Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Coomassie blue–stained gel showing the Wnt5a protein purification scheme. (B) The 24-h Wnt3a protein treatment activates the STF luciferase reporter in a dose-responsive manner. (C) Concomitant Wnt5a protein treatment inhibits Wnt3a-induced STF reporter activation. (D) Time-course analysis of Wnt5a-mediated inhibition of Wnt3a. At approximately 40 h post-transfection, cells were treated with Wnt3a protein for 0, 2, 4, 6, or 8 h alone or in conjunction with Wnt5a at t = 0, t = 1 h, or t = 3 h. (Filled diamonds indicate Wnt3a treatment alone; open diamonds indicate Wnt3a treatment at t = 0 plus Wnt5a protein treatment at t = 0; filled triangles indicate Wnt3a treatment at t = 0 plus Wnt5a protein treatment at t = 1; open circles indicate Wnt3a treatment at t = 0 plus Wnt5a protein treatment at t = 3.) (E) Wnt5a pretreatment does not enhance its inhibitory activity. At 24 h post-transfection, cells were pretreated with vehicle or Wnt5a protein (200 ng/ml) for 8 h. Cells were then treated with vehicle, Wnt3a alone, or Wnt3a concomitantly with Wnt5a for an additional 24 h, and luciferase assay was performed. (Filled squares indicate cells that were pretreated with vehicle for 8 h prior to Wnt addition; open squares indicate cells pretreated with Wnt5a protein for 8 h prior to Wnt addition.) Unless stated in text, luciferase activity was measured 48 h after transfection, approximately 24 h after Wnt treatment, and results are shown as the average fold change ± SD in luciferase activity over transfected, untreated negative controls.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Staining, Protein Purification, Luciferase, Activation Assay, Inhibition, Transfection, Activity Assay

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a protein treatment does not affect cytosolic β-catenin protein levels. The 293 cells were treated for 3 h with the indicated concentrations of Wnt proteins and then hypotonically lysed for Western blot analysis of cytosolic fractions. (B) Wnt5a protein treatment does not alter β-catenin cellular localization. Cells grown on coverslips were treated with the indicated concentrations of Wnt proteins for 6 h and then fixed and stained as described in the text. Membrane localized β-catenin is indicated by arrowheads; membrane and cytosolic staining is indicated by arrows.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Western Blot, Staining, Membrane

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a-mediated inhibition is PTX insensitive. At 24 h post-transfection, 293 cells were pretreated with vehicle or 100 ng/ml PTX for 24 h. Cells were then treated with indicated Wnt proteins concomitantly with vehicle or PTX for an additional 24 h, and luciferase assay was performed. (B) PTX is active in 293 cells as PTX pretreatment of cells inhibits LPA-induced Erk1,2 activation. The 293 cells were pretreated with 100 ng/ml PTX for 24 h and then treated with 10 μM LPA or vehicle for 10 min. Western blot analysis was then performed on total cell lysates using anti–phospho-Erk1,2 antibody. Membrane was stripped and reprobed for total Erk1,2 as a loading control. (C) Wnt5a protein does not directly stimulate intracellular Ca 2+ flux. The 293 and 293Fz4 cells were loaded with Fura-2-dextran and then monitored for changes in intracellular Ca 2+ concentration as determined by the change in 340/380 excitation wavelength ratio following Wnt5a (500 ng/ml) and subsequent ionomycin treatment. Data represent the average 340/380 excitation wavelength ratio of three or more independent cells within the same field ± SD. Wnt5a has little effect on the ratio over the 15-min time course, whereas ionomycin treatment rapidly and robustly induces Ca 2+ flux in these cells. Prolonged treatment with Wnt5a (up to 1 h) did not result in changes in intracellular Ca 2+ levels (unpublished data).

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Inhibition, Transfection, Luciferase, Activation Assay, Western Blot, Membrane, Control, Concentration Assay

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a treatment leads to β-catenin stabilization specifically in cells expressing mFz4. The 293 and 293Fz4 cells were treated with Wnt proteins and then assayed for cytosolic β-catenin protein accumulation via Western blot analysis. (B) Wnt5a treatment activates the STF reporter when LRP5 is coexpressed. At 24 h post-transfection with STF reporter and LRP5 or empty vector, 293Fz4 cells were treated with increasing concentrations of Wnt5a protein for an additional 24 h, and luciferase assay was performed as described in . (C) Wnt5a does not inhibit Wnt3a-mediated reporter activation in 293Fz4 cells. The 293 and 293Fz4 cells were treated with Wnts as indicated 24 h post-transfection with reporters, and luciferase assay was performed. (D) Loss of Wnt5a-mediated reporter inhibition is specific to mFz4 overexpression. The 293, 293Fz4, and 293Fz8 cells were treated with the indicated Wnts for 24 h, 24 h post-transfection, and then luciferase assay was performed. Wnt5a maintains inhibitory activity when mFz8, but not mFz4, is overexpressed.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activation Assay, Inhibition, Over Expression, Activity Assay

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a synergizes with mRor2 to inhibit Wnt3a-mediated reporter activation. The 293 cells stably expressing exogenous mRor2 (293Ror2) were treated with Wnts as indicated, 24 h post-transfection with reporters, and luciferase assay was performed as outlined in . (B) Wnt5a binds directly to the Fz4 and mRor2 CRD domains. Wnt5a protein specifically binds to the purified mFz4 and mRor2 CRDs; only background binding to the Smoothened CRD negative control is observed. While it may appear that Wnt5a binds to a similar extent to the Ror2 CRDs and Smo CRD, the input levels of the Smo CRD-IgG exceeded the Ror2 CRD-IgG levels, accounting for the increased background binding Smo CRD-IgG. (C) The mRor2 CRD domain is required for mediating Wnt5a inhibitory activity. The 293 cells were transiently transfected with reporters along with empty vector, mRor2, or mRor2ΔCRD constructs, treated with Wnts as described in text 24 h post-transfection for an additional 24 h, and then luciferase assay was performed as outlined in .

