MicroRNA-196a-5p (miR-196a-5p) has been reported to be involved in the metastatic process of several cancers. In present work, we aimed to investigate the effects of miR-196a-5p and its potential target IκBα on migration, invasion and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells.CCK-8 assay, wound healing assay and cell invasion assay were performed to evaluate the cell proliferation, migration and invasion. In vivo metastasis models were used to investigate the tumor metastasis ability. Real-time PCR, immunofluorescence staining or western blot were utilized to detect the expression of miR-196a-5p, IκBα, p-IκBα, nuclear p65 and EMT markers including E-cadherin, N-cadherin and fibronectin. Dual luciferase reporter assay was carried out to determine whether there is a direct interaction between miR-196a-5p and IκBα mRNA.Using SW480 cell with miR-196-5p over-expressed plus SW620 and HCT116 cells with miR-196a-5p knockdown, we found that miR-196a-5p promoted cell proliferation, migration and invasion in vitro and facilitated liver metastasis in vivo. We also observed that miR-196a-5p knockdown or NF-κB pathway inhibition up-regulated E-cadherin while down-regulated N-cadherin and fibronectin. By contrast, miR-196a-5p over-expression promoted EMT process of CRC. Data of dual luciferase reporter assay indicated that miR-196a-5p targeted the IκBα. Moreover, miR-196a-5p down-regulated IκBα expression while up-regulated nuclear p65 expression. Additionally, over-expression of IκBα in CRC cells attenuated the effects of miR-196a-5p on cell migration, invasion and EMT.miR-196a-5p may play a key role in EMT, invasion and metastasis of CRC cells via targeting the IκBα.The online version of this article (10.1186/s12885-018-5245-1) contains supplementary material, which is available to authorized users. Colorectal cancer is one of the most common malignant tumors in the gastrointestinal tract, and there are about 1.2 million new CRC cases have been reported worldwide every year . It has The microRNAs (miRNAs) are a class of small non-coding RNAs with about 22 nucleotides length . A growiNF-κB proteins are transcriptional factors which binding to the target genes to regulate their expression . ActivatIn present work, we employed the SW480 cells with miR-196-5p over-expressed plus SW620 and HCT116 cells with miR-196a-5p knockdown to explore the role of miR-196a-5p on CRC migration, invasion and liver metastasis. We further investigated whether miR-196a-5p exert its pro-metastatic effects via facilitating the EMT by a direct inhibition of the IκBα.2. In order to evaluate the influence of NF-κB pathway on the migration, invasion and EMT of CRC cells, cells were treated with NF-κB pathway inhibitor BAY 11–7082  for 48 h before following experiments.The human colorectal cancer (CRC) cell lines of SW480, SW620 and HCT116 were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. and cultured in DMEM medium  supplemented with 10% fetal bovine serum  at 37 °C in 5%COIn order to construct the IκBα expression plasmid, the full-length cDNA of IκBα was amplified and inserted into pcDNA3.1 vector with HindIII and BamHI sites. Lipofectamine 2000  was employed to transfect IκBα plasmid into SW480 cells. Further, miR-196a-5p mimics, NC mimics, miR-196a-5p inhibitor and NC inhibitor purchased from Shanghai GenePharma Co., Ltd. were transfected into CRC cells using the Lipofectamine® RNAiMAX  following the manufacturer’s protocol.5/well. 24 h latter, the lentivirus was added into the culture medium to infect the CRC cells. After screening with Purine toxin , CRC cells with stable miR-196a-5p over-expressed or knockdown were obtained.The lentiviral (LV) particles that over-expressing miR-196a-5p (LV-miR-196a-5p) and its negative control (LV-NC) or silencing miR-196a-5p (LV-anti miR-196a-5p) and its negative control (LV-anti NC) were purchased from Wanleibio Co., Ltd. CRC cells were seeded in 6-well plates at a density of 4 × 10The 3′-UTR region of wide-type of IκBα was amplified and inserted into pmirGLO plasmid at Nhe I and Sal I sites. Furthermore, miR-196a-5p mimics or the miR-196a-5p mutant was cotransfected with Luc-IκBα or the control into HEK 293 T cells using the Lipofectamine 2000 . Luciferase assays were carried out with the Dual-Luciferase Reporter Assay System  referring to the instructions.-ΔΔCt or 2-ΔCt method and sequences information was shown in Table The total RNA from CRC cells in different conditions was extracted using the TRIpure kit . After quantization of concentration, RNA was reversely transcribed into cDNA by the Super M-MLV reverse transcriptase (BioTeke). Quantitative PCR was carried out to determine the expression levels of miR-196a-5p and IκBα using 2 × Power Taq PCR MasterMix (BioTeke) and SYBR Green . The procedure for IκBα was as follows: first cycle including 94 °C for 5 min, 94 °C for 10 s, 60 °C for 20 s, 72 °C for 30 s, and then 40 cycles of 72 °C for 2 min 30 s, 40 °C for 1 min 30 s, melting from 60 °C to 94 °C for 1 s every 1 °C. While the miR-196a-5p expression level was measured under following condition: 94 °C for 2 min, 94 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s followed with 40 cycles of 72 °C for 2 min 30 s, 40 °C for 5 min 30 s, melting from 60 °C to 94 °C for 1 s every 1 °C. The data were calculated by 23/well. The cell viability was measured at 12 h, 24 h, 48 h and 72 h after transfection. CCK-8 regent  was added into culture medium 10 μl per well. After 1 h incubation, the optical density of solution was measured using a microplate reader at 450 nm.CRC cells were seeded into 96-well plates at 3 × 10The transfected or infected CRC cells were cultured in serum-free DMEM containing mitomycin C  for 1 h. Subsequently, a sterile 200 μl pipette tip was employed to cause scratches. Representative images of cells migrating into wounds were taken at 0 h, 24 h or 12 h after the scratch.4 CRC cells and the lower chambers were filled with 800 μl culture medium containing 30% FBS. After 24 h incubation, migrated cells were fixed with 4% paraformaldehyde for 20 min and stained with the crystal violet  for 5 min. Cells on lower surface were counted with an inverted microscope.Cell invasion assay was performed on 24-well transwell chambers coated with Matrigel . In brief, the upper chamber was added into 200 μl DMEM containing 1 × 10n = 6); SW480-LV-miR-196a-5p group (n = 6); HCT116-LV-anti NC group (n = 6); HCT116-LV-anti miR-196a-5p group (n = 6). According to the experimental group dividing, 2 × 106 transfected CRC cells were injected into spleens of the anesthetized nude mice and the spleens were resected 48 h latter. All mice were sacrificed 7 weeks after the injection and the hepatic metastases have been evaluated.To assess the effects of miR-196a-5p on tumor metastasis, we employed 4-week-old BALB/c nude mice to establish in vivo metastasis models. The thymic aplasia of the nude mice results in immunodeficiency and could avoid interference from host immune system. The nude mice used in present study were obtained from Beijing HFK Bioscience Co., Ltd.  2014–0004) and housed in a standard laboratory environment . During the experiments, mice were free access to the water and food and no adverse events were observed. In present work, 24 mice were randomly divided into four groups: SW480-LV-NC group  and eosin . The metastatic foci were observed under the OLYMPUS DP73 microscope.CRC cells or liver metastatic tissues were lysed with RIPA Lysis Buffer  containing 1% PMSF (Beyotime). Besides, the nuclear protein was isolated by the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein from different conditions were separated by 6–14% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk or 1% BSA, the membranes were incubated with following primary antibodies at 4 °C overnight: anti-E-cadherin , anti-N-cadherin , anti-fibronectin , anti-p-IκBα , anti-IκBα , anti-p65 , anti-β-actin  and Histone H3 . After rinsed with TSBT, the membranes were incubated with their corresponding secondary antibodies at 37 °C for 45 min. The protein bands were visualized using the ECL reagent (Beyotime).The CRC cells were seeded on slides and then fixed in 4% paraformaldehyde for 15 min. After permeabilized with 0.1% tritonX-100  for 30 min at room temperature, the slides were blocked with goat serum . Subsequently, cells were incubated with antibodies of E-cadherin , N-cadherin , Fibronectin  overnight at 4 °C. Then CRC cells were incubated with the goat anti-rabbit Cy3-conjugated IgG  or goat anti-mouse Cy3-conjugated IgG . Lastly, cells were counterstained with DAPI  before capturing images using the fluorescence microscope .P < 0.05 was seen as statistically significant.The data were presented as mean ± SD and were obtained from at least three individual experiments. One-way or two-way ANOVA test followed with post hoc Bonferroni’s test were used to analyze data. While the statistical difference between two groups were analyzed by student t test. Correlations were analyzed using Pearson’s correlation test. A We firstly performed Real-time PCR to detect the expression level of miR-196a-5p in various CRC cell lines. As shown in Fig. We then assessed whether the expression level of miR-196a-5p in CRC cells influence their proliferation. With this objective, we used miR-196a-5p mimic and inhibitor to induce or silence the miR-196a-5p expression in CRC cells , but no significance (p = 0.54). We performed dual luciferase reporter assay to determine whether there was an interaction between IκBα mRNA and miR-196a-5p. As shown in Fig. By analyzing the database of Tumor metastasis seriously affects the prognosis of CRC patients . While sThe miR-196a-5p was initially identified as an oncogenic miRNA which direct targeted annexin A1 to promote proliferation and prevent apoptosis of esophageal cancer cells . With fuEpithelial-mesenchymal transition is a reversible process which is been considered as a critical program during the metastatic cascade in several types of cancers including the CRC , 23. DurIt is worth mentioning that several literatures demonstrated that IκBα may work as a downstream target of miR-196a-5p during cancer progression. One study from Yang et al showed that miR-196a took an oncogenic role in glioblastoma multiforme by targeting IκBα . The IκBConsideration of above observations, we speculated miR-196a-5p may also facilitate the EMT of CRC via regulating the IκBα expression. By performing the dual luciferase reporter assay, we verified that miR-196a-5p directly bound to the IκBα gene. We also investigated the effects of miR-196a-5p on the expression of IκBα pathway. As expected, miR-196a-5p over-expression decreased the expression of IκBα and increased the level of nuclear p65, while miR-196a-5p knockdown up-regulated the IκBα and down-regulated the nuclear p65 in CRC cells. The data suggested that IκBα may be a target of the miR-196a-5p in CRC cells.We further evaluated that whether inhibition of the NF-κB pathway attenuated the EMT of CRC. The results demonstrated that deactivation of NF-κB pathway increased epithelial markers E-cadherin but decreased the mesenchymal markers N-cadherin and fibronectin. As IκBα is the natural inhibitor of NF-κB signaling pathway, these findings implied that miR-196a-5p and IκBα may take opposite roles in the EMT of CRC. Additionally, over-expression of IκBα in CRC cells attenuated miR-196a-5p induced-migration, invasion and EMT of CRC cells, which further confirmed that miR-196a-5p exerted its pro-metastatic effects by facilitating the EMT process via targeting IκBα. In order to further clarify the pro-metastatic effects and the potential mechanisms of miR-196a-5p in CRC, we will perform clinical studies to analyze the correlation between miR-196a-5p levels and liver metastasis. The relationship between the expression of miR-196a-5p and IκBα in the clinical CRC tissues will also be investigated in future.In conclusion, our data demonstrates that miR-196a-5p facilitates the proliferation, metastasis and invasion of CRC cells. Additionally, the pro-metastatic property of miR-196a-5p is exerted through regulating the EMT process via targeting the IκBα. Our findings suggest that regulating miR-196a-5p may be a potential strategy to ameliorate the tumor metastasis of CRC.Additional file 1:***P < 0.001 compared to LV-NC or LV-anti-NC group. (TIF 1803 kb)Western blot assay was employed to evaluate the expression levels of IκBα, E-cadherin and N-cadherin in liver metastatic nodules induced by (a) miR-196a-5p overexpressing or (b) miR-196a-5p downregulated CRC cells. Additional file 2:(a) The mRNA level of IκBα in CRC cell lines. (b) The correlation between miR-196a-5p and IκBα mRNA levels in CRC cells. (TIF 718 kb)Additional file 3:***P < 0.001 compared to vector group. (TIF 1284 kb)Transfection of IκBα plasmid induced the expression of IκBα in SW480 cells. (a) The protein level of IκBα in SW480 cells. (b) The mRNA level of IκBα in SW480 cells.


