Journal: Journal of Microbiology and Biotechnology

Article Title: Bifidobacterium bifidum DS0908 and Bifidobacterium longum DS0950 Culture-Supernatants Ameliorate Obesity-Related Characteristics in Mice with High-Fat Diet-Induced Obesity

doi: 10.4014/jmb.2210.10046

Figure Lengend Snippet: ( A ) Schematic representation of C3H10T1/2 MSCs differentiation timeline. ( B ) mRNA expression levels of the major thermogenic markers Ucp1 , Pgc1α , Prdm16 and Pparγ after DS0908 and DS0950 treatment in differentiated C3H10T1/2 mesenchymal stem cells (MSCs). ( C ) Thermogenic marker UCP1, PPARγ, PGC1α and PRDM16 protein expression levels after DS0908 and DS0950 treatments in differentiated C3H10T1/2 MSCs. ( D and E ) mRNA expression levels of beige ( Cd137 , Fgf21 , P2rx5 and Tbx1 ), brown ( Cox2 ) and white ( aP2 , Psat1 , Resistin and Serpina3k ) adipocyte-specific markers after DS0908 and DS0950 treatments in differentiated C3H10T1/2 MSCs. Tbp was used as an internal control gene and β‐actin as a protein loading control. The data from three individual experiments are expressed as the average ± standard error mean (SEM). *, **, *** and ns indicate p < 0.05, < 0.01, < 0.001 and non-significant, respectively, to express the statistically significant differences between the control (MDI) and the treatment groups in the figures. The protein band intensities were measured using ImageJ. Adipogenic differentiation medium, MDI: 0.5mM IBMX, 1 μM dexamethasone and 10 μg/ml insulin; 1 μM Rosiglitazone (Rosi); DS0908 = B. bifidum DS0908; DS0950 = B. longum DS0950.

Article Snippet: Antibodies against aP2 , AMPK, p-AMPK (Thr172), p38 MAPK, p-p38 MAPK (Thr180/Tyr182), p-CREB (Ser133), p-PKA substrate and anti-rabbit IgG anti-mouse IgG antibodies conjugated with horseradish peroxidase were acquired from Cell Signaling Technology (USA).

Techniques: Expressing, Marker

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 (panels a and b), and then were induced to differentiate in MDM for 4 (panels c,d,i,j) or 6 days (panels e and f and panels g and h). At the different time points cells were fixed and incubated with a monoclonal antibody against Troponin-T (panels a to f) or stained with Oil-red-O (panels g and h). At day 4, cells were also analyzed with a monoclonal antibody against Fabp4/aP2 (panels i and j). Cells were observed at a magnification of 20x, ( __ ): 60 µm. B: At day 1, C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h and then were induced to differentiate in MDM. Cells were then collected at the indicated time points and expression of specific mRNAs was analyzed by RT-PCR amplification. PCR products were analyzed on 1.2% agarose gels. Photographs of the gels are shown. RNase L protein expression was analyzed as in . Autoradiographs of these gels are shown (RNase L-P). C: Quantification of lipids accumulated in C2-RNase L cells treated ( ) or not ( ) with 5 mM IPTG for 6 h and then induced to differentiate in MDM for 6 days. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in untreated C2-RNase L cells was set to 1. Data were expressed as mean±SD of three different experimental points. (*) P<0.01 compared to non-induced cells.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Incubation, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 and then induced to differentiate in ADM. Cells were fixed and stained with Oil-red-O to reveal lipids accumulation at day 0, 3 and 6. Cells were observed at a magnification of 20x, ( __ ): 60 µm. B: Quantification of lipids in control ( ) or IPTG-treated C2-RNase L cells ( ) at day 0, 3 and 6 after induction of differentiation in ADM. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in C2-RNase L cells at T = 0, before addition of IPTG, was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.01 compared to untreated cells at T = 3. C: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 and then induced to differentiate in ADM. Cells were fixed at day 0, 3 and 6 and then incubated with an antibody against Fabp4/aP2 (Red). DNA was stained with Dapi (Blue). Cells were observed at 20x, ( __ ): 50 µm. D: Analysis of mRNA expression by RT-PCR amplification at different time points during adipocyte differentiation. RNase L was induced (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 2 and then cells were switched to ADM to induce adipocyte differentiation. PCR products were analyzed by gel electrophoresis. Photographs of the gels are shown.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Staining, Standard Deviation, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated, or not (0), with 5 mM IPTG for (2) or (6)h. Cells were then harvested and analyzed for RNase L 2-5A binding activity with the 2-5A radio-covalent binding assay. Proteins were separated on 10% polyacrylamide gels. A representative autoradiography of the gel and a densitometry of the gel are shown. The amount of 2-5A binding activity in C2-RNase L cells in the absence of IPTG was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.0 compared to non- induced cells (**) P<0.01 compared to control, untreated cells. B: RNase L was induced by 5 mM IPTG for 0, 2 or 6 hours, and then mRNA expression was analyzed by RT-PCR amplification and agarose gel electrophoresis. Photographs and densitometric analysis of the gels are shown. The level of the different mRNAs in untreated cells was set at 1. Quantification of EEF1α mRNA was used as a control of semi-quantitative RT-PCR experiment. Error bars refer to the standard deviation obtained in three independent experiments. C: C2-RNase L cells were treated, or not, with 5 mM IPTG for 2 or 6 h and then induced to differentiate in ADM. At T = 0, T = 2, T = 4 and T = 6 days after induction of differentiation, cells were fixed and stained with Oil-red-O to reveal lipid accumulation (panel on the right) or incubated with an antibody against Fabp4/aP2 (Red) (panel on the left); DNA was stained with Dapi (Blue). Cells were observed at 20x, ( __ ): 50 µm. D: Quantification of lipids in control or IPTG-treated C2-RNase L cells (for 2 or 6 h) at T = 0, T = 2, T = 4 and T = 6 days after induction of differentiation with ADM. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in C2-RNase L cells at T = 0, before IPTG treatment, was set to 1. Error bars refer to the standard deviation obtained in three independent experimental points. (*) P<0.01 compared to untreated cells at the same time during adipocyte differentiation.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Binding Assay, Activity Assay, Autoradiography, Standard Deviation, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Quantitative RT-PCR, Staining, Incubation