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Activation Assay, Stable Transfection, Expressing, Transfection, Luciferase, Purification, Binding Assay, Negative Control, Activity Assay, Plasmid Preparation, Construct

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) In 293 cells, mRor2 overexpression inhibits Wnt5a-mediated activation of the STF reporter. The 293 and 293Fz4 cells were transiently transfected with the reporters and LRP5 with or without mRor2 and then treated with Wnt proteins for 24 h, 24 h post-transfection. Overexpression of wild-type mRor2 inhibits Wnt5a-mediated activation of the STF reporter as well as allowing Wnt5a to inhibit Wnt3a-mediated STF reporter activation in 293Fz4 cells. (B) mRor2 overexpression inhibits Wnt5a-mediated activation of the STF reporter and allows Wnt5a to block Wnt3a-induced STF reporter activation in mouse L cells. L and LFz4 cells were transiently transfected with the reporters and LRP5 with or without mRor2 and then treated with Wnt proteins for 24 h, 24 h post-transfection. (C) Quantitative real-time PCR analysis of Ror2 expression in 293 and L cells. Reverse transcription followed by 45 cycles of quantitative real-time amplification of 293 cell RNA reveals a robust Ror2 product that is over 700 times more abundant than that produced from RNA derived from mouse L cells. Hprt quantification serves as an internal normalization control and RNA derived from 293Ror2 and LRor2 cells serves as a positive control for the reaction. The products from the PCR analysis are displayed below the charted quantitative data. (D) The cytoplasmic domain of mRor2 is required for its inhibitory function. Expression of a membrane-tethered variant of mRor2 possessing the extracellular domain of mRor2 fused to a GPI linkage (mRor2-GPI) reduces Wnt5a's ability to inhibit Wnt3a induced canonical Wnt signaling as well as inhibits wild type mRor2′s ability to enhance Wnt5a-mediated inhibition. (E) Expression of a membrane-tethered variant of mRor2 (mRor2-TM) that contains the extracellular and transmembrane domains of mRor2, but lacks the cytoplasmic domain, does not enhance Wnt5a mediated inhibition of canonical Wnt signaling. Expression of mRor2-TM reduces Wnt5a's ability to inhibit Wnt3a-induced canonical Wnt signaling when wild-type mRor2 is overexpressed. Western blot analysis shows that wild-type mRor2 levels do not change when the truncated mRor2 variants are coexpressed.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Over Expression, Activation Assay, Transfection, Blocking Assay, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription, Amplification, Produced, Derivative Assay, Control, Positive Control, Membrane, Variant Assay, Inhibition, Western Blot

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: In the presence of Fz4 and LRP5, Wnt5a can activate β-catenin/TCF signaling. However, when Ror2 is expressed, Wnt5a inhibits canonical Wnt signaling downstream of β-catenin stabilization, at the level of TCF-mediated transcription.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques:

Journal: eLife

Article Title: Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells

doi: 10.7554/eLife.105690

Figure Lengend Snippet: ( A, B ) Analysis of GBM patient versus control samples focused on the level of expression of human homologs of Drosophila asymmetric cell division (ACD) regulators by hierarchical clustering ( A ) and the Gene Distance Matrix ( B ). Color-coded scale bar indicates the fold level of expression. ( C ) Kaplan–Meier survival curves corresponding to patients in The Cancer Genome Atlas (TCGA) (IDHwt) and Gravendeel (IDHwt) cohort in GBM datasets. Low RAP2A expression levels in human GBM are associated with a poor prognosis.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A Plasmid #19311, Addgene) or eGFP (pLJMEGFP Plasmid #19319, Addgene) as infection control.

Techniques: Control, Expressing

Journal: eLife

Article Title: Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells

doi: 10.7554/eLife.105690

Figure Lengend Snippet: ( A ) RAP2A expression levels analysis according to IDHwt glioblastoma (GBM) subtypes in The Cancer Genome Atlas (TCGA) cohort. ( B–D ) Kaplan–Meier survival curves for each GBM subtype, proneural ( B ), mesenchymal ( C ), and classical ( D ), based on high and low RAP2A expression levels.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A Plasmid #19311, Addgene) or eGFP (pLJMEGFP Plasmid #19319, Addgene) as infection control.

