The bromodomain and extra-terminal domain (BET) family of proteins, especially BRD4 play an important role in epigenetic regulation, and are essential for cell survival and also are promising anticancer targets. This study aims to analyze the effect of BRD4 on the cell growth and progression of pancreatic cancer and novel mechanisms involved.Expression of BRD4 in pancreatic cancer and paired adjacent noncancerous tissues from 76 patients was analyzed by western blotting, immunohistochemistry, and real time PCR. Its correlation with the clinicopathological characteristics and prognosis of pancreatic cancer patients was analyzed. The effects of BRD4 on the cell proliferation were detected by colony formation assay and sulforhodamine B assay. Migration and invasion were determined by Transwell assays, and the effect of BRD4 on subcutaneous tumor formation was verified in nude mice. Cell cycle analysis was detected by flow cytometry. The potential downstream targets of BRD4 and related molecular mechanisms were clarified by RNA sequencing, chromatin immunoprecipitation and dual luciferase reporter assay.BRD4 was overexpressed in pancreatic cancer. Biological results showed that BRD4 functioned as tumor promoter, facilitated cell proliferation, migration and invasion in vitro and in vivo. Further, caveolin-2 was selected as the downstream gene of BRD4 by RNA sequencing. Caveolin-2 overexpression can partially reverse the decreased cell growth ability caused by BRD4 knockdown, but did not affect cell migration and invasion. Chromatin immunoprecipitation assay and dual luciferase reporter assay revealed BRD4 could bind to the promoter region of caveolin-2 and upregulate caveolin-2 expression. Clinical data further indicated a positive correlation between BRD4 and caveolin-2 expression. BRD4 (high)/caveolin-2 (high) correlated with shorter overall survival of patients with pancreatic cancer. Multivariate analysis revealed that both BRD4 and caveolin-2 were independent factors.Our findings reveal the oncogenic effects of BRD4 in pancreatic cancer and elucidate a possible mechanism by which BRD4 and caveolin-2 act to enhance cell growth. Targeting the BRD4-caveolin-2 interaction by development of BET inhibitors will be a therapeutic strategy for pancreatic cancer. Pancreatic cancer (PC) is a highly lethal disease, for which mortality closely parallels incidence. It is expected to become the second leading cause of tumor-related mortality in the United States by 2030 . DespiteEpigenetic regulation has become an alternative target for pancreas development and disease , 5. By iWritten consents were obtained from all subjects. This study was approved by the Research Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiaotong University and the first affiliated hospital with Nanjing Medical University. Written informed consent was obtained from all subjects.Seventy-six PC tissue samples and adjacent normal paraffin-embedded tissue samples were collected from the Department of Pathology, the first affiliated hospital with Nanjing Medical University, between January 2012 and December 2016. All of the patients were diagnosed and confirmed by histology. Tissue microarray (TMA) constructed by Shanghai Outdo Biotech Company (China), contained well-documented clinicopathological information, including age, gender, clinical stage, invasion depth, lymph nodes metastasis, distant metastasis, tumor differentiation, tumor location, nervous invasion, vessel invasion, and follow-up data . Detailed information can be found in Additional file Additionally, liquid nitrogen frozen PC tissue samples and adjacent normal tissues were obtained by surgical resection from ten patients at the department of bile and pancreatic surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University .2O2 block, and antigen retrieval was performed after heating in citrate buffer. The sections were incubated with an antibody against BRD4  and CAV-2  at 4 °C overnight and were incubated with biotinylated secondary antibodies.TMA were deparaffinized in xylene and rehydrated in a graded alcohol. 3% HA double-blind scoring was performed independently by two investigators. BRD4 and CAV-2 expression were scored according to staining intensity and the percentage of positive cells as previously described . The per2 and saturated humidity.Human normal pancreatic ductal cells HPDE and PC cell lines AsPC-1, BxPC-3, CFPAC-1, PANC-1, MIAPaCa-2, SW1990 and SU.86.86 were all obtained from Chinese Academy of Sciences Cell Bank . All cell lines were last authenticated in January 2018 by polymorphic short tandem repeat analysis after a series of experiments, and used for a maximum of fifteen passages. SW1990 was cultured in L-15 medium supplemented with 10% fetal bovine serum , and grown in room temperature air. PANC-1 was cultured in DMEM supplemented with 10% FBS, MIAPaCa-2 in DMEM supplemented with 10% FBS and 5% horse serum, the other cells in RPMI-1640 supplemented with 10% FBS, grown at 37 °C in 5% COBRD4 nc/siRNA/control/overexpression, as well as CAV-2 nc/siRNA/control/overexpression were all designed and purchased from GeneChem . SiRNA targeting BRD4  was used to knockdown the expression of BRD4. And siRNA targeting CAV-2  was used to downregulate the CAV-2 expression. Negative control siRNA  and BRD4/CAV-2 siRNA (4 µg) were transiently transfected using 8 µl Lipofectamine 2000  according to manufacturer’s instructions. PANC-1 was infected with lentiviral particles containing specific or negative control vectors  and the polyclonal cells with puromycin resistance (1 µg/ml) were selected for further experiments. The short hairpin RNA sequences for targeting the BRD4 gene were as follows. BRD4-sh1, sense 5′-CcggCACGGTACCAAACACAACTcTCAAGAGAAGTTGTGTTTGGTACCGTGTTTTTTg-3′, antisense 5′-aattcaaaaaaCACGGTACCAAACACAACTTCTCTTGAgAGTTGTGTTTGGTACCGTG-3′. BRD4-sh2, sense 5′-CcggCTACACGACTACTGTGACAcTCAAGAGATGTCACAGTAGTCGTGTAGTTTTTTg-3′, antisense 5′-aattcaaaaaaCTACACGACTACTGTGACATCTCTTGAgTGTCACAGTAGTCGTGTAG-3′.According to the above steps, BRD4-nc and BRD4-si1 were transfected into PANC-1 cells. After verifying the siRNA efficiency of transient transfection, each group with three repetitions was sent to Shanghai Bio Technology Corporation for purification and sequencing analysis .Sequencing raw reads were preprocessed by filtering out rRNA reads, sequencing adapters, short-fragment reads and other low-quality reads. We used Tophat v2.1.0  to map t® reagent . The PrimeScript RT reagent kit  was used to synthesize complementary DNA, which was used in the qPCR  with a 7500 Fast Real-time PCR system . The RT reactions were performed as follows: 37 °C for 15 min and 85 °C for 5 s. PCR amplification was performed in a thermal cycler for 40 cycles at the following cycle conditions: 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. The GAPDH expression was detected as the endogenous control. Primer sequences are shown in Additional file Total RNA from cells or tissues was extracted using TRIzolWhole-cell lysates were prepared in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fuoride  and protease inhibitor cocktail . The concentration of protein was quantified with the Bradford Method using Coomassie Blue Staining Solution . The protein lysates were separated on 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Following blocking with 5% BSA, the membrane was incubated with primary antibodies against BRD4 , CAV-2 , c-Myc , PCNA , CDK2 , pRb , β-actin  and GAPDH  at 4 °C overnight followed by incubation with secondary antibodies.Briefly, ~ 800 cells were added to each well of a 6-well culture plate. After 2 weeks of incubation, cell colonies were washed twice with PBS, fixed with 4% para -formaldehyde for 15 min and then stained with 0.5% crystal violet for 30 min. The excess stained was removed. Individual clones with ≥ 50 cells were counted.Cells seeded in 96-well plates under different treating conditions were cultured for 24, 48, 72, and 96 h. Optical density (OD) values were measured at 560 nm by microtiter plate reader (VERSMax).The migration assay was detected by Transwell chambers  with or without pre-coated thin layer of Matrigel (BD Biosciences). Cells were allowed to migrate towards the bottom wells. After 24 h of incubation, the migrating cells were stained with the crystal violet solution.For cell apoptosis analysis, cells were harvested at 70–80% confluence under different treating conditions, and incubated with reagent containing Annexin V-FITC and propidium iodide (BD Biosciences) for 15 min in darkness at room temperature. Apoptotic cells were analyzed using FACSCalibur flow cytometer (BD Biosciences). For cell cycle analysis, cells were fixed in 70% ethanol at 4 °C overnight and then treated with RNase A (50 µg/ml) and stained with propidium iodide (25 µg/ml) for 30 min at 37 °C. Distribution of cell-cycle phases was determined using ModFit software (BD Biosciences).ChIP was performed following the manufacturer’s protocol for Simple Chip plus Enzymatic Chromatin IP Kit . The aforementioned anti-BRD4 antibody  was used for the ChIP assay. The purified DNA was analyzed by RT-qPCR. ChIP primers of CAV-2 were described in Additional file 3 cells/well, and cotransfected with BRD4-nc/BRD4-si1 and plasmid containing CAV-2 promoter regions  using Lipofectamine™ 2000. After 48 h, Dual Luciferase® reporter assay system (Promega Corporation) was used to detect fluorescence intensity. Renilla luciferase gene was used as the internal reference to verify the transfection efficiency and calculate the relative luciferase activity as follows: Relative luciferase activity = firefly luciferase activity/Renilla luciferase activity.Cells were plated in 96-well plates at 4 × 106 cells/each) were subcutaneously implanted into BALB/c nude mice, respectively. A total of 15 nude mice were used with 5 mice in each group. Tumor size was measured every 3 days and recorded by using the following equation: Volume (mm3) = (length × width2)/2. After 30 days, the mice were sacrificed, and the tumors were excised, weighed, harvested and embedded in paraffin. The BRD4 , ki-67  and cleaved Caspase 3  expressions were detected by immunohistochemical staining respectively.The animal experiments were conducted according to the guidelines for animal experimentation and approved by the Experimental Animal Ethics Committee of Shanghai Jiaotong University. Stable transfected cell lines PANC-1-BRD4-nc, PANC-1-BRD4-sh1, or PANC-1-BRD4-sh2 . Univariate and multivariate COX regression analysis was performed. Those parameters with a p-value < 0.05 in the univariate analyses were included in a COX multivariate proportional hazards regression model. All statistical analyses were carried out using SPSS 17.0. Differences were deemed statistically significant at p < 0.05.Data were presented as the mean ± one standard deviation (SD) from at least three separate experiments. The Student’s test, one-way analysis of variance, χTen fresh samples validated that high BRD4 mRNA expression in PC compared to adjacent normal tissues Fig. a. The exGiven the fact that CFPAC-1 and PANC-1 have relatively higher endogenous BRD4 expression, we chose them for BRD4 silencing experiments. Western blot was used to confirm the efficacy of BRD4 knockdown Fig. a. SulforTo further investigate whether interference with BRD4 could inhibit tumor growth in vivo, we constructed stable inhibition of BRD4 cell line in PANC-1. Subcutaneous tumor transplantation test showed that, compared with PANC-1/BRD4-nc, PANC-1/BRD4-sh1 and PANC-1/BRD4-sh2 groups had slower growth rate Fig. a and tumRNA sequence was employed to identify the downstream target of BRD4. We found that there were 27 differentially expressed gene, 12 up regulations and 15 down regulations . ChIP results showed that BRD4 can directly bind to the promoter region  of CAV-2 Fig. a. FurtheThe correlation between BRD4 and CAV-2 protein levels and clinicopathological characteristics of PC were further evaluated. As shown in Table Kaplan–Meier analysis revealed that patients with higher BRD4 Fig. a and CAVThe bromodomain and extra-terminal domain (BET) family of proteins, especially BRD4 has emerged as regulating multiple genes expression involved in cell cycle, cell growth and inflammation due to their important roles in epigenetic regulation , 20. UnbHowever, how BRD4 regulates cell growth, migration and invasion is not clear. Sahai et al.  found thWe further identified the downstream targets of BRD4 by RNA sequencing. There were 27 differentially expressed genes, 12 up-regulated and 15 down-regulated. Among them, the downregulation of CAV-2 was shown to be the second most. CAV-2 is a member of the caveolae family and plays a vital role in intracellular cell transport and signal transduction . Higher The correlation between BRD4 and CAV-2 protein levels and clinicopathological characteristics of PC were further evaluated. BRD4 was positively correlated with clinical stage, lymph nodes metastasis, nervous invasion, and vessel invasion. And CAV-2 was positively correlated with clinical stage, invasion depth, lymph nodes metastasis, and tumor differentiation. Survival analysis results showed that patients with higher BRD4 and CAV-2 expression possessed worse prognosis. Multivariate COX analysis revealed that BRD4 and CAV-2 were independent factors in determining prognosis of patients with PC. Moreover, there was a positive correlation between BRD4 and CAV-2 expression.In sum, our findings reveal the oncogenic effects of BRD4 in PC and elucidate a possible mechanism by which BRD4 and CAV-2 act to enhance PC growth. Given that overexpression of both BRD4 and CAV-2 are observed in PC, targeting the BRD4-CAV-2 interaction by developed BET inhibitors may be applicable to prevent tumor growth in PC.Additional file 1: Table S1. Detailed clinical information and follow-up data of 76 patients with PC. Table S2. The primer sequence of BRD4, CAV-2 and GAPDH. Table S3. The ChIP primer sequence of CAV-2. Table S4. Differentially expressed genes following BRD4 knockdown.Additional file 2: Fig. S1. The diagram of gene oncology enrichment.Additional file 3: Fig. S2. The diagram of gene oncology classification.


