Journal: Oncotarget

Article Title: Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling

doi: 10.18632/oncotarget.9967

Figure Lengend Snippet: BEAS-2B cells were exposed to increasing concentrations (0–5 μm) of Cr(VI) for 24 h. ( A ) The relative miR-21 level was determined by Taqman real-time PCR. ( B ) Immunoblot analysis of PDCD4 protein levels after acute Cr(VI) treatment. ( C ) Representative images of fluorescence immunostaining of PDCD4 ( D ) Cr(VI) increases the binding of miR-21 to the 3′-UTR of PDCD4. BEAS-2B cells were transfected with renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), and pGL3-promoters and treated with 5 μM Cr(VI) for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods. The results are expressed as a relative activity (relative luminescence units (RLU)) normalized to the luciferase activity in the vector control cells without treatment. ( E ) Immunoblot analysis demonstrates that acute treatment of Cr(VI) decreases E-cadherin levels associated with an increase in β-catenin and TCF4 protein levels in BEAS-2B cells. Data presented in the bar graphs are the mean ± SD of three independent experiments. *indicates a statistically significant difference from control cells with p < 0.05.

Article Snippet: Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Immunostaining, Binding Assay, Transfection, Construct, Negative Control, Luciferase, Activity Assay, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling

doi: 10.18632/oncotarget.9967

Figure Lengend Snippet: BEAS-2B cells were maintained in a medium containing various concentrations of Cr(VI) (0.125, 0.25 and 0.5 μM) for 6 months. ( A ) Total cell lysates were prepared for Western blot analysis using specific antibodies against E-Cadherin, Active-β-catenin, β-catenin, TCF4 and GAPDH. Representative images of fluorescence immunostaining of ( B ) E-cadherin ( C ) β- catenin and ( D ) TCF4.

Article Snippet: Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Western Blot, Fluorescence, Immunostaining

Journal: Oncotarget

Article Title: Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling

doi: 10.18632/oncotarget.9967

Figure Lengend Snippet: BEAS-2B cells were exposed to various concentrations of Cr(VI) (0.125, 0.25 and 0.5 μM) for 6 months. ( A ) Expression levels for uPAR and c-Myc were analyzed by Western blot. To evaluate invasive capacity ( B – C ) 1 × 10 5 cells from each group were seeded on the top chamber of 24-well plate culture inserts coated with 20 μl of matrigel in duplicate. Cells were cultured for additional 48 h. Invaded cells on the bottom of insert were stained and quantified. ( D – E ) For the ChIP assay, TCF4 binding regions on uPAR and c-Myc promoters were identified. Chromatin isolated from chronic Cr(VI) exposed BEAS-2B cells were immunoprecipitated with an anti-TCF4 antibody or control mouse IgG. The TCF4 binding to the uPAR and c-Myc promoters was analyzed by RT PCR with specific primers. Data presented in the bar graphs are the mean ± SD of three independent experiments. *indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Cell Culture, Staining, Binding Assay, Isolation, Immunoprecipitation, Control, Reverse Transcription Polymerase Chain Reaction

Journal: medRxiv

Article Title: ARF1 prevents aberrant type I IFN induction by regulating STING activation and recycling