Techniques: Expressing

Journal: eLife

Article Title: Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells

doi: 10.7554/eLife.105690

Figure Lengend Snippet: ( A ) Different GBM cell lines show similar RAP2A mRNA levels and significantly lower levels than in control Astros. ( B ) RAP2A mRNA levels are significantly higher in the GB5 line after infecting this line with RAP2A (GB5-RAP2A). Data shown in the scaled bar graphs in (A) and (B) was analyzed with an ANOVA and a t -test, respectively; error bars show the SD; n=2 (in A) and 3 (in B) different experiments. ( C ) Immunofluorescences of the glioblastoma stem cell (GSC) stem cell markers CD133, SOX2, and Nestin reveal a significant reduction in the protein intensity in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the box plots was analyzed with a Mann–Whitney U for CD133 and Nestin and with a t -test for Sox; the central lines represent the median and the box limits the lower and upper quartiles, as determined using R software; crosses represent sample means; error bars indicate the SEM; n=number of sample points; *p<0.05, **p<0.01, ***p<0.001, ns, not significant; scale bar: 10 µm. Figure 3—source data 1. Source data of (CD133) analysis. Figure 3—source data 2. Source data of (SOX2) analysis. Figure 3—source data 3. Source data of (Nestin) analysis.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A Plasmid #19311, Addgene) or eGFP (pLJMEGFP Plasmid #19319, Addgene) as infection control.

Techniques: Control, Expressing, MANN-WHITNEY, Software

Journal: eLife

Article Title: Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells

doi: 10.7554/eLife.105690

Figure Lengend Snippet: RT-PCRs and western blots show a significant decrease in the mRNA and protein expression levels, respectively, of the glioblastoma stem cell (GSC) markers CD133, SOX2, and Nestin in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student‘s t -test; error bars show the SD; n=3 different experiments; *p<0.05, **p<0.01. Figure 4—source data 1. Original files for the blots displayed in . Figure 4—source data 2. Original files for the blots displayed in , labeling the relevant bands.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A Plasmid #19311, Addgene) or eGFP (pLJMEGFP Plasmid #19319, Addgene) as infection control.

Techniques: Western Blot, Expressing, Control, Two Tailed Test, Labeling

Journal: eLife

Article Title: Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells

doi: 10.7554/eLife.105690

Figure Lengend Snippet: ( A ) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significantly lower number of Ki67-expressing cells per neurosphere than control GB5 neurospheres. Data shown in the box plots was analyzed with a t -test; the central lines represent the median and the box limits the lower and upper quartiles, as determined using R software; crosses represent sample means; error bars indicate the SEM; n=number of sample points. ( B ) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significant decrease in their size compared to control GB5 neurospheres of the same stage. Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s t -test; error bars show the SD; n=total number of neurospheres of three different experiments; *p<0.05; scale bars: 10 µm (in A) and 100 µm (in B). Figure 5—source data 1. Source data of analyisis. Figure 5—source data 2. Source data of analysis.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A Plasmid #19311, Addgene) or eGFP (pLJMEGFP Plasmid #19319, Addgene) as infection control.

Techniques: Expressing, Control, Software, Two Tailed Test

Journal: eLife

Article Title: Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells

doi: 10.7554/eLife.105690

Figure Lengend Snippet: ( A ) Early-stage GB5 neurospheres expressing RAP2A (GB5-RAP2A) show an odd number of cells ( ; ; ) significantly more frequently than control GB5 neurospheres, which show more frequently an even number of cells ( ; ; ). ( B ) GB5-RAP2A dividing cells show a significant increase in the number of asymmetric NUMB localization in the progeny compared to control GB5 dividing cells. Data shown in the bar graphs was analyzed with a chi-square test with Yates correction; n=number of neurosphere cell clusters analyzed. **p<0.01; ***p<0.001, scale bar: 20 µm. Figure 6—source data 1. Source data of analysis. Figure 6—source data 2. Source data of analysis.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A Plasmid #19311, Addgene) or eGFP (pLJMEGFP Plasmid #19319, Addgene) as infection control.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: Lack of HIF-P4H-1 affects the expression of several genes involved in inflammatory and apoptotic pathways. ( a ) Heatmap analysis of microarray data shows that expression of several genes involved in proinflammatory pathways is downregulated in Hif-p4h-1 −/− MEFs relative to wt after 24 h culture in normoxia (21% O 2 ) and hypoxia (1% O 2 ). A comparison between one set of samples is shown in the heatmap. The heatmap was generated by Chipster using Pearson correlation as a distance measure and average linkage for constructing the dendrogram. The red color represents upregulated genes and the green color represents downregulated genes. The values in the color key represent the expression levels between the samples. ( b ) qPCR analysis verifies that the expression of genes for the low affinity III receptor of the Fc fragment of IgG ( Fcgr3 ), the α and γ chains of the complement C1q subcomponent ( C1qa , C1qc ) and the C-X-C motif chemokine 10 ( Cxcl10 ) is downregulated in Hif-p4h-1 −/− MEFs relative to wt in normoxia and hypoxia. ( c ) qPCR analysis shows that expression of the pro-apoptotic genes for the p53 apoptosis effector related to peripheral myelin protein 22 ( Perp ) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 ( Bnip3 ) is upregulated in Hif-p4h-1 −/− MEFs relative to wt. ( b , c ) data are presented as mean ± s.d., n = 3–4 individual Hif-p4h-1 −/− and wt MEF isolates analysed in triplicates. * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test.

Article Snippet: For liquid chromatography-mass spectrometry (LC-MS) overexpressed Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. The cells were lysed in RIPA buffer and the cell lysate was pre-cleared with Protein A/G plus agarose beads (Santa Cruz Biotechnologies).