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: RNase L activity in C2-RNase L cells, C2-RNase L cells treated with IPTG and C2-RLI cells. C2-RLI cells (1, ), C2-RNase L cells (2, ) and C2-RNase L treated with 5 mM IPTG (3, ) for 6 h were analyzed for RNase L 2-5A binding activity with the 2-5A radiocovalent binding assay. Proteins were separated in 10% polyacrylamide gels. A representative autoradiography and a densitometry of the gel are shown. The amount of 2-5A binding activity in C2-RLI cells was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. B: C2-RLI differentiation. Cells were fixed and incubated with Oil-red-O to reveal lipid accumulation (i = day 0, j = day 6 after induction of differentiation with ADM) and expression of Troponin T and Fabp4/aP2 was analyzed using monoclonal antibodies against Troponin T (Green, c,d) and Fabp4/aP2 (Red e,f) at day 0 (a,c,e,g,i) and day 6 after induction of differentiation with ADM (b,d,f,h,j). DNA was stained with Dapi (Blue a,b,g,h). A merge of Dapi, Troponin T and Fabp4/aP2 labeling is shown (g,h). Cells were observed at 20x, ( __ ): 20 µm. C: Quantification of lipids in C2-RLI cells at day 0 ( ) and day 6 after induction of differentiation with ADM ( ). A value of 1 corresponds to the amount of lipids in C2-RLI cells at T = 0, before differentiation. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.01 compared to untreated C2-RNase L cells and (**) P<0.01 compared to IPTG-treated C2-RNase L cells at the same time point during adipocyte differentiation. D: Analysis of mRNA expression by RT-PCR amplification at different time points during adipocyte differentiation. C2-RLI cells were switched to ADM to induce adipocyte differentiation at day 1. PCR products were analyzed by gel electrophoresis. Photographs of the gels are shown.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Activity Assay, Binding Assay, Autoradiography, Standard Deviation, Incubation, Expressing, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) RT-PCR analysis of ZFP36L1 expression in human malignant B cells representing different stages of B cell differentiation. For comparison BLIMP1 expression was also measured. β ACTIN expression levels are shown as a loading control. (B) Comparison of ZFP36L1 protein levels in human Ramos B cells before and after 3 h PMA stimulation and in two myeloma cell lines RPMI-8226 cells and KMS-11. BLIMP1 expression is also shown in RPMI-8226 cells and KMS-11 cells. HSP90 levels are shown as a loading control. ZFP36L1/L2, HSP90 and BLIMP1 proteins were detected by anti-BRF1/2, anti-HSP90 and anti-BLIMP1 antibodies respectively. Anti-BRF1/2 antibody cross-reacts with ZFP36L2 (approx. 60 kDa) and ZFP36L1 appears typically as a constellation of induced bands (approx. 40 kDa) in human cells.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Differentiation

