Journal: PLoS ONE

Article Title: Role of T198 Modification in the Regulation of p27 Kip1 Protein Stability and Function

doi: 10.1371/journal.pone.0017673

Figure Lengend Snippet: A . U87MG cells were transduced with the indicated Ads, included in 3D Matrigel matrix and images were taken 24 hours later (20× objective). In the graph the length of cell protrusions is reported. Data represents the mean value (± standard deviation) obtained measuring protrusions length in at least 20 cells for each type of transduction in 3 independent experiments. B . Western blot analysis of p27 expression in SCC9 cells transiently transfected with different p27 mutants cloned in the pDNR vector and analyzed 48 hours after transfection. Vinculin was used as the loading control. (A = alanine, G = glycine, S = serine, V = valine, D = aspartic acid, E = glutamic acid, F = phenylalanine). C . RT-PCR on RNA extracted from SCC9 treated as in B. GAPDH was used as internal control. NT = Non Transfected cells, C RNA = amplification of non retro-transcribed RNA. D . SCC9 cells were transfected with pDNR p27 WT and with the indicated mutants and 48 hours later RNA and protein extracts were prepared from each plate. Results represent the ratio between the fentomoles of p27-mRNA evaluated by qRT-PCR analysis and the amount of p27 protein expression evaluated by densitometric analysis of the blots and normalized to the loading control vinculin. Data represents the mean value (± standard deviation) of three independent experiments. Statistical analysis was carried out using Student's t test. E . p27 wt and the indicated T198 mutants cDNAs were in vitro transcribed and translated, loaded into SDS-PAGE gel and subjected to autoradiography. Inset shows control of plasmidic DNA quantification used in this experiment. Luc = luciferase positive control vector, E.V. = empty vector.

Article Snippet: Different amounts of recombinant p27 mutants were incubated with 40 ng of recombinant cyclin A 2 -CDK2 complex (SignalChem) or with 300 ng of cyclin D 1 -CDK4 (SignalChem) complex, on ice for 1,5 hour.

Techniques: Transduction, Standard Deviation, Western Blot, Expressing, Transfection, Clone Assay, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, RNA Amplification, Quantitative RT-PCR, In Vitro, SDS Page, Autoradiography, Luciferase, Positive Control

Journal: PLoS ONE

Article Title: Role of T198 Modification in the Regulation of p27 Kip1 Protein Stability and Function

doi: 10.1371/journal.pone.0017673

Figure Lengend Snippet: A . Western Blot analysis of p27 expression in SCC9 cells were transfected with the indicated p27 mutants and 48 hours later treated with cycloheximide (CHX) for 3, 6 and 9 hours. The densitometric analysis of the blots is reported in the graph (right) and is expressed as percentage of remaining protein respect to untreated cells. B . Western Blot analysis of p27 recombinant proteins in in vitro degradation assay using bacterial His-tagged recombinant p27 proteins incubated with proteasome extracts for 3, 6 and 9 hours. Quantification of this degradation assay is reported in the right graph and expressed as described in A. C . Western blot analysis of p27 and Skp2 expression in MDAH cells transduced with AdSkp2 or with AdNeg (Control) shRNAs in which the different p27 proteins were expressed and treated or not with CHX for 6 and 9 hours. Total protein extracts were analyzed by western blot using the indicated antibodies (left). The densitometric analysis of a representative Skp2 silencing experiment is reported in the graph (right) and expressed as in A. D . Western blot of 3T3 p27 KO fibroblasts expressing the different p27 mutants as indicated and treated with MG132 (50 µM) or DMSO for 6 and 9 hours. A typical experiment is shown.

Article Snippet: Different amounts of recombinant p27 mutants were incubated with 40 ng of recombinant cyclin A 2 -CDK2 complex (SignalChem) or with 300 ng of cyclin D 1 -CDK4 (SignalChem) complex, on ice for 1,5 hour.

