 mg/kg/d ABT199 (n = 5), 100 mg/kg/d ABT263 (n = 5), or vehicle (n = 6) for 21 days. Tumor sizes were measured by an external caliper. See Female NMRI in vitro Western blotting.Per mouse sample, 10 tumor sections of 50 μm were homogenized in 2% Chaps buffer as previously described. Western blot detection of protein levels of BCL-2 like family members was carried out as described for Sections of treated and untreated KCNR tumors harvested at 4 hours after administration of the last dose were homogenized using the Ultra Turrax T25 tissue homogenizer (Janke & Kunkel) and lysed (overnight at 4°C) in 2% Chaps buffer. Co-immunoprecipitation was carried out as described above.The following antibodies were used: rabbit anti-human Ki-67 (clone SP6) monoclonal antibody , rabbit anti-human cleaved caspase 3 (Asp175) polyclonal antibody  and BrightVision horseradish peroxidase-conjugated goat anti-rabbit polyclonal secondary antibody . See http://r2.amc.nl).RNA was extracted from tumors with TRIzol  following the manufacturers protocols. RNA concentration and quality were determined using the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Fragmentation of cRNA, hybridization to hg-u133 plus 2.0, microarrays and scanning were carried out according to the manufacturers protocol . The mRNA gene expression data were normalized with the MAS5.0 algorithm within the GCOS program of Affymetrix Inc. Target intensity was set to 100. All data were analyzed using the R2 genomic analysis and visualization platform (


C. elegans . Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5–GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in  in vivo and how phosphorylation affects protein function. Recent developments in CRISPR/Cas9-based genetic engineering make it possible to substitute individual amino acids, which allows investigating the role of single and multi-site phosphorylation of substrates in vivo. Here, we focus on an intensively phosphorylated cell division protein, LIN-5NuMA. C. elegans LIN-5 participates in chromosome segregation and is essential for positioning the spindle and cell cleavage plane during asymmetric cell division. Previously, we demonstrated that the polarity kinase PKC-3 phosphorylates LIN-5 to inhibit its function. Our current analysis reveals four additional phosphorylated residues that are critical for LIN-5 function. Two of these residues contribute to the interaction of LIN-5 with its binding partner GPR-1/2, whereas the other two residues are critical in dynein motor recruitment by LIN-5. Together, our results reveal that multisite phosphorylation of LIN-5 is essential to ensure proper chromosome segregation and spindle positioning.Protein kinases control biological processes by phosphorylating specific amino acids of substrate proteins. It remains a major challenge to identify which phosphorylation events are critical  Animal development and tissue homeostasis depend critically on cell divisions that create cells with specific shapes and functions, in the right numbers and at the proper positions. The spindle apparatus plays a central role in the cell division process, as it segregates the chromosomes in mitosis and determines the plane of cell cleavage during cytokinesis –3. PlaceCaenorhabditis elegans and Drosophila melanogaster revealed that the position of the spindle responds to polarity cues during asymmetric cell division  mutation by introducing a single nucleotide alteration in the endogenous lin-5 locus. This resulted in a typical lin-5 loss-of-function phenotype, with homozygous sterile, thin and uncoordinated larvae that fail to undergo chromosome segregation but continue abortive mitoses [lin-5[L663S] mutants, both tissues contained severely reduced numbers of nuclei compared to the wild type, consistent with a failure to undergo chromosome segregation in most post-embryonic divisions , aspartic acid  or glutamic acid , all resulted in typical lin-5 mutant offspring. Regardless of the mutant combination, homozygous animals derived from heterozygous parents developed into sterile, thin and uncoordinated larvae, and showed severely impaired cell division during larval development  did not lead to any detectable phenotype or defects in cell division   