Amyloid-beta oligomers (AβO) have been proposed as the most potent neurotoxic and inflammation inducers in Alzheimer’s disease (AD). AβO contribute to AD pathogenesis by impairing the production of several cytokines and inflammation-related signaling pathways, such as the Janus kinases/signal transducer of transcription factor-3 (JAK/STAT3) pathway. STAT3 modulates glial activation, indirectly regulates Aβ deposition, and induces cognitive decline in AD transgenic models. However, in vivo studies using an AβO microinjection rat model have not yet explored STAT3 role. The main purpose of this study was to elucidate if a single microinjection of AβO could promote an increased expression of STAT3 in glial cells favoring neuroinflammation and neurodegeneration. We designed a model of intrahippocampal microinjection and assessed glial activation, cytokines production, STAT3 expression, and neurodegeneration in time. Our results showed robust expression of STAT3 in glial cells (mainly in astrocytes) and neurons, correlating with neuronal death in response to AβO administration. A STAT3 inhibition assay conducted in rat primary hippocampal cultures, suggested that the induction of the transcription factor by AβO in astrocytes leads them to an activation state that may favor neuronal death. Notwithstanding, pharmacological inhibition of the JAK2/STAT3 pathway should be focused on astrocytes because it is also essential in neurons survival. Overall, these findings strongly suggest the participation of STAT3 in the development of neurodegeneration. Alzheimer’s disease (AD) is a neurodegenerative condition characterized by the presence of intracellular neurofibrillary tangles and extracellular neuritic plaques (NP), induced by beta-amyloid (Aβ) peptide accumulation. For years, the Aβ aggregation hypothesis has been proposed as the central mechanism underlying the development of the disease, yet it is still controversial ,2,3. Aβ Neuroinflammation is another indirect mechanism of Aβ fibrils and oligomers that leads to neurotoxicity and precedes cognitive decline. Aβ is recognized through different receptors located in glial cells, such as the T-cell ligand receptors, NOD-like receptors, formyl peptide receptors, and scavenger receptors . This reSeveral models have assessed the AβO toxic effects. Studies using astrocytes and microglial cultures have revealed that AβO are potent proinflammatory inducers in glial cells in comparison to fibrils, but the latter could be responsible for chronic inflammation establishment ,26. NeveSignal transducers and transcription activators (STAT) are a family of cytoplasmic factors activated by phosphorylation through the Janus kinases (JAK) pathway, primed by several cytokines. Cytokines such as IL-6 induce the JAK2/STAT3 pathway and regulate several transcription factors linked to inflammation . FurtherWe investigated the effects of AβO on neurodegeneration linked to neuroinflammation along 18 h, 72 h, and 7, 15 and 30 days, after its intrahippocampal microinjection. We aimed to identify STAT3 expression, and our findings suggest an increase of STAT3 expression in glial cell, induced by AβO. For this, two JAK2/STAT3 inhibitors were tested in primary cell culture, demonstrating the relevance of the pathway in astrocytes activation, a process linked to neurodegeneration development. The JAK2/STAT3 pathway modulation could be a promising strategy to control neuronal death, only directed towards astrocytes because if it carried out in neurons could contribute neurodegeneration. Future pharmacological research could focus on the modulation of the signaling pathways that regulate the glial phenotype, this would allow elucidating the neuroinflammation and neurodegeneration process present in AD. p < 0.05), compared with saline solution treatment at 18 h in the CA1 region. After 72 h, the number of FJB positive cells decreased in AβO and SS groups, remaining discretely higher (p < 0.05) in rats injected with AβO. Although the CA1 region showed an apparent increase of FJB cells in the AβO group at day 7, the difference was not significant. In contrast, FJB positive cells were evident in the dentate gyrus (DG) region of AβO microinjected rats sacrificed at 72 h and 7 days post-injection. Even though there is an apparent reduction of FJB cells in this time window, the statistic analysis did not show differences between the two sacrifice time points.Neuronal degeneration was evaluated with the Fluoro-Jade B (FJB) staining in rats sacrificed at 18 h, 72 h, and 7, 15, and 30 days after AβO microinjection. As shown in p < 0.05) . For this reason, we quantified the neuronal nuclei protein (NeuN) positive cells in the hippocampus A, at the < 0.05) B. p < 0.05). AβO-injected rats showed a higher neuronal loss over time. The spaces in the preparations left by dead cells were detected mainly in DG of rats treated with oligomers and sacrificed at day 15, correlating with a significant decrease of NeuN positive cells in AβO group (p < 0.05) (p < 0.05). This observation may be related to a recovery phenomenon in the DG region [Furthermore, immunohistochemistry (IHC) evidenced neuronal death, some dystrophic neurites and pyramidal cell disarray promoted mostly by AβO compared with the SS injection. Also, we found a moderate de crease of NeuN positive cells between 15 and 30 days in control groups ( < 0.05) B. Such nG region , which iA theory for AβO toxicity is by means of glial activation, which induces an inflammatory microenvironment and thus neurodegeneration. We therefore sought to identify the presence of mature neurons with an anti-NeuN antibody, microglial cells with an anti-Ionized calcium-binding adapter molecule 1 (Iba1) antibody, and an anti-glial fibrillary acidic protein (GFAP) antibody to detect astrocytes.In The immunofluorescence at day 15 showed a reduction of astrocyte hypertrophy in the control group as compared to day 7 C. SimilaTransmission electron microscopy allowed us to analyze the time course of ultrastructural changes induced by AβO. AβO favored neuronal loss in rats at 18 h when compared to the control group, where the cellular structure was conserved as shown with FJB staining A.Despite the mechanic injury in the control group, cellular ultrastructure and density remained conserved after 72 h. Edema and vacuoles in the neuropil were evident in oligomer-treated rats, suggesting neuronal loss. Also, neurons suffered severe cytoplasm disruption followed by loss of organelles, such as endoplasmic reticulum and mitochondria. Moreover, we detected swollen astrocytes and vacuolated microglial cells surrounding neurons on the degeneration process, correlating with the activated morphology observed by immunofluorescence. Other observations at this time after AβO injection were the presence of swollen mitochondria, axon degeneration, and amyloid-like fibrils, whereas these were absent in the control group B. At day 7, the AβO group showed the presence of vacuoles in the neuropil, endoplasmic reticulum disruption, axonal degeneration, abundant neuronal loss spaces, vacuolated microglia, and cytoplasm disruption A. In addAfter 15 days, we found an apparent structural recovery in the SS and AβO rats B. The coInterestingly at day 30, microstructural recovery was observed in the cytoplasm and mitochondria of neurons in AβO-injected rats. However, activated microglia and axonal degeneration persisted, none of these changes were found in the control group C.p < 0.01 for IL-1β and IL-10, p < 0.05 for IL-6) was noticed in AβO rats sacrificed at day 30. Glial cell activation by AβO leads to the release of several inflammatory mediators. For this reason, we quantified the proinflammatory cytokines IL-1β, IL-6, and the anti-inflammatory IL-10 in rat hippocampus homogenates using ELISA. No significant differences of IL-1β and IL-10 between the rats microinjected with AβO nor saline solution at 18 h, 72 h, and 7 and 15 days A were obp < 0.001), 72 h (p < 0.001), 7 days (p < 0.001), and 30 days (p < 0.01).IL-6 levels in AβO-injected rats decreased in 18 h, 72 h, and 7 d. Next, we evaluated IL-6 transcripts by qPCR B. In conWe next aimed to identify if the IL-6 signaling downstream transcription factor STAT3 was activated. Previously has been reported that STAT3 participate inducing neuronal death , for thiAt 72 h after microinjection B, an eviThe 7-day control group rats C exhibitHigh IL-6 mRNA levels B correlaFurthermore, results from AβO rats at day 15 suggest a high glial STAT3 expression in CA1 and DG, similarly with day 7. These cells display a hypertrophic morphology, not presented by saline solution rats D.Few STAT3 glial cells with resting morphology were detected in the cortex of control rats at day 30 E. AβO-inTo identify STAT3 localization, we performed immunofluorescence at 72 h and 7 days, where we detected consistent neuronal death. The vast majority of STAT3 positive cells were astrocytes and presented nuclear stain . InteresAn inhibition assay was conducted to evaluate the STAT3 expression in neurons and astrocytes exposed to AβO. Considering our previous results, we decided to expose a primary mixed hippocampal culture for 72 h to a 20 µM AβO solution from the human peptide. We initially used AG490 (25 and 50 µM), a JAK2 inhibitor, without detecting significant changes in total STAT3 expression in cultures both exposed to AβO and to AG490 25 µM+AβO . HoweverNext, we decided to probe Stattic, a small-molecule inhibitor of STAT3 expression  at diffep < 0.05) in comparison to vehicle, Stattic, and Stattic+AβO (p < 0.05). In contrast, in neurons, we identified a significant reduction in STAT3 integrated density in AβO treated cultures (p < 0.05).On 5 µM Stattic incubation, STAT3 B was expttic+AβO E. The Asttic+AβO  presenteSeveral AD genetic models have been developed to investigate Aβ toxicity. It has been thought that only Aβ fibrils or plaques could induce neuronal death in transgenic mice . HoweverAnother way of AβO-induced neuronal death comes from astrocytes and microglia proliferation and activation. It has been proposed that both cells could mediate the Aβ aggregates phagocytosis, particularly fibrils, displaying a protective role. In contrast, AβO could induce a deleterious inflammatory microenvironment that exacerbates neurodegeneration, with impaired phagocytic activity ,25,46,47Results show that our AβO preparation promoted astrocyte and microglia proliferation. The relation between glial activation and neurotoxicity was the presence of hypertrophic astrocytes and microglial cells with swollen or amoeboid morphology, and neuronal loss. The highest number of activated glial cells was observed at 72 h, correlating with extensive degeneration visualized in DG with the FJB staining. Interestingly, the