This study describes the interaction between Cav2 calcium channels and Rabconnectin-3, a di-subunit protein that is associated with synaptic vesicles. Immunostaining reveals that both Rabconnectin-3α (RB-3α) and Rabconnectin-3β (RB-3β) are colocalized in mouse hippocampal neurons. Co-immunoprecipitations from brain tissue is consistent with the formation of a protein complex between RB-3α and RB-3β and both Cav2.2 and the related Cav2.1 calcium channel. The coexpression of either RB-3α or RB-3β with Cav2.2 calcium channels in tsA-201 cells led to a reduction in Cav2.2 current density without any effects on the voltage-dependence of activation or inactivation. Coexpression of both Rabconnectin-3 subunits did not cause an additive effect on current densities. Finally, the presence of Rabconnectin-3 did not interfere with μ-opioid receptor mediated Gβγ modulation of Cav2.2 channels. Altogether, our findings show that Rabconnectin-3 has the propensity to regulate calcium entry mediated by Cav2.2 channels. Cav2 channels are essential mediators of synaptic communication in the mammalian and invertebrate nervous systems –5. CalciRabconnectin-3 is a poorly characterized synaptic protein that is formed by α and β subunits, encoded respectively by DMXL2 and WDR7 –21. RabcHere, we present evidence that RB-3α and RB-3β form a molecular complex with Cav2.1 and Cav2.2 channels in mouse brain. Furthermore, we show that both subunits affect Cav2.2 current density, suggesting that Rabconnectin-3 may contribute to regulating brain function via alteration of presynaptic calcium influx.We first examined the co-localization of Rabcconnectin-3 subunits with Cav2.2 calcium channels expressed endogenously in cultured mouse hippocampal neurons (10 days in vitro). Fig. 1b + Cavα2δ-1) channels expressed in tsA-201 cells in the absence or presence of Rabconnectin-3. Representative whole-cell Cav2.2 current recordings are shown in Fig. 1 subunit with RB-3β and performed a co-immunoprecipitation we found an interaction between these proteins : -30.95 ± 8.1; P > 0.05 compared to RB-3α), and that the formation of a complete Rabconnectin-3 complex is still capable of modulating Cav2.2 current density.To ascertain whether Rabconnectin-3 can alter Cav2.2 channel function, we recorded whole cell barium currents through recombinant Cav2.2  and subcloned into pcDNA3.1 vectors. Human WDR7 cDNA was purchased from Thermo Scientific and human DMXL2 cDNA was obtained from Origene™.After splitting and seeding to 50% confluence, tsA-201 cells were transfected with 3 μg of each plasmid encoding Cav2.2α1, Cavβ1 and Cavα2δ-1, respectively, in the presence or absence of WDR7, DMXL2 or both cDNAs. In addition, 0.5 μg of cDNA encoding green fluorescent protein was added to the transfection mixture to identify and select transfected cells. For G protein studies 3 μg of the μ-opioid receptor cDNA were added. Cells used for electrophysiology experiments were moved to 30 °C after transfection, whereas those used for Western blotting were maintained at 37 °C.2, 10 TEA-Cl, 1 MgCl, 128 NaCl, 5 KCl, 10 HEPES and 10 glucose (pH 7.4). Patch pipettes were filled with a solution containing (in mM) 110 CsCl, 2.5 MgCl, 10 EGTA, 10 HEPES, 3 ATP, and 0.5 GTP (pH 7.4). Current densities were obtained by dividing peak current by the whole cell capacitance. Current density–voltage relationships were generated from the peak current obtained during 250 ms pulses between − 50 to + 40 in 5 mV increments from a holding potential of − 80 mV. Steady-state inactivation was measured by applying 2 s conditioning pulses from − 100 to + 20 mV in 10 mV increments followed by a 100-ms test pulse to + 10 mV. I-V relationships were fitted with a Boltzmann equation of the form: I = Gmax*(Vm − Vr)/(1 + exp.(−(Vm − V1/2act)/k)), where I is the peak current, Vm is the membrane voltage, V1/2act is the half activation potential, Vr is the reversal potential, and k is the slope factor. Steady-state inactivation curves were fitted with the equation: I/Imax = 1/(1 + exp.((Vp − V1/2inac)/k)), where I/I max is the normalized peak current, Vp is the conditioning pre-pulse, V1/2inac is the half-inactivation potential and k is the slope factor.Electrophysiological recordings were performed using whole cell patch-clamp at room temperature (22–24 °C). Currents were recorded using an Axopatch 200B amplifier linked to a computer with pCLAMP9.2 software. The external recording solution contained (in mM): 5 BaClG protein modulation induced by μ-opioid receptor activation was assessed as described by us previously . In brieCells were detached from culture dishes, washed with phosphate-buffered saline, and lysed in single-detergent lysis buffer . Protein concentration was determined using the Bradford assay. Rat brain proteins were lysed in RIPA buffer (25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and Complete 1×). Fifty micrograms of protein samples were boiled for 5 min in protein-loading buffer . Co-immunoprecipitation assays were performed using rat brain proteins or lysates from transfected tsA-293 cells. One milligram of proteins was incubated with 3 μg of specific or irrelevant (as an isotype control) antibodies. Immune complexes were resolved in 8–15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes then immunoblotted using the following antibodies: Cav2.1, Cav2.2 , WDR7 , and DMXL2 .Transfected cells were washed with ice-cold HEPES-based saline solution (HBSS) and incubated on ice for 15 min to stop trafficking of proteins. Surface proteins were biotinylated for 1 h on ice with 1 mg/ml of EZ- Link Sulfo-NHS-SS-Biotin (Thermo Scientific). The reaction was quenched with 100 mM glycine for 15 min, and cells were lysed in modified RIPA buffer  for 45 min. Protein quantification was performed using a Bio-Rad protein assay dye, and 2 mg of lysates was incubated with 100 μl of Neutravidin beads (Thermo Scientific) for 1.5 h at 4 °C. Beads were washed three times with lysis buffer and proteins eluted with 2× Laemmli sample buffer. Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using anti-Cav2.2  and 1/5000 anti-Na/K-ATPase  antibodies. For each experiment, background was subtracted and integrated density of bands was measured and normalized to the Na/K-ATPase signal as loading control.Hippocampal neurons (10 DIV) were blocked for 1 h with 0.5% BSA + 10% NGS + 0.3% Triton X-100 in 1X PBS. Sections were then incubated with the following antibodies: Cav2.2 , WDR7  and DMXL2  overnight at 4 °C. Neurons were washed 3 times with PBS before incubation with the following secondary antibodies: anti-rabbit , anti-mouse  and anti-goat  for 1 h at room temperature. Images were taken using a Zeiss LSM 510 confocal microscope.p < 0.05, ** p < 0.01, ***p < 0.001.All error bars reflect standard errors. Statistical analysis was conducted with Student’s t-tests. Significance was set at 0.05. Asterisks denote significance as follows: *


Alzheimer’s disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.We prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells.Monomers, oligomers, and fibrils of Aβ were successfully prepared. Aβ oligomers and fibrils interacted with NLRP3. Aβ oligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβ monomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1β was not secreted according to the cell-based assay.Oligomerized Aβ originating from non-toxic Aβ monomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer’s disease.The online version of this article (10.1186/s41232-018-0085-6) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by neuronal cell death accompanied by the interstitial deposition of amyloid β (Aβ) plaque and cytoplasmic phosphorylated tau protein, and neurofibrillary tangles (NFTs) . RecentlThe inflammasome has been identified as an intracellular complex that plays an important role in innate immune responses against pathogens and toxic metabolites. NACHT, LRR, and PYD domain-containing protein 3 (NLRP3), an intracellular pattern recognition receptor, has been reported as a component of the intracellular innate immune receptor complexes with adopter protein ASC that forms inflammasome, an interleukin (IL)-1β-processing platform –4.We previously developed the NLRP3 inflammasome in a cell-free system to detect intrinsic and extrinsic directly interacting ligands . The NALThere have been several reports that Aβ oligomerization, which is thought to play an important role in the innate immune response and cell death, is required for the pathogenesis of AD , 8. It hCurrently, numerous ligands, including Aβ, have been reported to activate the NLRP3 inflammasome; however, none were confirmed at the molecular level in previous reports. Thus, the purpose of our study was to clarify whether Aβ oligomers directly or indirectly activate the NLRP3 inflammasome. In order to confirm direct interactions, we used the cell-free system in this study.Aβ42 was purchased form the Peptide Institute . Anti-Aβ mouse monoclonal antibody (6E10) and anti-mouse immunoglobulin G (IgG) were purchased from BioLegend  and R&D Systems , respectively. Thioflavin T (ThT) was obtained from Sigma-Aldrich . Anti-FLAG monoclonal antibody M2 (F1804) was purchased from Sigma-Aldrich . Human IL-1β ELISA Set II was purchased from BD Biosciences . Anti-cleaved caspase-1 rabbit monoclonal antibody (Asp297) (D57A2) (S4199) was purchased from Cell Signaling Technology . MCC950 (PZ0280) was purchased from Sigma-Aldrich . Isoliquiritigenin (I0822) was purchased from Tokyo Chemical Industry Co., Ltd. .Aβ42 stock solution (400 μM) was prepared by dissolving the lyophilized peptide in 0.1% ammonia solution. The oligomerization reaction was initiated by diluting the stock solution in 10-times diluted phosphate-buffered saline (PBS)  at room temperature. For oligomer and fibril formation, Aβ42 monomers (45 μM) were incubated at room temperature for 14 h (oligomers) or at 37 °C for 48 h (fibrils).The Aβ samples (7.5 μL) were mixed with the native-PAGE sample buffer (2.5 μL) and used for native-PAGE with an 8% gel. A NativeMark Unstained Protein Standard (Invitrogen) comprising 1236 IgM Hexamer (1236 kDa), Apoferritin band 1 (720 kDa), Apoferritin band 2 (480 kDa), and bovine serum albumin (66 kDa) was used as a molecular mass marker. Following transfer to a nitrocellulose membrane, the membrane was blocked with 5% skim milk in Tris-buffered saline including 0.05% Tween20 (TBST) for 1 h at room temperature. Then, the membrane was incubated with anti-Aβ  for 1 h at room temperature. After washing with TBST, it was incubated with the secondary antibody  for 1 h at room temperature. After washing with TBST, proteins were visualized using the ECL Plus Western Blotting Detection System . Luminescence was detected by the LAS4000 mini luminescent Image Analyzer .The 10 μM Aβ samples were dropped onto a silicon wafer and allowed to air-dry. Samples were observed at an acceleration voltage of 15 kV using a field-emission scanning electron microscope .The Aβ samples (5 μM) were mixed with a ThT solution (50 μM) in PBS. Then, ThT fluorescence was measured using a microplate reader  at an excitation wavelength of 450 nm and emission wavelength of 490 nm .pDONR221-NLRP3 and pDONR221-ASC were inserted into pEU-E01-GW-bls-STOP and pEU-E01-GW-STOP, respectively, for cell-free protein expression, as previously reported . The entforward primer S1-GFP_F: 5′-CCACCCACCACCACCAATGGTGAGCAAGGGCGAGG-3′ with reverse primer GFP-T1(F)_R: 5′- TCCAGCACTAGCTCCAGACTTGTACAGCTCGTCCATGC -3′. The second step was carried out using the following primer sets: attB1-S1: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCACCCACCACCACCAATG-3′ and attB2-T1: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCCAGCACTAGCTCCAGA-3′. PCR products were inserted into a Gateway® pDONR™221 Vector (pDONR221)  using the Gateway® BP Clonase™ II Enzyme mix  to generate entry clones. The GFP entry clones pDONR221-GFP was inserted into pEU-E01-GW-bls-STOP using the Gateway™ LR Clonase™ II Enzyme mix  for cell-free protein expression. The constructed plasmids were used to synthesize specific proteins with a WEPRO1240 Expression Kit , followed by Western blotting to confirm prompt protein synthesis.v/v) Tween20], 1 mg/mL of BSA, 17 μg/mL of streptavidin-conjugated ALPHA donor beads , 17 μg/mL of protein-A-conjugated ALPHA acceptor beads, and 5 μg/mL of anti-FLAG mAb M2 and incubated in a 1/2ALPHAPlate-96 shallow well  at 25 °C for 24 h. The fluorescence emission signals of each well were measured using an EnSpire™ Multimode Plate Reader .Synthesized protein-protein interactions were assessed using the amplified luminescent proximity homogeneous assay (ALPHA). A total of 100 ng of each protein was added to ALPHA buffer [100 mM Tris-HCl (pH 8.0), 0.01% (The interaction between NLRP3 and Aβ was assessed by incubating 100 ng of NLRP3-FL-Btn with 5 μg/mL of anti-Aβ mAb (6E10) , 17 μg/mL of protein-A-conjugated ALPHA acceptor beads, and 17 μg/mL of streptavidin-conjugated ALPHA donor beads for 24 h with the indicated concentrations of Aβ and incubated in a 1/2ALPHAPlate-96 shallow well  at 25 °C for 24 h. The fluorescence emission signals of each well were measured using an EnSpire™ Multimode Plate Reader .5 HEK 293T cells were co-transfected with the indicated amount of non-tagged expression plasmids such as pcDNA3-NLRP3, pcDNA3-ASC, pcDNA3-caspase-1, and pcDNA3-IL-1β. After hours, the transfection medium was changed to fresh medium together with Aβ monomers, oligomers, or fibrils at the indicated concentrations. IL-1β concentrations in the culture supernatants were measured 16 h after medium change using the enzyme-linked immunosorbent assay (ELISA) with specific human IL-1β antibodies  according to the manufacturer’s instructions.Human embryonic kidney (HEK) 293T cells were maintained in DMEM (Gibco) with 10% heat-inactivated FBS, penicillin, and streptomycin. Transfection was carried out with the calcium phosphate method. A total of 1 × 105/mL in a volume of 1 mL of RPMI1640, including 10% FBS with Aβ oligomers, and lipopolysaccharide (LPS) or left untreated for 16 h at 37 °C in a humidified atmosphere with 5% CO2. Concentrations of IL-1β in the culture supernatants were measured by ELISA with specific human IL-1β antibodies  according to the manufacturer’s instructions.Human peripheral blood mononuclear cells (MNCs) were separated by Ficoll gradient centrifugation  according to the manufacturer’s instructions. The cells were cultured in 24-well flat-bottom plates  at a final cell density of 5 × 10The levels of endotoxin contamination in Aβ monomers, oligomers, and fibrils were quantified by the Limulus amebocyte lysate assay using Endospecy, a chromogenic endotoxin-specific assay kit, according to the manufacturer’s instructions .U test. A p value < 0.05 was considered significant.Results are presented as the mean ± standard deviation of triplicate data acquisition, and the significance of differences was evaluated using the Mann-Whitney Aβ oligomerization reactions successfully progressed, as confirmed by native PAGE followed by Western blotting analysis, the ThT assay, and SEM analysis Fig. . As showC-terminal biotinylated full-length NLRP3 (NLRP3-FL-Btn), N-terminal FLAG-tagged full-length ASC (FLAG-ASC-FL), and FLAG-tagged CARD-only ASC (FLAG-ASC-CARD) are schematically indicated in Fig. Next, we investigated whether Aβ monomers, oligomers, and fibrils interacted with NLRP3 under cell-free conditions with ALPHA. Direct interactions between NLRP3-FL-Btn and Aβ oligomers or Aβ fibrils were observed in a dose-dependent manner Fig. .Next, we tested whether Aβ induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL. Both Aβ oligomers and Aβ fibrils significantly induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL in the cell-free system   DMSO were not reduced compared with 1% (v/v) DMSO alone (p = 0.275)  and found that NLRP3 was able to more specifically recognize Aβ as compared with GFP Additional file 2:Figure S2. MCC950 (A) and isoliquiritigenin (B), known NLRP3 inhibitors, did not affect the interaction between FLAG-NLRP3-FL and Aβ oligomers or fibrils in the cell-free system. (PPTX 51 kb)


The crystal structures of the fumarate salts of the psychomimetics MiPT and 4-HO-MiPT are reported. The extended structure of both compounds feature two-dimensional networks of ions connected through N—H⋯O and O—H⋯O hydrogen bonds. N-isopropyl-N-methyltryptaminium (MiPT) fumarate {systematic name: [2-(1H-indol-3-yl)ethyl](methyl)propan-2-ylazanium 3-carboxyprop-2-enoate}, C14H21N2+·C4H3O4−, and 4-hydroxy-N-isopropyl-N-methyltryptaminium (4-HO-MiPT) fumarate monohydrate {systematic name: [2-(4-hydroxy-1H-indol-3-yl)ethyl](methyl)propan-2-ylazanium 3-carboxyprop-2-enoate monohydrate}, C14H21N2O+·C4H3O4−·H2O, are reported. Both salts possess a protonated tryptammonium cation and a 3-carboxyacrylate (hydrogen fumarate) anion in the asymmetric unit; the 4-HO-MiPT structure also contains a water molecule of crystallization. Both cations feature disorder of the side chain over two orientations, in a 0.630 (3):0.370 (3) ratio for MiPT and a 0.775 (5):0.225 (5) ratio for 4-HO-MiPT. In both extended structures, N—H⋯O and O—H⋯O hydrogen bonds generate infinite two-dimensional networks.The solid-state structures of the salts of two substituted tryptamines, namely  N,N-dimethyltryptamine (DMT) and its derivatives have been used by humans for centuries because of their psychoactive, entheogenic, or hallucinogenic effects, or combinations thereof  was reported in 1981  is the most potent based upon qualitative effects on humans  cation and one 3-carboxyacrylate (C4H3O4−) anion. The indole ring system of the cation is near planar with an r.m.s. deviation from planarity of 0.006 Å. The singly protonated fumarate anion is in the trans configuration and is slightly distorted from planarity with an r.m.s. deviation of 0.133 Å and a carboxylate twist angle of 18.370 (5)°. The N-methyl-N-isopropylammonium group is disordered over two orientations in a 0.630 (3):0.370 (3) ratio.The molecular structure of MiPT fumarate is shown on the left of Fig. 1N-methyl-N-isopropyltryptammonium (C14H21N2O+) cation, one 3-carboxyacrylate anion and one water molecule of crystallization. The indole ring system of the cation is close to planar with an r.m.s. deviation of 0.021 Å. The singly protonated fumarate anion is also near planar with an r.m.s. deviation of 0.049 Å. The N-methyl-N-isopropylammonium group shows a similar disorder to the MiPT structure over two orientations in a 0.775 (5):0.225 (5) ratio.The molecular structure of 4-HO-MiPT fumarate monohydrate is shown on the right of Fig. 1N-methyl-N-isopropylamine and fumarate ions are linked into infinite two-dimensional networks lying parallel to the (010) plane through N—H⋯O and O—H⋯O hydrogen bonds , which has been structurally characterized .Single crystals suitable for X-ray analysis were obtained from the slow evaporation of aqueous solutions of commercial samples of Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). The following restraints were applied: C—N = 1.54±0.01, N—H = 0.87±0.01, O—H = 0.86±0.01 Å. N- and O-bound H atoms were refined with Uiso(H) = 1.5Ueq. In the MiPT fumarate, the N-methyl-N-isopropylaminium group is disordered. It is modeled as two components: N2 and C11–C14 with an occupancy of 0.630 (3) and N2A and C11A–C14A with an occupancy of 0.370 (3). 4-HO MiPT fumarate exhibits a similar disorder of the N-methyl-N-isopropylaminium group that is modeled as two components: N2, C11--C14 with an occupancy of 0.775 (5) and N2A, C11A–C14A with an occupancy of 0.225 (5).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019011253/hb7844sup1.cifCrystal structure: contains datablock(s) MiPT, 4-HO-MiPT, I. DOI: 10.1107/S2056989019011253/hb7844MiPTsup2.hklStructure factors: contains datablock(s) MiPT. DOI: 10.1107/S2056989019011253/hb78444-HO-MiPTsup3.hklStructure factors: contains datablock(s) 4-HO-MiPT. DOI: Click here for additional data file.10.1107/S2056989019011253/hb7844MiPTsup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019011253/hb78444-HO-MiPTsup5.cmlSupporting information file. DOI: 1946718, 1946717CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