did not reveal significant divergence from the wild type   Videos. lin-559A,S662Ames-1(bn74ts) mutant strain expressing GFP-β-tubulin, we observed normal spindle rotation in lin-5 mutant embryos, with only one of 13 embryos showing a tilted spindle angle in the EMS blastomere (mes-1(bn74ts); lin-5 mutant embryos showed an occasional failed rotation or tilted spindle angle. In control mes-1(bn74ts); gsk-3(RNAi) mutants, the EMS spindle failed to rotate in 9/11 embryos  / egfp::lin-5), LIN-5 localized normally, but the eGFP staining was lost , while two D residues and two phosphorylated serines are negatively charged in the LIN-5 peptide.mes-1/src-1 signaling  mutation and mes-1 knockdown did not interfere with A-P positioning of the spindle in EMS. Moreover, we could functionally replace serine 659 and 662 with glutamic and aspartic acid, suggesting that charge, rather than phosphoregulation, is critical for GPR-1/2 interaction.Phosphorylation offers the opportunity to regulate LIN-5–GPR-1/2 binding. In fact, dual GSK3 and CK1 phosphorylation of the LRP6 Wnt-co-receptor regulates the interaction of LRP6 with axin ,51. We dignaling . Even thXenopus and human NuMA through phosphorylation of the C-terminus  ATP. Reactions were terminated by the addition of SDS (5x sample buffer). For mass spectrometry analysis, no [γ-32P] ATP was added to the kinase assays and incubation time was prolonged to 2 hours at 30°C.For in vitro GSK3 and CK1 kinase assays, peptides (RRRIRCGSPDSLPDFLADN) containing either unphosphorylated, phosphorylated S659 or phosphorylated S662 were used. Kinases were incubated for 30 min at 25°C with synthetic peptide in kinase buffer containing 200 μM ATP, 50 mM HEPES at pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, supplemented with 20 μCi [γ-32P] ATP for radioactive kinase assays. Reactions were terminated by the addition of SDS (4x sample buffer).For in vitro kinase assays were performed as previously described  ATP for radioactive kinase assays. Reactions were terminated by the addition of SDS (4x sample buffer). For mass spectrometry analysis, no [γ-32P] ATP was added to the kinase assays and incubation time was prolonged to 2 hours at 25°C. Kinases used in this study were: recombinant C. elegans PAR-1 , and mammalian Aurora B (a kind gift from Susanne Lens), CK1 (New England Biolabs), CK2 (New England Biolabs), and GSK3 (New England Biolabs).In short, kinases were incubated for 30 min at 25°C with bacterially produced GST or GST–LIN-5 in kinase buffer containing 200 μM ATP, 50 mM HEPES at pH 7.5, 10 mM MgCl2 chromatography  / egfp::lin-5 embryos treated with egfp RNAi and stained with LIN-5 antibodies. Graphs indicate single values for centrosomes and cytoplasm in one- and two-cell embryos.(TIF)Click here for additional data file.S6 Fig(A) Averages of quantification of intestinal nuclei and P-cells plus juvenile motor neurons (P2-P10 region) by propidium iodide staining in wild type, homozygous LIN-5 phosphorylation mutants, and homozygous lin-5(e1348) null animals. Statistical analysis in s.e.m., analyzed by Graphpad PRISM. (B) Statistical averages of DIC microscopy imaging of hallmarks of the first 2 embryonic divisions in LIN-5 phosphorylation mutants. Oscillations are plotted in percentage of embryo height, elongation and AB size as a percentage of embryo width, flattening and rotation as a total fraction of analyzed embryos. Statistical analysis in s.e.m., analyzed by Graphpad PRISM. (C) Quantification of EMS spindle rotation in mes-1(bn74 ts); lin-5 phosphorylation mutant and mes-1(bn74 ts); gsk-3(RNAi) embryos. Spindle rotation was quantified by live-imaging of the gfp::tubulin marker. Wt indicates number of embryos with wild type rotation, defective A-P indicates number of embryos with a failure to fully align in the anterior-posterior direction, defective L-R indicates number of embryos with a failure to rotate in the left-right direction.