doi: 10.1101/2023.04.28.23289152

Figure Lengend Snippet: a , Induction of the ISRE promoter in THP-1-Dual WT (grey) and THP-1-Dual KO-cGAS (pink) cells transduced with lentiviral vectors expressing ARF1 WT or R99C as indicated, quantified by Lucia luciferase (LLuc) activity 72 h post transduction. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n=3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING, anti-cGAS and anti-GAPDH. b , Exemplary electron microscopy analysis of HEK293T cells transiently transfected with ARF1 WT or ARF1 R99C as indicated. Mitochondria (m) are highlighted in insets in bottom panels. Electron-dense granules and inflated cristae are highlighted by black and white arrows, respectively. Annotations: cp, cytoplasm; er, endoplasmic reticulum; g , Golgi apparatus; lv, large vesicle; ly, lysosome; m, mitochondria; nc, nucleus. c, Exemplary immunoblots showing fractionation of ARF1 WT, R99C, Q71L and vector transfected HEK293T cells as indicated. WCLs and fraction blots stained by anti-FLAG, anti-TFAM (mitochondria), anti-LAMIN B1 (nucleus) and anti-GAPDH (cytosol). d, qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of ( c ) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 3 ± SEM. e , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of primary fibroblasts from healthy donors (n2, f1, I7) or a patient (Patient 1) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 5 ± SEM. f, qPCR of representative ISG OAS1 in U2OS cells stably expressing STING and depleted of mtDNA by ddC, or untreated (NT), upon transfection with empty vector, ARF1 WT or R99C n=3 ± SEM. g , Exemplary immunoblot of WCLs of HEK293T cells transiently expressing ARF1 WT, R99C or vector. Blots were stained with anti-MFN1, anti-RHOT1, anti-FLAG and anti-GAPDH. Quantification of the band intensities for MFN1 normalized to the band intensities of GAPDH. Bars represent mean of n = 6 ± SEM (biological replicates). h , Exemplary immunoblots showing fractionation of HEK293T cells expressing ARF1 WT, R99C or vector control as well as VCP. WCLs and fraction blots stained by anti-FLAG, anti-HA, anti-TFAM (mitochondria), anti LAMIN B1 (nucleus) and anti-GAPDH (cytosol). i , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of (h) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using 725 the ΔΔCT method. n = 5 ± SEM.

Article Snippet: Proteins were stained with primary antibodies mouse anti-FLAG M2 (1: , Sigma-Aldrich), sheep anti-STING (1:, Bio-Techne), rabbit anti-pTBK1 (1:, Cell Signaling), rabbit anti-TBK1 (1:, Cell Signaling), rabbit anti-IRF3 (1:, Cell Signaling), rabbit anti-HA (1:, Cell Signaling), rabbit anti-ARF1 (1:300, Proteintech), rabbit anti-cGAS (1:, Proteintech), rabbit anti-RFP (1:, Abcam), mouse anti-turboGFP (1:, Origene), rabbit anti-TFAM (1:, Proteintech), mouse anti-Lamin B1 (1:10,000, Proteintech), rat anti-GAPDH (1:, BioLegend), rabbit anti-MFN1 (1:, Cell Signaling), mouse anti-RHOT1 (1:, Abnova), mouse anti-Drp1 (1:, Cell Signaling), rabbit anti-pDrp1 (1:, Cell Signaling), rabbit anti-SURF4 (1., Novus Biologicals) and subsequently Infrared Dye labelled secondary antibodies (LI-COR), diluted in 0.05% Casein in PBS.

Techniques: Transduction, Expressing, Luciferase, Activity Assay, Western Blot, Staining, Electron Microscopy, Transfection, Fractionation, Plasmid Preparation, Stable Transfection

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Association between LEF1 mRNA expression with survival status of MM patients. A, The overall survival curve exhibited the overall survival of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software ( www.genomicscape.com ). B, The progression-free survival (Left) and overall survival (Right) status of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software ( www.genomicscape.com ). Red line: high expression group, blue line: low expression group. P values were denoted on the plot.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Expressing, Software

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Knockdown of LEF1 suppressed growth of U266 and RPMI8226 myeloma cells. A, The mRNA expression level of LEF1 in U266 and RPMI8226 cells infected with LEF1 shRNA (shLEF1) or scramble shRNA as the control (shCon) for 48 h was assessed by quantitative real-time RT-PCR (qRT-PCR). B, The protein expression level of LEF1 in U266 and RPMI8226 cells infected with shLEF1 or shCon for 72 h was detected by Westernblot analysis. C, The cell viability of U266 and RPMI8226 cells following transduction with shLEF1 or shCon was determined by CCK-8 assay. D, Brdu incorporation was determined in U266 and RPMI8226 cells transduced with shLEF1 or shCon. * P < 0.05 and ** P < 0.01 versus shcon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Knockdown, Expressing, Infection, shRNA, Control, Quantitative RT-PCR, Transduction, CCK-8 Assay, BrdU Incorporation Assay