All dimethoxybenzaldehyde molecules in the crystal structure are nearly planar.The crystal structures of four dimethoxybenzaldehyde (C 9H10O3) isomers, namely the 2,3-, 2,4-, 2,5- and 3,5- isomers, are reported and compared to the previously reported crystal structures of 3,4-dimethoxybenzaldehyde and 2,6-dimethoxybenzaldehyde. All dimethoxybenzaldehyde molecules in the crystal structures are nearly planar. The largest deviation (1.2 Å) from the aromatic plane is found for one of the methoxy groups of 2,3-dimethoxybenzaldehyde. Upon rapid cooling of 3,4-dimethoxybenzaldehyde and 3,5-dimethoxybenzaldehyde, a metastable polymorph is formed. The crystal studied for the 3,5- isomer was refined as a two-component twin.The crystal structures of four dimethoxybenzaldehyde (C Schiff base compounds are versatile ligands in numerous metal–organic complexes that are used as a catalyst. Examples include C—O coupling reactions  Å]. Similarly, in the crystal structure of 2,5-DMBz, aromatic π–π stacking interactions are present along the a-axis direction [centroid–centroid separation = 3.8780 (3) Å], as shown in Fig. 7et al., 2014et al., 2016In the crystal structure of 2,3-DMBz, one of the methoxy groups lies in the plane of the aromatic ring  was dissolved in 4 mL of isopropyl ether. Slow evaporation of a 1:1 mixture of this solution and heptane yielded colorless block-shaped crystals suitable for single crystal X-ray diffraction.25 mg of 2,4-dimethoxybenzaldeyhyde  was dissolved in a 1:1 ratio of heptane/acetone (1.5 mL). Slow evaporation yielded colorless block-shaped crystals suitable for single crystal X-ray diffraction.1 g of 2,5-dimethoxybenzaldeyhyde  was dissolved in a mixture of heptane (1 mL) and acetone (1 mL). Slow evaporation yielded colorless needles suitable for single crystal X-ray diffraction.et al., 2016It was noted that 3,5-dimethoxybenzaldehyde  oils out from solution, therefore the same method was used as had previously been employed for 3,4-dimethoxybenzaldehyde (de Ronde Uiso(H) = 1.2–1.5Ueq(C). The crystal of 3,5-DMBz studied was refined as a two-component twin.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018017152/zp2031sup1.cifCrystal structure: contains datablock(s) 2,3DMBz, 2,4DMBz, 2,5DMBz, 3,5DMBz. DOI: 10.1107/S2056989018017152/zp203123DMBzsup2.hklStructure factors: contains datablock(s) 2,3DMBz. DOI: 10.1107/S2056989018017152/zp203124DMBzsup3.hklStructure factors: contains datablock(s) 2,4DMBz. DOI: 10.1107/S2056989018017152/zp203125DMBzsup4.hklStructure factors: contains datablock(s) 2,5DMBz. DOI: 10.1107/S2056989018017152/zp203135DMBzsup5.hklStructure factors: contains datablock(s) 3,5DMBz. DOI: Click here for additional data file.10.1107/S2056989018017152/zp203123DMBzsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018017152/zp203124DMBzsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018017152/zp203125DMBzsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018017152/zp203135DMBzsup9.cmlSupporting information file. DOI: 1882761, 1882760, 1882759, 1882758CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


I compound based on 3-adamantane-1-carboxylate (−tr-ad-COO) is a three-dimensional coordination framework with channels along the c-axis direction. Hydrogen bonding between water molecules of crystallization and carboxylate groups is realised in form of right- and left-handed helical motifs.The title Ag −tr-ad-COO), was employed for the synthesis of the title silver(I) coordination polymer, {[Ag(C13H16N3O2)]·2H2O}n, crystallizing in the rare orthorhombic C2221 space group. Alternation of the double μ2-1,2,4-triazole and μ2-η2:η1-COO−  bridges between AgI cations supports the formation of sinusoidal coordination chains. The AgI centers possess a distorted {N2O3} square-pyramidal arrangement with τ5 = 0.30. The angular organic linkers connect the chains into a tetragonal framework with small channels along the c-axis direction occupied by water molecules of crystallization, which are interlinked via O—H⋯O hydrogen bonds with carboxylate groups, leading to right- and left-handed helical dispositions.The heterobifunctional organic ligand, 3-adamantane-1-carboxylate ( The packig is shown in Fig. 5The water guest molecules inside the [001] channels are responsible for the extended hydrogen-bonding network Table 2. Togethetr-ad-COOH) was synthesized by refluxing 3-amino-adamantane-1-carboxylic acid -3-carboxyadamantane , tr-ad-COOH  and 5 ml of water was added into a Teflon vessel, which was sealed and heated at 413 K for 24 h and slowly cooled to room temperature over 48 h, yielding colourless needles of I .The synthesis of A⋯O5B = 0.77 Å). CH hydrogen atoms were positioned geometrically and refined as riding, with C—H = 0.94 Å (triazole); C—H = 0.98 Å (adamantane CH2); C—H = 0.99 Å (adamantane CH) and with Uiso(H) = 1.2Ueq(C). OH hydrogen atoms were located and then refined with O—H = 0.85 Å (H2O) and with Uiso(H) = 1.5Ueq(O). For one of the disordered water molecules, the H atoms were not located.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019009708/hb7837sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019009708/hb7837Isup2.hklStructure factors: contains datablock(s) I. DOI: 1939097CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Oncogenic Notch1 is known to activate the NF-κB pathway in T cell acute lymphoblastic leukemia (T-ALL) and to up-regulate the transcription of Asb2α, a specificity factor for an E3 ubiquitin ligase complex that plays an important role in hematopoietic differentiation. Therefore, we hypothesize that Notch1 might regulate the NF-κB pathway through Asb2α.Asb2 or Asb2-shRNA. Experiments using real-time PCR, western blot and co-immunoprecipitation were performed to evaluate the expression levels of related genes. Cell proliferation and apoptosis were measured while the expression of Asb2 was enhanced or inhibited.The study involved down-regulation of Notch1 in T-ALL cell lines (CCRF-CEM cells and MOLT-4 cells) through treatment with gamma-secretase inhibitor (GSI) as well as the modulation of Asb2 in CCRF-CEM cells and MOLT-4 cells through transduction with lentivirus carrying Here, we demonstrated for the first time that Notch1 can activate the transcription of Asb2α, which then stimulates activation of NF-κB in T-ALL cells. Asb2α exerts its effects by inducing degradation and dissociation of IκBα from NF-κB in T-ALL cells. Moreover, specific suppression of Asb2α expression can promote apoptosis and inhibit proliferation of T-ALL cells.Notch1 modulates the NF-κB pathway through Asb2α, indicating that Asb2α inhibition is a promising option for targeted therapy against T-ALL. The NF-κB protein functions as a transcription factor that mediates a broad range of biological processes, including cell survival, proliferation and differentiation . In the Asb2) gene is of particular interest due to its vital function in hematopoietic differentiation. Moreover, some studies have shown that Notch signaling initiates the degradation of Jak2, Jak3 and E2A proteins by up-regulating the expression of Asb2 [Asb2 gene encodes two different isoforms, Asb2α and Asb2β, which are involved in hematopoietic differentiation and myogenic differentiation, respectively [Considering its role in cell survival and proliferation, abnormal or uncontrolled activation of NF-κB is frequently encountered in several lymphoid malignancies and solid tumors –7. There of Asb2 , 15. As  of Asb2 , 17. Theectively , 18. Asbectively . Recentlectively , 21. ConAsb2 transcription and NF-κB activation in T-ALL cells. Inhibition of Asb2α expression can significantly decrease Notch-induced NF-κB activation, suggesting that Notch signaling mediates NF-κB activation through Asb2α. In addition, we explore the mechanism whereby Asb2α promotes NF-κB activation. Our results demonstrate that Asb2α is able to target IκBα for destruction and thus is able to free NF-κB from an inhibitory status. Our findings are the first to reveal that Asb2α is an important regulator between Notch and the NF-κB signaling pathway in T-ALL cells, indicating that Asb2α might play a vital role in T-ALL formation and shedding light on a therapeutic target for T-ALL disease.In