(TIF)Click here for additional data file.S7 Figlin-5 and endogenously tagged egfp::gpr-1. Representative images of four-cell embryos, stained with anti-LIN-5 (red) and anti-eGFP (green) antibodies, and DAPI to visualize DNA. All images taken with same exposure time, objective and magnification. Anterior to the left, ventral up.Immunohistochemical staining of embryos expressing wild type or phosphomutant (TIF)Click here for additional data file.S8 Figegfp::lin-5 C. elegans embryos with anti-LIN-5 (red) and anti-GFP (green) antibodies, DNA stained with DAPI. Two representative embryos are shown for every condition. All images same objective and magnification, anterior to the left, scale bars 10 μm.Immunohistochemical staining of heterozygous and homozygous (TIF)Click here for additional data file.S9 Fig(A) Representative snapshots of live imaging of GFP::tubulin in gfp::tubulin one-cell embryos treated with or without perm-1 RNAi + 1 μM nocodazole, and imaged by spinning disk confocal microscopy. Scale bars, 10 μm, all images with same objective and magnification. (B) Representative snapshots of live imaging of mCherry::DHC-1 in mcherry::dhc-1; egfp::lin-5 one-cell embryos in prophase and metaphase treated with or without perm-1 RNAi in the presence or absence of 1 μM nocodazole, and imaged by spinning disk confocal microscopy. All images taken with same objective and magnification, anterior to the left, scale bars 10 μm.(TIF)Click here for additional data file.S1 VideoVideo of DIC time-lapse microscopy imaging of hallmarks of the first 2 embryonic divisions in a wild type embryo as quantified in (MP4)Click here for additional data file.S2 Videolin-5; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S3 Videolin-5[S397A]; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S4 Videolin-5[S397E]; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S5 Videolin-5; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S6 Videolin-5; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S7 Videolin-5; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S8 Videolin-5; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S9 Videolin-5; gfp::tubulin embryo as quantified in Representative video of UV laser spindle ablation experiments in a (AVI)Click here for additional data file.S10 Videomcherry::dhc-1; gfp::tubulin embryo as analyzed in Representative video of GFP::tubulin in a (AVI)Click here for additional data file.S11 Videomcherry::dhc-1; gfp::tubulin embryo as analyzed in Representative video of mCherry::DHC-1 in a (AVI)Click here for additional data file.S12 Videomcherry::dhc-1; gfp::tubulin embryo as analyzed in Representative video of GFP::tubulin (magenta) and mCherry::DHC-1 (gray) in a (AVI)Click here for additional data file.S13 Videomcherry::dhc-1; gfp::tubulin; egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of GFP::tubulin in a (AVI)Click here for additional data file.S14 Videomcherry::dhc-1; gfp::tubulin; egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of mCherry DHC-1 in a (AVI)Click here for additional data file.S15 Videomcherry::dhc-1; gfp::tubulin; egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of GFP::tubulin (magenta) and mCherry::DHC-1 (gray) in a (AVI)Click here for additional data file.S16 Videomcherry::dhc-1; gfp::tubulin; lin-5 / egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of GFP::tubulin in a (AVI)Click here for additional data file.S17 Videomcherry::dhc-1; gfp::tubulin; lin-5 / egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of mCherry::DHC-1 in a (AVI)Click here for additional data file.S18 Videomcherry::dhc-1; gfp::tubulin; lin-5 / egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of GFP::tubulin (magenta) and mCherry::DHC-1 (gray) in a (AVI)Click here for additional data file.S19 Videomcherry::dhc-1; gfp::tubulin; lin-5 / egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of GFP::tubulin in a (AVI)Click here for additional data file.S20 Videomcherry::dhc-1; gfp::tubulin; lin-5 / egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of mCherry::DHC-1 in a (AVI)Click here for additional data file.S21 Videomcherry::dhc-1; gfp::tubulin; lin-5 / egfp::lin-5 embryo treated with egfp RNAi as analyzed in Representative video of GFP::tubulin (magenta) and mCherry::DHC-1 (gray) in a (AVI)Click here for additional data file.S1 TableOverview of all strains used in this study, including the corresponding figure. CRISPR/Cas9 genome engineered strains are indicated in the comment section.(PDF)Click here for additional data file.