Techniques: Expressing, Microarray, Generated, Two Tailed Test

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: Lack of HIF-P4H-1 increases cell death. ( a – c ) Analysis of relative caspase 3/7 activity ( a ), caspase-3 and cleaved caspase-3 ( b ), and p53 ( c ) protein amounts in wt and Hif-p4h-1 −/− MEFs treated with or without 100 ng/ml LPS for 24 h. ( d ) Analysis of viability of wt and Hif-p4h-1 −/− MEFs treated with or without 5 mg/ml of LPS for 48h. Quantitation of cell viability based on 7-actinomycin D staining and FACS analysis is shown. ( e ) Analysis of relative caspase 3/7 activity in Hif-p4h-1 −/− MEFs transfected with empty vector (EV) or a vector encoding V5-tagged human recombinant HIF-P4H-1 (OE). ( f ) Analysis of viability of wt and Hif-p4h-1 −/− MEFs treated with or without 2 μM staurosporine for 24 h. ( g ) Analysis of viability of Hif-p4h-1 −/− MEFs transfected with empty vector (EV) or a vector overexpressing (OE) V5-tagged human recombinant HIF-P4H-1 and treated with 2 μM staurosporine for 24 h. Quantitation of cell viability based on 7-actinomycin D staining and FACS analysis is shown. ( h ) Western blot analysis of p53, caspase-3, cleaved caspase-3 and HIF-P4H-1 in wt and Hif-p4h-1 −/− MEFs treated with or without 10 μg/ml cisplatin for 6 h. ( i ) Western blot analysis of caspase-3, cleaved caspase-3 and HIF-P4H-1 in wt MEFs treated with or without 10 μg/ml cisplatin and 50 µM FG4497 for 6 h. ( j ) Analysis of viability of Hif-p4h-1 −/− and wt MEFs treated with or without 50 μg/ml cisplatin and 100 μM PFT-α for 24 h. Quantitation of cell viability based on dynamic imaging of live and dead cells in the IncuCyte TM zoom live-cell imaging system is shown. ( k ) Western blot analysis of HIF-P4H-1 in wt HCT-116 cells treated with or without 100 ng/ml LPS and 50 µM FG4497 for 6 h. Data are presented as representative Western blots and as mean ± s.d., n = 3 individual MEF isolates or experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: For liquid chromatography-mass spectrometry (LC-MS) overexpressed Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. The cells were lysed in RIPA buffer and the cell lysate was pre-cleared with Protein A/G plus agarose beads (Santa Cruz Biotechnologies).

Techniques: Activity Assay, Quantitation Assay, Staining, Transfection, Plasmid Preparation, Recombinant, Western Blot, Imaging, Live Cell Imaging, Two Tailed Test

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: Lack of HIF-P4H-1 increases the amount of p53. ( a ) Western blot analysis of p53 in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h. ( b , c ) Western blot analysis of p53 in Hif-p4h-1 and scrambled (Scrm) siRNA transfected HEK293 cells ( b ) and wt MEFs treated with 50 µM FG4497 for 24 h ( c ). ( d ) qPCR analysis of p53 mRNA in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h. ( e) Analysis of p53 protein turnover rate. Hif-p4h-1 −/− and wt MEFs were treated with 200 μg/ml of cycloheximide for the indicated time points and whole cell lysates were blotted for p53. ( f ) Western blot analysis of MDM2 in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h. ( g ) Western blot analysis of p53 and MDM2 in wt MEFs treated with 10 μM nutlin-3a for 24 h with or without overexpression of human HIF-P4H-1 (OE). ( h , i ) Western blot analysis of HIF1α in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h ( h ) and in scrambled and Hif-p4h-1 siRNA transfected HEK293 cells ( i ). ( j ) Western blot analysis of ubiquitination of p53 in Hif-p4h-1 and scrambled siRNA transfected HEK293 cells. The cells were transfected with HA-ubiquitin and endogenous p53 was immunoprecipitated followed by Western blotting with anti-HA and anti-P53 antibodies. ( k ) Western blot analysis of p53 in Hif-p4h-1 −/− and wt MEFs were treated with or without 10 μM MG132 for 5 h. Data are presented as representative Western blots and as mean ± s.d., n = at least 3 individual MEF isolates or experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: For liquid chromatography-mass spectrometry (LC-MS) overexpressed Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. The cells were lysed in RIPA buffer and the cell lysate was pre-cleared with Protein A/G plus agarose beads (Santa Cruz Biotechnologies).

Techniques: Western Blot, Cell Culture, Transfection, Over Expression, Immunoprecipitation, Two Tailed Test

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: HIF-P4H-1 interacts with p53. ( a ) Immunoprecipitation of endogenous p53 from wt MEFs treated with 10 μM MG132 for 4 h before cell lysis. Co-immunoprecipitation of endogenous HIF-P4H-1 was analyzed by Western blotting with an anti-HIF-P4H-1 antibody. ( b ) HEK293 cells were transfected with a plasmid encoding full-length p53 together with a plasmid encoding V5-tagged human HIF-P4H-1 (OE) or an empty vector (EV) and analysed by Western blotting with anti-p53 and anti-V5 antibodies. ( c ) HEK293 cells were transfected as in ( b ) treated with 10 μM MG132 for 4 h and immunoprecipitated with an anti-V5 antibody. Co-immunoprecipitation of p53 was analysed by Western blotting with an anti-p53 antibody. ( d ) HEK293 cells were transfected with a plasmid encoding a deleted form of p53 that lacks amino acid residues 75–207 (p53Δ) together with a plasmid encoding V5-tagged human HIF-P4H-1 (OE) or an empty vector and analysed by Western blotting with anti-p53 and anti-V5 antibodies. ( e ) HEK293 cells were transfected as in d , treated with 10 μM MG132 for 4 h and immunoprecipitated with an anti-V5 antibody. Co-immunoprecipitation of p53 was analysed by Western blotting with an anti-p53 antibody. Data are presented as representative Western blots and as mean ± s.d., n = at least 3 individual experiments. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: For liquid chromatography-mass spectrometry (LC-MS) overexpressed Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. The cells were lysed in RIPA buffer and the cell lysate was pre-cleared with Protein A/G plus agarose beads (Santa Cruz Biotechnologies).