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: downregulation of ZFP36L1 is associated with plasmacytoid differentiation of B cells. (A) Western Blot analysis of ZFP36L1 expression in IL-2/5 treated murine leukemic BCL1 cells. Protein lysates were made from unstimulated cells (lane 1) or from cells 96 h after stimulation with cytokines (20 ng/ml IL-2 and 5 ng/ml IL-5) (lane 2). ZFP36L1 and HSP90 proteins were detected by anti-BRF1/2 and anti-HSP 90 antibodies respectively. (B) qRT-PCR analysis of zfp36l1 and blimp1 mRNA expression in day 0 versus 48 h IL-2/5 stimulated BCL1 cells. (C) qRT-PCR analysis of time course of zfp36l1 and blimp1 mRNA expression over 3 days in LPS (10 µg/ml) stimulated murine splenic B cells. The 2 –ΔΔCT method of relative quantification was used to determine the fold change in mRNA expression. The results shown were normalized to β-actin mRNA expression. The results show mean ±SD from one representative experiment.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: IgM production. Levels of IgM production in ZFP36L1 transfected BCL1 stimulated with IL-2/5 (20 ng/ml IL-2 and 5 ng/ml IL-5) over 4 days compared to levels produced by empty vector BCL1 cells cultured under the same conditions. Cells were co-transfected with either pcDNA3.ZFP36L1 or empty pcDNA3 vector and pcDNA3.EGFP and then sorted on the basis of EGFP expression before been set up in culture in the absence or presence of cytokines. Cells were cultured at 4.0×10 4 /ml and ELISA measurements were made in duplicate. The results shown are representative of 3 similar experiments.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Transfection, Produced, Plasmid Preparation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) qRT-PCR analysis of zfp36l1 mRNA levels in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 lentivirus infected cells compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. The results represent mean ±SD (n = 3) of zfp36l1 mRNA levels in three independent cells lines generated by three independent rounds of lentiviral infection for each cell type (apart from wild-type). * = p<0.05 as determined by t-test. (B) Western blot analysis of ZFP36L1 protein expression in wild-type, empty vector, scramble, pSicoR.scramble.RNAi1and pSicoR.scramble.RNAi2 cells. HSP90 levels are shown as a loading control.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, Generated, Western Blot, Expressing

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: Cell numbers in the absence (A) and presence (B) of cytokines (IL-2 and IL-5) in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Cells numbers were counted in quadruplicate 4 days after seeding 2×10 5 /ml cells on day 0 in flasks in the absence or presence of cytokines (IL-2 20 ng/ml and IL-5 5 ng/ml). Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. IgM secretion (ng/ml) measured by ELISA, 4 days after the start of the culture, in the absence (C) and presence (D) of cytokines (IL-2 20 ng/ml and IL-5 5 ng/ml) in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. ** = p<0.01, * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Plasmid Preparation, Infection, Generated, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) Heat map expression profile of known ZFP36L1 target genes in terminal B cell differentiation. The profiles are compared with the reference genes, ZFP36L1, BLIMP1 and XBP1 (first three rows). GMCSF, VEGFA and IL-3 are validated ZFP36L1 targets; the remaining genes have been validated for ZFP36 . Data was taken from GEO Accession number GSE 6691 . Red indicates high and blue, low expression. Key: CLL: B chronic lymphocytic leukemia, MM: multiple myeloma, WM-BL: Waldenstrom’s macroglobulinemia B cells, WM-PB: Waldenstrom’s macroglobulinemia plasma cells, NBC: normal B cells, PC: normal plasma cells. (B) Graphical representation of the ARACNe network for BLIMP1 and ZFP36L1. Nodes representing the BLIMP1 and ZFP36L1 hubs are shown enlarged. Only first neighbours of these hubs are shown in the module. Nodes representing inferred BLIMP1 targets (significantly up-regulated in normal B cells) are blue; nodes representing inferred ZFP36L1 targets (significantly up-regulated in normal plasma cells) are red. Network graphics were generated as group attribute layout using Cytoscape version 2.6.0. .