These data were correlated with clinical parameters and compared with peripheral blood mononuclear cells (PBMCs) (n = 10). Increased frequencies of T cells expressing co-inhibitory receptors LAG-3, PD-1, TIM-3 or CTLA-4, and co-stimulatory receptors CD28, OX40 or 4-1BB were observed in BPH tissue compared to PBMCs. These findings are consistent with chronic activation and possible functional exhaustion of PILs that may be further augmented by several identified pro-inflammatory factors, such as IL-8 and MCP-1, promoting inflammation and chemotaxis of immune cells to the prostate. Prostate size and plasma prostate-specific antigen levels positively correlated with IL-8 and MCP-1 concentrations, and frequencies of T cells expressing CTLA-4 and TIM-3. It remains to be established whether the link between inflammation and BPH progression supported by our findings reflects a progressive failure of the immune system leading to decreased immune surveillance and development of prostate cancer.Benign prostatic hyperplasia (BPH) is a common chronic non-malignant condition whose prevalence substantially increases with age. Immune cell infiltration and pro-inflammatory mediators have been implicated in the pathogenesis. Here, we characterized 21 extracellular markers on prostate-infiltrating lymphocytes (PILs) and analyzed expression of 26 soluble proteins in prostate tissue obtained from BPH patients ( Benign prostatic hyperplasia (BPH) refers to a non-malignant stromal and epithelial cell propagation resulting in enlargement of the prostate. BPH is a chronic progressive condition affecting approximately 10% of men at age of 30, gradually reaching prevalence of 80-90% for 70-80 year olds , 3. TreaKnowledge regarding the pathogenesis of BPH still remains fragmentary but its progression may result from multiple factors including changes in epithelial-stromal interactions, local endocrine and autonomous nerve system deregulations . Increasin situ immune activation.We performed a detailed comparative phenotypic characterization of prostate-infiltrating lymphocytes (PILs) freshly isolated from BPH tissue and their counterparts isolated from peripheral blood (PB). In addition, soluble factors in the prostate tissue were analyzed to identify pro-inflammatory components which may affect the microenvironment of BPH lesions. The overall goal of the study was to gain knowledge regarding the immunological involvement in BPH progression by correlating clinical parameters of the disease and n = 31) (Table n = 10) of BPH patients. Cell viability of PILs was maintained during processing with a median of 83.9% living cells  cells in BPH tissue compared to PBMCs  (Table n = 23), there were more CD8+ T cells present than CD4+ T cells.Comparing peripheral blood mononuclear cells (PBMCs) with PILs from BPH patients showed major differences in composition of analyzed immune cell subsets Figure . There wCs Table . Subdivi7) Table . In appr+CD45RO−) and central memory T cells (CCR7+CD45RO+) in BPH tissue as compared to PBMCs. The proportion of effector memory T cells (CCR7−CD45RO+) was increased while there was no difference in terminally differentiated T cells (CCR7−CD45RO−) (Table +/CD25highCD127−/low) in BPH tissue as compared to PBMCs  (Table There were significantly reduced proportions of naïve (CCR7−) Table . Further+ T cells, CD4+ and CD8+ T cells) as compared to PBMCs. CD8+ T cells expressing TIM-3 were almost completely absent in both PB and BPH tissue , programmed cell death-1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) or cytotoxic T lymphocyte-associated protein-4 (CTLA-4) were increased in frequency in BPH tissue in all T cell compartments  while CD8+/CD27+ were increased in BPH tissue  , and CD69 was expressed more frequently by CD8+ T cells than CD4+ T cells (76.5% vs. 55.5% in BPH tissue)    Figure .Patients were subdivided based on clinical parameters to elucidate potential reasons for the observed significant heterogeneity in T cell subset composition in BPH tissue between the patients. A positive correlation was found between prostate size and plasma prostate-specific antigen (p-PSA) level .Patients with larger prostate size (>61 g) had an increased frequency of all T cell compartments expressing CTLA-4 compared to those with smaller prostate size (≤61 g) Figure . There w+ T cells, γδ T cells and 4-1BB+ T cells . Representative plots are presented in Figure In contrast, frequencies of all compartments of T cells expressing CD69 were decreased in patients with larger prostate size Figure  and a nes Figure  and 2D as Figure . A negat+ T cells expressing CD69 was decreased in patients with high p-PSA (>10 μg/L) compared to patients with low p-PSA (<4.5 μg/L) . Frequency of total T cells expressing 4-1BB was limited in patients with low and intermediate (>4.5-10 μg/L) p-PSA but statistically increased in patients with high p-PSA  Figure , also shA Figure , and a p+ T cells expressing TIM-3 were increased in frequency in patients with high p-PSA compared to those with low p-PSA . Representative plots are presented in Figure