proportion of activated astrocytes was higher than microglia, though this myeloid cell was located near to neuron loss spaces. Several reports have suggested that activated astrocytes could contribute to Aβ production and aggregation ,52. A reThe ultrastructural analysis allowed us to validate the close relation between neuronal alterations and glial activation. At 18 h, neuropil alterations and the low number of neurons correlated with the increased neurodegeneration observed by FJB staining. Also, significant changes in neuron morphology are reflected by cytoplasm disruption and loss of organelles, accompanied by surrounding hypertrophic astrocytes. Moreover, a high degree of disruption of myelin sheaths also suggests axon degeneration. Changes of the inner membrane and cristae of mitochondria are synonymous of mitochondrial dysfunction. Both abnormalities induced by Aβ aggregates have been previously described ,54. SpacThe presence of amyloid-like fibrils, as well as of intracellular AβO, should be confirmed in future experiments of immunoelectron microscopy in the AβO rats. There is a high probability that a part of the AβO preparation could form fibrils due to the physiological temperature and pH conditions of rat brain tissue. This is supported by the fact that we were able to identify activated microglia near those fibrils and that they were not observed at later times, probably due to phagocytosis.Although at day 30 in AβO rats, a reduction in cytoplasmic alteration was observed, Aβ continued exerting neurotoxicity effects and promoting inflammation, due to the presence of axonal degeneration, as well as microglia with amoeboid morphology and organelle phagocytosis, suggesting that Aβ is capable of inducing chronic neuronal death, altering DG neurogenesis, and decreasing cell proliferation .Considering the presence of glial activation in all the evaluated times, we quantified IL-1β, IL-6, and IL-10, which have been associated with beneficial or detrimental activity related to Aβ aggregates neurotoxicity ,57,58. IAbout the IL-1β and IL-6 significant increase in AβO rats at day 30, we suggest these proinflammatory cytokines mostly derive from the high number of activated microglial cells. Previous reports have indicated the IL-1β role in the induction of glial activation and neuron damage . DespiteIL-10 is a cytokine, which reduces inflammation and apoptosis. For this reason, it has been proposed as a possible therapeutic target in several CNS disorders, such as AD . HoweverIn the present study, IL-10 expression was observed in AβO rats after 30 days and could be favoring neuron density as well as decreased neuron alterations aforementioned. Despite IL-10 expression, confocal an electron microscopy showed that phagocytosis was not diminished in microglia, which may be due to high levels of IL-1β and IL-6. This data suggests the need for a balance in the cytokine microenvironment, rather than a predominant inflammatory profile. IL-1 and IL-6 are proinflammatory cytokines; however, the role of both plays in the inflammatory process may be different. It is known that IL-6 is a pleiotropic cytokine. Reported studies indicate that the overexpression of IL-6 in CNS generates the presence of reactive gliosis that can lead to neurodegeneration. On the other hand, it is known that IL-10 has a fundamental role in limiting chronic and acute inflammation. However, under pathological conditions, it is known that the levels of pro- and anti-inflammatory cytokine may be elevated. This has been demonstrated in the CSF of patients with AD . HoweverAn interesting finding of our experiments was a reduction of IL-6 in AβO rats from 18 h to 7 days. For this reason, we contrasted these results with relative IL-6 mRNA expression, detecting high mRNA levels in AβO administrated rats compared with the control group. It has been documented that the underestimation of cytokine production is possibly due to cellular consumption in cytokine quantification by ELISA , which cWe think that 18 h to 7 days, exists the possibility that IL-6 could be bound to its soluble receptor, making difficult the protein determination by ELISA multiplex assay. Considering that IL-6 was activating the trans-signaling pathway we identified STAT3 expression, a key transcription factor of the IL-6 signaling. A previous report of Aβ aggregated microinjection in mice and cell culture exposition showed that Aβ induces STAT3 activation through tyrosine phosphorylation (p-Tyr STAT3), leading to apoptosis only in neurons . Some olAfter 72 h, when AβO mediated neurodegeneration and glial activation is observed, a moderate STAT3 glial increased expression was evident. The predominant cell population consisted of astrocytes exhibiting nuclear activation. Neurodegeneration persisted closely to glial activation in AβO rats on day 7, where STAT3 expression increased in glial cells with activated morphology; some of them displayed nuclear expression. Moreover, complementary immunofluorescence exposed nuclear STAT3 expression in neurons of the CA1 region. In contrast, cytoplasmic STAT3 neuronal expression was detected in control rats. This should be studied in the future due to its possible relevance in neuronal recovery after traumatic injury. Supposing, IL-6 trans-signaling in previous acute times, it would justify the high STAT3 astrocyte expression, mainly at day 7 after AβO administration. STAT3 expression remained 15 days after surgery correlating with high glial activation and neuronal death. Besides, the classical IL-6 signaling pathway could induce anti-inflammation and a proinflammatory response by trans-signaling; it has been recently studied because of its implication in neurodegenerative disorders, such as amyloid pathology in AD ,69. CytoSTAT3 is an important transcription factor activated by stimulation of several proinflammatory  and anti-inflammatory (IL-10) molecular mediators. In the CNS, controversial functions of STAT3 has been reported, which could be due to a cell-specific response or the inflammatory microenvironment. For example, STAT3 has been involved in axon regeneration . BesidesRegarding apoptosis, STAT3 activation by proinflammatory cytokines enhances cytochrome C release and caspase activation . In an eTo clarify the JAK2/STAT3 role, we designed an inhibition assay in rat primary hippocampal mixed culture, using a JAK2 (AG490) and a STAT3 specific inhibitor (Stattic). Cultures were exposed to a cytotoxic concentration of AβO for 72 h, a time that course with high neurodegeneration in the animal model. Both assays obtained results suggesting that low-moderate inhibition of the JAK2/STAT3 pathway decreased glial activation, which helped to prevent a neuron death promoted by AβO. However, when we tried to increase the inhibition state of STAT3, the results showed a reduced survival of neurons and astrocytes. The above pointed out that the JAK2/STAT3 pathway plays an essential role in AβO toxicity since it promotes the reactivity of astrocytes, which could secrete different inflammatory mediators that lead neurons to the neurodegeneration process. In this sense, studies have consistently demonstrated the pathogenic JAK2/STAT3 pathway activation in glial cells is primed by the presence of aggregated Aβ. The JAK2 stimulation in AD transgenic mice (APP/PS1 or 3xtgAD) indicated that STAT3 activation promotes astrogliosis and synaptic dysfunction. Conversely, astrocyte-specific inhibition of the JAK2/STAT3 pathway in APP/PS1 mice control astrocyte reactivity restores synaptic deficits, and produces a Aβ deposition reduction, not linked to a microglial phagocytosis increase . MoreoveThe JAK/STAT3 inhibition is a promising strategy to modulate glial reactivity to favor neuron survival under AβO insults. Nonetheless, as observed in our assays, high or total inhibition of the JAK2/STAT3 pathway could induce neurodegeneration due to its crucial role in cell proliferation, axon regeneration, and synaptic plasticity. Thus, the ideal strategy would be to modulate the pathway only in astrocytes, as stated by the previously mentioned experiments ,30. HoweFuture studies should clarify both cytokines’ role in the dynamic of STAT3 activation, considering that both resulted increase on day 30. Another issue that should be explained is the possibility of density recovery due to the reduction of Aβaggregates. If so, it would be interesting to evaluate the administration of a second AβO dose.AβO were obtained from synthetic Aβ 1-42 peptide  using a method previously described [escribed . The pepg of body weight were purchased from Bioinvert® bioterium, Estado de México, México (SENASICA AUT-B-A-1016-028). Animal experiments were conducted according the Official Mexican Norms (NOM-062-ZOO-1999 and NOM-033-SAG/ZOO-2014) and the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures. Also, the experimental protocol (no. 113/09) was approved by the Ethics Committee of National Neurology and Neurosurgery Institute “Manuel Velasco Suárez” (April 2009).Male Wistar rats (Rattus novergicus) aged 12–14 weeks and 230–250 V:-0.30 mm, L: ± 0.25 mm y AP: −0.42 mm from bregma) [Housing conditions were 22–24 °C of temperature, 50–60% humidity, 12/12 h light/dark cycle, water and food ad libitum. Rats were anesthetized with an intramuscular injection of xylazine (12 mg/kg) and ketamine (85 mg/kg), pain were managed with tramadol. A bilateral microinjection was performed in a stereotaxic surgery apparatus  in the coordinates corresponding to CA1 hippocampus region ( bregma) . The ratRats were anesthetized as previously mentioned and perfused with cold and sterile saline solution 0.9%, followed by 4% Phosphate-Buffered Paraformaldehyde. Brains were removed and stored for 1 week in a solution with 4% Phosphate-Buffered Paraformaldehyde and 4% of sucrose. Brain hemispheres were divided and dehydrated in gradual alcohols , followed by xylene treatment and paraffin embedded. 