Techniques: Western Blot, Expressing, Transfection, Recombinant, In Vitro, Degradation Assay, Incubation, Transduction, Control

Journal: PLoS ONE

Article Title: Role of T198 Modification in the Regulation of p27 Kip1 Protein Stability and Function

doi: 10.1371/journal.pone.0017673

Figure Lengend Snippet: A . Western blot analysis of p27 expression in HT1080 transfected with the indicated vectors and analyzed 48 hours after transfection. B . Western blot analysis of p27 and CDK2 expression in SCC9 cells transfected with the indicated FLAG-p27 or control (pFLAG-BAP) vectors analyzed 48 hours after trasfection. The amount of CDK2 bound to the different p27 mutants is shown in the right blots (IP anti-FLAG). C and D . Western blot analysis of p27 expression in HT1080 transfected with the indicated vectors treated with CHX for 3, 6 and 9 hours. In the graph is reported the densitometric analysis of the blots.

Article Snippet: Different amounts of recombinant p27 mutants were incubated with 40 ng of recombinant cyclin A 2 -CDK2 complex (SignalChem) or with 300 ng of cyclin D 1 -CDK4 (SignalChem) complex, on ice for 1,5 hour.

Techniques: Western Blot, Expressing, Transfection, Control

Journal: PLoS ONE

Article Title: Role of T198 Modification in the Regulation of p27 Kip1 Protein Stability and Function

doi: 10.1371/journal.pone.0017673

Figure Lengend Snippet: A Western blot analysis of p27 and Zs green expression in SCC9 cells transduced with the indicated AdZsgreen viruses and analyzed 24 hours post-transduction. B . Typical image of colony assay analysis in SCC9 cells transduced as in A and allow to grow for 15 days. C . Quantification of growth inhibition induced by the different p27 proteins respect to control cells evaluated by colony assay. Data represents the mean value of three independent experiments performed in quadruplicate. The mean transduction efficiency (% of green cells over the total) is reported. D . Percentage of colonies expressing the Zs Green protein 15 days post-transduction as determined counting at least 80 colonies for each mutants. A representative experiment is shown.

Article Snippet: Different amounts of recombinant p27 mutants were incubated with 40 ng of recombinant cyclin A 2 -CDK2 complex (SignalChem) or with 300 ng of cyclin D 1 -CDK4 (SignalChem) complex, on ice for 1,5 hour.

Techniques: Western Blot, Expressing, Transduction, Colony Assay, Inhibition, Control

Journal: PLoS ONE

Article Title: Role of T198 Modification in the Regulation of p27 Kip1 Protein Stability and Function

doi: 10.1371/journal.pone.0017673

Figure Lengend Snippet: A . Immunoprecipitation analysis of 3T3 p27 KO fibroblasts transduced with the indicated Ad Zs-green p27 and lysed 48 hours post-transduction. IP and lysates were analyzed by western blot using anti-CDK2, anti-CDK4 and anti-p27 antibody. B . Quantification of p27/CDK2-4 interaction as evaluated by IP and western blot analysis. Data represents the mean value (± standard deviation) of three independent experiments and are expressed as fold increase respect to the p27 WT /CDK binding value. C . In vitro kinase assay of recombinant cyclin A 2 /CDK2 complex in the presence of increasing amounts (0,5, 1 and 2 µg) of recombinant His-tagged p27 WT and mutants proteins as indicated using histone H1 as substrate. In the upper panel the expression of p27 in each reaction is shown. In the lower panel the phosphorylated Histone H1 is shown. A typical assay is reported. In the lower graph the amount of p27 proteins present in each reaction as evaluated by densitometric scanning of the blots is shown. In the lower table the percentage of kinase activity inhibition by the different p27 mutants respect to control kinase activity is reported. D . Same as in C using as kinase the recombinant cyclin D 1 /CDK4 complex and pRB fragment as substrate.

Article Snippet: Different amounts of recombinant p27 mutants were incubated with 40 ng of recombinant cyclin A 2 -CDK2 complex (SignalChem) or with 300 ng of cyclin D 1 -CDK4 (SignalChem) complex, on ice for 1,5 hour.