CD158b-expressing total T cells were increased in frequency in patients with intermediate p-PSA and high p-PSA compared to patients with low p-PSA Figure  and therA Figure . A positn = 27) (p=0.006). The same pattern was observed with CD4+ T cells expressing LAG-3  showed an increased frequency of total T cells expressing LAG-3 compared to patients without histological signs of malignancy  had an increased frequency of CD4t Figure   were excluded as being undetectable in the analyzed samples, and the remaining are presented in Figure Granulocyte-colony stimulating factor (G-CSF), monocyte chemotactic protein-1 (MCP-1), IL-7, IL-8 and IL-15 were increased in BPH tissue of patients with larger prostate size, while the concentration of eotaxin was reduced compared to patients with lower prostate size Figure . ProstatIL-8, MCP-1 and interferon gamma-induced protein-10 (IP-10) were elevated in BPH tissue of patients with high p-PSA compared to patients with low p-PSA Figure . There wThe role of the immune system in the pathogenesis in BPH is uncertain, but prostatic inflammation is commonly found in BPH patients , 8. EarlWe found indications of chronic activation supported by presence of T cells expressing activation-induced co-inhibitory receptors, co-stimulatory receptors and high frequency of potential regulatory T cells in BPH tissue. Previous studies have shown that BPH-derived epithelial and stromal cells produce pro-inflammatory cytokines and chemokines , 11, 12.+ T cell subgrouping into CD4+ and CD8+ T cells, we found a clear predominance of CD8+ T cells in BPH tissue consistent with previous findings in a large cohort of BPH specimens [+ T cells [In the current study, lymphocytes isolated from BPH tissue differed greatly in subset composition compared to lymphocytes isolated from PB. This is to some extent expected as the role of the immune system differs in these different bodily environments. Regarding CD3pecimens . In the pecimens , possibl T cells , 10.Our results revealed increased frequencies of T cells expressing co-inhibitory receptors  in BPH tissue compared to PBMCs; T cells expressing PD-1 being the most abundant subset. These co-inhibitory receptors are up-regulated in response to activation, and through different pathways they dampen and control an active immune response –18. IncrInterestingly, frequencies of several lymphocyte subsets expressing co-inhibitory receptors correlated with clinical parameters. Increasing prostate size and/or p-PSA can be seen as signs of long BPH development  and, spen = 4), T cells expressing LAG-3 were significantly elevated in frequency in these patients. Speculatively, this could be a mechanism of immune evasion by tumor cells and warrants further studies elucidating the prevalence of LAG-3 expression in prostate tissue of PC patients. In a study by Sfanos and colleagues, high frequency of PD-1+CD8+ T cells was reported [www.ClinicalTrials.gov, NCT01968109). Additional profiling of co-inhibitory receptors in PC is warranted for predictive purposes in future treatments utilizing these immunotherapeutic approaches.Despite the low number of patients diagnosed with malignancy (reported . Antibod+ T cells expressing CD25 (α chain of the IL-2 receptor) and CD8+ T cells expressing co-stimulatory receptor CD27.This medication effectively limits the amount of available dihydrotestosterone (DHT), a more potent form of testosterone, in blood and prostate. Based on functional assays, it has been suggested that DHT acts as an anti-inflammatory factor for CD4+ T cells, inhibiting their IL-2 production [Androgen-depleting treatment with 5-ARI was associated with increased frequency of CD4oduction . With ouin vitro assays, IL-8 and MCP-1 can be produced by epithelial and/or stromal cells of the prostate [The concentrations of several pro-inflammatory factors were found to be correlated to clinical parameters, in particular prostate size and p-PSA. Elevated serum-PSA levels have previously been shown to positively correlate with presence and extent of inflammation , 24. Herprostate , 11, 12.prostate . In suppprostate , 25. Theet al. [+ T cells resulted in secretion of multiple soluble proteins; IL-6, IL-8, IP-10, MCP-1, G-CSF and eotaxin being found most abundant. In addition, Penna et al. [in vitro culturing studies. Here, we confirm presence of these proteins in vivo and correlated them with prostate size and/or p-PSA.Vignozzi et al.  have shoa et al.  have sugIn conclusion, BPH tissue was found to be a pro-inflammatory, chemotactically attractive site in which chronic activation could induce exhaustion of prostate-infiltrating T cells. There is currently no direct evidence that BPH is a precursor of malignant transformation , 27. HowIdeally, the results of the current study should have been strengthened by comparative analysis of healthy prostate tissue. This was, however, not possible due to ethical constrains and logistic limitations. Nevertheless, this matter needs to be addressed in follow-up studies. Detailed characterization of infiltrating immune cells and the microenvironment