8 µm-thick sagittal serial slices were obtained, and the slides were deparaffinized and hydrated. Antigen recovery was performed with 0.1 M citrate buffer, pH 6 and 0.2% Triton at 60 °C for 12 min. For IHC, endogenous peroxidase was inactivated with blocking reagent  for 10 min. Additionally, the tissue was incubated with bovine serum albumin (BSA) 2%-Triton 0.2% for 15 min. Slides were blocked with Background Sniper  for 15 min. Primary antibodies were diluted in BSA 1% and incubated at 4 °C overnight, followed by incubation Mach2 Universal HRP-Polymer Detection  for 30 min. 3, 3′-diaminobenzidine (Dako K3468) was used for color developing. Slides were mounted and analyzed on a Leica DM LS microscope . STAT-3 images were captured with the ScanScope CS digital processor .Primary antibodies used for IHC were mouse monoclonal anti-NeuN , rabbit polyclonal anti-STAT3 .After antigen retrieval in immunofluorescence, sections were permeabilized as we previously mentioned and blocked in 2% BSA for 30 min. Primary antibodies diluted in BSA 1% were incubated at 4 °C overnight, followed by secondary antibody incubation for 1 h at room temperature. In some cases, nuclei were stained with 4′, 6-diamidino-2-phenylindole  for 5 min. Slides were mounted with Vectashield antifade mounting medium . Images were captured on a Leica TCS SP8 confocal microscope and processed with LAS AF software (Leica Microsystems). Primary antibodies used in IF were mouse monoclonal anti-NeuN , chicken polyclonal anti-GFAP , rabbit polyclonal anti-GFAP , rabbit polyclonal anti-Iba1 , and anti-STAT3. The secondary antibodies used were goat anti-chicken IgY Alexa Fluor 488 , goat anti-mouse IgG Cy5 , anti-rabbit IgG Alexa 555 . 2MnO4 for 20 min, after distilled water washes, slides were incubated at 4 °C overnight in 0.001% Fluoro-Jade B  previously diluted in 0.1% acetic acid [FJB was used to stain brain cells under degeneration . Tissue tic acid . Slides n = 4, per group and time). For degenerated neurons, the FJB staining was evaluated in control and AβO groups at 18 h, 72 h, and 7 days post-surgery . Mature neurons were identified with Anti-NeuN antibody in control and AβO microinjected rats sacrificed on days 15 and 30 after stereotaxic surgery , one out every 10 sections was evaluated. Cell count was performed on the Leica Application Suite software version 4.0  on a 265 × 365 µm frame at 40× magnification, analyzing 3 adjacent fields on CA1 region and the same for dentate gyrus. The experimental groups were blinded in during the count and we considered positive cells with whole cell bodies within the counting frame. Average of positive cells per mmreported ,81. Rats were perfused with 4% Paraformaldehyde-2.5% Glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.4). Brains were removed and post-fixed in the same solution for 24 h. Tissues were processed in a LEICA EM TP  and embedded in Epon resin . A 90 nm slices were obtained, mounted in 200 mesh copper grids, and contrasted with 2% uranyl acetate and lead citrate. Images were captured on a TEM  ,83.n = 4, for each time and treatment evaluated) was dissected and homogenized in sterile phosphate-buffered saline containing 0.01% Triton X-100 and protease inhibitors  [™ Ultrasonic Liquid Processors . Samples were centrifuged at 1000 g and 4 °C for 10 min, to assure remaining debris remove, the resulted supernatant was centrifuged at 20,000 g for 40 min at 4 °C [® MAP Rat Cytokine/Chemokine magnetic bead panel kit (Millipore), according to the supplier’s protocol . The panel kit was reading with the LUMINEX MAGPIX® detection system with xPONET® software and the results were analyzed in MILLIPLEX® Analyst version 5.1 . The cytokines sensitivity ranges were 2.4–10,000 pg/mL for IL-1β, 73.2–300,000 pg/mL for IL-6, and 7.3–30,000 pg/mL for IL-10.Rat hippocampus for each subgroup of study  , using t at 4 °C . The cytn = 4, for each time and treatment evaluated) with TRIzol® reagent, considering the supplier’s protocol . cDNA was synthesized from 500 ng of total RNA using AMPIGENE® cDNA Synthesis kit . The reverse-transcribed product was diluted five times and 2.0 μL of cDNA was mixed with 2× TaqMan Master Mix and 0.5 µL of 20× Gene Expression Assay, the reaction was conducted in 7500 Fast Real-Time PCR system . The TaqMan® gene-specific probes  selected in this work were IL-6 (cat. no. Rn01410330_m1) and β-actin (cat. no. Rn00667869_m1). The 2ΔΔCt− method was used to analyze the relative target gene expression respect the β-actin gene expression [Total RNA was isolated from rat hippocampus , using a significance level (α) of 0.05. Before any statistical analysis, equality of variances was assessed by Levene’s test. Data were analyzed with MiniTabt-test was conducted. The integrated density of total STAT3 was calculated in Fiji software  [Differences between five evaluated times for each treatment (SS or Aβ) were identified with a one-way ANOVA test and by specific comparisons. Subsequently, to determine statistical significance between the different study groups (SS versus Aβ) at equivalent time intervals, a MD, USA) .The temporal study of AβO rat intrahippocampal microinjection suggested, oligomers mediate indirect neurotoxicity and neurodegeneration through glial activation and the consequent inflammatory response. Based on our present results, AβO induces the JAK/STAT3 pathway stimulation in astrocytes promoting it activation state and in neurons favor surveillance. However, the constant activation of the pathway by AβO, leads to hypertrophic astrocytes, which may promote an inflammatory microenvironment and increased neurodegeneration. A therapy of JAK/STAT3 pathway inhibition, mainly directed to regulate astrocytes reactivity, could be an excellent strategy to diminish neuron death and cognitive decline in AD. The use of JAK/STAT3 inhibitors should be considered with caution because a high dosage would favor the neuronal death in addition to the generated by Aβ aggregates. Considering the direct relationship between inflammation and neurodegeneration at short times, it would be useful to try different drugs that modulate astrocyte activation states. The challenge is to probe if the return to a quiescent state could improve other AβO toxic effects such as mitochondrial dysfunction, LTP impairment, oxidative damage, membrane pore formation, neurogenesis, and plasticity impairments.


The coordinated and sequential actions of lineage-specific transcription factors and epigenetic regulators are essential for the initiation and maintenance of cellular differentiation. We here report KDM4D histone demethylase as a key regulator of adipogenesis in C3H10T1/2 mesenchymal stem cells. The depletion of KDM4D results in impaired differentiation, which can be rescued by exogenous KDM4D, PPARγ, and C/EBPα, but not by C/EBPβ. In addition, KDM4D interacts physically and functionally with both NFIB and MLL1 complex to regulate C/EBPα and PPARγ expression upon adipogenic hormonal induction. Although KDM4D is dispensable for the binding of both NFIB and MLL1 complex to the target promoters, the demethylation of tri-methylated H3K9 by KDM4D is required for NFIB and MLL1 complex to deposit tri-methylated H3K4 and activate PPARγ and C/EBPα expression. Taken together, our data provide a molecular framework for lineage-specific transcription factor and histone modifiers to cooperate in adipogenic differentiation, in which KDM4D removes repressive histone marks at genes with a bivalent chromatin domain and allows NFIB and MLL1 complex to promote the expression of key adipogenic regulators. Peroxisome proliferator activator receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are in the center of the regulatory network for adipocyte differentiation and play critical roles in the expression of genes associated with adipogenesis and lipid metabolism5. A number of pro-/anti-adipogenic transcription factors have been identified, which include CCAAT/enhancer binding proteins (C/EBPs), early B-cell factors (EBFs), Krüppel-like factors (KLFs), sterol regulatory element-binding protein-1 (SREBP-1), and nuclear factor I family of transcription factors (NFI)8. These factors have been shown to regulate the expression or function of PPARγ either directly or indirectly.The differentiation of mesenchymal stem cells (MSCs) to adipocytes is initiated and maintained by the coordinated actions of signal transduction pathways and a series of transcription programs operating at different stages of differentiation9. Di- and tri-methylation of histone H3 lysine 9 (H3K9) as well as the tri-methylation of histone H3 lysine 27 (H3K27) are recognized as the hallmarks of gene repression10, whereas the methylation of histone H3 lysine 4 (H3K4) and lysine 36 (H3K36) are associated with gene activation13. Histone lysine methyltransferases (KMTs) have been implicated in the regulation of adipogenic differentiation14. KMTs mediating the di- and tri-methylation of H3K9 act as negative regulators of adipogenesis. G9a and SUV39H1 inhibit adipogenesis by repressing the expression of key adipogenic regulators, PPARγ and C/EBPα, respectively16. In addition, SETDB1 has recently been reported to contribute to the establishment of the H3K4me3/H3K9me3 bivalent chromatin domain, which is believed to make MSCs and preadipocytes paused for adipocyte differentiation17. In contrast, KMTs responsible for the methylation of H3K4 is associated primarily with the positive regulation of adipogenesis. MLL3 (KMT2C) and MLL4 (KMT2D), which are members of the MLL family responsible for the mono- and di-methylation of H3K4, are required for the expression of PPARγ and C/EBPα in cells as well as adipogenesis in vivo19. On the other hand, the function of MLL1/MLL2 (KMT2A/KMT2B) in adipogenesis, which mediates tri-methylation of H3K420, has not been studied in detail and remains elusive.Lysine methylation of histones is a prominent posttranslational modification that has been implicated in both the activation and repression of genes depending on the sites and status of modification21, whereas H3K27 specific histone demethylase JMJD3 (KDM6B) is essential for the expression of brown fat (BAT)-selective genes22. The KDM4/JMJD2 family proteins are JmjC domain-containing histone demethylases responsible for the removal of H3K9me3 as well as H3K36me324 and have been implicated in the regulation of adipogenesis28. KDM4A plays critical role in the PPARγ-mediated regulation of a specific transcription program, and KDM4B has been shown to interact with C/EBPβ to promote mitotic clonal expansion during the differentiation of 3T3-L1 preadipocytes27. On the other hand, the roles of KDM4D in adipogenic differentiation, which targets H3K9 only not H3K36, have not been defined yet29.In addition to KMTs, members of histone demethylase (KDM) family of proteins have been suggested to be regulators of adipogenesis. Lysine-specific demethylase 1 (LSD1) promotes adipogenesis by demethylating H3K4 on the promoter regions of the Wnt signaling components31. Both MLL3/MLL4 and an H3K27 specific histone demethylase, UTX, are associated with the Pax transactivation domain-interacting protein (PTIP), and they cooperate to regulate the expression of PPARγ and C/EBPα in adipogenic differentiation32. In addition, MLL2 and KDM4B cooperate in estrogen receptor α (ERα)-regulated transcription31. In this report, we have identified KDM4D as a critical regulator of the adipogenic differentiation of C3H10T1/2 MSCs. In addition, KDM4D interacts physically with MLL1 complex as well as the NFIB transcription factor, and that all of these