Techniques: Immunoprecipitation, Lysis, Western Blot, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: p53 is hydroxylated at Pro142. ( a ) Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h, separated by SDS-PAGE, trypsinized and analyzed by LC-MS. Mass signals extracted from the high energy trace of the MS e measurement assigned to the peptide TCPVQLWVDSTPPPGTR (140–156) are shown. Low molecular weight area for the oxidized (i) and unmodified (ii) peptide with the b4 ions was used to assign hydroxylation to Pro142. Detailed information on all found fragment ions are provided in Supplementary tables and . ( b ) [2,3,4,5- 3 H] proline-labeled Flag-tagged p53 was produced in wt HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 with or without 50 μM FG4497 for 6 h. Flag-p53 was immunoprecipitated and the amount of 4-hydroxy[ 3 H]proline formed was measured by a radiochemical method. The data are given as the amount of 4-hydroxyproline residues/50 000 proline residues. ( c ) HCT-116 cells were transfected with scrambled or HIF-P4H-1 siRNA and treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. Endogenous p53 was immunoprecipitated and analysed by Western blotting with antibodies agains hydroxyproline and p53. ( d ) Wt and HIF1A −/− HCT-116 cells were treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h with or without 50 μM FG4497. Endogenous p53 was immunoprecipitated and analysed by Western blotting with antibodies against hydroxyproline, P(Ser15)-p53 and p53. ( e , f ) Flag-tagged wt, Pro12Ala/Pro13Ala and Pro142Ala p53 were overexpressed (OE) in HCT-116 cells with or without V5-tagged HIF-P4H-1 ( e ) and with or without 50 μM FG4497 for 6 h ( f ) and the amount of p53 and HIF-P4H-1 was analysed by Western blotting with anti-Flag and anti-V5 antibodies, respectively. ( g ) Flag-tagged wt and Pro142Ala p53 and V5-tagged HIF-P4H-1 were overexpressed in HEK293 cells and treated with 10 μM MG132 for 6 h. Flag-tagged p53 proteins were immunoprecipitated and co-immunoprecipitation of HIF-P4H-1 was analysed by Western blotting with an anti-V5 antibody. ( h ) Flag-tagged wt and Pro142Ala p53 were overexpressed in HCT-116 cells and treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. Flag-p53 was immunoprecipitated and hydroxylation was analysed by Western blotting with an anti-hydroxyproline antibody. ( i ) V5-tagged HIF-P4H-1 was overexpressed (OE) in wt and HIF1A −/− HCT-116 cells treated with 10 μM MG132 for 6 h. Endogenous p53 was immunoprecipitated and co-immunoprecipitation of HIF-P4H-1 was analysed by Western blotting with an anti-V5 antibody. Data are presented as representative Western blots and as mean ± s.d., n = at least 3 individual experiments in ( b – i ). * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: For liquid chromatography-mass spectrometry (LC-MS) overexpressed Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. The cells were lysed in RIPA buffer and the cell lysate was pre-cleared with Protein A/G plus agarose beads (Santa Cruz Biotechnologies).

Techniques: Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Molecular Weight, Labeling, Produced, Transfection, Western Blot, Two Tailed Test

Journal: The EMBO Journal

Article Title: A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease

doi: 10.1038/s44318-024-00328-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse anti-Mical-L1 , 1:100 , Novus , H00085377.

Techniques: Recombinant, Transduction, CRISPR, Gene Knockout, Expressing, Concentration Assay, Immunofluorescence, Western Blot, Flow Cytometry, Sequencing, Red Blood Cell Lysis, Cell Recovery, Plasmid Preparation, SYBR Green Assay, Reverse Transcription, Software, Microscopy, Magnetic Beads, Transmission Assay

Journal: OncoTargets and therapy

Article Title: Circular RNA circLPAR3 Facilitates Esophageal Squamous Cell Carcinoma Progression Through Upregulating HMGB1 via Sponging miR-375/miR-433

doi: 10.2147/OTT.S244699

Figure Lengend Snippet: HMGB1 was a target of miR-375/miR-433. ( A ) The binding sites and mutant binding sites between high-mobility group box 1 (HMGB1) and miR-375/miR-433 were shown. ( B, C ) Dual-luciferase reporter assay was used to detect the interaction between HMGB1 and miR-375/miR-433 in KYSE30 and KYSE150 cells. ( D ) The expression levels of miR-375 and miR-433 were determined by qRT-PCR to evaluate the transfection efficiency of miR-375 and miR-433 mimics. ( E, F ) The mRNA and protein levels of HMGB1 were measured by qRT-PCR and WB analysis to evaluate the effect of miR-375/miR-433 overexpression on HMGB1 expression. KYSE30 and KYSE150 cells were co-transfected with (short hairpin) sh-circLPAR3#1 and anti-miR-375 or anti-miR-433. ( G ) The protein level of HMGB1 was tested by Western blot (WB) analysis in KYSE30 and KYSE150 cells. * P < 0.05.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting circLPAR3 (sh-circLPAR3#1/2) and its negative control (sh-NC), miR-375/miR-433 mimic and inhibitor (miR-375/miR-433 and anti-miR-375/miR-433) or their negative controls (miR-NC and anti-NC), HMGB1 overexpression plasmid (HMGB1) and its negative control (vector) were all synthesized by Ribobio (Guangzhou, China).