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Depletion of LEF1 induced apoptosis of U266 and RPMI8226 myeloma cells. A-B, The cellular apoptosis was detected in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 5 days by flow cytometry analysis following Annexin V/7AAD staining. C, The protein levels of PARP and cleaved caspase 3 was detected in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 5 days by Westernblot analysis. D, Intracellular ROS production was determined by CM-H2DCFDA staining in U266 and RPMI8226 cells following transduction with shLEF1 or shCon for 72 h. * P < 0.05 and ** P < 0.01 versus shCon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Transduction, Flow Cytometry, Staining

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: LEF1 inhibited transcription of CYLD gene through promoter binding. A, The protein levels of CYLD in U266 and RPMI8226 cells following transduction with shLEF1 or shCon for 72 h was measured by Westernblot analysis. B, The CYLD mRNA expression level in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 72 h was determined using qRT-PCR. C, Schematic representation of potential LEF1-binding sites in CYLD promoter and the location of the primers used for ChIP assay. D, U266 cells transduced with shLEF1 or shCon were co-transfected with either wild-type CYLD luciferase reporter construct (pGL3-CYLD-wt) or the plasmid containing the mutation in the putative LEF1 binding site (pGL3-CYLD-mt) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined with dual-luciferase reporter assay kit. E, ChIP assay was performed to show binding of LEF1 on CYLD promoter in U266 cells. Anti-LEF1 antibody or control IgG was used to precipitate sonicated chromatin from U266 cells. The purified DNA was analyzed by real-time PCR with primers flanking potential LEF1 binding site. Data showed fold enrichment relative to control IgG. * P < 0.05 and ** P < 0.01 versus shCon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Binding Assay, Transduction, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Construct, Plasmid Preparation, Mutagenesis, Activity Assay, Reporter Assay, Control, Sonication, Purification, Real-time Polymerase Chain Reaction

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Depletion of LEF1 repressed NF-κB signaling pathway through modulation of CYLD in U266 cells. A, The protein levels of p65 and p-p65 in U266 cells following LEF1 knockdown for 72 h was measured by Westernblot analysis. B, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. C, The mRNA expression levels of Cyclin D1, Bcl-2 and Bcl-xL in U266 cells transduced with shLEF1 or shCon for 72 h were determined using qRT-PCR. * P < 0.05 and ** P < 0.01 versus shCon group. D, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) in the presence of CYLD siRNA or the control siRNA together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. * P < 0.05 and ** P < 0.01.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Knockdown, Transduction, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Id2 expression is enhanced during a conversion to T H 17 phenotype from iT reg cells. a In vitro polarization scheme of T H 0, T H 17, iT reg and ex-Foxp3 T H 17 cells. b RT-qPCR analysis of Id2 mRNA in in vitro generated T H 0, T H 17, iT reg and ex-Foxp3 T H 17 cells; results are presented relative to Hprt expression ( n = 3) c , d Western blot analysis to determine the kinetics of indicated proteins during T H 17 ( c , top panel) and iT reg ( d , top panel) cell differentiation and maintenance conditions. β-actin is used as loading control. RT-qPCR analysis of Il17a , Il17f ( c , lower panel) and Il10 ( d , lower panel) mRNA during each T H 17 and iT reg cell differentiation conditions; results are presented relative to Hprt expression ( n = 3). e Sorted CD4 + naive T cells were activated in vitro under iT reg cell differentiation condition for 3 days. After 3 days, iT reg cells were re-stimulated in vitro in ex-Foxp3 T H 17 polarizing conditions for additional 3 days. Western blot analyses for the indicated proteins were performed after harvesting the cells at indicated time points ( top panel ). RT-qPCR analysis of Il10 and Il17a mRNA under similar experimental conditions is shown in the lower panel; results are presented relative to Hprt expression ( n = 3). * P < 0.05 (Student’s t -test). Data are representative two independent experiments (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Generated, Western Blot, Cell Differentiation, Control