three factors are required for adipogenesis. RNA-seq analysis and chromatin immunoprecipitation analysis support the idea that KDM4D, MLL1, and NFIB function as a complex and promote the expression of adipogenic genes including PPARγ and C/EBPα. Finally, we demonstrate coordinated actions of KDM4D, NFIB, and MLL1 complex in the expression of PPARγ and C/EBPα, in which KDM4D functions as a ‘gate-opener’ by removing the repressive H3K9me3 and allows NFIB and MLL1 complex to mediate the tri-methylation of H3K4 and activate the transcription of PPARγ and C/EBPα.KMTs and KDMs are often found in the same complex, and it is believed that they cooperate to regulate gene expressionKdm4d mRNA and KDM4D protein increased gradually, which is similar to other members of the Kdm4 family of genes and is correlated with the expression patterns of PPARγ and C/EBPα , the levels of lls Fig. . In contlls Fig. . These f33. Because there have been reports suggesting that KMT and KDM are involved in adipogenic differentiation through regulating the expression of Wnt ligands34, we examined whether KDM4D regulates the expression of Wnt ligands . The activation of Wnt signaling facilitates the osteogenic differentiation of MSCs and its inhibition allows MSCs to differentiate into adipocytes, presumably via activation of C/EBPβlls Fig. . In partlls Fig. . These r26. Therefore, we examined whether KDM4D interacts with the adipogenic transcription factors, such as C/EBPβ, PPARγ, and C/EBPα by a co-immunoprecipitation assay, but no evidence to support the physical interaction of KDM4D with those adipogenic regulators was found (data not shown). Next, mass spectrometry of proteins co-purified with FLAG-KDM4D in C3H10T1/2 cells was carried out and a list of KDM4D interacting protein candidates was identified, which included NFIB, Wdr5, Ash2l, and members of the NuRD nucleosome remodeling complex; namely Mta1/2 and Rbbp7  resulted in inhibited differentiation. Importantly, the knockdown of MLL1 (shMLL1) led to severely hampered adipogenic differentiation upon MDI induction, which has not been previously reported . We first identified 1481 up-regulated and 963 down-regulated genes during the adipogenic differentiation of C3H10T1/2 MSCs and designated them as adipogenesis-associated genes (AAGs)  was next conducted to determine if the genes affected by KDM4D depletion overlapped with those deregulated by NFIB or MLL1 knockdown, particularly those associated with adipogenic differentiation, such as ion Fig. . GO analion Fig. , upper, ion Fig. , lower. ion Fig. . Althougion Fig. . Next, wion Fig. . Surprision Fig. . Chromatter Fig. . Consistter Fig. . In addiPparg and Cebpa, suggesting that NFIB is essential for both histone modifying enzymes to be recruited to the target genes  or purchased from Origene , Addgene , and Korea Human Gene Bank . The construction of FLAG- or HA- epitope tagged genes was carried out using the standard protocols by PCR. Complete sequences of all PCR-amplified products were verified by sequencing. The detailed information regarding the plasmids and primer sets used to construct the epitope-tagged genes is provided in Supplementary Table The full-length cDNAs used in this study were either amplified directly from mouse cDNA  supplemented with 10% fetal bovine serum  and antibiotics in a humidified atmosphere with 5% CO2 at 37 °C. Adipogenesis was induced by treatment of two day post confluent C3H10T1/2 MSCs (designated by day 0 or D0) with 10  µg/ml insulin , 1  µM dexamethasone , and 0.5  mM isobutylmethylxanthine  in the presence of 10% FBS until day two. The cells were then fed with DMEM supplemented with 10% FBS and 10  µg/ml insulin for two days, after which they were fed every other day with DMEM containing 10% FBS. Differentiation was monitored initially by the appearance of lipid droplets in the cells and then confirmed by Oil Red O (ORO) staining. For ORO staining, the cells were washed three times with PBS  and fixed for 1  h with 10% formaldehyde . Subsequently, 0.35% Oil red O  in isopropanol was diluted with water (3:2), filtered through a 0.22  µm filter, and added to the fixed cells for 20  min at room temperature. The cells were then washed with water, and stained lipid droplets were visualized using an Olympus BX50 optical microscope. All images were taken using a 30× objective and processed using the Adobe Photoshop CC 2015 software.TM PLUS Reagent  according to the manufacturer’s recommendations. For a retroviral infection, replication-defective retroviruses were made in ecotropic phoenix retroviral packaging cells  by transfection of the relevant retroviral vectors using the WelfectEXTM PLUS Reagent. The transfected cells were selected with 5  µg/ml of puromycin  for 24  h and the cells were then replenished with fresh medium without puromycin. Twenty-four hours after replacing with the medium, the viral supernatants were collected and filtered through a 0.45  µm filters. C3H10T1/2 MSCs were seeded one day before infection, and infected with the viral supernatants for two consecutive days. To enhance the infection efficiency, 8  µg/ml of polybrene  was added to the viral supernatants.The cells were transfected using polyethyleneimine  or WelfectEXKdm4d, Nfib, Mll1, or Ash2l and then selected in the presence of puromycin (5  µg/ml) for two weeks. Puromycin-resistant clones were isolated and tested for knockdown efficiency by RT-qPCR. For each target, at least two independent clones were selected, and the expression of targeted genes was confirmed by immunoblot analysis before being used for further study. Sequence information regarding the shRNAs used in this study is provided in the supplemental information  in the presence of a protease inhibitor cocktail  followed by 20  s of sonication using a VCX130  sonicator (power setting 20% amplitude), and centrifugation at 12,000 × g for 20  min. For FLAG immunoprecipitation, proteins were incubated at 4 °C for 16  h in RIPA buffer-equilibrated FLAG-M2 agarose . For HA immunoprecipitation, proteins were precleared using protein A/G sepharose 4 Fast Flow  for 2  h, after which the precleared proteins were incubated at 4 °C for 16  h with anti-HA antibody followed by the immobilization of antibodies for 3  h with protein G sepharose 4 Fast Flow. Immuno-complexes were washed four times with RIPA buffer and eluted with SDS running buffer in the presence of β-mercaptoethanol  for 5  min at 94 °C. The following antibodies were used for immunoblot assays: monoclonal anti-Flag M2 , monoclonal anti-Actin , monoclonal anti-HA (16B12) , monoclonal anti-PPARγ , polyclonal anti-C/EBPα , polyclonal anti-aP2 , polyclonal anti-KDM4D , polyclonal anti-NFIB , polyclonal anti-Wdr5 , polyclonal anti-MLL1 , polyclonal anti-Ash2l , polyclonal anti-Menin , polyclonal anti-PTIP , and polyclonal anti-Rbbp5 . Detailed information regarding the antibodies, including working concentrations, is provided in the supplementary information (Supplementary Table For quantitative reverse transcription PCR (RT-qPCR) analysis, the total RNA was isolated using an RNeasy plus mini kit  according to the manufacturer’s instructions. cDNA synthesis was then carried out using a GoScript reverse transcription system  as recommended by the manufacturer. qPCR was conducted using an ABI 7300 Real-Time PCR system  with SYBR Green I  and i-StarTaq DNA polymerase . The relative mRNA level was first quantified using the standard curve method, after which the data were normalized to GAPDH mRNA. The primer sets used in this study can be found in the supplementary information  on an ABI 7300 Real-Time PCR system with SYBR Green I and iStarTaq DNA polymerase. For quantification, the % input value per sample was calculated using a standard curve method. The data were presented as % input or relative ChIP signal as indicated in the figures. The primers used for ChIP-qPCR analysis are listed in Supplementary Table Sample preparation for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was conducted as follows. C3H10T1/2 MSCs expressing full length FLAG-KDM4D were lysed with a hypotonic buffer , then passed 10 times through a 26 G 1/2 needle. After incubating on ice for 10  min, the nuclei were harvested by centrifugation and the pellets were suspended with RIPA buffer followed by 150  s of sonication using a VCX130 sonicator (power setting 40% amplitude). The nuclear extracts were then incubated with the FLAG-M2 agarose for 16  h at 4 °C. The immunoprecipitated proteins were separated by 10% SDS-PAGE and visualized with Coomassie brilliant blue G-250. LC-MS/MS analysis, and protein identification using the MS/MS spectra were performed at the Korea Basic Science Institute .The total RNA was isolated from eight different samples  using an RNeasy plus mini kit according to the manufacturer’s instructions. The RNA quality was assessed with an Agilent Bioanalyzer-2100 using the RNA 6000 Nano Chip  and RNA quantification was performed using an ND-2000 Spectrophotometer . Library preparation with SMARTer Stranded RNA-Seq Kit , HiSeq 2500 sequencing, and raw data generation were conducted by eBiogen . The high-quality sequence reads from each sample were assembled and mapped to the annotated mouse genomic DNA  using a CLC Genomic Workbench 11.0.1  with the following parameters: mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.8, and similarity fraction 0.8. Only protein coding genes with RPKM (Reads Per Kilobase of exon model per Million mapped reads) >1.0 were regarded as expressed and the genes with RPKM > 1.0 in at least one sample were kept for subsequent analysis. For differentially expressed gene (DEG) analysis, the total read counts were normalized in quantiles by RPKM before calculating gene expression levels and fold changes (FC). For cluster analysis, the genes with expression changes during adipogenic differentiation of C3H10T1/2 MSCs (between days 0 and 5) that were >2-fold, and FDR < 0.05 were selected for further analysis. The expression changes in these selected genes in shKDM4D, shMLL1, and shNFIB cells were calculated and subjected to cluster analysis using hclust within the R package. A heat map was generated by Pheatmap within the R package. Gene ontology (GO) analysis and KEGG pathway analysis were conducted using DAVID bioinformatics resources 6.8.p values were determined by Student’s t-tests of the indicated paired groups.The data shown for all qPCR-based experiments are representative of at least three independent experiments as indicated in the figure legends and are presented as the means ± SD. Statistical significance and The RNA-seq data were deposited to the Gene Expression Omnibus (GEO) repository (NCBI) under the accession number: GSE131369.Supplementary information.Dataset 1.Dataset 2.Dataset 3.