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Expressing, Cell Differentiation, Generated

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: qRT-PCR analysis of blimp1, xbp1, irf4 and bcl6 mRNA expression in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Cells were cultured in medium alone for 48h. Total RNA was extracted from 5×10 6 cells, 1 µg RNA was reverse transcribed and the resulting cDNA was used as template for qRT-PCR assay with mouse gene specific primers for blimp1, xbp1, irf4 and bcl6. The 2 –ΔΔCT method of relative quantification was used to determine the fold change in mRNA expression compared to levels in wild-type cells. Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Infection, Cell Culture, Generated

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) Western blot analysis of BLIMP1 levels in control and ZFP36L1 knockdown cells. BLIMP1 expression levels are upregulated in ZFP36L1 knockdown cells compared to controls. βACTIN levels are shown as a loading control. (B) ZFP36L1 mediates degradation of the BLIMP1 3′UTR. HEK 293 T cells were transfected with pMIRBLIMP1 3′UTR construct alone (control) or with either ZFP36L1 or a zinc finger domain mutant, ZFP36L1 Mut. Renilla luciferase was also included in all transfections as a normalization control. 24 hours later cell lysates were harvested and firefly and renilla luciferase levels measured using a Fluorstar Optima plate reader. Relative levels of luciferase activity were measured. Mean ±SD, are shown for four independent experiments (n = 4), * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Western Blot, Expressing, Transfection, Construct, Mutagenesis, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Mechanisms of GnRH-Induced Extracellular Signal-Regulated Kinase Nuclear Localization

doi: 10.1371/journal.pone.0040077

Figure Lengend Snippet: (A) HeLa cells were seeded in 12 well plates, transduced with Ad mGnRHR and kept in reduced (0.1%) serum for 16 hours prior to stimulation for varied periods with 100 nM GnRH or 1 µM PDBu. Whole cell protein extracts were then immunoblotted for phospho Ser217/221 MEK1/2 (ppMEK1/2), phospho Thr183/Tyr185 ERK (ppERK) and for total ERK (ERK1 and/or 2) as indicated. (B) HeLa cells were seeded in 96-well imaging plates, transduced with Ad mGnRHR and kept in reduced (0.1%) serum for 16 hours prior to stimulation for 5 min with 1 or 100 nM GnRH as indicated. Control cells (Ctrl.) were treated with medium alone. The cells were then fixed and stained for endogenous ppERK, ERK and DAPI before image acquisition and analysis (as described in the ). The figure shows representative images and the lower images show “segmented” ERK staining, with lines indicating the perimeters of the nuclei and cells obtained using automated image analysis algorithms, as described in the . Each of the image panels corresponds to a width of approximately 250 µm and represents approximately 1/20 th of the area captured in each field of view.

Article Snippet: Ser217/221 phosphorylated MEK1/2 were detected using a rabbit anti-ppMEK1/2 monoclonal antibody (clone 41G9, 1∶1000, Cell Signaling Technology).

Techniques: Transduction, Imaging, Staining

Journal: Arthritis Research & Therapy

Article Title: Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

doi: 10.1186/ar2991

Figure Lengend Snippet: Oligonucleotide sequences

Article Snippet: Immunolabeling and immunoprecipitation were performed using anti-type I collagen (SouthernBiotech, Birmingham, AL, USA), anti-MMP-1 (Chemicon International Inc., Temecula, CA, USA), anti-phospho-c-Jun, anti-phospho-Smad2, and anti-c-Jun (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-AP2, anti-SP1, anti-p300, anti-Ets1, and anti-TFIIEα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Smad2/3 (Upstate, now part of Millipore Corporation, Billerica, MA, USA), anti-β-tubulin (Sigma-Aldrich) primary antibodies, and anti-goat (The Binding Site, Birmingham, UK) or anti-rabbit or anti-mouse (DakoCytomation, Baar, Switzerland) IgG antibodies coupled to horseradish peroxidase.

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Pull Down Assay

Journal: Arthritis Research & Therapy

Article Title: Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

doi: 10.1186/ar2991

Figure Lengend Snippet: Proteasome inhibition abolishes the increased binding of SP1 to COL1A2 but not of AP2 to COL1A1 promoter induced by TGF-β . (a) Schematic representation of the COL1A1 and COL1A2 promoter regions and the deleted COL1A1 construct. (b) Dermal fibroblasts were transiently transfected with luciferase reporter gene constructs carrying the SV40 promoter, full-length COL1A1 promoter, deleted COL1A1 promoter, or no promoter. Luciferase activity was measured after TGF-β treatment (16 or 24 hours) and normalized to the levels obtained with cells transfected with the promoter-free construct. Histograms show the increase in COL1A1 promoter activity in treated cells relative to untreated cells. The results represent the mean ± standard deviation (SD) of two independent experiments. (c, d) Fibroblasts were treated with TGF-β (5 ng/mL) for 4 hours or bortezomib (1 μM) for 16 hours or both (TGF-β was added 1 hour after bortezomib) for 4 hours or were left untreated. Crosslinked chromatin was extracted, sonicated, and immunoprecipitated with anti-AP2 or anti-SP1 antibodies. Transcription factor-bound DNA fragments were quantified by real-time polymerase chain reaction using the primers indicated in Table 1. The increase in treated cells relative to untreated cells is shown. The bars represent the mean ± SD of two independent experiments; * P < 0.05 and *** P < 0.0005 in comparison with untreated cells. Bor, bortezomib; COL1A , collagen 1A; ND, not determined; TF, transcription factor; TGF-β, transforming growth factor-beta; UT, untreated.