this report, we show that Notch signaling can up-regulate Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen . Propidium iodide was obtained from Sigma . FITC-conjugated annexin V was purchased from BD Biosciences . The Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Institute of Biotechnology (China). DMSO, GSI and MG132 were also purchased from Sigma .2 in air. For the chemical treatment experiments, exponentially grown CCRF-CEM cells and MOLT-4 cells were harvested, resuspended (at 4 × 105 cells/ml) in fresh culture medium and incubated for 24 h before treatment with 5 μM MG132 or 10 μM GSI for 24 h. DMSO-treated cells served as the control. For viral infection experiments, exponentially grown CCRF-CEM cells and MOLT-4 cells were harvested, resuspended (at 1 × 105 cells/ml) in fresh culture medium and incubated for 12 h before being infected with 4 × 106 TU of lentivirus for 72 h.Human embryonic kidney (HEK) 293 cells were cultured in DMEM supplemented with 10% FBS. The CCRF-CEM human immature T cell line was obtained from Shanghai Bioleaf Biotech . The human leukemia T-cell line (MOLT-4 cells) was purchased from Procell . CCRF-CEM and MOLT-4 cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 °C in a humidified atmosphere of 5% COAsb2 shRNA2 were as follows: sense 5’-CAGGCAGGCTGATTAGATATTCAAGAGATATCTAATCAGCCTGCCTGTTTTTTCTCGAGG-3’ and antisense 5’-GATCCCTCGAGAAAAAACAGGCAGGCTGATTAGATATCTCTTGAATATCTAATCAGCCTGC CTG-3’. Plasmids pLVX-shRNA2-m and PLVX-mcmv-ZsGreen1 were purchased from Biowit Technologies, Ltd. (China). pLVX-shRNA2-m was first digested with PstI and then filled in with Klenow. The larger fragment was then extracted and cleaved with BamHI. Finally, the Asb2 shRNA oligonucleotides were synthesized, annealed and ligated into the pLVX-shRNA2-m vector to obtain pLVX-shRNA2-hASB2. pCMV-ASB2-HA and Asb2 deletion constructs were kindly provided by Dr. Jay L. Hess . The full-length HA-tagged hAsb2 sequence was then cloned into the pLVX-mcmv-ZsGreen1 vector through EcoRI and NotI digestion and ligation. The sequences for the Asb2 shRNA1 were as follows: sense 5’-CACCCGAACATCGACGCCTATATTTCAAGACGATA TAGGCGTCGATGTTCG TTTTTTG-3′ and antisense 5’-AGCTCAAAAAACGAACATCGACGCCTATATCGTCTTGAAA TATAGGCGTCGATGTTCG-3’. The sequences for the Asb2 shRNA3 were as follows: sense 5’-CACCGGCTGATTAGATACCTGAA TTCAAGACGTTCAGGTATCTAATCAGCCTTTTTTG-3’ and antisense 5’-AGCTC AAAAAAGGCTGATTAGATACCTGA ACGTCTTGAATTCAGGTATCTAATCAGCC-3’.The sequences for the .6 293 T cells were seeded in 10-cm tissue culture plates in 10 ml of growth medium and then incubated at 37 °C with 5% CO2 overnight. The cells were 80–90% confluent at the time of transfection. Approximately 2–4 h before transfection, the medium was replaced with 5 ml of fresh complete growth medium. The 293 T cells were transfected with a highly efficient transfection reagent  according to the manufacturer’s instructions. Approximately 12–16 h after transfection, the transfection medium was replaced with 10 ml of fresh complete growth medium, and the cells were incubated at 37 °C for an additional 48 h. The cells were then harvested, and the lentiviral supernatant was filtered through a 0.45-μm low-protein-binding filter to remove cellular debris.The 293 T cell line was used to obtain lentivirus from packaging plasmids and the lentiviral vector. Approximately 24 h before transfection, 6–8 × 10HEK293 cells were lysed with Cell Lysis Buffer for Western and IP (Beyotime) at 4 °C for 15 min. The cell extracts were incubated with anti-HA antibody (1:5000) overnight at 4 °C. Agarose affinity beads were then added and incubated with the extracts for 1 h at room temperature. The beads were washed 3 times with RIPA buffer . Proteins were eluted by boiling in SDS loading buffer, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and detected by Western blot. The primary antibody directed against the HA tag was obtained from Santa Cruz.The cells were lysed in Cell Lysis Buffer for Western and IP (Beyotime) and incubated at 4 °C for 15 min. Protein extracts were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes before overnight incubation with primary antibodies directed against Asb2 (Santa Cruz), IκBα , NF-κB-p65 (BioWorld), Caspase 3 , NICD (abcam), Hes1 (abcam), lamin  or GAPDH  at 4 °C. The membrane was washed with 0.1% Tween-20 in Tris-buffered saline and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG secondary antibody  for 1 h at room temperature. The immunoreactive bands were visualized using an ECL Western blotting detection kit  with light-sensitive film.RNA was extracted from cells using TRIzol reagent (Invitrogen), and cDNA was synthesized using a First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions. Real-time quantitative reverse-transcription PCR (RT-qPCR) was performed using 2X SYBR Green/Fluorescein qPCR Master Mix (Fermentas) on an ABI 7900 Sequence Detection System (Applied Biosystems). The data were analyzed using the comparative CT method (ABI User Bulletin number 2). The primer sets were as follows: Asb2, forward 5’-CGTGGTGCAGTTCTGTGAGT-3′ and reverse 5’-GTGAGCCAGAGGTCTTGGAG-3’; IκBα, forward 5’-GCAAAATCCTGACCTGGTGT-3′ and reverse 5’-GCTCGTCCTCTGTGAACTCC-3’; Actin, forward 5’-AGCGAGCATCCCCCAAAGTT-3′ and reverse 5’-GGGCACGAAGGCTCATCATT-3’.3 CCRF-CEM cells were resuspended in 100 μl of RPMI 1640 medium in a 96-well plate. Lentiviruses harboring empty vector, Asb2 or Asb2-shRNA were then incubated with the cells for 72 h. After the cells were infected, 10 μl of CCK-8 solution was added to each well, and the 96-well plate was continuously incubated at 37 °C for 2.5 h. The OD value for each well was then read at 450 nm on a microplate reader  to determine cell viability. The assay was repeated three times. Cell viability was calculated as follows:Cell viability was determined using a CCK-8. Briefly, 5 × 106) were washed in ice-cold PBS and resuspended in 500 μl of annexin V binding buffer  containing annexin V-FITC and PI (1 μg/ml) before being incubated for 30 min at 4 °C. FACS analysis was performed with Cell Quest-Pro software, and cells negative for both annexin V and PI were considered viable.ALL cell apoptosis was detected using an annexin V/propidium iodide (PI) staining assay. Cells (1 × 10p < 0.05 was considered to indicate a significant difference.All the experiments were performed three times. The data are expressed as mean ± standard deviation (SD). Differences between the control and experimental results were tested by Student’s t-test (two-tailed) and one-way analysis of variance (ANOVA). All statistical analyses were carried out using SPSS version 22.0  and Asb2 gene transcription in NIH 3 T3 cells (a mouse embryonic fibroblast cell line) and in lymphoid cells [Asb2 transcription was significantly decreased 24 h after GSI treatment of CCRF-CEM cells and MOLT-4 cells  E3 ubiquitin ligase complex, Asb2-induced IκBα degradation may be proteasome dependent. To verify this hypothesis, CCRF-CEM cells and MOLT-4 cells were treated with the proteasome inhibitor MG132. Proteasome inhibition significantly restored the protein level of IκBα in CCRF-CEM cells and MOLT-4 cells Fig. , suggestTo test whether IκBα physically interacts with Asb2, 293 cells were transfected with HA-tagged Asb2 or with empty vector. A co-immunoprecipitation (co-IP) assay was then performed using an antibody directed against the HA tag. This experiment showed that IκBα was pulled down by HA-tagged full-length Asb2 Fig. , suggestAsb2 expression can suppress the tumorigenesis of T-ALL cells, CCRF-CEM cells and MOLT-4 cells were transduced with lentiviruses containing wild-type Asb2, Asb2-shRNA or empty vector. MTT assay results demonstrated that the expression of wild-type Asb2 could significantly increase the viability of CCRF-CEM cells, whereas Asb2 knockdown could decrease the viability of CCRF-CEM cells (Fig. Asb2 led to a lower number of apoptotic CCRF-CEM cells, whereas Asb2 knockdown resulted in an elevated number of apoptotic CCRF-CEM cells through annexin V binding (Fig. Asb2 knockdown resulted in elevated expression of cleaved caspase-3 in MOLT-4 cells (Fig. Enhanced activation of NF-κB results in the occurrence of cancer by promoting cellular proliferation and inhibiting apoptosis . To testlls Fig. . In addiing Fig. . Consistlls Fig. . These rHes1, which then suppresses the expression of CYLD, a negative regulator of IKK activity in T-ALL cells [A previous study found that Notch stimulates NF-κB activation by initiating the transcription of LL cells ; howeverAsb2 is expressed during all-trans retinoic acid-induced differentiation of promyelocytic cell lines . Asb2α iAs the substrate-binding subunit of the ECS ligase complex, Asb2 mediates the ubiquitination of certain substrates, such as the actin-binding protein filamin, as well as Jak2, Jak3 and E2A , 24–26. Asb2 belongs to a large protein family that has 18 members in humans . Each ofWe demonstrated that Notch1 is able to up-regulate the expression of Asb2α and activate NF-κB in T-ALL cells. Furthermore, we found that Notch1 regulates the NF-κB pathway through Asb2α, which is capable of interacting with IκBα and then inducing degradation of IκBα. We also showed that suppression of Asb2α expression can promote apoptosis and inhibit proliferation of T-ALL cells, suggesting that Asb2α may play an important role in the pathogenesis of T-ALL. Taken together, our findings might provide a promising option for targeted therapy against T-ALL.


Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the p57Kip2 imprinting domain, is expressed exclusively from the paternal allele and participates in the cis-silencing of the neighboring imprinted genes through chromatin-level regulation. In light of our previous evidence of a functional interaction between myogenic factors and imprinting control elements in the regulation of the maternal p57Kip2 allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of p57Kip2 expression in muscle cells.The cell-cycle inhibitor p57p57Kip2 allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel p57Kip2 regulatory region, located intragenically and containing two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion demonstrated that the LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal p57kip2 intragenic region. Finally, upon differentiation, the binding of MyoD to this region and its physical interaction with Kcnq1ot1, analyzed by ChIP and RNA immunoprecipitation assays, correlate with the loss of EZH2 and H3K27me3 from chromatin and with p57Kip2 de-repression.We found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional p57Kip2 during differentiation and, possibly, its aberrant silencing observed in several pathologic conditions.These findings highlight the existence of an imprinting-independent role of Kcnq1ot1, adding new insights into the biology of a still mysterious LncRNA. Moreover, they expand our knowledge about the molecular mechanisms underlying the tight and fine regulation of The online version of this article (10.1186/s13072-019-0253-1) contains supplementary material, which is available to authorized users. Kcnq1ot1 expression levels were analyzed 24 h after transfection in proliferation conditions, while 24 h after the shift to differentiation conditions (DMEM containing 0.5% FCS) samples were collected to analyze p57 and p21 expression levels.8 × 10Kcnq1ot1 expression and 24 h after the shift to differentiation medium to determine p57 and p21 expression levels.Polymorphic fibroblasts (C57BL/6 × SD7) expressing MyoD were transfected 48 h after infection, with 200 nM siRNAs. Samples were collected 24 h after transfection to check the reduction of 6 cells were fixed with 1% formaldehyde for 10 min at 37 °C. Glycine was added to a final concentration of 0,125 M in phosphate-buffered saline (PBS) for 5 min at 4 °C and 5 min at room temperature with gentle shaking. Cells were washed with cold PBS, scraped and nuclei were isolated in buffer A , 10 mM NaCl, and 0.5% NP-40). After centrifugation and washing once in an equal volume of buffer A, nuclei were lysed in buffer B , and incubated on ice for 10 min. An equal volume of buffer C  was added, and nuclear chromatin was sonicated to obtain fragment sizes ranging from 300 to 700 bp in length. Chromatin was quantified for DNA, and 150 μg aliquots were prepared for each sample. Protein A Sepharose was blocked in an equal volume of buffer D  containing 400μg/ml yeast RNA and 800 μg/ml of bovine serum albumin (BSA) for 4 h. Beads were washed three times with buffer D and incubated overnight at 4 °C with 5 μg of anti-biotin antibody . The sonicated chromatin was incubated overnight at 4 °C with 25 pmol of biotin-labeled oligo antisense to Kcnq1ot1 (biotin 5′-CCAAAAGAACTGTGGACAAATATGCTGAGGCTG-3′), or biotin-labeled Scrambled oligo (biotin 5′-GCCAAGTGTTAAAGGCCAAACTACGTTGAGAGA-3′), and incubated overnight at 4 °C. 50 μl of beads with bound antibody was added to samples containing the oligonucleotides and incubated for 4 h at 4 °C. Before washing, the supernatant of the scrambled samples was taken as Input. Each sample was washed twice with Low Salt buffer  and with High Salt buffer ; once time with LiC Buffer  and TE Buffer . Samples were eluted with 300μl of Elution Buffer , treated with 1 μg of RNAse A (Sigma-Aldrich) for 10 min at room temperature and with and 240 μg of Proteinase K (Sigma-Aldrich) for 5 h at 62 °C. The DNA was extracted with phenol–chloroform, precipitated with isopropanol and re-suspended in 50 μl of nuclease-free water (Promega). After the immunoprecipitation, the DNA concentration of each sample was measured. The enrichment of Kcnq1ot1-interacting genomic DNA regions was analyzed by qPCR using the following primers:Maternal KvDMR1F: 5′-ACTCGGAATTCAGGTGTGGG-3′R: 5′-GGTTGGAGGTCACCACAACAT-3′Paternal KvDMR1F: 5′-ACTCGGAATTCAGGTGTGGA-3′R: 5′-GGTTGGAGGTCACTACAACAT-3′p57 promoterF: 5′-ACTGAGAGC-AAGCGAACAGG-3′R: 5′-ACCTGGCTGATTGGTGATGG-3′KvDMR1F: 5′-GCACAAGTCGCAAGTCCGCG-3′R: 5′-ATGGAGCCCAGCCGCGAAAG-3′p57iMaternal F: 5′-CAGATCTGACCTCAGACCCA-3′R: 5′-GACCTGTTCCTCGCCATCCT-3′p57iPaternal F: 5′AACTTCCAGCAGGATGTGCC-3′R: 5′-GACCTGTTCCTCGCCATCCT-3′p57iF: 5′-AACTTCCAGCAGGATGTGCC-3′R: 5′-CATCCACTGCAGACGACCAG-3′Dppa2 promF: 5′-TGCCCTGGATTTAAAACGTC-3′R: 5′-CGAGCTTTGTCCTCCTGGTA-3′Nap1l4 promF: 5′-AGGTGTTGGGATTGAAGGTG-3′R: 5′-CACCCAATACAAAGGCTGCT-3′ChOP assays were carried out according to the published protocol , 75 withKcnq1ot1F: 5′-TTCTGGAGGCGATTGAGGC-3′R: 5′-AGCAACCAGAACCAGGTGAGAG-3′HOTAIRF: 5′-GCGCCAACGTAGACCAAAAG-3′R: 5′-TACCGATGTTGGGGACCTCT-3′Undifferentiated and differentiated C2.7 muscle cells were harvested in PBS supplemented with 0,2 mM PMSF. Nuclei were isolated with buffer A , incubated at 4 °C for 30 min. Nuclei were extracted with NT2 buffer . Nuclear protein extracts were quantified and 1,5 mg of protein were used for each sample. 100μl of protein A Sepharose were incubated with 5 μg of anti-MyoD antibody , anti-EZH2 antibody, anti-LSD1 antibody (ab 17721 Abcam), normal rabbit IgG ([12-370] Merk Millipore) or normal mouse IgG ([12-371] Merk Millipore) for 6 h at 4 °C. Lysate pre-clearing was performed using 150μl of protein A Sepharose and keeping samples for 6 h at 4 °C. 100 μl of beads with bound antibody were added to pre-cleared samples and incubated overnight at 4 °C. Before washing, the supernatant of the IgG sample was taken as Input. Each sample was washed four times with 500 μl of NT2 buffer, treated with 10U of DNase (Roche), RNA was extracted with Trizol Reagent (Invitrogen) and precipitated overnight with isopropanol. RNA was re-suspended in 25 μl of nuclease-free water (Promega), and Kcnq1ot1 enrichment was analyzed by RT-qPCR using the following primers:t test. Statistical significance is shown as p < 0.05 (*) or p < 0.01 (**) or p < 0.001 (***).For statistical analysis, comparisons were performed using parametric paired Student’s Additional file 1.Kcnq1ot1 knockdown boosts p57 induction in MyoD-converted fibroblasts. Polymorphic fibroblasts  infected with the MyoD retroviral vector were transfected with Kcnq1ot1 or control siRNAs and analyzed by RT-qPCR for p57 and p21 expression 24 h after the shift to differentiation medium. Values, relative to those of Tbp RNA, are the mean ± SEM of three independent experiments. Statistical significance: p value < 0.05 (*).Additional file 2. Kcnq1ot1 knockdown affects maternal but not paternal p57 expression. RNAs from siCTR and siKcnq1ot1 samples  and differentiated (D) C2.7 muscle cells; p57 and the muscle-specific gene Myogenin (MyoG) were used to follow the differentiation process. Values, relative to those of Tbp RNA, are the mean ± SEM of four independent experiments. Statistical significance: p value < 0.05 (*); p value < 0.01 (**).Additional file 4. Differential epigenetic status of the maternal and paternal p57 intragenic regions. Left: Allele-specific ChIP-qPCR analysis of H3K4me3 accumulation at Maternal and Paternal p57 intragenic regions  in polymorphic fibroblasts; Amylase promoter (Amy p) was used as negative control. Values are the mean ± SEM of three independent experiments performed and were expressed as percentages of Input. Statistical significance: p value < 0.05 (*). Right: qPCR analysis of the MeDIP assays performed in polymorphic fibroblasts  using allele-specific primers for the p57 intragenic region . Translocase of inner mitochondrial membrane 17 promoter (Timm p) was used as a negative control. The results shown represent one of two independent experiments performed. Values were expressed as percentages ± SEM of Input DNA for each sample analyzed in triplicate.Additional file 5. Verification of Kcnq1ot1 depletion in cells used for the ChIP assays reported in Fig. p value < 0.001 (***)Additional file 6.H3K27me3 association to the p57 intragenic region decreases after Kcnq1ot1 depletion. Polymorphic fibroblasts  infected with the MyoD retroviral vector were transfected with Kcnq1ot1 or control siRNAs as in Fig. p57 intragenic region (M-p57i), β-Globin promoter (β-Glob p) used as an invariant control and β-Actin promoter (β-Act p) as a negative control. Values obtained are expressed as percentages of Input chromatin and normalized to those of Albumin promoter, used as an additional invariant control. The results shown represent one of two independent experiments and error bars represent the mean ± SEM of each sample analyzed in triplicate.Additional file 7. H3K27me3 association to the p57 intragenic region decreases during differentiation. ChIP-qPCR analysis of H3K27me3 association to the p57 intragenic region (p57i) and p57 promoter (p57 p) in undifferentiated (U) and differentiated (D) C2.7 muscle cells. β-Actin promoter (β-Act p) was used as a negative control. Values obtained were expressed as percentages of Input chromatin and normalized to those of Albumin promoter, used as an invariant control. The results are the mean ± SEM of three independent experiments. Statistical significance: p value < 0.05 (*).Additional file 8. Kcnq1ot1 and HOTAIR are differentially associated with LSD1. Cell extracts of differentiated C2.7 muscle cells were immunoprecipitated using anti-LSD1 antibody or control IgG. Immunopurified materials were subjected to RT-qPCR with specific primers for Kcnq1ot1 and HOTAIR transcripts. Values, relative to a representative experiment, were expressed as fold enrichment respect to IgG.Additional file 9. Additional methods.