Saccharina japonica, obtaining three glucuronomannan oligosaccharides  and found that GMn ameliorated behavioral deficits in Parkinsonism mice and downregulated the apoptotic signaling pathway, especially with GM2 showing a more effective role in neuroprotection. However, the neuroprotective mechanism is unclear. Therefore, in this study, we aimed to assess the neuroprotective effects of GM2 in vivo and in vitro. We applied GM2 in 1-methyl-4-phenylpyridinium (MPP+)-treated PC12 cells, and the results showed that GM2 markedly improved the cell viability and mitochondrial membrane potential, inhibited MPP+-induced apoptosis, and enhanced autophagy. Furthermore, GM2 contributed to reducing the loss of dopaminergic neurons in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice through enhancing autophagy. These data indicate that a possible protection of mitochondria and upregulation of autophagy might underlie the observed neuroprotective effects, suggesting that GM2 has potential as a promising multifunctional lead disease-modifying therapy for PD. These findings might pave the way for additional treatment strategies utilizing carbohydrate drugs in PD.Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is caused by dopamine depletion in the striatum and dopaminergic neuron degeneration in the substantia nigra. In our previous study, we hydrolyzed the fucoidan from  Parkinson’s disease (PD), a chronic neurodegenerative disease named after James Parkinson, is caused by dopamine depletion in the striatum and dopaminergic neuron degeneration in the substantia nigra ,2,3. TheIncreasing evidence has shown that an impairment of autophagy plays a fundamental role in the development and progression of PD . AutophaSaccharina japonica exhibited neuroprotective effects in H2O2-induced SH-SY5Y cells through the inhibition of apoptosis ..23].2 and maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum (FBS), 100 U·mL−1 penicillin, and 100 μg·mL−1 streptomycin. Neuronal differentiation of PC12 cells was induced by (nerve growth factor) NGF  in DMEM containing 1% FBS every other day for 3 days.PC12 cells, a rat adrenal pheochromocytoma cell line (between 15–25 passage) obtained from iCell Bioscience Inc, Shanghai , were cultured in T-25 flasks at 37 °C in 95% air/5% CO+ for 24 h to detect the cytotoxicity. PC12 cells were treated with GM2 for 12 h and, after, were exposed to 2 mmol·L−1 MPP+ for 24 h. The control cells were treated with media containing the same volume of PBS only. The cell viability was measured by the CCK-8 regent and LDH leakage.Cells were treated with different concentrations of GM2 or MPP5 cells∙mL−1) was incubated with fluorescein isothiocyanate (FITC)-conjugated anti-annexin V and propidium iodide (PI) for 15 min in the dark. The fluorescence spectra of annexin V and PI were detected using 527/32 nm and 586/42 nm filters, respectively.The cells were treated as above. After trypsinization, the cells were suspended in annexin-binding buffer. The cell suspension  was measured at the excitation wavelengths of 514 nm. The JC-1 aggregates  were measured at the excitation wavelengths of 585 nm. The colored images were obtained using a laser scanning confocal microscope . The quantification of fluorescence intensity was measure with a microplate reader MMP was quantified using a JC-1 fluorescent probe . PC12 cells were treated as described above. PC12 cells were incubated with 1 mL of the 10 µg•mL5 cells·well−1. Cells were treated as described above. After rinsing three times, cells were incubated with 10 µmol·L−1 dichlorofluorescein diacetate (DCFH-DA) at 37 °C for 40 min. Images were obtained using a laser scanning confocal microscope (Olympus Fluoview FV1000). Quantification of the fluorescence intensity was measured with a microplate reader .Intracellular ROS levels were analyzed using a ROS assay kit . PC12 cells were seeded into 6-well plates at 2 × 105 cells·well−1. Cells were homogenized in 0.4 mL of 0.2% Triton X-100 in PBS and centrifuged at 12,000 rpm·min−1 for 3 min, and supernatants were collected for use in a subsequent assay. SOD and GSH were measured using commercial assay kits  according to the manufacturer’s instructions. Total protein concentration was detected using the bicinchoninic acid (BCA) kit.Cells were seeded into 6-well plates at 2 × 10−1) in the assay buffer. The mixtures were incubated at 37 °C for 3 h, and the absorbance was read at 405 nm.The activities of caspase-3 and caspase-9 were measured using a colorimetric caspase-3/-9 assay kit according to the manufacturer’s protocol . The reaction mixture contained 30 μL of cell lysate and 10 μL of the caspase-3 or caspase-9 substrate  2015 0003. The date of approval was 27 Feburary 2019.n = 12): (1) control, (2) MPTP only, (3) MPTP + Madopar (70 mg·kg−1), (4) MPTP + GM2 (10 mg·kg−1), and (5) MPTP + GM2 (20 mg·kg−1). The mice in groups (2–5) received MPTP hydrochloride intraperitoneally at a dose of 25 mg·kg−1·day−1 from the 8th day to the 14th day. Group (1) received the same volume of saline. Then, groups (3–5) were treated intraperitoneally with GM2 or Madopar from the 15th day to the 19th day.The mice had 7 days to adapt to the environment and were randomly assigned into five groups (n = 5) on the 20th day, 25th day, and 30th day after GM2 treatment  (PVDF) membrane and blocked with 5% bovine serum albumin for 2 h. Membranes were immunoblotted with specific antibodies  overnight at 4 °C and then applied to appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:2000) for 1 h. Signals were detected using an enhanced chemiluminescence.®® 488 goat anti-rabbit IgG  for 1 h. Positive neurons were detected in five to six sections using a scanning confocal microscope (Olympus Fluoview FV1000). Fluorescence intensity was quantified using Image J software. The average fluorescence intensity was calculated from 6–8 distributed areas in the sample images.Five mice from each group were anesthetized and perfused with an intracardial infusion of saline, followed by 4% paraformaldehyde. Brains were subsequently post-fixed in the same fixative for 48 h and then dehydrated in 20% and 30% sucrose, successively, at 4 °C. Brains were sectioned at a thickness of 16 μm on a sliding microtome for immunofluorescence . Sections were incubated with 0.2% Triton X-100 for 15 min at 25 ± 5 °C and blocked in 5% goat serum for 30 min at 37 °C. Then, the sections were stained with a primary antibody for against tyrosine hydroxylase (TH) (1:100) overnight at 4 °C. After washing three times with PBS, sections were incubated with Alexa Fluor p-values < 0.05 were considered significant .Each experiment was performed independently at least three times, and the data are presented as mean ± SEM for the indicated number of separate experiments; 17.0 SPSS software  was applied to analyze data, and one-way ANOVA was used to compare the mean differences among the groups. In this study, we applied GM2 with the single and definite structure to investigate potential neuroprotective effects in a PD model. The results demonstrated that treatment with GM2 reduced apoptosis and enhanced mitochondrial function in PC12 cells. Furthermore, GM2 prevented dopaminergic neuron loss in PD mice through an enhancement of autophagy. These results suggest that GM2 might represent a promising agent to fight PD through enhancement of mitochondrial function and autophagy.


MR1A/MR1B transcript is associated with the prevalence of MR1 + CD4/CD8 cells in the thymus. Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand(s).Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full-length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are incompletely understood. In this report, we sought to characterize the expression and function of these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. However only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A decreases MAIT cell activation following bacterial infection. Additionally, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Here, we find that relative expression of  MAIT cells produce the effector cytokines IFN-γ and TNF-α following encounter with infected cells3. MAIT cells respond to a wide array of microbes including, but not limited to, Mycobacterium tuberculosis (Mtb), Escherichia coli (E. coli), Salmonella enterica serovar Typhimurium, Candida albicans , Legionella pneumophila , and Streptococcus species6. While the prevalence and phenotype of MAIT cells in mice is distinct from that in humans, mice lacking MAIT cells had reduced capacity to control infection with F. tularensis, Mycobacteria bovis BCG (BCG), Klebsiella pneumoniae, and Legionella longbeachae9. In some cases, MAIT cells have been associated with the maturation of dendritic cells (DC), which in turn play a role in the recruitment of activated Major Histocompatibility Complex (MHC) Class I and Class II restricted T cells9. Therefore, MAIT cells represent an important mechanism by which the immune system senses and responds to microbial infection.MR1-restricted T (MAIT) cells are a subset of CD8 αβ T cells that represent 1–10% of circulating T cells. They are enriched in mucosal sites, including the skin, intestinal mucosa, and lung, and they can serve to sense intracellular microbial infection10. MR1, though similar in many ways to canonical Class I molecules, has distinct features that render it uniquely suited to present microbial ligands. MR1 transcript is expressed in all nucleated cells; however unlike MHC Class I molecules, which are constitutively detected on the cell surface, MR1 resides in the endoplasmic reticulum (ER) as well as late endosomal vesicles12. Following infection, MR1 binds microbial ligand, and this complex is thought to traffic to the cell surface to stimulate MAIT cells12. We have previously shown that MR1 mediated antigen presentation is dependent on the vesicular trafficking proteins Syntaxin18 and VAMP413. More recently, we have observed that distinct trafficking pathways exist to present endogenous and exogenous mycobacterial antigen by MR1 to stimulate MAIT cells14.MAIT cells recognize microbial antigen processed and presented by the MHC Class I related molecule, MR115. These antigens were first described as intermediates in the riboflavin synthesis pathway, but recent reports have highlighted the increasing diversity of the MAIT ligand repertoire17. For example, MR1 also presents antigen(s) from S. pyogenes, a bacteria that cannot synthesize riboflavin4. In silico screens showed that MR1 can bind a range of synthetic compounds, including commonly prescribed pharmaceuticals16, We have recently shown through metabolomics analysis that MAIT stimulatory antigens include ligands distinct from those generated in the riboflavin synthesis pathway5.While MHC Class I molecules traditionally present peptides to stimulate CD8 + T cell responses, MR1 binds and presents microbial small molecule metabolites to MAIT cellsMR1 is non polymorphic, highly conserved across species and individuals, with the transcript ubiquitously expressed20. MR1 pre-mRNA undergoes alternative splicing to produce multiple isoforms, which have been demonstrated at the transcript level to be expressed in human tissues and cell lines21. The structure of MR1 is similar to that of MHC Class I molecules, with α1 and α2 domains that bind ligand, an α3 domain that interacts with β2-microglobulin, and a transmembrane domain for surface expression22. The full length isoform, MR1A, contains all encoded exons and can stimulate MAIT cells. The shorter isoform MR1B lacks the α3 domain but does encode the ligand binding and transmembrane domains. The function of MR1B is remains incompletely understood. Overexpression of MR1B in a fibrosarcoma model suggested a functional role for MR1B in stimulating MAIT cells following infection with E. coli23. MR1C is a putative soluble isoform, lacking both the α3 and transmembrane domains, however, there are no reports on its expression or function.The underlying gene organization of MR1 is distinct from canonical MHC molecules. MR1A and MR1B transcripts are detectable across human tissues, with considerable variation in isoform expression