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Overexpression of Id2 in vitro fails to stabilize iT reg lineage commitment, and convert them into T H 17-like cells. a Naive CD4 + T cells were sorted from wild-type C57BL/6 (B6) mice, and transduced with control vector (Empty RV) or vector encoding Id2 cDNA (Id2 RV) under iT reg differentiation condition. After 3 days cells were harvested and Id2 expression was measured by RT-qPCR ( n = 3, per group) and Western blot analysis. b Comparison of mRNA expression for T-bet , Gata3 , Rorγt and Foxp3 between Empty RV and Id2 RV transduced T cells after 3 days post spinfection under iT reg differentiation condition ( n = 3, per group). c Flow cytometry analysis of Foxp3 expression between Empty RV or Id2 RV transduced (GFP + ) and non-transduced (GFP − ) iT reg cells ( n = 3, per group). d Comparison of mRNA expression for genes encoding the indicated cytokines between Empty RV and Id2 RV transduced T cells after 3 days post spinfection under iT reg differentiation condition ( n = 3, per group). e Flow cytometry analysis of IL-17A, IL-17F and IL-22 from Empty RV or Id2 RV transduced CD4 + GFP + iT reg cells ( n = 3, per group). NS, not significant, * P < 0.05, ** P < 0.005 (Student’s t -test). All data are representative two or three independent experiments (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Over Expression, In Vitro, Transduction, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Comparison, Flow Cytometry

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Mice with T reg -specific ectopic expression of Id2 displayed enhanced conversion to ex-Foxp3 T H 17 cells from T reg cells after induction of EAE. a Schematic representation the mouse model employed. Converging arrows in the lower panel indicate genotyping primers. b Genotyping PCR to detect the presence of Id2-EmGFP transgene in Id2 EmGFP Foxp3 YFP−Cre mice. c Flow cytometry analysis of YFP and EmGFP expression in CD4 + CD25 + T cells from spleen (SP) and peripheral lymph nodes (pLN) of Foxp3 YFP−Cre and Id2 EmGFP Foxp3 YFP−Cre mice (left panel). Id2 expression was assessed between non-T reg and T reg cells by intracellular staining (right panel). d Schematic of experimental EAE model. e Representative flow cytometry analysis of Id2 expression between tdTomato + YFP + (T reg ) and tdTomato + YFP − (ex-T reg ) cells in CD4 + T cells from 6 to 8 week-old R26T Foxp3 YFP−Cre mice on day 24 after induction of EAE. f Mean clinical scores in mice after induction of EAE (R26T Foxp3 YFP−Cre ; n = 12, Id2 EmGFP R26T Foxp3 YFP−Cre ; n = 9). g Representative Spinal Cord (SC) sections and hematoxylin and eosin (H&E) staining from EAE induced mice at day 24 after immunization (scale bar, 100 μm). h Flow cytometry analysis and percentages of Foxp3 sufficient (YFP + ) and deficient (YFP − ) populations among CD4 + tdTomato + in SP and SC from R26T Foxp3 YFP−Cre and Id2 EmGFP R26T Foxp3 YFP−Cre mice at day 22–24 after induction of EAE. i Representative flow cytometry analysis and percentages of IFN-γ + , IL-17A + and IFN-γ + IL-17A + in CD4 + tdTomato + YFP − or CD4 + tdTomato + YFP + T cells in total SP and SC cells stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h. NS, not significant, *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t -test). All data are representative of two independent experiments (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: T reg -cell-specific overexpression of Id2 results in systemic autoimmunity in older mice. a A representative picture of 12–16 week-old Foxp3 YFP−Cre and Id2 EmGFP Foxp3 YFP−Cre mice ( left ) and spleen (SP) and peripheral lymph nodes (pLN) derived from them (right). b Representative hematoxylin and eosin (H&E)-stained skin, lung, liver sections from 12–16 weeks old Foxp3 YFP−Cre and Id2 EmGFP Foxp3 YFP−Cre mice (scale bar, 100 μm). c Representative FACS plots and percentages of CD4 + and CD8 + T cells from SP and pLN of 12–16 weeks old Foxp3 YFP−Cre and Id2 EmGFP Foxp3 YFP−Cre mice. d , e Representative FACS plots and percentages of CD62L hi CD44 lo (naive) and CD62L lo CD44 hi (effector/memory) cells among CD4 + and CD8 + T cells in SP and pLN 12–16 week-old mice. f Representative FACS plots and percentages of Foxp3 + T reg cells in thymus, SP and pLN from 12–16 week-old mice. NS, not significant, *P < 0.05, **P < 0.005 (Student’s t -test). All data are representative three independent experiments (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Over Expression, Derivative Assay, Staining