Techniques: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Transfection, Over Expression, Western Blot

Journal: OncoTargets and therapy

Article Title: Circular RNA circLPAR3 Facilitates Esophageal Squamous Cell Carcinoma Progression Through Upregulating HMGB1 via Sponging miR-375/miR-433

doi: 10.2147/OTT.S244699

Figure Lengend Snippet: Effect of HMGB1 overexpression on esophageal squamous cell carcinoma (ESCC) progression. ( A ) The protein level of high-mobility group box 1 (HMGB1) was detected by Western blot (WB) analysis to evaluate the transfection efficiency of HMGB1 overexpression plasmid. KYSE30 and KYSE150 cells were co-transfected with (short hairpin) sh-circLPAR3#1 and HMGB1 overexpression plasmid. ( B ) CCK-8 assay was performed to measure the viability of KYSE30 and KYSE150 cells. ( C ) Colony formation assay was used to test the number of colonies in KYSE30 and KYSE150 cells. ( D, E ) The number of migrated and invaded KYSE30 and KYSE150 cells was assessed by transwell assay. ( F ) The protein levels of proliferating cell nuclear antigen (PCNA), Ki-67, matrix metalloproteinase2 (MMP2) and MMP9 in KYSE30 and KYSE150 cells were determined by WB analysis. * P < 0.05.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting circLPAR3 (sh-circLPAR3#1/2) and its negative control (sh-NC), miR-375/miR-433 mimic and inhibitor (miR-375/miR-433 and anti-miR-375/miR-433) or their negative controls (miR-NC and anti-NC), HMGB1 overexpression plasmid (HMGB1) and its negative control (vector) were all synthesized by Ribobio (Guangzhou, China).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay, Colony Assay, Transwell Assay

Journal: Experimental Physiology

Article Title: Inhibitory role of long non‐coding RNA OIP5‐AS1 in rheumatoid arthritis progression through the microRNA‐448–paraoxonase 1–toll‐like receptor 3–nuclear factor κB axis

doi: 10.1113/ep088608

Figure Lengend Snippet: FIGURE 1 Overexpression of OIP5-AS1 alleviates RA severity in rats. (a) RA rat limbs became red and swollen from the second week after model establishment. (b,c) The scoring of limb swelling (b) and RA (c). (d) Infiltration of immune cells in rat synovial tissues measured using HE staining. (e) Protein levels of IL-1β, IL-6, IL-10 and TNF-α in rat serum measured using ELISA. (f) OIP5-AS1 expression in rat synovial tissues detected using RT-qPCR. (g) OIP5-AS1 expression in rat synovial tissues after Lv-OIP5-AS1 overexpressing vector transfection detected using RT-qPCR. Data are expressed as means ± SD. In (b,c) data were analysed using two-way ANOVA; in (d,e) data were analysed using one-way ANOVA with Tukey’s multiple comparison test for the post hoc test after ANOVA; and in (f,g) data were analysed by the unpaired t test. (b–f) *P < 0.05, **P < 0.01 vs. sham group; #P < 0.05 vs. Lv-NC group; (g) **P < 0.01 vs. Lv-NC group

Article Snippet: The sequences of OIP5-AS1 and PON1 transcripts were obtained from the GenBank Database (https://www.ncbi.nlm.nih.gov/), and the lentiviral (Lv) vectors for RNA overexpression and small interfering RNAs (siRNAs), as well as the negative control (NC) vector and the scramble siRNAwere synthesized by Sangon Biotech Co., Ltd (Shanghai, China). miR-448 mimic and the mimic control were purchased from GenePharma Co., Ltd (Shanghai, China).

Techniques: Over Expression, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Transfection, Comparison

Journal: Experimental Physiology

Article Title: Inhibitory role of long non‐coding RNA OIP5‐AS1 in rheumatoid arthritis progression through the microRNA‐448–paraoxonase 1–toll‐like receptor 3–nuclear factor κB axis

doi: 10.1113/ep088608

Figure Lengend Snippet: FIGURE 3 Overexpression of OIP5-AS1 decreases proliferation of RA-FLSs. (a) RA-FLS biomarkers CD55, CD90, CD44, CD14 and CD68 determined using flow cytometry. (b) Subcellular localization of OIP5-AS1 in FLS from RA patients determined by a FISH assay. (c) OIP5-AS1 Lv-overexpressing vectors and the corresponding empty vector, or siRNAs (siRNA-1, siRNA-2, siRNA-3) targeting OIP5-AS1 and the corresponding scramble siRNA were supplied to RA-FLSs, and then RT-qPCR was performed to measure OIP5-AS1 expression. (d) Number of cells in S stage (EdU-labelled cells) determined using EdU labelling assay. (e) Cell viability detected using a Cell Titer-Glo Kit. Three independent experiments were performed; data are presented as means ± SD; in (c,d) data were analysed using one-way ANOVA, and in (e) data were analysed using two-way ANOVA, and Tukey’s multiple comparison test was used for the post hoc test after ANOVA. *P < 0.05, **P < 0.01 vs. Lv-NC group; #P < 0.05, ##P < 0.01 vs. Scr group

Article Snippet: The sequences of OIP5-AS1 and PON1 transcripts were obtained from the GenBank Database (https://www.ncbi.nlm.nih.gov/), and the lentiviral (Lv) vectors for RNA overexpression and small interfering RNAs (siRNAs), as well as the negative control (NC) vector and the scramble siRNAwere synthesized by Sangon Biotech Co., Ltd (Shanghai, China). miR-448 mimic and the mimic control were purchased from GenePharma Co., Ltd (Shanghai, China).