in healthy, BPH and malignant prostate tissue could help to develop new approaches to prophylactic and therapeutic treatment of prostate cancer.This study has been conducted in accordance with the Declaration of Helsinki, according to national/international guidelines, and was approved by the Regional Ethical Review Board in Stockholm, Sweden . Patients were informed about the study and gave their consent if willing to participate.n = 25) or adeno enucleation (AE) (n = 6) surgical procedure at Karolinska University Hospital Huddinge, Sweden  . P-PSA level was measured in μg/L and patients were divided into three groups: low <4.5 μg/L (n = 10); intermediate >4.5-10 μg/L (n = 7); and high >10 μg/L (n = 6). Grouping based on androgen-depleting treatment with 5-ARI, created two groups; yes (n = 16) and no (n = 10). Grouping based on treatment with α-blockers created two groups; yes (n = 5) and no (n = 24), but did not result in any significances. Presence of urinary tract infection was determined by urine bacteria culture prior to surgery and created two groups; patients with a positive (n = 15) or negative (n = 14) culture. Based on pathology report, four patients were histologically diagnosed with BPH and PC . These patients were compared with patients without malignancy (n = 27).Clinical parameters were used to subgroup the patients Table . Prostatg for 7 min , and supernatant was decanted, collected and stored at −80°C until time of multiplex analysis. The cell pellet was resuspended in PBS and mononuclear cells were isolated using density gradient centrifugation with Lymphoprep  as previously described [Isolation of PILs was performed using a recently described protocol involving immediate tissue processing without any enzymatic treatment or long incubation steps, and performing acquisition of fresh cells . Brieflyescribed .PBMCs from blood samples were separated using Lymphoprep as described above. Cells were frozen in complete RPMI medium with 10% DMSO and stored in liquid nitrogen. At day of analysis, PBMCs were thawed in complete RPMI and washed twice with PBS. The complete 1640 RPMI medium  contained 2mM L-glutamine , 100 U/mL penicillin G (Thermo Scientific), 100μg/mL streptomycin (Thermo Scientific) and 10% human AB serum .Cells were stained with titrated antibodies in an extracellular nine-color flow cytometry panel and incubated for 15 min in the dark at 4°C. All included antibodies were monoclonal and of mouse origin. Fluorescein isothiocyanate (FITC)-conjugated anti-CD28 (CD28.1); anti-CD69 (FN50); anti-CD94 (HP-3D9); anti-CD134/OX40 (ACT35); anti-CD158b (CH-L); phycoerythrin (PE)-conjugated anti-CD25 (M-A251); anti-CD27 (M-T271); anti-CD56 (NCAM16.2); anti-CD279/PD-1 (MIH4); PE-CF594-labeled anti-CD197/CCR7 (150503); PE-Cy7-conjugated anti-CD3 (SK7); allophycocyanin (APC)-conjugated anti-CD19 (HIB19); anti-CD45RO (UCHL1); Alexa700 (A700)-conjugated anti-CD56 (B159); APC-Cy7-conjugated anti-CD8 (SK1); V500-conjugated anti-CD4 (RPA-T4); and isotype controls for FITC, PE and APC IgG1 (X40) were purchased from BD Biosciences. FITC-conjugated anti-TCRγδ pan (IMMU510); APC-A700-conjugated anti-CD127 (R34.34) and krome orange-conjugated anti-CD4 (13B8.2) were purchased from Beckman Coulter . FITC-conjugated anti-CD137/4-1BB (4B4-1), anti-CD152/CTLA-4 (A3.4H2.H12), anti-CD223/LAG-3 (17B4), and APC-conjugated anti-CD366/TIM-3 (F38-2E2) were purchased from LifeSpan Biosciences . Isotype control for FITC IgG2a (MOPC-173) was purchased from Biolegend .g and washed with PBS once. Viability dye 7-aminoactinomycin D  was added according to manufacturer's instructions and incubated at room temperature in the dark for 10 min. PBS was added to dilute the cell suspension 1:4.After cell surface staining, cells were centrifuged for 4 min at 700−/CD3+ cells and further subpopulations of CD3− and CD3+ T cells were defined (+ cells), CD4+ and CD8+ T cells (both gated from CD3+ cells).Cells were acquired on a BD Canto I SORP and BD FACSAria using BD FACSDiva Software v.7.0 and v.6.1.3 respectively (BD Biosciences). Data was analyzed and displayed using FlowJo v.10 . Single-color stained samples, fluorescence-minus-one samples and isotype controls were used for compensation, gating and for background correction . Gates t defined . Frequenn = 31). MILLIPLEX MAP Human Cytokine/Chemokine Premixed 26 Plex  was used according to manufacturer's protocol and as described before [Fluorescent bead-based multiplex immunoassay was used to measure 26 cytokine/chemokine concentrations in BPH tissue supernatant collected during processing  and Microsoft Excel 14.5 . Non-parametric Mann-Whitney U-test was used to determine significant differences between PB and BPH tissue and also when comparing patient subgroups. Significance levels were set to *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. Non-parametric Spearman's rank correlation coefficient was used to determine statistical correlation between different parameters. Nonlinear regression (least squares ordinary fit and robust fit straight line) was used to plot parameters.