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Id2 is induced by IL-1β and IL-6 mediated signaling and downstream transcription factors STAT3, IRF4 and BATF in T reg cells. a Strategy for sorting T Naive , iT reg , and ex-Foxp3 T H 17 cells from Foxp3 Thy1.1 reporter mice (left). Flow cytometry analysis of IL-1r1 and IL-6rα among the T Naive , iT reg , and ex-Foxp3 T H 17 cells (right). b , c FACS-sorted iT reg cells were re-stimulated with cytokines as indicated and analyzed for Id2 and Foxp3 mRNA expression by RT-qPCR ( b ) as well as for Id2 protein levels by flow cytometry ( c ). d Cartoon depicting highly putative STAT3, IRF4, and BATF binding motifs upstream of Id2 transcription start site (TSS). e Cartoon depicting Id2 promoter constructs used for luciferase reporter assay. f Id2 promoter-luciferase construct (−582/+36) were co-transfected with the combination of STAT3, IRF4 and BATF expressing vectors in HEK-293 T cells. Lysates were prepared 30 h after transfection, and luciferase activities were measured with the reporter activities normalized to renilla luciferase activity. *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t -test). All data are representative three independent experiments with similar results (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Binding Assay, Construct, Luciferase, Reporter Assay, Transfection, Activity Assay

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Id2 expression inhibits enrichment and transcriptional activity of E2A on Foxp3 promoter. a RT - qPCR analysis of Foxp3 , E2A and Id2 mRNA in in vitro generated T H 0, T H 17, ex-Foxp3 T H 17, and iT reg cells. b ChIP-qPCR analysis for E2A occupancy at three putative E-box sites on Foxp3 promoter regions (−1593 to −1584, −1295 to −1286, and −837 to −829) and negative control (−411 to −244; No E-box site) in iT reg and ex-Foxp3 T H 17 cells. Enrichments are calculated relative to the input chromatin for corresponding sites. c Jurkat cells were transfected with either Foxp3 promoter reporter construct alone or along with expression plasmids encoding E2A and Id2 as indicated, followed by luciferase assay. d Effect of mutagenesis on Foxp3 promoter reporter activity. Jurkat cells were transiently co-transfected with the indicated plasmids for luciferase assay as described. N = Normal E box, X = Mutated E box. NS, not significant, *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t -test). All data are representative of two or three independent experiments (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, In Vitro, Generated, ChIP-qPCR, Negative Control, Transfection, Construct, Luciferase, Mutagenesis