Techniques: Over Expression, Flow Cytometry, Plasmid Preparation, Quantitative RT-PCR, Expressing, Comparison

Journal: Experimental Physiology

Article Title: Inhibitory role of long non‐coding RNA OIP5‐AS1 in rheumatoid arthritis progression through the microRNA‐448–paraoxonase 1–toll‐like receptor 3–nuclear factor κB axis

doi: 10.1113/ep088608

Figure Lengend Snippet: FIGURE 4 Overexpression of OIP5-AS1 promotes RA-FLS apoptosis and inhibits expression of inflammatory factors in vitro. (a,b) Apoptosis of RA-FLSs determined using Hoechst 33258 staining (a) and flow cytometry (b), respectively. (c) Caspase-3 activity in RA-FLSs detected using a Caspase-3 Assay Kit. (d) Levels of IL-1β, IL-6, IL-10 and TNF-α in extracted RA-FLSs evaluated using ELISA its. Three independent experiments were performed; data are presented as means ± SD; in all panels data were analysed using one-way ANOVA and Tukey’s multiple comparison test. *P < 0.05, **P < 0.01 vs. Lv-NC group; #P < 0.05, ##P < 0.01 vs. Scr group

Article Snippet: The sequences of OIP5-AS1 and PON1 transcripts were obtained from the GenBank Database (https://www.ncbi.nlm.nih.gov/), and the lentiviral (Lv) vectors for RNA overexpression and small interfering RNAs (siRNAs), as well as the negative control (NC) vector and the scramble siRNAwere synthesized by Sangon Biotech Co., Ltd (Shanghai, China). miR-448 mimic and the mimic control were purchased from GenePharma Co., Ltd (Shanghai, China).

Techniques: Over Expression, Expressing, In Vitro, Staining, Flow Cytometry, Activity Assay, Caspase-3 Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Experimental Physiology

Article Title: Inhibitory role of long non‐coding RNA OIP5‐AS1 in rheumatoid arthritis progression through the microRNA‐448–paraoxonase 1–toll‐like receptor 3–nuclear factor κB axis

doi: 10.1113/ep088608

Figure Lengend Snippet: FIGURE 5 Overexpression of miR-448 (or overexpression of PON1) partially reverses the effect of OIP5-AS1 (or si-OIP5-AS1) on RA-FLS. (a) miR-448 mimic was further transfected into RA-FLSs with stably overexpressed OIP5-AS1, while Lv-PON1 overexpressing vector was transfected into RA-FLSs with silenced OIP5-AS1, and then expression of miR-448 and PON1 in cells was determined using RT-qPCR. (b) Number of cells in S stage (EdU-labelled cells) determined using EdU labelling assay. (c) Cell viability detected using a Cell Titer-Glo Kit. (d) Apoptosis of RA-FLSs determined using flow cytometry. (e) Caspase-3 activity in RA-FLSs detected using a Caspase-3 Assay Kit. (f) Levels of IL-1β, IL-6, IL-10 and TNF-α in RA-FLSs evaluated using ELISA Kits. Three independent experiments were performed; data are presented as meana ± SD. In (a) data were analysed using unpaired t test; in (b,d,e,f) data were analysed using one-way ANOVA; and in (c) data were analysed using two-way ANOVA, and Tukey’s multiple comparison test was used for the post hoc test after ANOVA. *P < 0.05, **P < 0.01 vs. Lv-OIP5-AS1 + mock group; #P < 0.05, ##P < 0.01 vs. si-OIP5-AS1 + Lv-NC group

Article Snippet: The sequences of OIP5-AS1 and PON1 transcripts were obtained from the GenBank Database (https://www.ncbi.nlm.nih.gov/), and the lentiviral (Lv) vectors for RNA overexpression and small interfering RNAs (siRNAs), as well as the negative control (NC) vector and the scramble siRNAwere synthesized by Sangon Biotech Co., Ltd (Shanghai, China). miR-448 mimic and the mimic control were purchased from GenePharma Co., Ltd (Shanghai, China).

Techniques: Over Expression, Transfection, Stable Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Flow Cytometry, Activity Assay, Caspase-3 Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A) Ccn3 expression by quantitative real-time qPCR in INS832/13 cells transduced with either Ad-β-Gal or Ad-CN-FoxO1 and cultured for 24 h. Results are means +/– SEM of 4 separate experiments. B) Ccn3 mRNA levels in INS832/13 cells treated with or without 10% serum and LY294002 (50 µM) for 4 h. Results represent means +/– SEM of 3 separate experiments. C) Ccn3 expression in isolated islets from WT and transgenic mice with CAFoxO1 overexpression in their β-cells (called “305 mice”) (n = 5 for each). *, p<0.05.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Transduction, Cell Culture, Isolation, Transgenic Assay, Over Expression