Article Snippet: Immunolabeling and immunoprecipitation were performed using anti-type I collagen (SouthernBiotech, Birmingham, AL, USA), anti-MMP-1 (Chemicon International Inc., Temecula, CA, USA), anti-phospho-c-Jun, anti-phospho-Smad2, and anti-c-Jun (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-AP2, anti-SP1, anti-p300, anti-Ets1, and anti-TFIIEα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Smad2/3 (Upstate, now part of Millipore Corporation, Billerica, MA, USA), anti-β-tubulin (Sigma-Aldrich) primary antibodies, and anti-goat (The Binding Site, Birmingham, UK) or anti-rabbit or anti-mouse (DakoCytomation, Baar, Switzerland) IgG antibodies coupled to horseradish peroxidase.

Techniques: Inhibition, Binding Assay, Construct, Transfection, Luciferase, Activity Assay, Standard Deviation, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

doi: 10.1186/ar2991

Figure Lengend Snippet: Bortezomib activates the MMP-1 promoter via binding of c-Jun . (a) Schematic representation of MMP promoter regions. Transcription factor (TF) binding sites and TATA boxes are shown (adapted from ). (b) Luciferase reporter gene experiments were performed as described in Figure 2b, except that the constructs carried the SV40, wild-type, or variant MMP-1 promoter or no promoter. Luciferase activity was measured after bortezomib (4 or 24 hours) or TNF-α (1 hour) treatment, normalized, and reported as in Figure 2b. The results represent the mean ± standard deviation (SD) of two independent transfections. (c) Fibroblasts were treated for 1 hour with TNF-α (10 ng/mL) or for 16 hours with bortezomib (1 μM). Chromatin immunoprecipitation was performed with anti-c-Jun or anti-AP2 antibodies, and the results were quantified by real-time polymerase chain reaction using the primers indicated in Table 1. The increase in binding of c-Jun or AP2 to the MMP promoters in treated cells relative to untreated cells is shown. The results represent the mean ± SD of three independent experiments; * P < 0.05 in comparison with untreated cells. (d, e) Nuclear extracts were prepared from fibroblasts that were treated for 16 hours with 1 μM bortezomib or 1 hour with 10 ng/mL TNF-α or that were left untreated. Binding of TF to a synthetic AP-1 site was assessed by electrophoretic mobility shift assay (d) using a specific radiolabeled probe in the presence (+) or absence (-) of anti-Ets or anti-c-Jun antibodies or cold probe (AP-1). A gray arrow indicates band shift, and a white arrow indicates supershifted band. Alternatively, TF-DNA binding was assessed by a DNA pull-down assay (e) using a biotinylated MMP-1 probe and anti-c-Jun antibodies. Total nuclear protein content was assessed using anti-TFIIEα antibodies on unbound fractions. Bor, bortezomib; MMP, matrix metalloproteinase; ND, not determined; NE, nuclear extract; TNF-α, tumor necrosis factor-alpha; UT, untreated; WT, wild-type.

Article Snippet: Immunolabeling and immunoprecipitation were performed using anti-type I collagen (SouthernBiotech, Birmingham, AL, USA), anti-MMP-1 (Chemicon International Inc., Temecula, CA, USA), anti-phospho-c-Jun, anti-phospho-Smad2, and anti-c-Jun (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-AP2, anti-SP1, anti-p300, anti-Ets1, and anti-TFIIEα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Smad2/3 (Upstate, now part of Millipore Corporation, Billerica, MA, USA), anti-β-tubulin (Sigma-Aldrich) primary antibodies, and anti-goat (The Binding Site, Birmingham, UK) or anti-rabbit or anti-mouse (DakoCytomation, Baar, Switzerland) IgG antibodies coupled to horseradish peroxidase.

Techniques: Binding Assay, Luciferase, Construct, Variant Assay, Activity Assay, Standard Deviation, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Pull Down Assay