Radix salvia miltiorrhiza, possesses anti-oxidant and anti-inflammatory actions, as well as neuroprotective effects. The present study aims to explore the possible mechanism by which Tan IIA attenuated Aβ-induced neurotoxicity. Exposure of SH-SY5Y cells to different concentrations of Aβ led to neurotoxicity by reducing cell viability, inducing cell apoptosis and increasing neuroinflammation in a dose-dependent manner. Moreover, Aβ treatment promoted cyclooxygenase-2 (COX-2) expression and Prostaglandin E2 (PGE2) secretion, and activated nuclear transcription factor kappa (NF-κB) pathway in SH-SY5Y cells. However, pretreatment of SH-SY5Y cells with Tan IIA prior to Aβ prevented these Aβ-induced cellular events noticeably. These data suggested that Tan IIA exerted its neuroprotective action by alleviating Aβ-induced increase in COX-2 expression and PGE2 secretion via inactivation of NF-κB pathway.Amyloid-β (Aβ)-induced neurotoxicity is a major pathological mechanism of Alzheimer’s disease (AD). Tanshinone IIA (Tan IIA), extracted from traditional Chinese herb  Neurodegenerative diseases represent the progressive dysfunctions and neuron losses in the central nervous system, pathologically identified by the accumulation and misfolding of disease-specific proteins in particular part of the brain . AlzheimRadix salvia miltiorrhiza (Danshen) [Many naturally derived antioxidants have been found to be able to induce decline in Aβ-induced neurotoxicity, such as Ginsenoside Rg1 , curcumiDanshen) . Tan IIADanshen) , cardiovDanshen)  and cereDanshen) . Tan IIADanshen)  revealedDanshen) . Tan IIADanshen) . These p2.The human neuroblastoma SH-SY5Y cells obtained from the American Type Culture Collection  were grown in DMEM  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Invitrogen). Cells were maintained at 37 °C in humidified 5% CO4OH/Water and stored in aliquots in tightly sealed vials at − 20 °C. The solution was equilibrated to room temperature for at least 1 h before use. To investigate the neurotoxicity of Aβ, SH-SY5Y cells were exposed to Aβ at various concentrations  for 48 h. To detect the neuroprotective effects of Tan IIA against Aβ-induced toxicity, SH-SY5Y cells were pretreated with Tan IIA (5 or 10 μM) for 1 h, followed by exposure to Aβ (25 μM) for 48 h.Tan IIA used in the study was purchased from Sigma Chemical Company . Tan IIA was dissolved in dimethyl sulfoxide (DMSO) before use. Aβ (1–42) peptide monomer (rat/mouse) was purchased from Sigma (Sigma). Aβ Peptide was dissolved in 1% NH−ΔΔCt method. The sequences used for the real-time PCR were as follows: IL-1β, sense: 5′-GCAATGAGGATGACTTGTTCTTTG-3′ and antisense: 5′-CAGAGGTCCAGGTCCTGGAA-3′; TNF-α, sense: 5′-ACCTCTCTCTAATCAGCCCTCT-3′ and antisense: 5′-GGGTTTGCTACAACATGGGCTA-3′; IL-6, sense: 5′-AGCCACTCACCTCTTCAGAAC-3′ and anti-sense: 5′-ACATGTCTCCTTTCTCAGGGC-3′ COX-2, sense: 5′-CCAGCACTTCACGCATCAGT-3′ and anti-sense: 5′-ACGCTGTCTAGCCAGAGTTTCAC-3′; mPGES-1, sense: 5′-CCAAGTGAGGCTGCGGAAGAA-3′ and anti-sense: 5′-GCTTCCCAGAGGATCTGCAGA-3′ β-actin, 5′-CCTGACTGACTACCTCATGAAG-3′ and anti-sense: 5′-GACGTAGCACAGCTT’CTCCTTA-3′.Total RNA was extracted from SH-SY5Y cells using Trizol (Invitrogen). ImProm-II reverse transcription system  was applied to generate the first strand cDNA. qRT-PCR was performed using SYBR Premix Ex Taq™  on the 7500 Real Time PCR System . β-actin was used as endogenous controls. The relative level of mRNA was analyzed using the 2Western blot analysis was performed following previous protocols . Briefly3/well. Cell viability was analyzed using MTT assay (Sigma) after treatments described as above. The absorbance at 450 nm was then determined using a microtiter plate reader .SH-SY5Y cells were seeded into the 96-well plate at a density of 3 × 10SH-SY5Y cells inoculated in 96-well plate were treated as stated above. Then, the culture supernatant was collected to measure the concentrations of TNF-α, IL-1β, IL-6 and PGE2 using Enzyme-Linked Immunosorbent Assay (ELISA) kits , according to the manufacturer’s instructions. Proteins were extracted from treated SH-SY5Y cells to detect the protein levels of caspase 3 using ELISA kits (Elisa biotech). Nuclear cell lysates were fractionated using the Active Motif Nuclear Extract Kit , according to the manufacturer’s instructions. Nuclear lysates (20 μg) were then assayed for NF-κB that bound to immobilized double-stranded DNA oligonucleotides containing NF-κB consensus sequences, using the 96-well TransAM NF-κB ELISA kit (Elisa biotech) according to the manufacturer’s instructions.Apoptosis was assessed using an Annexin-V-FITC apoptosis detection kit . Briefly, SH-SY5Y cells were seeded in 6-well plates. After indicated treatments as mentioned above, cells were harvested and stained with FITC-Annexin V and Propidium iodide (PI). Then, cells were subjected to the FACScan flow cytometer  with Cell Quest 3.2 (Becton–Dickinson).t-test or one-way ANOVA using SPSS 19.0 software . A p value < 0.05 was considered significant.All data were presented as mean ± SD from three independent experiments. Statistical significance was determined by Student’s To determine the neurotoxicity of Amyloid β-protein (Aβ), SH-SY5Y cells were exposed to different concentrations of Aβ  for 48 h. MTT assay results indicated that Aβ resulted in a decrease in the viability of SH-SY5Y cells in a dose-dependent manner  played pivotal functions in inflammation, which was catalyzed by a rate-limiting enzyme cyclooxygenase-2 (COX-2) , 23. TheA previous study reported that Aβ could activate the NF-κB pathway . Thus, wTo investigate whether Tan IIA could prevent Aβ-induced neurotoxicity, SH-SY5Y cells were pretreated with Tan IIA (5 or 10 μM) for 1 h prior to exposure to Aβ (25 μM) for 48 h. Subsequently, MTT and flow cytometry analysis were conducted to determine the cell viability and apoptosis rate in SH-SY5Y cells. As shown in Fig. To further confirm the mechanism of Tan IIA in Aβ-mediated neuroinflammation, SH-SY5Y cells were preconditioned with Tan IIA (5 or 10 μM) for 1 h followed by treatment with Aβ (25 μM) for 48 h. The concentration and mRNA levels of inflammatory factors including IL-1β, TNF-α and IL-6 were detected by ELISA assay and qRT-PCR analysis, respectively. As expected, pretreatment of SH-SY5Y cells with Tan IIA substantially prevented Aβ-induced increase in the concentrations of IL-1β, TNF-α and IL-6 in culture supernatant  or in combined with Tan IIA (5 or 10 μM) were determined by qRT-PCR analysis, western blot analysis and ELISA assay. As displayed in Fig. In the present study, we investigated the neuroprotective effects of Tan IIA against Aβ-induced neurotoxicity in SH-SY5Y cells. Our study found that pretreatment with Tan IIA prevented Aβ-mediated reduction in cell viability, suppressed Aβ-triggered apoptosis and attenuated Aβ-induced release and expression of pro-inflammatory cytokines. Mechanistically, Tan IIA alleviated Aβ-induced COX-2 expression, PGE2 synthesis, and activation of NF-κB pathway. These results demonstrate that the neuroprotective effects of Tan IIA are most likely mediated by the NF-κB pathway.Aβ is the major protein component of senile plaques, and the excess deposition of Aβ in the brain plays a central role in the etiology of AD. Therefore, to determine the mechanism by which Aβ induces neurotoxicity is important to identify potential molecular therapeutic targets for AD. Previous research efforts have reported that cultured neurons exhibited the characteristics of apoptosis when exposed to Aβ , 18. TheTan IIA has been reported to be effective in many diseases due to its anti-oxidant and anti-inflammation effects. Qian et al.  revealedIncreasing evidence suggests that NF-κB pathway contributed to the up-regulation of pro-inflammatory and cytotoxic genes during the degenerative process of disease , 32, indIn summary, our study demonstrated that Tan IIA attenuated Aβ-induced neurotoxicity by down-regulating COX-2 expression and PGE2 synthesis, and inactivation of NF-κB pathway in SH-SY5Y cells. These findings suggest that Tan IIA protect SH-SY5Y cells against Aβ-induced neurotoxicity most likely via NF-κB pathway.


II atoms to form the dinuclear complex, in which both PtII atoms are in a distorted square-planar arrangement.The hydrido and phosphido ligands bridge the two Pt P)platinum(II)] diethyl ether monosolvate, [Pt2Br2(C12H10P)H(C18H15P)2]·C4H10O or [Pt2(μ-H)(μ-PPh2)Br2(PPh3)2]·(C2H5)2O, the PtII atoms are coordinated in a distorted square-planar arrangement, with one hydrido and one phosphido ligand bridging in a trans position. In the lattice, C—H⋯·O and C—H⋯π interactions are present. This complex has a total number of 32 electrons, 16 electrons for each PtII atom. One of the Br atoms is disordered over two positions in a 0.92:0.08 ratio.In the title compound, μ-diphenylphosphido-μ-hydrido-bis[bromido(triphenylphosphane-κ One of the attractive features of this dinuclear complex is that it is doubly bridged by hydrido and phosphido ligands in a trans fashion. The bridging of metal–metal-bonded homodinuclear complexes with a phosphido ligand allows the stabilization of the metal–metal bond. Many phosphido complexes  have been well documented and both their structural and reactivity features investigated (μ-PPh2)Br2(PPh3)2], and one solvent ether molecule  and 0.0250 (2) Å from the plane. The Pt1—P1 and Pt2—P3 bond lengths  are comparable, and consistent with literature values for a platinum–phosphorus bond. The Pt—Br bonds   in freshly distilled THF (30 ml) was stirred at room temperature for three h with a 2M solution of HBr . The product, a yellow powder, was recrystallized from ether/pentane (v:v = 1:1) yielding pale-yellow crystals.In a 100ml schlenk, [PtSHELXL2014. C-bound H atoms were positioned geometrically and refined as riding atoms, with C—H = 0.95 (aromatic), 0.99 (CH2) and 0.98 Å (CH3) and with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl). Atom Br1 is disordered over two positions in a 0.92:0.08 ratio.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901800868X/xu5929sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901800868X/xu5929Isup2.hklStructure factors: contains datablock(s) I. DOI: 1849017CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Natural killer/T-cell lymphoma (NKTCL) is a highly aggressive non-Hodgkin lymphoma often resistant to chemotherapy. Serum level of soluble IL-2 receptor α (IL-2Rα) is elevated in NKTCL patients and correlates significantly with treatment response and survival. In the current study we examined the potential role of IL-2Rα by over-expressing IL-2Rα in representative cell lines.Levels of IL-2Rα were evaluated in the human natural killer cell line NK-92 and the NKTCL cell line SNK-6. Lentiviral vectors were used to express latent membrane protein 1 (LMP1) in NK-92 cells, and IL-2Rα in both NK-92 and SNK-6 cells. The biological effects of these genes on proliferation, apoptosis, cell cycle distribution, and chemosensitivity were analyzed.Expression of IL-2Rα was significantly higher in SNK-6 cells than in NK-92 cells. Expressing LMP1 in NK-92 cells remarkably up-regulated IL-2Rα levels, whereas selective inhibitorss of the proteins in the MAPK/NF-κB pathway significantly down-regulated IL-2Rα. IL-2Rα overexpression in SNK-6 cells promoted cell proliferation by altering cell cycle distribution, and induced resistance to gemcitabine, doxorubicin, and asparaginase. These effects were reversed by an anti-IL-2Rα antibody.Our results suggest that LMP1 activates the MAPK/NF-κB pathway in NKTCL cells, up-regulating IL-2Rα expression. IL-2Rα overexpression promotes growth and chemoresistance in NKTCL, making this interleukin receptor a potential therapeutic target.The online version of this article (10.1186/s40880-018-0334-8) contains supplementary material, which is available to authorized users. Due nt years –9. Asparnt years , 9, 10, nt years .Interleukin-2 (IL-2) is a pro-inflammatory cytokine that activates and maintains immune responses . IL-2 exIn the present study, we examined the potential effects of IL-2Rα over-expression on cell proliferation, cell cycle distribution and sensitivity to chemotherapeutic drugs in representative NKTCL cell lines. Mechanistic investigation identified latent membrane protein 1 as a down-stream effector.2. SNK-6 cells were cultured in RPMI 1640  supplemented with 10% fetal bovine serum  and recombinant human IL-2 . NK-92 cells were maintained in RPMI 1640 containing 10% FBS.The human NKTCL cell line SNK-6 and human natural killer cell line NK-92  were incubated at 37 °C in an atmosphere of 5% CONotI/BamHI sites of the LV5 vector . Vector encoding IL-2Rα and the packaging vectors pGag/Pol, pRev, and pVSV-G (GenePharma) were co-transfected into HEK293T cells using Lipofectamine 2000 . Supernatant containing recombinant lentivirus was collected 72 h after transfection. NK-92 and SNK-6 cells (1 × 104 cells/well) were separately infected for 24 h with the viruses in the presence of polybrene (5 μg/ml). The medium was replaced with fresh medium, and cells were cultured another 48 h. Transfected cells were selected using puromycin (1.0 μg/ml) for 7–10 days prior to expansion.Lentivirus vectors expressing latent membrane protein-1 (LMP1) were constructed as described previously . Brieflyg for 10 min at 4 °C, the supernatant (10 μg protein per lane) was fractionated using 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were incubated at room temperature for 1 h with one of the following primary antibodies: IL-2Rα, B-Raf, p-B-Raf, p38, p-p38, ERK, p-ERK ; LMP1, JNK, p-JNK ; p65 and cyclins A1, A2, B1, and D (Boster); cyclin E ; cyclin-dependent protein kinase (CDK) 1 (Abcam); and CDK2 and 4 (Boster). After extensive washing, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG  at room temperature for 40 min. Signals were detected with an enhanced chemiluminescence kit . Results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sample protein concentration was determined using a BCA method.Western blot analysis was performed as described previously . Briefly® SybrGreen qPCR Master Mix (DBI Bioscience), cDNA derived from 0.2 μg of input RNA, 5 pM of each primer, and 7 μl double-distilled H2O. PCR reactions were carried out using a Stratagene Mx3000P Real-Time PCR system  with the following steps: pre-denaturation at 95 °C for 2 min, followed by 40 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s. Each reaction was performed three times. Fold differences in cDNA level relative to the GAPDH level were calculated using the 2−ΔΔCt method. The following primers were used: IL-2Rα sense, 5′-AAATGACCCACGGGAAGAC-3′; IL-2Rα antisense, 5′-TTGTGACGAGGCAGGAAGT-3′; LMP1 sense, 5′-CAACAACGGCAAGACTCCC-3′; LMP1 antisense, 5′-CCTCAAAGAAGCCACCCTC-3′).Quantitative RT-PCR was performed as described previously . Brieflyg for 5 min. sIL-2Rα concentration in the supernatant was measured using a sandwich enzyme-linked immunosorbent assay .NK-92, SNK-6 and NK-92 transduced with lentivirus encoding LMP1, and NK-92 and SNK-6 transduced with lentivirus encoding IL-2Rα or negative control lentivirus were centrifuged at 382×50 values.Cell proliferation was assessed using the Cell Counting kit-8 . For cytotoxicity assay, SNK-6 cells were exposed to doxorubicin, gemcitabine, or asparaginase of varying concentrations for 24 or 48 h prior to CCK-8 assay. Optical density was measured at a wavelength of 450 nm using a Multiskan microplate reader . Relative fold drug resistance was calculated using IC®, Shanghai, China). The suspension was passed through a 300-mesh filter, and DNA content of stained nuclei was analyzed using a BD FACS Calibur flow cytometer . Each experiment was performed in triplicate.Flow cytometry was used to determine cell cycle distribution and detect apoptosis. Upon 85% confluence, culture medium was removed and cells were suspended, centrifuged and fixed in precooled 70% ethanol for 1 h. The suspension was centrifuged again, the supernatant was removed, and the cells were washed with ice-cold PBS and stained with propidium iodide  in the presence of RNase A . The percentage of apoptotic cells was determined by flow cytometry on a BD FACS Calibur flow cytometer. All experiments were performed in triplicate.t-test. Differences were defined as statistically significant at P < 0.05 (2-sided).Results are expressed as mean ± SD. Statistical analysis was performed using SPSS 17.0 . Inter-group differences were assessed for significance using Student’s IL-2Rα expression was significantly higher in SNK-6 cells than in NK-92 cells at both the mRNA Fig. a and proLentivirus encoding LMP1 and negative control lentivirus infected cells efficiently at a multiplicity of infection of 300 . This last treatment achieved partial remission and its toxicity was tolerable  and who did not respond to two cycles of EPOCH  and two cycles of SMILE  was then given two cycles of pegaspargase and anti-IL-2Rα antibody basiliximab  has made radiotherapy the primary therapy , 21, 22. failure , 9, 10. In previous clinical work, our group reported that sIL-2Rα levels in serum were significantly elevated in NKTCL, and that these levels correlated with chemotherapy response and prognosis . The preElevation of various cytokines has been observed in both non-Hodgkin and Hodgkin lymphoma –25. IL-2We provide here evidence that IL-2Rα up-regulation in NKTCL may be mediated by the Epstein–Barr viral protein LMP1. The Epstein–Barr virus plays a pivotal role in NKTCL pathogenesis, and LMP1 regulates several cytokines and cytokine receptors in malignancies associated with the Epstein–Barr virus –30. We fElevated levels of sIL-2R predict inferior outcomes in several types of lymphoma, including B-cell and T-cell non-Hodgkin lymphomas , 32, 33.l-Asp. As further evidence for the involvement of IL-2Rα, this chemoresistance was reversed by anti-IL-2Rα antibody. This suggests the potential of targeting IL-2Rα for treating NKTCL. Indeed, we combined the anti-IL-2Rα antibody basiliximab with pegaspargase to successfully treat a patient with relapsed/refractory NKTCL. Both basiliximab and the humanized monoclonal anti-IL-2Rα antibody daclizumab have been used to treat autoimmune diseases such as multiple sclerosis as well as prevent transplant rejection. We plan to conduct a prospective phase 2 clinical trial to evaluate the efficacy of combination of basiliximab and pegaspargase in patients with relapsed/refractory NKTCL [Resistance to chemotherapy is the major cause of treatment failure in NKTCL, especially in advanced disease . While ory NKTCL .Our results should be interpreted with caution given several limitations. We did not investigate which IL-2-associated signaling pathways may help mediate the tumor-promoting effects of IL-2Rα in NKTCL, nor did we examine the immunomodulatory effects of IL-2Rα in NKTCL. The role of serum sIL-2Rα in lymphomagenesis needs to be further investigated. Thorough study of these questions will improve our understanding of the role of IL-2Rα in the pathogenesis and progression of NKTCL.In NKTCL, LMP-1 acts via the NF-κB pathway to up-regulate IL-2Rα, and promotes tumor growth. Further studies are warranted to understand how IL-2Rα exerts its oncogenic effects and to explore IL-2Rα as a therapeutic target.Additional file 1: Figure S1. Levels of several cell cycle proteins were increased in SNK-6 cells when IL-2Rα was overexpressed, as detected by Western blot. Control samples came from uninfected cells, while negative control samples came from cells infected with NC lentivirus.Additional file 2: Figure S2. Positron emission tomography-computed tomography of a 42-year-old man with refractory NKTCL before and after treatment with anti-IL-2Rα antibody (basiliximab) and pegaspargase. Before treatment, diffuse infiltration of lymphoma with high metabolic activity was observed (right panel). After two cycles of treatment, partial remission was observed (left panel).