Techniques: Immunoprecipitation, Transduction, Western Blot, Standard Deviation, Binding Assay, In Vitro, Kinase Assay, Recombinant, Expressing, Activity Assay, Inhibition, Control

Journal: PLoS ONE

Article Title: Role of T198 Modification in the Regulation of p27 Kip1 Protein Stability and Function

doi: 10.1371/journal.pone.0017673

Figure Lengend Snippet: A . HT1080 cells were transduced with the indicated Ad Zs-green p27s and 24 hours later allowed to migrate on FN-coated Fluoroblok. A typical image of Fluoroblok bottom (Migrated cells) is shown. In the graph the quantification of migrated cells is reported. Results are expressed as fold induction respect to control cells. B . IP analysis on cytoplasmic fractions of HT1080 expressing pEGFP-p27 mutants and DsRed-stathmin as indicated and adhered on FN for one hour. Cytosolic lysates and IP proteins were then evaluated by western blot analysis using anti-p27 and anti-Stathmin antibodies. C . Orthotopical projection of cell paths of U87MG cells transfected with DsRed-Stathmin, EGFP-p27 T198V and EGFP-p27 T198E allowed to adhere on FN and followed for two hours (left). In the figures yellow dashed lines delimitate the cytoplasm and blue lines the nucleus positions at time 0. In the graph (right) the speed of the transfected cells is reported and represents the mean (±SD) of three independent experiments in which at least 10 cells/experiment were tracked. Statistical analysis was carried out using Mann-Whitney U-Test.

Article Snippet: Different amounts of recombinant p27 mutants were incubated with 40 ng of recombinant cyclin A 2 -CDK2 complex (SignalChem) or with 300 ng of cyclin D 1 -CDK4 (SignalChem) complex, on ice for 1,5 hour.

Techniques: Transduction, Control, Expressing, Western Blot, Transfection, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-766-3p Contributes to Anti-Inflammatory Responses through the Indirect Inhibition of NF-κB Signaling

doi: 10.3390/ijms20040809

Figure Lengend Snippet: Generalizability of the suppression of inflammatory stimuli in miR-766-3p-transfected cells. ( A ) NHMCs were transfected with miRNA mimics (30 nM) and exposed to TNF-α or IL-1β for 24 h. The expression of the indicated genes was examined by a qPCR. ( B , C ) C28/I2 cells ( B ) and NHMCs ( C ) were co-transfected with pNF-κB-Luc along with miRNA mimics (C28/I2: 5 nM, NHMC: 30 nM). After incubation, cells were treated with TNF-α or IL-1β for 6 h and subjected to a luciferase assay to evaluate the activity of NF-κB. The luciferase activity was normalized by the number of viable cells and then normalized to the respective values in the vehicle samples (upper bar graphs). The lower scatterplots show the frequency of NF-κB inhibition by miR-766-3p. Assays were performed in quadruplicate. Data are expressed as the mean ± SEM. Asterisks indicate statistically significant differences ( p < 0.05).

Article Snippet: The human synovial fibroblast cell line MH7A was obtained from Riken Cell Bank (Ibaraki, Japan) [ ], the human chondrocyte cell line C28/I2 was obtained from Merck Millipore (Darmstadt, Germany), and normal human mesangial cells (NHMCs) were obtained from Lonza (Basel, Switzerland).

Techniques: Transfection, Expressing, Incubation, Luciferase, Activity Assay, Inhibition

Journal: Immunity

Article Title: T cell intrinsic Receptor Interacting Protein 2 regulates pathogenic T helper-17 cell differentiation

doi: 10.1016/j.immuni.2018.08.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse C28 (clone 37.51) , BioXcell , Cat # BE001-1.

Techniques: Virus, Recombinant, Red Blood Cell Lysis, Isolation, Mutagenesis, Cell Isolation, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Plasmid Preparation, Software