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Id2 dependent T reg plasticity enhances antitumor immunity. a Schematic for doxycycline (Dox)-inducible Id2 overexpression in T reg cells in TetR- Id2 EmGFP Foxp3 YFP−Cre mice. b Id2 expression was assessed by intracellular staining in SP and pLN from both PBS or Dox treated groups. c Experimental scheme of mouse melanoma model. Females, 8–10 week-old, TetR-Id2 EmGFP Foxp3 YFP−Cre mice were injected subcutaneously with B16.F10 cells. Mice were treated intraperitoneally either with PBS or Dox from day 0–9, every 72 h. d Tumor progression, expressed as mean tumor volume (mm 3 ) in both treatment groups (PBS; n = 8, Dox; n = 8). e Representative tumor size in PBS and Dox treated groups on d 23. f Difference in tumor weight, as measured at end point of analysis (PBS; n = 8, Dox; n = 8). g Flow cytometry analysis of CD4 + Foxp3 + T cells in tumor-draining lymph nodes (dLN), non-draining lymph nodes (non-dLN) as well as within tumor infiltrated lymphocytes. h Intracellular staining of cytokines in CD8 + T cells isolated from dLN, non-dLN and tumor infiltrated lymphocytes, stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h. NS, not significant, *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t -test). All data are representative of three independent experiments (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Over Expression, Expressing, Staining, Injection, Flow Cytometry, Isolation

Journal: Cell Death & Disease

Article Title: Linc-DYNC2H1-4 promotes EMT and CSC phenotypes by acting as a sponge of miR-145 in pancreatic cancer cells

doi: 10.1038/cddis.2017.311

Figure Lengend Snippet: Gemcitabine-resistant pancreatic cancer cells exhibit enhanced EMT potential. ( a ) MTT assay after 72 h treatment with gemcitabine in BxPC-3-Gem and parental BxPC-3 cells. ( b – e ) The expression levels of EMT markers, ZEB1, E-cadherin and vimentin were detected by RT-qPCR ( b , d ) and western blotting ( c and e ) in BxPC-3, BxPC-3-Gem, AsPC-1 and PANC-1 cells. GAPDH was used as a loading control ( c and e ). Bands intensities normalized to GAPDH were shown ( f ) The migration and invasion abilities of BxPC-3 and BxPC-3-Gem cells were measured by Transwell assay (Scale bar, 200 μm). The data shown were from three independent experiments. ** P <0.01; *** P <0.001 versus BxPC-3

Article Snippet: After 1 h of blocking, membranes were incubated with the following primary antibodies: mouse anti-GAPDH (KC-5G4) (KANGCHEN, Shanghai, China), β -actin (Santa Cruz, CA, USA), and MMP3, Oct4, Sox2, Nanog, E-cadherin, vimentin, ZEB1, Lin28 (Proteintech, Wuhan, China) at 4 °C over night.

Techniques: MTT Assay, Expressing, Quantitative RT-PCR, Western Blot, Control, Migration, Transwell Assay

Journal: Cell Death & Disease

Article Title: Linc-DYNC2H1-4 promotes EMT and CSC phenotypes by acting as a sponge of miR-145 in pancreatic cancer cells

doi: 10.1038/cddis.2017.311

Figure Lengend Snippet: miR-145 targets EMT and CSC markers which are upregulated by linc-DYNC2H1-4. ( a ) BxPC-3-Gem cells were transfected with p-miR-145 overexpression vector or empty vector for 24 h, linc-DYNC2H1-4 upregulated genes, Oct4, Lin28, Nanog Sox2 and ZEB1 as well as its target genes E-cadherin and vimentin were detected by western blotting. ( b – d ) MiR-145 inhibits MMP3 in pancreatic cancer cells. Luciferase reporter activity in BxPC-3-Gem cells co-transfected with miR-145 expression or empty vector together with a luciferase reporter vector containing the wild-type MMP3 3′ UTR ( b ). BxPC-3-Gem cells were transfected with empty or miR-145 overexpression vector for 24 h, expressions of miR-145 and MMP3 were analyzed by RT-qPCR ( c ) and western blotting ( d ). BxPC-3-Gem cells ( e ) and PANC-1 cells ( f ) were transfected with linc-DYNC2H1-4 overexpression vector (linc-DYNC2H1-4), or miR-145 expression vector (miR-145) alone or together for 24 h, the protein expression levels of EMT- and CSC-associated genes were detected by western blotting. The data shown were from three independent experiments. * P <0.05; ** P <0.01 and *** P <0.001 versus control