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A) A consensus Forkhead binding site (shown in bold) is conserved in the rat, mouse and human Ccn3 promoters. Distance from the respective transcription start sites is indicated. B) FoxO1 was immunoprecipitated from cross-linked chromatin extracted from INS832/13 cells transduced with Ad-βGal or Ad-CAFoxO1 and cultured in the presence or absence of serum using an anti-FoxO1 antibody. Eluted DNA was PCR-amplified using oligonucleotides flanking the indicated forkhead site in the rat Ccn3 promoter shown in (A). The figure shows duplicates for each condition. C) FoxO1 increased Ccn3 promoter activity. INS cells were transiently transfected with a plasmid containing a Ccn3 promoter-driven CAT construct concomitantly with either a plasmid encoding CAFoxO1 or control βGal. Lysates were prepared 24 h post-transfection for CAT assays. CAT activity was assigned an arbitrary value of 1 in βGal (control) samples. Results are means +/- SEM of 3 separate experiments. *, p<0.05.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: Binding Assay, Immunoprecipitation, Transduction, Cell Culture, Amplification, Activity Assay, Transfection, Plasmid Preparation, Construct, Control

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A) We determined CCN3 protein levels in different genetic models of insulin resistance. We prepared paraffin sections from WT, CAFoxO1 transgenics (305), db/db , and Irs2 –/– mice and performed CCN3 immunostaining (at least n = 3 for each). Representative images are shown. B) Ccn3 expression in both the exocrine and the endocrine fraction was determined by PCR in 2 months old male animals. Amylase and insulin were used as specific exocrine and endocrine markers. Actin was used as a control in each sample. Representative images of 3 separate experiments are shown.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: Immunostaining, Expressing, Control

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A–B) Western blot of CCN3 proteins in the media (A) or whole cell extracts (B) of either AdGFP or AdCCN3 infected cells and control uninfected cells. In (B) the top arrow indicate full-length CCN3 at a molecular weight of 47 KDa, whereas the bottom arrow indicate a CCN3 fragment of 35 KDa that have both been described in the literature. C) Immunohistochemical analysis of endogenous CCN3 protein localization in INS832/13 cells. We performed triple immunohistochemistry with CCN3 (blue), insulin (green) and Vamp/synaptobrevin (red) antibodies. D) Simple immunohistochemistry for CCN3 combined with DAPI staining demonstrate both nuclear and cytoplasmic localization of CCN3 in untransfected cells (Control, top) and cells transfected with a scrambled siRNAs (SiCont, middle). No CCN3 expression could be detected in cells transfected with Ccn3 siRNAs after 48 h (SiCcn3, bottom). Representative images of 3 separate experiments are shown.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: Western Blot, Infection, Control, Molecular Weight, Immunohistochemical staining, Immunohistochemistry, Staining, Transfection, Expressing

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A) The effect of CCN3 protein (1 nM) on β-cell proliferation was evaluated by BrdU incorporation in INS832/13 cells incubated at 5 mM glucose (G5), 25 mM glucose (G25) or in the presence of 10% serum. B) We studied the effects of 1 nM CCN3 protein on cAMP levels in INS cells by ELISA. C–D) Dose-dependent effects of CCN3 on cAMP levels in isolated rat islets (C) and INS cells (D). E) We measured Ccn3 expression in cells transduced with increasing concentrations of siRNA specifically targeting Ccn3 mRNA or scrambled siRNA as control by qPCR. F) Proliferation of INS832/13 cells transduced with either 50 pmol CCN3 or control siRNA. Results represent mean ± SEM of three separate experiments carried out at least in duplicate. *, p<0.05; **, p<0.01.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: BrdU Incorporation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Transduction, Control

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A) mRNA levels of cells treated with or without CCN3 for 24 h. B) Reported changes in gene expression following CCN3 treatment. Results represent means +/– SEM of 3 separate experiments. *, p<0.05; **, p<0.01; ns = not significant.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: Gene Expression

Journal: PLoS ONE

Article Title: Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

doi: 10.1371/journal.pone.0064957

Figure Lengend Snippet: A) We evaluated the effects of Ccn3 overexpression on insulin secretion by the hGH co-transfection system. In brief, we measured the hGH levels in the media of INS832/13 cells co-transduced with hGH and CCN3 or the empty plasmid and incubated at 2.8 mM (G2.8) or 16 mM glucose (G16). KCl (35 mM) was used as control. B) We studied the effects of CCN3 protein on insulin secretion. Insulin released in the culture medium was measured following incubation of INS832/13 cells in 2.8 mM or 16 mM glucose/KRBH medium or in the presence of 35 mM KCl. Secreted insulin levels were normalized to protein content. C) Measurements of intracellular calcium in living INS832/13 cells treated as described in (B) using the Fura-2 dye. We measured the ratio of fluorescence signals produced by excitation of 340 nm and 380 nm and detected at 510 nm to determine intracellular calcium concentrations. D) Glucose oxidation was measured as 14 CO 2 production from [U- 14 C]-glucose in cells treated as described in (A) and the results were normalized to protein content. E) Cellular insulin content was measured by Elisa after lysis of the cells in acidic ethanol. Results represent mean ± SEM of three separate experiments carried out in triplicate. *, p<0.05.

Article Snippet: Full-length human CCN3 protein were purchased from R&D systems (Minneapolis, MN).

Techniques: Over Expression, Cotransfection, Transduction, Plasmid Preparation, Incubation, Control, Fluorescence, Produced, Enzyme-linked Immunosorbent Assay, Lysis