among donors and tissues. We developed a lung epithelial cell line deficient in MR1 and utilized this system to show that MR1B can antagonize MR1A in the presentation and/or processing of mycobacterial antigen(s). While MR1A is observed in the ER and vesicular compartments, MR1B appears to reside primarily in intracellular vesicles. Finally, we show that surface expression of MR1A on CD4 + CD8 + MR1 expressing thymocytes is associated with relative abundance of MAIT cells in the thymus. Taken together, our results suggest that the splice variant MR1B can regulate the response of MAIT cells to intracellular infection.Here, we sought to determine the role of MR1 isoforms in the presentation of microbial ligand to MAIT cells. We show 25. We analyzed exon-exon junctions corresponding to inclusion or exclusion of exon 4 to distinguish MR1A from MR1B, using nearly 10,000 RNA-seq samples from 31 non-diseased tissue sites in over 500 deceased individuals from the Genotype Tissue Expression (GTEx) project 26. Here, we show that both MR1A and MR1B are expressed across human tissues, as seen in prior, non-quantitative studies  samples12. Using quantitative real-time PCR (qRT-PCR), we generated a standard curve for each splice variant by using plasmids designed to be specific to each isoform based on unique exon-exon junctions  and macrophages, both MR1A and MR1B transcripts were readily detected  expression of MR1B than MR1A, relative to the LAEC prepared from the remaining donors which expressed 2–5 fold higher MR1A relative to MR1B. Taken together, these results confirm that both MR1A and MR1B transcript are expressed, but highlights heterogeneity both within tissues and across donors with regard to the expression of MR1 splice variants.As primary large airway epithelial cells (LAEC) are capable of presenting mycobacterial antigen to stimulate MAIT cells, we prepared LAEC from human airway tissue, and confirmed that these cells expressed for EpCam Fig. E2. LAEC MR1 (Beas2B:MR1_KO)29. To identify MR1 deficient cells, we performed limiting dilution cloning, and then screened for the absence of 6FP induced MR1 surface stabilization  that lacked the gene for ion Fig. A30. FuncMR1A, MR1B, or MR1C under the control of the CMV promoter. To distinguish MR1A from MR1B or MR1C following transfection, we tagged MR1A with GFP and MR1B and MR1C with RFP . We then transfected these plasmids individually into Beas2B:MR1_KO and A549:MR1_KO cells and infected the cells with M. smegmatis overnight in order to stimulate MAIT cells  and sorted the transduced cells based on GFP expression following overnight administration of doxycycline Fig. A14. We cRFP Fig. B-left. IM. smegmatis  thymocytes constitutively express high levels of cell-surface MR132. It has been previously reported that the percentage of MR1-expressing thymocytes varies from 0.5 to 15% of DP thymocytes32. The role of MR1 expressing DP thymocytes in the selection of MAIT thymocytes is not well understood33. To determine whether MR1 + DP thymocytes are capable of presenting mycobacterial antigen to activate MAIT cells, we enriched for MR1-expressing DP thymocytes from human thymus using positive selection following surface staining for MR1A (Table M. smegmatis (MOI = 3) and tested for their ability to activate MAITs. As shown in Fig. Amongst PBMC, surface expression of MR1 has been difficult to observe in the absence of either exogenous or bacterially-derived antigen(s)MR1A and MR1B mRNA by qRT-PCR, which we then compared to surface MR1A expression in these donors , but there was a significant association between MR1B transcript and the frequency of MR1 + DP thymocytes . Intriguingly, we observed a strong positive association  between relative MR1A/MR1B transcript and the frequency of MR1 + DP thymocytes.To assess whether relative expression of the MR1 isoforms is associated with prevalence of MR1A expressing thymocytes, we sorted MR1 expressing DP thymocytes from nine donors Fig. B. We theors Fig. C. We obs34. Among CD3 thymocytes, we observed frequencies of MAIT cells, with variation across donors . However, the absolute amounts of MR1A and MR1B mRNA did not appear to be significantly correlated with frequency of MR1/5-OP-RU + thymocytes  to identify MAITs Fig. D34. Amontes Fig. F. These In this report, we show that MR1B, an alternatively spliced form of MR1, inhibits mycobacterial antigen presentation to MAIT cells by the full-length variant, MR1A. Both MR1A and MR1B are ubiquitously expressed, but with considerable variation between both discrete cell and tissue types as well as individual donors. These results suggest that MR1B can serve to modulate the recognition of MR1 ligands by MR1A. We postulate that in addition to microbial recognition, MR1B could play a role in the prevention of unwanted MAIT cell activation, and in the development of MAIT cells.These results raise a number of questions about the mechanism(s) by which MR1B mediates this inhibitory function. As we found that MR1B did not traffic to the cell surface following exposure to exogenous ligand, we believe that MR1B is not directly blocking TCR engagement with MR1A. Additionally, as MR1A and MR1B reside in the same intracellular compartments, we postulate that the inhibitory effect of MR1B could depend upon its proximity to MR1A. For example, MR1B may be competing for ligand, limiting the ability of MR1A to bind and present antigen to MAIT cells, or reducing MR1A folding and stability. Alternatively, MR1B could compete for binding to a molecular chaperone which is necessary for the loading and/or stabilization of the MR1A molecule following exposure to bacterial ligands, or for subsequent translocation of the loaded MR1A-ligand complex to the cell surface.While coexpression of MR1A and MR1B resulted in decreased MAIT activation, we did not observe a decrease in MR1A protein abundance or surface expression. However, the co-transfection approach cannot replicate the variations in MR1A and MR1B expression and relative expression that we observed in our analysis of gene expression across cells and tissues. To address this, we have developed an MR1A expression system dependent on doxycyline, that in turn allows to vary the timing and abundance of MR1A relative to MR1B. Here, we found that expression of MR1B prior to MR1A resulted in a decrease in total MR1A protein, further inhibiting the presentation of ligands to MAIT cells. Further studies involving analysis of MR1B protein structure, ligand binding, or possibly its associated chaperones could would be logical next steps to elucidate the mechanism by which MR1B is acting to inhibit MR1A.MR1A/MR1B, while uterus, breast, and colon expressed lower MR1A/MR1B transcript. As these data were derived from mRNA from the whole tissue rather than specific cell types, the full significance of these data will require further investigation. Importantly, we observe that in unfractionated single cells prepared from the small intestine, we observed relative expression of MR1A/MR1B transcript that was similar to what we observed in our analysis of large datasets. This would suggest that different cell types within a given tissue can express markedly different levels of MR1 isoforms. However, the wide variation would lead us to speculate that the relative abundance of MR1B could be associated with diminished activation of tissue resident MAITs. This could have significance with regard for the ability to recognize microbial infection or be associated with a propensity for autoimmunity.We found that both the abundance and relative expression of MR1A and MR1B varies considerably across tissues and donors. At present, the significance of this variation is not clear with regard to MAIT cell prevalence in tissues, their state of activation, and their ability to respond to MR1 antigens. Preliminarily, we note that tissues such as blood and spleen expressed relatively higher levels of 36. Recently, an intronic SNP in MR1 was shown to be associated with susceptibility to tuberculosis37. These results raise the possibility that regulation of MR1A and MR1B presentation is controlled at the level of gene expression. Alternately, we postulate microbial infection could alter expression of MR1 splice variants.Alternative splicing is often regulated by genetic modifications such as changes in DNA methylation or single nucleotide polymorphisms (SNPs)40. However, the role of alternative splicing in the regulation of MHC and subsequent acquisition of adaptive immunity is incompletely explored. An α3 domain deletion mutant of HLA-A11 can promote evasion of NK cell mediated cytotoxicity of the HIV-infected cell41. Alternative splicing of the non-classical molecule HLA-G has been reported and is hypothesized to be associated with protection of the developing fetus43. These reports suggest that a putative role for alternative splicing is to control immune cell activation and therefore target immune responses to microbial infection. A recent study suggested that viral infection downregulates surface expression of MR1 and thereby presentation of MR1-bound antigen to MAIT cells, through the HSV-1 protein US344. The mechanism of this is unknown but suggests that impeding MR1 function can further regulate MAIT cell activity. Here, the relationship of MR1 splice variants in the context of both infection and autoimmunity remains to be explored.Alternative splicing is common and represents an important means of controlling gene expression. For instance, dysregulated splicing has been associated with both autoimmunity and inflammationMR1A/MR1B expression and prevalence of MR1A-expressing CD4 + CD8 + thymocytes. CD1d expressing DP thymocytes have been shown to contribute to the selection of iNKT cells, and MR1 + DP thymocytes have been suggested to play a similar role in the selection of MAIT cells in the thymus46. Furthermore, we find that the prevalence of MAIT cells in the thymus was associated with MR1A-expressing DP thymocytes. While the frequency of MR1A expressing DP thymocytes varied, cell surface of MR1 protein was relatively constant between donors. Therefore, it is clear that the cell surface expression of MR1 is not strictly dependent on the relative abundance of the MR1A and MR1B splice variants. Recent work by Legoux and colleagues demonstrated that intrathymic development of murine MAIT cells could be governed, in part, by metabolites produced by commensal bacteria presented by MR147. Therefore, mechanisms regulating MR1 expression and capacity to bind and present antigen, could potentially also contribute to thymic development of MAIT cells. We observed that relative MR1A/MR1B expression on DP thymocytes was associated with the prevalence of MR1 + DP thymocytes. Additionally, the prevalence of MR1 + DP thymocytes was associated with that of MAIT precursors in the thymus. How DP MR1 expressing thymocytes function in MAIT development and the role of alternative splicing in this development will require detailed in vitro modeling. One limitation of our study is that we did not have matched PBMC from our thymus donors, and therefore, we were not able to establish the relationship of intrathymic MR1 expressing cells and the prevalence of MAITs in the blood. Taken together, our results suggest a potential role of alternative splicing in modulating presentation of microbial ligand to MAIT cells.Our results demonstrate an association between the relative All experiments and methods were performed in accordance with relevant guidelines and regulations.. Antibodies utilized are listed in Table 2Doxycycline  was resuspended to 2 mg/mL in sterile water and was used at 2 µg/mL. 6-formylpterin  was resuspended to 3 mg/mL in 0.01 M NaOH. Control vehicle used was 0.01 M NaOHM. smegmatis and M. bovis Bacillus Calmette-Guérin (BCG) were utilized from frozen glycerol stock. For short-term M.smegmatis infection, cells were infected for 1 h in a 96 well ELISPOT MSHA nitrocellulose plate at the MOI indicated in the figure. For overnight M.smegmatis infection, cells were infected for 16 h at an MOI of 3. For thymocyte infection with M. smegmatis, thymocytes were infected at an MOI of 3 for 1 h. For BCG infection, adherent cells were infected overnight at a MOI of 15. BCG infected cells were harvested and added to the ELISPOT assay at the amount indicated in the figure. To generate M. smegmatis supernatant, M. smegmatis was cultured with shaking for 24 h then pelleted. The supernatant was passed through a 0.22 µm filter to remove any bacteria. The supernatant was utilized at the indicated volume in an ELISPOT assay.−1; Orthoclone OKT3, eBioscience, San Diego, USA) in RPMI 1,640 media with 10% human serum in a T-25 upright flask in a total volume of 30 ml. The cultures were supplemented with IL-2 on days 1, 4, 7 and 10 of culture. The cell cultures were washed on day 5 to remove soluble anti-CD3 monoclonal antibodies.T-cell clones were cultured in the presence of X-rayed  allogeneic PBMCs, X-rayed allogeneic LCL  and anti-CD3 monoclonal antibody  in a buffer solution of 0.1 M Na2CO3, 0.1 M NaHCO3, pH = 9.6). Then, the plate was washed three times with sterile PBS and blocked for 1 h at room temperature with RPMI 1,640 media containing 10% heat-inactivated HS pool. Then, the APCs and T cells were prepared as described above and co-incubated overnight. Antigen presenting cells and antigen used are noted in the relevant figure. T-cell clones were added at 5 × 103 per well. The plate was incubated overnight at 37 °C and then washed six times with PBS containing 0.05% Tween. The plate was then developed as previously described and analyzed using an AID ELISPOT reader 5.A MSHA S4510 96 well nitrocellulose-backed plate  was coated overnight at 4 °C with 10 μg mlAll samples were collected, and all experiments were conducted under protocols approved by the institutional review board at Oregon Health and Science University. PBMCs were obtained by apheresis from healthy adult donors with informed consent. De-identified lungs, upper airway, or small intestine were obtained from the Pacific Northwest Transplant Bank  Table  with inf29. All cell lines were cultured in DMEM  supplemented with L-glutamine and 10% heat inactivated FBS. All cell lines were confirmed to be mycoplasma free.BEAS-2B and A549 cell lines was obtained from ATCC. A549:MR1_KO were generated as described previously48. Briefly, lentiviruses were produced by co-transfection of HEK 293T cells with the lentiviral vectors that contained the gene of interest (pCDH-sgRNA or pCW-Cas9) and the packaging plasmids (psPAX2 and pMD2.G). Transfection was performed with Lipofectamine 3,000  according to manufacturer’s instructions. Cells were cultured in DMEM supplemented with 10% FBS. 48 h after transfection, lentivirus-containing supernatants were harvested, centrifuged for 5 min at 1,250 rpm and filtered through a 0.45 µm filter. The Beas2B_MR1KO cell line was transduced with a lentivirus expressing pDMLV_Tet_MR1AGFP by spinoculation. Transduced cells were exposed to doxycycline overnight to promote expression of MR1AGFP and subsequently sorted on an InFlux cell sorter based on GFP expression. Cells were subsequently cultured, validated for MR1AGFP expression by flow cytometry, and utilized for further experiments.Lentivirus production was performed as described previously48. Wildtype Beas2B cells were seeded in a 6 well plate and transduced by spinoculation with a lentivirus encoding the CRISPR/Cas9 gene with guide RNA specifically targeted to MR1 that had been previously used to generate an A549 MR1 knockout cell line29. Following this, limiting dilution was performed on transduced cells. Cells were grown in DMEM media containing 10% fetal bovine serum and 1% gentamicin antibiotic. Three clones were validated as knocked out for MR1 protein expression, as well as functionally unable to stimulate MAIT clones, and cryopreserved in 90% fetal bovine serum/10% DMSO. One clone was selected and utilized for all future experiments (Beas2B:MR1_KO).CRISPR/Cas9-mediated genome-editing for MR1 was performed as described previouslyPBMCs were isolated from the peripheral blood of healthy donors using Ficoll-Paque gradients.28. PBMCs obtained by apheresis were resuspended in 2% human serum in RPMI and were allowed to adhere to a T-75 flask at 37 °C for 1 h. After gentle washing twice with PBS, nonadherent cells were removed and 10% human serum in RPMI containing 30 ng ml−1 of IL-4  and 30 ng ml−1 of granulocyte–macrophage colony-stimulating factor  was added to the adherent cells to generate dendritic cells. For generation of macrophages cells, adherent cells were incubated in 10% human serum in RPMI for 1 h. The cells were X-rayed with 3,000 cGray using X-RAD320  to prevent cell division. After 5 days, cells were harvested with cell-dissociation medium  and used as APCs in assays.Monocyte-derived DCs and macrophages were generated by the method of RomaniLung and small intestine single cell suspensions were prepared from recently deceased donor tissue. Characteristics of deceased donors are listed in Table 2.Large airway epithelial cells were generated as described previously from the upper airway of human donors and cryopreserved in heat-inactivated fetal bovine serum with 10% DMSO8 cells/ml in a 90% FBS/10% DMSO freezing solution with a post-thaw viability of approximately 50%Thymocytes: Thymus tissue was ground in a GentleMACS dissociator with of DMEM plus 10% FBS to form a single cell suspension. The suspension was cryopreserved at 2–3 × 10Total RNA was isolated using TRIZOL  and phenol chloroform extraction followed by an RNAeasy Kit . cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit . qPCR was performed using SYBRGreen Power Master Mix  on a Step One Plus Real-Time PCR System . Primers were designed to be specific for each gene assay and generated by the DNA services core and IDT Technologies. The absolute quantification method was used using by generating a standard curve with a plasmid specific for each gene assayed. Primer sequences are noted in Table 12. A gene encoding MR1B_RFP or MR1C_RFP was ligated into this construct using EcoRI and KpnI, to create pCI_MR1B_RFP or pCI_MR1C_RFP. Confirmatory sequencing was performed. Restriction enzymes and ligation kit were obtained from New England Biolabs . PCR and gel purification kits were obtained from Qiagen .We utilized our previously described pCI construct for all transient transfectionsTransfection of Beas2B or A549 cells using plasmids was done using an Amaxa Nucleofector, Kit T solution , and programs G-016 according to manufacturer’s protocol. For co transfections, a 2:1 ratio of MR1B:MR1A was used unless otherwise specified, and the amount of MR1A was kept fixed at 2.5 ug. For single transfections, 2.5 ug of plasmid was transfected. A pCI empty vector was used as a control for all transfections. Confirmation of transfection was performed using flow cytometry on a BD Fortessa to detect either GFP (pCI_MR1AGFP) or RFP (pCI_MR1BRFP or pCI_MR1CRFP). All analyses were performed using FlowJo software .BEAS-2B:MR1_KO cells, either untransfected, or transfected with pCI empty vector, pCI_MR1AGFP, and/or pCI_MR1BRFP, were plated into 6 well tissue culture plates overnight. The next day, the cells were treated with 100 µM 6-FP versus an equivalent volume of 0.01 M NaOH. After 16 h, the cells were harvested on ice and split into two groups for primary staining and isotype control staining. Primary staining was done with an antibody against MR1  at 1:100 for 40 min on ice in the presence of 2% human serum, 2% goat serum, and 0.5% FBS. Biotinylated mouse anti IgG2A  served as the isotype control. After washing, streptavidin-Alexa 647  was added for 40 min on ice. Cells were washed, fixed in 1% PFA and analyzed by flow cytometry on a BD Fortessa, or BD Symphony and data were analyzed on FlowJo .2. After 48 h, live images were acquired on a high-resolution wide field CoreDV system  with a Nikon Coolsnap ES2 HQ. For 6FP surface stabilization assay, cells were transfected as above, and administered 50 uM 6FP or an equivalent volume of 0.01 M NaOH for 16 h. Cells were fixed for 20 min in 4% PFA and stained with DAPI  to visualize nucleus before acquisition on a wide field CoreDV system. Each image was acquired as Z-stacks in a 1,024 × 1,024 format with a 60 × objective (NA 1.42).BEAS-2B:MR1_KO cells were transfected with MR1A_GFP or MR1B_RFP and plated into 1.5 mm glass bottom chamber slides  and incubated at 37° C and 5% CO3/well) in an ELISPOT assay following incubation with an MAIT clone (5 × 103/well). The ELISPOT was set up and developed as described above.pCI_MR1BRFP or pCI_MR1CRFP was transfected into Beas2B_MR1KO cells overexpressing a Tet_MR1AGFP lentivirus either 24 h prior to or 16 h post induction of MR1A expression. Flow cytometry was performed as described earlier to measure MR1AGFP expression or MR1BRFP expression using a BD Symphony and data were analyzed using FlowJo software. For functional analyses, cells were harvested on ice, infected 1 h with a titrating dose of M. smegmatis, and utilized as antigen presenting cells , CD14, and CD20, for 30 min on ice in the dark, as well as Live/Dead Fix Aqua for viability. Cells were harvested on ice, washed in PBS, resuspended in FACS buffer, and sorted on a BD InFlux sorter for Live, CD45- CD14-, and CD20- CD326 + cells in order to isolate CD326 + epithelial cells. Cells were sorted into TRIZOL for immediate RNA isolation and subsequent quantitative real time PCR analysis. Sorting of MR1 + DP thymocytes: Thymocytes were thawed and blocked with FACS buffer, and 2e06 cells were stained with antibodies for the surface markers CD3, CD4, CD8, and MR1-biotin (clone 26.5), or the isotype control (IgG2a-biotin) for 30 min on ice in the dark. Cells were washed twice with cold PBS and stained with streptavidin-PE (1:2000 dilution) for 15 min in the dark. Cells were washed twice and resuspended in FACS buffer and sorted on a BD InFlux Sorter for Live, CD3 + CD4 + CD8 + MR1 expressing thymocytes, which was confirmed based on an isotype control for MR1. Cells were sorted into TRIZOL for immediate RNA isolation and subsequent quantitative real time PCR analysis.For quantifying the frequency of MAIT thymocytes, thymocytes were thawed, blocked with FACS buffer, and 2e06 cells were incubated with the MR1/5-OP-RU tetramer or the MR1/6FP tetramer , for 45 min at RT in the dark. Following this, antibodies to the surface markers CD3, CD4, and CD8 and Live/Dead Fix Aqua viability stain were added at manufacturer recommended concentrations. Cells were incubated at RT in the dark for 15 min, washed twice with PBS, and fixed with 1% paraformaldehyde. Acquisition was performed on a BD Symphony flow cytometer and data were analyzed using FlowJo software . All antibodies utilized are listed in Table MR1 across normal tissue RNA-seq samples comprising the GTEx project26. To generate this figure, we first used Snaptron to query GTEx RNA-seq samples for numbers of reads spanning each of three exon-exon junctions, whose hg38 coordinates are (a) chr1:181,050,287–181,052,234, (b) chr1:181,052,511–181,053,572, and (c) chr1:181,050,287–181,053,572. Junctions (a) and (b) point to inclusion of exon 4, while junction (c) points to exclusion of exon 4. For a given sample, we averaged the numbers of reads spanning junctions (a) and (b) and divided the result by the number of reads spanning junction (c) to estimate the ratio of exon 4 inclusion to exclusion25. We subsequently used Mathematica 10.4 to plot results across samples. Scripts to reproduce Fig. https://github.com/nellore/mr1.Figure t-test, unless otherwise indicated. For comparison of DP MR1 + thymocyte gene expression with frequency of DP thymocytes or of MAIT thymocytes, Pearson’s correlation calculation was performed. For comparisons of functionality between transfected antigen presenting cells, linear regression analysis was performed and significant differences between slopes and y-intercepts were measured and reported. Error bars in the figures indicate the standard deviation, standard error of the mean, or the data set range as indicated in each figure legend. P values < 0.05 were considered significant .Data were analyzed and plotted using Prism 7 GraphPad Software . Statistical significance was determined using unpaired Student’s two-tailed Supplementary Information.