Article Snippet: After 1 h of blocking, membranes were incubated with the following primary antibodies: mouse anti-GAPDH (KC-5G4) (KANGCHEN, Shanghai, China), β -actin (Santa Cruz, CA, USA), and MMP3, Oct4, Sox2, Nanog, E-cadherin, vimentin, ZEB1, Lin28 (Proteintech, Wuhan, China) at 4 °C over night.

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: TNIK inhibition abrogates colorectal cancer stemness

doi: 10.1038/ncomms12586

Figure Lengend Snippet: ( a ) Structure of NCB-0846 (left) and its diastereomer NCB-0970 having the opposite configuration at the terminal hydroxyl group (right). ( b ) Inhibition of TNIK enzyme activity by the indicated concentration of NCB-0846 or NCB-0970. ( c ) Inhibitory profiles of NCB-0846 and NCB-0970 across the human kinome. The degree of inhibition is presented schematically by the size of the circles. ( d ) Inhibition of TCF4 phosphorylation by NCB-0846. The recombinant human TCF4 protein was incubated with the recombinant human TNIK protein in the presence of the indicated concentration of NCB-0846 or NCB-0970 and analysed by immunoblotting. ( e ) Inhibition of TNIK auto-phosphorylation by NCB-0846. HCT116 cells were cultured in the presence of DMSO (vehicle), 1 μM NCB-0846, or 1 μM NCB-0970 for 4 or 24 h and then analysed by immunoblotting with anti-phosphorylated TNIK, anti-TNIK, and anti-γ-tubulin (loading control) antibodies. ( f ) Suppression of TCF/LEF transcriptional activity by NCB-0846. HEK293 cells were transfected with pGL4.49[luc2P/TCF-LEF-RE/Hygro] and then treated with the indicated concentration of NCB-0846 or NCB-0970 for 24 h. The cells were then incubated with or without recombinant Wnt3a (10 ng ml −1 ) for 5 h before measurement of luciferase activity. Data are represented as ratios relative to the DMSO control (set at 100%)±s.e.m. ( g ) HCT116 (left) and DLD-1 (right) cells were transfected with one of the luciferase reporter plasmids (SuperTOP-flash or SuperFOP-flash) and then treated with 20 mM LiCl and the indicated concentration of NCB-0846 or NCB-0970 for 24 h. Data are presented as the ratios of SuperTOP-flash to SuperFOP-flash (TOP/FOP ratio). Error bars represent s.d. ( h ) HCT116 cells were treated with DMSO (control) or the indicated concentration of NCB-0846. Twenty-four hours later, the expression levels of the TNIK , AXIN2 , CCND1 and MYC genes (normalized to ACTB ) were quantified in triplicate by RT–PCR. The values of cells treated with DMSO were set to one. Error bars represent s.d. ( i ) HCT116 and DLD-1 cells were treated with DMSO (vehicle) or the indicated concentration of NCB-0846 or NCB-0970 for 48 h. The expression of TNIK, AXIN2, c-MYC, LRP6, LRP5 and β-actin (loading control) proteins was analysed by immunoblotting.

Article Snippet: HEK293 cells were transiently transfected with pGL4.49[luc2P/TCF-LEF-RE/Hygro] reporter vector (Promega) using Fugene HD Transfection Reagent (Promega; ).

Techniques: Inhibition, Activity Assay, Concentration Assay, Recombinant, Incubation, Western Blot, Cell Culture, Transfection, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction