Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A and B, mRNA expression levels of LEDGF/p75 ( black bars ) and Sp1 ( gray bars ) were analyzed by real time PCR. Total RNA was isolated from LECs separated from lenses of human subjects of different age groups and reverse transcribed cDNA was subjected to real time PCR analysis with specific primers as detailed in . Age group 1 (n = 4, 16–26 years); Age group 2 (n = 3, 34–42 years); Age group 3 (n = 7, 52–75 years). n; denotes number of subjects. The data represent the mean ± S.D. from three independent experiments (** p<0.001 ). C, Western analysis of LEDGF/p75 and Sp1 protein using their corresponding specific antibodies. hLECs isolated from eye lenses of 24- and 64-year-old human subjects were cultured as described in . Cellular proteins from confluent cells were extracted, and equivalent amounts were loaded onto SDS-PAGE, transferred to a PVDF membrane and processed for immunoblotting. Western analysis showed the expression levels of LEDGF/p75 ( upper panel) and Sp1 ( middle panel). Lower panel, membrane probed with β-actin antibody as loading/internal control. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Each band of blot was quantified using densitometer shown at the right . Images are representatives from three independent experiments. D and E, Sp1 upregulated expression of LEDGF/p75 protein and mRNA in hLECs in dose dependent fashion. hLECs were transfected with either pCMV-vector or increasing amounts of pCMV-Sp1 (2, 4 and 8 µg) as indicated and described in section. Total Protein and RNA were extracted after 48 h of transfection and were used for Western analysis (D) and real time PCR (E) respectively, using specific probes. D, left , Western analysis data showing the expression levels of LEDGF/p75 ( upper panel) in cells transfected with plasmid encoding Sp1 at different concentrations ( middle panel). Lower panel, membrane probed with β-actin antibody. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Right , Histogram displaying relative protein band density indicated as values ± S.D. of three independent experiments. E, Histogram showing the values (mean ± S.D.) of Sp1 concentration-dependent expression of LEDGF/p75 mRNA (black bars vs gray bars) obtained from three independent experiments (** p <0.001). F, A Sp1 inhibitor, artemisinin, reduced expression of LEDGF/p75 in LECs in dose-dependent manner. Cultured cells were treated with either increasing concentrations of artemisinin (50, 150 and 300 µM) or with vehicle control. Cell lysates were resolved onto SDS-PAGE and analyzed by Western blot for the effects of artemisinin on expression of LEDGF/p75 and Sp1 protein. Relative band density in pixels is shown below the Western blot images (* p <0.01, ** p <.001). β-actin was used as internal control. G and H, Representative immunoblots showing depletion of Sp1 using Sp1 Knockdown assay. Sp1-specific shRNA constructs were transiently (G) and stably (H) transfected as described in section. Protein lysate was prepared and Western analysis was carried out. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1 or LEDGF/p75 or β-actin. Relative band density in pixels is shown below the Western blot images (** p <.001).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Reverse Transcription, Western Blot, Cell Culture, SDS Page, Membrane, Control, Stripping Membranes, Transfection, Plasmid Preparation, Concentration Assay, Knockdown, shRNA, Construct, Stable Transfection

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Left half, diagrams showing the 5′-deletion constructs of LEDGF/p75 promoter linked to CAT reporter gene used for transient transfections. Right half, CAT activity of the LEDGF/p75 promoter deletion constructs and empty CAT vector in hLECs. 5′-deletion mutant constructs and pGFP were cotransfected into hLECs. 48 h later, protein was extracted and CAT activity was measured. CAT activity ( right ) was normalized to GFP readings (O.D.). The data represent the mean ± S.D. from three independent experiments. B, Point mutation analysis showing Sp1 site-dependent transcriptional activity of the LEDGF/p75 gene promoter in hLECs. Left half, schematic representation of Sp1-site-directed mutants of LEDGF/p75 promoter linked to CAT. Right half, CAT activity of the wild-type (WT) and its mutant constructs (Mut-3, Mut-2, Mut-1 and Mut- 1+2+3) and empty CAT vector in hLECs. All data are presented as the mean ± S.D. derived from three independent experiments (* p <0.01, ** p <0.001).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Construct, Transfection, Activity Assay, Plasmid Preparation, Mutagenesis, Derivative Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Representative gel-shift mobility assays showing Sp1 binding to radiolabeled oligonucleotide probes containing consensus Sp1 sites as indicated. Nuclear extracts isolated from hLECs were incubated with 32 p-labeled probes containing Sp1 binding sites (WT-probes) or their corresponding mutants (Mut probes). Nuclear extracts bound to oligos containing Sp1 sites and yielded to complex, Sp1/DNA (Cm1) (A, lanes 1, 3 and 5). No complex occurred with mutant probes (A, lanes 2, 4, and 6). The oligonucleotide probes of both wild-type and mutated sequence used in assay are shown adjacent to image. B, Gel-shift assay showing the binding of Sp1 in nuclear extract of Sp1 overexpressed with hLECs to 32 p-labeled probes with its site. Nuclear extract isolated from cells transfected with plasmid encoding Sp1 or its corresponding vector was incubated with WT-probe1 or standard control probe (sc-2502; Santa Cruz Biotech). The DNA-protein complex was resolved on a 5% acrylamide gel. A discrete Sp1 expression-dependent DNA-protein complex was observed (B; lanes 1 vs 3) in comparison to vector transfected cells (lane 1), while the mutated probe failed to generate the complex (B, lanes 2 and 4). B, Right (lanes 5 and 6) , depletion of endogenous Sp1 with its specific antibody. Nuclear extracts were incubated with either anti-Sp1 antibody (lane 6) or normal rabbit IgG (lane 5), and recovered nuclear extracts were incubated with the same probes (lanes 5 and 6). Lanes 7 and 8, standard control containing • Sp1 site (sc-2502, Santa Cruz Biotech) or its @ mutant (sc-2503) processed for gel-shift assay using the same nuclear extracts. Extreme right , Depletion assay using anti-Sp3 antibody with nuclear extract showing no change in Sp1/DNA complex (lane 10) and the complex was indistinguishable from Lane 9. Images are representatives from three independent consistent observations.

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Gel Shift, Binding Assay, Isolation, Incubation, Labeling, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Control, Acrylamide Gel Assay, Expressing, Comparison, Depletion Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Interrupting Sp1 activity by artemisinin interrupted LEDGF/p75 promoter activity in a concentration-dependent manner. Upper panel, a diagram of the LEDGF/p75 promoter representing three Sp1-binding sites (−170/+35) used for CAT activity. A selective Sp1 inhibitor , artemisinin, reduced the activity of LEDGF/p75 promoter in LECs in dose-dependent fashion. Artemisinin or its diluents (control) were added to culture medium of LEDGF/p75 promoter constructs or empty vector transfected cells monolayer. Cells were disrupted and CAT activities were measured as described in the section. Data are the mean of three experiments, and error bars indicate standard deviation (** p<0.001 ). B and C, Influence of Sp1 overexpression on transcriptional activity of LEDGF/p75 promoter. Plasmid encoding pCAT- LEDGF/p75 (−170/+35) or pCAT-V was cotransfected into hLECs ( B ) and Cos7 cells ( C ) with indicated amounts of pCMV-Sp1. Following CAT assay, CAT values were analyzed and represented as histograms, with Sp1 (black bar) or without Sp1 (open bar) overexpression. Empty CAT vector shows insignificant CAT activity (gray bar). Transfections were carried out as described in and level of Sp1 protein was evaluated using Western analysis (B and C, Upper panel). The data are representative of at least three independent experiments. Each value represents the mean ± S.D. (** p<0.001 ).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Activity Assay, Concentration Assay, Binding Assay, Control, Construct, Plasmid Preparation, Transfection, Standard Deviation, Over Expression, Western Blot

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Repression of LEDGF/p75 transcription by Sp1 Sumoylation. Cells were transfected or cotransfected with pCAT- LEDGF/p75 (pCAT-LED) or pCAT vector (pCAT-V) and/or with increasing amounts of a plasmid encoding Sumo1 (pEGFP-Sumo1) as indicated. Cells were disrupted at predefined times and processed for CAT assay. Data indicate CAT activity in cells overexpressing different amounts of Sumo1 (A, gray bars) and without Sumo1 (black bars). Experiments were performed three times, and data are presented as mean ± S.D. B, ChIP assay coupled with desumoylation and DNA-protein complex dissociation experiments showed that the effect of Sumo1 on the abundance of Sp1 was concentration-dependent. hLECs were transfected with either pCMV-Sp1 alone or cotransfected with pEGFP-Sumo1 or pFLAG-Senp1. ChIP assay was performed in duplicates from each sample with anti-Sp1 or anti-Sp3 antibody or control IgG. Following processing, one set of precipitated samples was submitted for PCR analysis of Sp1 responsive region of LEDGF/p75 promoter (B) as described in . In another set of experiments, DNA bound proteins were eluted with high salt solution, and Western analysis was performed on elutes to measure Sp1 prevalence by anti-Sp1-antibody (C: lane 1, pCMV-Sp1; lane 2, pEGFP-Sumo1; lane 3, pFLAG-Senp1; lane 4, pCMV-Sp1 plus pEGFP-Sumo1; lane 5, pCMV-Sp1 plus pFLAG-Senp1; p-vector). Images shown in the panel (B) are of representatives of Sp1-3 (site 3) region. Similar results were obtained with Sp1-1 (site 1) and Sp1-2 (site 2) in the LEDGF/p75 promoter when ChIP-PCR analysis was done (data not shown). Following stripping of Sp1 immunoblotted membrane, the same membrane was reprobed with Sp3 specific antibody, and no bands were observed (D).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Transfection, Plasmid Preparation, Activity Assay, Concentration Assay, Control, Western Blot, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, hLECs were transfected with either mock, negative control siRNA or LEDGF/p75 siRNA. Following transfection, cell extracts were prepared and expression was examined by Western analysis using Anti-LEDGF/p75 antibody (A). Relative density in pixels is shown on the right . B, Control siRNA (siControl) or LEDGF/p75 siRNA (siLEDGF/p75) transfected cells were seeded in 12-well plates and submitted to UVB exposure as described in . A survival assay-MTS assay was conducted, and data shown are mean ± S.D. values of three independent experiments. ** p<0.001 compared with control siRNA. C, Sp1 overexpression in cells with siRNA LEDGF/p75 conferred resistance against UVB stress. LEDGF/p75 siRNA transfected hLECs were transiently re-transfected with pCMV-Sp1 and then exposed to UVB stress. MTS assay was performed to evaluate vulnerability. * p<0.01 compared with respective controls. D, Sp1 or LEDGF/p75 overexpression in hLECs provided cytoprotection against stress induced by UVB. Cells were cultured and exposed to different doses of UV stress as indicated. Cell viability was analyzed using MTS assay as described in . * * p<0.001 compared with respective controls. Data represent mean ± S.D. from three independent experiments.

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Transfection, Negative Control, Expressing, Western Blot, Control, Clonogenic Cell Survival Assay, MTS Assay, Over Expression, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: EpoR surface modulation is a critical component of the erythroid iron deprivation response. (A) Immunoblots of surface-biotinylated proteins from erythroid progenitors untreated or subjected to 16 h of iron deprivation or FA treatment and densitometry from multiple experiments for relative levels of surface EpoR associated with treatments, with normalization to total biotinylated protein levels ( n = 4, one-way ANOVA; IB, immunoblot; PD, pull-down). (B) Immunoblots of total membrane fractions from erythroid progenitors, untreated or subjected to 16 h of iron deprivation or 50 µM FA treatment and densitometry from multiple experiments for relative levels of EpoR associated with treatments, with normalization to ATP1A1 ( n = 3, one-way ANOVA). (C) Immunoblot analysis of iron-replete and -deprived erythroid progenitors subjected to cytokine starvation and Epo stimulation for 0, 10, and 30 min and densitometry from multiple experiments for fold change in STAT5a/b phosphorylation at 10 min associated with iron deprivation ( n = 3, two-way ANOVA). Unt, untreated; +FA, FA treated; −Iron, iron deprived; +Iron, iron-replete. (D) Circulating RBC count and RBC mean corpuscular volume (MCV) values in WT and EpoR-H mutant mice subjected to dietary iron deprivation for the indicated number of days ( n = 12 per group, intragroup comparisons between day 7 and day 42 values, repeated measures two-way ANOVA; CBC, complete blood count). (E) Flow cytometry of splenic Lin − Kit + progenitors from WT and EpoR-H mutant mice cultured in erythroid medium with transferrin saturations (TSATs) of 100% or 10% ± isocitrate. (F) Summary of multiple flow cytometry studies as in E, showing fold change in the percentage of CD71 + cells normalized to WT progenitors cultured in medium with 100% TSAT, fold change in the CD71 + percentage associated with iron deprivation, and fold increase in the CD71 + percentage associated with isocitrate treatment of iron-deprived cells (right; n = 3; left: two-way ANOVA; middle and right: Student’s t test). Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. IC, isocitrate; ns, not significant.

Article Snippet: The cells were then incubated with a biotinylated antilineage cocktail (130-090-858; Miltenyi Biotec) and lineage depleted on a column (AutoMACS Pro; Miltenyi Biotec).

Techniques: Western Blot, Membrane, Phospho-proteomics, Mutagenesis, Flow Cytometry, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: Scribble is regulated by the erythroid iron deprivation response and controls surface EpoR display. (A) Heat map of SCRIB expression levels in hematopoietic hierarchy from BloodSpot server using normal human hematopoietic DMAP dataset. (B) Immunoblot of cytosolic (Cy), membranous (Me), and residual insoluble (In) fractions from progenitors cultured in erythroid medium at indicated TSATs ± isocitrate and densitometry from multiple experiments for relative, normalized levels of membranous and cytosolic Scribble ( n = 3 for each, one-way ANOVA). (C) Immunofluorescence localization of Scribble in progenitors cultured in erythroid medium with the indicated TSATs ± isocitrate (confocal microscopy). Representative results from three independent experiments. Bar, 15 µm. (D) Quantitative RT-PCR measurements of relative, normalized SCRIB transcripts in human progenitors cultured in erythroid medium with indicated TSATs ± isocitrate ( n = 3). IC, isocitrate. (E) Immunoblots of cytosolic and membrane fractions from progenitors cultured in erythroid medium with the indicated TSATs ± cathepsin inhibitor (CA074me). Representative results from three independent experiments (Fig. S2 E). (F) Immunoblot of surface-biotinylated proteins (streptavidin pull-down) and of input lysates (Input) from primary progenitors transduced with lentiviral shRNA control or Scribble-targeting constructs and densitometry from multiple experiments for relative levels of total EpoR, expressed as fold change associated with Scribble knockdown, with normalization to tubulin ( n = 3, Student’s t test). (G) Immunoblot of surface-biotinylated proteins (streptavidin pull-down), input lysates (Input) from HUDEP-2 erythroblasts transduced with lentiviral shRNA control and Scribble-targeting constructs, and densitometry from multiple experiments as in left panel for fold change in surface EpoR associated with Scribble knockdown normalized to total surface-biotinylated proteins ( n = 3, Student’s t test). EV, empty vector, lentiviral shRNA control; PD, pull-down; shScrib, Scribble-targeting lentiviral shRNA constructs. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; ***, P < 0.001.

Article Snippet: The cells were then incubated with a biotinylated antilineage cocktail (130-090-858; Miltenyi Biotec) and lineage depleted on a column (AutoMACS Pro; Miltenyi Biotec).

Techniques: Expressing, Western Blot, Cell Culture, Immunofluorescence, Confocal Microscopy, Quantitative RT-PCR, Membrane, Transduction, shRNA, Control, Construct, Knockdown, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: Blockade of isocitrate production impairs TfR2 surface delivery. (A) Pulse-chase analysis of surface-biotinylated TfR2 in erythroid progenitors, with assessment of the effects of aconitase inhibition. Cells cultured in erythroid medium ± 50 µM FA underwent surface biotinylation and were returned to culture for the indicated durations. The cells were then harvested for streptavidin pull-down and immunoblot. (B) Graph of densitometry from multiple experiments as in A for relative TfR2 levels, with normalization to TfR1 ( n = 3, no intergroup difference by two-way ANOVA). (C) Analysis of the surface delivery of TfR2, with assessment of the effects of aconitase inhibition. Cells cultured in erythroid medium ± FA underwent trypsin-mediated stripping of surface TfR2 followed by recovery culture for 0–60 min. The cells were then harvested for streptavidin pull-down and immunoblot. Also shown are control cells not subjected to trypsinization (−Tryp). (D) Graph of densitometry from multiple experiments similar to C for surface TfR2 levels at 0–3 h after trypsinization normalized to total biotinylated protein ( n = 4). (E) Quantitation from multiple experiments as in C of the effect of FA treatment on surface TfR2 recovery at 10 min after trypsinization, showing TfR2 levels normalized to total biotinylated protein and fold change in normalized TfR2 from 0 to 10 min of recovery ( n = 3, Student’s t test, *, P < 0.05). Graphs depict mean ± SEM from the indicated number of independent experiments.

Article Snippet: The cells were then incubated with a biotinylated antilineage cocktail (130-090-858; Miltenyi Biotec) and lineage depleted on a column (AutoMACS Pro; Miltenyi Biotec).

Techniques: Pulse Chase, Inhibition, Cell Culture, Western Blot, Stripping Membranes, Control, Quantitation Assay

Journal: BMC Cell Biology

Article Title: Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

doi: 10.1186/1471-2121-5-2

Figure Lengend Snippet: A) Effect of AMSH-2 on TGF-β induced transcriptional activity of 3TP driven luciferase. HepG2 cells were cotransfected with 3TP-lux reporter together with the empty vector or increasing amounts of AMSH-2 Myc construct. Twenty-four hours posttransfection, cells were serum-starved and left untreated (white bars) or stimulated (black bars) with 5 ng/ml TGF-β1 for 16–20 h before the luciferase and β-galactosidase activities were measured. B) Effect of AMSH-2 on TGF-β induced transcriptional activity of PAI-1 luciferase. HepG2 cells were cotransfected with PAI-1 reporter together with the empty vector or 0.9 μg/ml of AMSH-2 Myc construct. A similar procedure as described in A was followed. C) AMSH-2 does not stimulate NF-kB signaling pathway. 293 cells were cotransfected with ELAM luciferase reporter construct together with an empty vector, 0.9 μg/ml of AMSH-2 Myc construct or 0.9 μg/ml of TRAF-2, used as a positive control. Forty-eight hours posttransfection, luciferase and β-galactosidase activities were measured.

Article Snippet: Twenty-four h postransfection, the cells were depleted from serum and stimulated for 16–20 h with 5 ng/ml of purified recombinant human TGF-β1 (R&Dsystems.

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Construct, Positive Control

Journal: BMC Cell Biology

Article Title: Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

doi: 10.1186/1471-2121-5-2

Figure Lengend Snippet: A) AMSH-2 interacts with Smad2 and Smad7, but not with Smad3 or Smad4. Vector or AMSH-2 V5 construct were coexpressed in 293T cells together with the Smad proteins as indicated on the top of the figure. Lysates were immunoprecipitated with anti-V5 antibody and western blotted with anti-Flag or anti-HA antibodies. To control for Smad and AMSH-2 protein expression part of the lysates were immunoprecipitated with anti-Flag, anti-HA or anti-V5 antibodies and western blotted with the same antibodies. B) Smad7 inhibitory effect on TGF-β signaling pathway is rescued by AMSH-2. HepG2 cells were cotransfected with 3TP-lux reporter together with the empty vector or 10 ng/ml of Smad7 with or without 0.3 μg/ml of AMSH-2 Myc construct. Twenty-four hours posttransfection, cells were serum-starved and left untreated (white bars) or stimulated (black bars) with 5 ng/ml TGF-β1 for 16–20 h before luciferase and β-galactosidase activities were measured.

Article Snippet: Twenty-four h postransfection, the cells were depleted from serum and stimulated for 16–20 h with 5 ng/ml of purified recombinant human TGF-β1 (R&Dsystems.

Techniques: Plasmid Preparation, Construct, Immunoprecipitation, Western Blot, Expressing, Luciferase

Journal: BMC Cell Biology

Article Title: Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

doi: 10.1186/1471-2121-5-2

Figure Lengend Snippet: AMSH-2 RNA interference has a negative effect on TGF-β signaling pathway. HepG2 cells were transfected with 3TP-lux reporter and scramble, laminA/C, AMSH-2 or Smad7 siRNA duplexes as indicated in the bottom of the graph. Forty-eight hours posttransfection cells were serum-starved and left treated (black bars) or not (white bars) with 5 ng/ml TGF-β1.

Article Snippet: Twenty-four h postransfection, the cells were depleted from serum and stimulated for 16–20 h with 5 ng/ml of purified recombinant human TGF-β1 (R&Dsystems.

Techniques: Transfection

Journal: The FEBS journal

Article Title: Aberrant Sumoylation Signaling Evoked by Reactive Oxygen Species Impairs Protective Function of Prdx6 by Destabilization and Repression of its Transcription

doi: 10.1111/febs.12866

Figure Lengend Snippet: Sumo1 suppressed expression of Prdx6 and its transregulator Sp1 protein and mRNA in dose-dependent fashion. hLECs (8X105) were transfected with different concentrations of pEGFP-Sumo1 (1, 2 and 4μg). After 48h, cells were processed for Western and real-time analysis by using specific probes to measure protein (A) and mRNA (B) expression. An inverse relation was evident between Sumo1 expression levels and Prdx6 and Sp1, suggesting aberrant expression of Sumo1 adversely affected Prdx6 and Sp1 protein and mRNA abundance. Tubulin antibody was used as an internal control. Histogram values represent mean ± SD of three independent experiments (*p<0.001 vs control).

Article Snippet: The following antibodies were used: Prdx6 monoclonal (Lab Frontier, Seoul, Korea), Prdx6 monoclonal (IP grade, Abcam, Cambridge, MA, USA), Prdx6 polyclonal (sc-134478; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 monoclonal (sc-5308; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 polyclonal (sc-9060; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP monoclonal (sc-69779; IP grade, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP polyclonal (Invitrogen, USA), Sp1 monoclonal (sc-17824; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and Senp1 monoclonal (sc-271360; Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Western Blot, Control

Journal: The FEBS journal

Article Title: Aberrant Sumoylation Signaling Evoked by Reactive Oxygen Species Impairs Protective Function of Prdx6 by Destabilization and Repression of its Transcription

doi: 10.1111/febs.12866

Figure Lengend Snippet: Senp1 upregulated Prdx6 and Sp1 protein and mRNA expression. hLECs (8X105) were transfected with different concentrations of pFlag-Senp1 (0.5, 1 and 2). After 48h, protein and mRNA expression were measured by Western (A) and real-time PCR (B) analysis using specific probes. Senp1 increased the expression levels of both Prdx6 and its transregulator Sp1. Tubulin antibody was used as an internal control. Histogram values represent mean ± SD of three independent experiments (*p<0.001 vs control).

Article Snippet: The following antibodies were used: Prdx6 monoclonal (Lab Frontier, Seoul, Korea), Prdx6 monoclonal (IP grade, Abcam, Cambridge, MA, USA), Prdx6 polyclonal (sc-134478; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 monoclonal (sc-5308; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 polyclonal (sc-9060; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP monoclonal (sc-69779; IP grade, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP polyclonal (Invitrogen, USA), Sp1 monoclonal (sc-17824; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and Senp1 monoclonal (sc-271360; Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: The FEBS journal

Article Title: Aberrant Sumoylation Signaling Evoked by Reactive Oxygen Species Impairs Protective Function of Prdx6 by Destabilization and Repression of its Transcription

doi: 10.1111/febs.12866

Figure Lengend Snippet: Prdx6 gene transcription was regulated by Sumoylation and deSumoylation mechanism. (A) Schematic diagram of wild-type Prdx6 gene promoter construct (−839/+109) containing three Sp1-binding elements linked to CAT reporter plasmid vector. (B) Sumo1 significantly repressed Prdx6 promoter activity in dose-dependent manner. hLECs were transiently cotransfected with pCAT-Prdx6 (−839/+109) with different concentrations of pEGFP-Sumo1 (gray bars; 1, 2 and 4 μg). After 72h, protein was extracted and CAT-ELISA was performed to measure the effects of Sumo1 on Prdx6 promoter activity. Sumo1 downregulated (gray bars) the promoter activity. (C) Senp1 dramatically enhanced Prdx6 promoter activity in concentration-dependent fashion. hLECs were cotransfected with pCAT-Prdx6 plasmid (−839/+109) with increasing concentrations of pFlag-Senp1 (gray bars; 0.1, 0.5, 1 and 2μg). After 72h, extracted cell lysates from these transfectants were analyzed for CAT activity (shown as a histogram). The data represent the mean ± SD from three independent experiments (** p<0.05;* p<0.001). DNA concentration was maintained by cotransfecting pcDNA3 plasmid, and transfection efficiency was normalized with SEAP value as described [40, 47].

Article Snippet: The following antibodies were used: Prdx6 monoclonal (Lab Frontier, Seoul, Korea), Prdx6 monoclonal (IP grade, Abcam, Cambridge, MA, USA), Prdx6 polyclonal (sc-134478; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 monoclonal (sc-5308; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 polyclonal (sc-9060; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP monoclonal (sc-69779; IP grade, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP polyclonal (Invitrogen, USA), Sp1 monoclonal (sc-17824; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and Senp1 monoclonal (sc-271360; Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Construct, Binding Assay, Plasmid Preparation, Activity Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection

Journal: The FEBS journal

Article Title: Aberrant Sumoylation Signaling Evoked by Reactive Oxygen Species Impairs Protective Function of Prdx6 by Destabilization and Repression of its Transcription

doi: 10.1111/febs.12866

Figure Lengend Snippet: Sumoylation and deSumoylation of Sp1 was involved in regulating Prdx6 transcription. (A) Schematic illustration of wild-type Prdx6 gene promoter construct (−839/+109) containing three Sp1-binding elements (WT) and its mutant (all three Sp1 sites disrupted using site-directed mutagenesis) linked to CAT vector. (B) Prdx6 promoter activity was down regulated in cells overexpressing Sumo1 compared to cells overexpressing Senp1. hLECs were co-transfected with wild-type Prdx6 promoter linked to CAT vector or mutated construct (at all three Sp1 sites) along with either pEGFP-Sumo1 or pFlag-Senp1. The effects of pEGFP-Sumo1 (dark gray bar) or pFlag-Senp1 (black bar) on CAT activity are shown. Empty CAT-vector served as control (open bar). We did not observe significant changes in pCAT-Prdx6 mutant coexpressed with either pEGFP-Sumo1 or pFlag-Senp1, suggesting that Prdx6 transcription was linked to Sumoylation/deSumoylation of Sp1. The transfection efficiencies were normalized using cotransfected pEGFP-vector. Data represent the mean ± SD from three independent experiments (*, p<0.001). (C) Sp1-siRNA assay confirming the involvement of Sumo1 and Senp1 in modulating Prdx6 transcriptional activity through Sp1. hLECs were transiently cotransfected with pCAT-Prdx6 reporter plasmid and pFlag-Senp1 with or without siRNA specific to Sp1, as indicated. CAT activity was monitored. The results are represented as a histogram showing that promoter activity was significantly reduced in Sp1-siRNA transfected cells compared to untransfected cells (lined bar vs black bar). Transfection efficiencies were normalized using pEGFP vector. Data represent the mean ± SD from three independent experiments (*, p<0.001). (D) Silencing of Sp1 by specific siRNA was validated by Western blotting. The membrane was striped and stained with β-actin antibody.

Article Snippet: The following antibodies were used: Prdx6 monoclonal (Lab Frontier, Seoul, Korea), Prdx6 monoclonal (IP grade, Abcam, Cambridge, MA, USA), Prdx6 polyclonal (sc-134478; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 monoclonal (sc-5308; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 polyclonal (sc-9060; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP monoclonal (sc-69779; IP grade, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP polyclonal (Invitrogen, USA), Sp1 monoclonal (sc-17824; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and Senp1 monoclonal (sc-271360; Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Construct, Binding Assay, Mutagenesis, Plasmid Preparation, Activity Assay, Transfection, Control, Western Blot, Membrane, Staining

Journal: The FEBS journal

Article Title: Aberrant Sumoylation Signaling Evoked by Reactive Oxygen Species Impairs Protective Function of Prdx6 by Destabilization and Repression of its Transcription

doi: 10.1111/febs.12866

Figure Lengend Snippet: Prdx6-deficient LECs facing oxidative stress showed decreased expression and activity of Senp1, and reduced expression of Sp1. Prdx6+/+ and Prdx6−/− cells were cultured in either 10% FBS or 0.2% BSA. (A) Cell lysate was prepared and immunoblotted. Reduced Sp1 expression was observed in Prdx6−/− cell cultures in 0.2% BSA (lane 4). Also in these cells, Senp1 was found to be reduced and dimerized (lane 4) with increased expression of Sumo1. (B) RNA was isolated from Prdx6+/+ and Prdx6−/− cells cultured in either 10% FBS or 0.2% BSA and subjected to real-time PCR to measure mRNA level using specific probes corresponding to Prdx6, Sumo1 and Senp1 as indicated. The expression level of Sumo1 was increased in Prdx6−/− cells (b: Sumo1, −/−; black bar); in contrast, Senp1 expression was suppressed (b: Senp1, −/−; black bar) in cells under serum depletion stress (0.2% BSA) compared to LECs cultured at normal physiological conditions (a: Senp1, −/−; black bar and a: Sumo1, −/−; black bar). **p<0.05 and *p<0.001 showed statistically significant.

Article Snippet: The following antibodies were used: Prdx6 monoclonal (Lab Frontier, Seoul, Korea), Prdx6 monoclonal (IP grade, Abcam, Cambridge, MA, USA), Prdx6 polyclonal (sc-134478; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 monoclonal (sc-5308; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 polyclonal (sc-9060; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP monoclonal (sc-69779; IP grade, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP polyclonal (Invitrogen, USA), Sp1 monoclonal (sc-17824; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and Senp1 monoclonal (sc-271360; Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Serum Depletion

Journal: The FEBS journal

Article Title: Aberrant Sumoylation Signaling Evoked by Reactive Oxygen Species Impairs Protective Function of Prdx6 by Destabilization and Repression of its Transcription

doi: 10.1111/febs.12866

Figure Lengend Snippet: Quantitative real-time PCR

Article Snippet: The following antibodies were used: Prdx6 monoclonal (Lab Frontier, Seoul, Korea), Prdx6 monoclonal (IP grade, Abcam, Cambridge, MA, USA), Prdx6 polyclonal (sc-134478; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 monoclonal (sc-5308; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Sumo1 polyclonal (sc-9060; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP monoclonal (sc-69779; IP grade, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), GFP polyclonal (Invitrogen, USA), Sp1 monoclonal (sc-17824; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and Senp1 monoclonal (sc-271360; Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Sequencing

Journal: Cancer cell

Article Title: Integrin αvβ6-TGFβ-SOX4 Pathway Drives Immune Evasion in Triple-Negative Breast Cancer.

doi: 10.1016/j.ccell.2020.12.001

Figure Lengend Snippet: Figure 3. Efficacy of Integrin avb6/8 mAb in Metastatic Murine TNBC Models Resistant to PD-1 Blockade (A and B) (A) Py8119GFP+ TNBC (n = 10 mice/group) or (B) 4T1 TNBC (n = 12 mice/group) primary tumor volume shown at indicated time points. Mice with similar tumor burden were treated with indicated antibodies (intraperitoneal [IP], 0.2 mg/dose, twice weekly) until tumor volume in any group reached 1,000 mm3. To deplete CD8+ T cells, mice were treated with anti-CD8b antibodies (0.1 mg/dose) on days 1, 1, and weekly thereafter. (C and D) Kaplan-Meier analysis of survival for mice described in (A) and (B), respectively. (E) Primary tumor volume shown at indicated time points for Py8119 model following monotherapy with integrin avb6 or isotype control mAbs; in the indicated groups, CD8+ T cells (CD8b mAb) and/or NK cells (NK1.1 mAb) were also depleted by administration of the respective antibodies (0.1 mg/dose) on days 1, 1, and weekly thereafter.

Article Snippet: In vivo experiments were performed with the following mAbs: inVivoMAb mouse anti-PD1 (BioXcell RMPI-14 clone), inVivoMAb rat IgG2a, isotype control (BioXcell 2A3 clone) and CD8a depletion antibody (BioXcell, 2.43 clone).

Techniques: Control

Journal: Cancer cell

Article Title: Integrin αvβ6-TGFβ-SOX4 Pathway Drives Immune Evasion in Triple-Negative Breast Cancer.

doi: 10.1016/j.ccell.2020.12.001

Figure Lengend Snippet: Figure 5. Relevance of SOX4 to the Efficacy of Integrin avb6/8 mAb Treatment (A) Immunoblot showing levels of SOX4 and GAPDH proteins in GFP+ 4T1 murine TNBC cells containing a DOX-inducible Sox4 cDNA construct (4T1Sox4Dox). Cells were treated with the indicated concentrations of DOX for 48 h. (B) Cells from (A) were co-cultured with murine GFP-specific CD8+ T cells (red) (E/T = 1:1), and the fraction of surviving cells was quantified (y axis) after 24 h of co- culture. (C) 4T1Sox4Dox tumor cells were treated with either DOX (500 ng/mL) alone or in combination with integrin avb6/8 mAb for 48 h followed by co-culture with murine GFP-specific CD8+ T cells (red) for 18 h. (D) 4T1Sox4Dox TNBC (n = 10 mice/group) primary tumor volume shown at indicated time points. Mice with similar tumor burden were fed either a regular diet or a DOX-containing diet (625 ppm, Envigo Teklad) starting on day 7 to induce the expression of SOX4 in tumor cells. Mice receiving either diet also received monotherapy with integrin avb6/8 or isotype control mAbs (IP, 0.25 mg/dose, twice weekly) until tumor volume in any group reached 1,000 mm3. Data are summarized as mean ± SD of tumor volume. (E) Number of lung surface metastases in mice treated as described in (D) following staining with picric acid for 24 h. Summary of number of lung surface metastases (left) and representative images (right) on day 21 following tumor inoculation. (F) Quantification of tumor-infiltrating CD8+ T cells per gram of tumor (right) following treatment as described in (D) on day 21 following tumor inoculation. (G) Immunoblot showing levels of SOX4 and GAPDH proteins in 4T1Sox4Dox tumors (n = 3 per group) derived from mice treated as described in (D) on day 21. Numbers represent relative quantification of SOX4 to GAPDH, normalized to the average expression in vehicle and IgG-treated controls. Data in (A), (B), (C) and (G) are representative of at least two independent experiments with technical triplicates and summarized as mean ± SEM (B and C). Data in (D), (E), and (F) are an average of two independent experiments and summarized as mean ± SD. To determine statistical significance, a one-way ANOVA with Dunnett’s (B–E) post hoc test or an unpaired Student’s t test (F) was used. ***p < 0.001; **p < 0.01; *p < 0.05; n.s., not significant.

Article Snippet: In vivo experiments were performed with the following mAbs: inVivoMAb mouse anti-PD1 (BioXcell RMPI-14 clone), inVivoMAb rat IgG2a, isotype control (BioXcell 2A3 clone) and CD8a depletion antibody (BioXcell, 2.43 clone).

Techniques: Western Blot, Construct, Cell Culture, Co-Culture Assay, Expressing, Control, Staining, Derivative Assay

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Aging/aged hLECs displayed increased accumulation of ROS, which was associated with progressive decline in Prdx6, Cat and Nrf2 expression. ( A ) Excessive accumulation of ROS in aging/aged hLECs. Primary hLECs isolated from lenses of different ages were divided into six groups: 16–21 y (n = 6); 24–26 y (n = 6); 34–36 y (n = 4); 52–58 y (n = 6); 62–68 y (n = 12); 75 y (n = 4). Cells were cultured in 96 well plate (5000/well), and ROS were quantified using H2-DCF-DA dye assay as shown. Data represent the mean ± S.D. of two independent experiments. 16–21 y vs 24–26 y, 34–36 y, 52–58 y, 62–68 y and 75 y (aging samples); *p < 0.001. ( B – D ) Aging/aged hLECs showing a significant loss of Prdx6, Cat and Nrf2. Total RNA was isolated from hLECs and human lenses of different ages as indicated and was processed for real-time PCR analysis. # LECs directly detached from lenses and were used for assays to avoid cell culture effects. The data represent the mean ± S.D. from three independent experiments. p values were determined for younger vs aging samples. * p < 0.001.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Aging hLECs displayed a significant loss in Nrf2 binding to ARE and in transactivating Prdx6 promoter activity. ( A ) Gel-shift with nuclear extract from lenses of variable ages shows age-related loss of Nrf2 binding to ARE in Prdx6 promoter. Nuclear fraction directly isolated from hLECs of different ages containing equal amounts of protein were incubated with 32 p-labeled wild-type ARE probe from Prdx6 promoter and processed for gel-shift assay. An apparent age-related reduction in Nrf2/ARE binding was observed (A, Nrf2/DNA). ( B ) Nrf2-specific antibody depletion assay revealing depletion of Nrf2/ARE complex, demonstrating specificity of Nrf2 binding to ARE probe. Equal amounts of nuclear protein were incubated with antibody specific to Nrf2 to deplete Nrf2. No Nrf2/DNA band was detected with Nrf2-depleted extracts (B, lanes: left panel; 26 y, 52 y and 66 y vs right panel; 26 y, 52 y and 66 y). Wild-type probe, underlined bases denote ARE sequences (−357/−349) present in Prdx6 regulatory region. ( C ) Age-related transcriptional activity of Prdx6 promoter in primary hLECs of variable ages. Top panel, diagrammatic sketch showing the 5′- constructs of human Prdx6 promoter ranging from −918/+30 bps linked to CAT reporter gene. Lower panel, histogram showing CAT activity of Prdx6 promoter and empty CAT vector. Cells were transiently transfected with Prdx6 promoter plasmid along with pGFP-vector plasmid. 48 h later, CAT activity was monitored (Methods section). Transfection efficiency was normalized with GFP O.D. recorded at Ex485/Em530nm. Data represent the mean ± S.D. from two independent experiments. Younger age (18 y and 24 y) vs aging sample; * p < 0.001.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: Binding Assay, Activity Assay, Gel Shift, Isolation, Incubation, Labeling, Antibody Depletion Assay, Construct, Plasmid Preparation, Transfection

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: SFN enhanced expression of antioxidants Prdx6, Cat and Phase II protein GST π mRNA and protein in dose-dependent manner in SRA-hLECs. ( A ) Viability assay showing the concentration-dependent effects of SFN on survival of SRA-hLECs. Cultured SRA-hLECs were treated with different concentrations of SFN to determine a nontoxic concentration of SFN using MTS assay. DMSO vs SFN treated; *p < 0.001. ( B and C ) SFN significantly enhanced Prdx6 mRNA and protein expression. Cells were treated with DMSO vehicle or different concentrations of SFN for 6 h and 24 h. mRNA and protein were extracted, and subjected to real-time PCR and immunoblotting using probes specific to Prdx6. SFN produced a concentration-dependent increased pattern of Prdx6 mRNA ( B ) and protein ( C ) expression. ( D and E ) As noted in B and C, mRNA and cellular extract isolated from cells treated or untreated with SFN were submitted to real time-PCR (D) and immunoblot (E) analyses using primers and antibody specific to Cat, respectively. ( F and G ) SFN also significantly augmented levels of GST π , a phase II enzyme. In parallel experiments the expression level of GST π was examined in SFN-treated cells by real-time PCR ( F ) and Western analysis ( G ). ( C,E and G ); Upper panel shows a representative of immunoblot; lower panel; protein bands were quantified using a densitometer, and levels were normalized to corresponding β-actin levels with values presented as histograms. ( B–G ), Data represent means ± S.D. of three independent experiments. Open vs gray and black bars; gray vs black bar; * p < 0.001, ** p < 0.05.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: Expressing, Viability Assay, Concentration Assay, Cell Culture, MTS Assay, Real-time Polymerase Chain Reaction, Western Blot, Produced, Isolation

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Rat LECs treated with SFN displayed increased levels of antioxidant genes/proteins, Prdx6, Cat, and phase II protein GST π . (A) Determination of noncytotoxic concentration of SFN in primary culture of rLECs. Primary cultures of rLECs were treated with different concentrations of SFN as indicated for 24 h. Cells were subjected to MTS assay to measure viability. Histogram reflects values; nontoxic concentrations were 2.4 μM and 4.8 μM. DMSO vs SFN treated samples; *p < 0.001, **p < 0.05. ( B and C ) rLECs treated with SFN showed enhanced expression of Prdx6 mRNA and protein. Cells were treated with 2.4 μM or 4.8 μM of SFN or DMSO as indicated. Total RNA and protein were isolated. Real-time PCR and Western analysis with Prdx6 specific probes revealed a concentration-dependent increased expression of Prdx6 mRNA ( B ) and protein ( C ). ( D and E ) Expression assays showing SFN enhanced expression of Cat in rLECs. Experiments and parameters were similar to those noted above ( B and C ). RNA and protein extract were processed for real-time PCR and Western analyses using Cat specific primers and antibody, respectively. ( F and G ) SFN-treated rLECs displayed significantly increased levels of GST π mRNA and protein in time- and concentration-dependent fashion. mRNA and cellular extracts were isolated from SFN-treated primary rLECs, and were processed for real-time PCR ( F ) and Western analysis ( G ) assays. ( C,E and G ); Upper panel, representative Immunoblot. Lower panel, densitometric analysis of protein band level; levels were normalized to corresponding β-actin levels and values are presented as histograms (C and G, dotted line shows marking of boundary of bands). ( B–G) , Data represent means ± S.D. of three independent experiments. Open vs gray and black bars; gray vs black bar; * p < 0.001, ** p < 0.05.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: Concentration Assay, MTS Assay, Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: SFN enhanced physical and functional binding of Nrf2 to ARE present in the regulatory region of human Prdx6 promoter in SRA-hLECs. ( A ) Gel-shift and antibody depletion assay showed SFN enhanced Nrf2 binding to oligo probes containing ARE derived from Prdx6 promoter in concentration-dependent fashion. Nuclear fraction extracted from SRA-hLECs was incubated with 32 p radiolabeled wild-type or mutant probes containing ARE sites. SFN concentration-dependent binding activity of Nrf2 to ARE (Nrf2/DNA; lanes 1 vs 2, 3 and 4) compared to mutant probe (lanes 9, 10, 11 and 12). Antibody depletion assay showed disruption of Nrf2/DNA complex (lanes 5, 6, 7 and 8), suggesting that Nrf2 in nuclear extract selectively bound to ARE. (However, antibody did not entirely deplete Nrf2 in nuclear faction of SRA-hLECs, so some residual interaction can be seen in all lanes.) ( B ) SFN rapidly stimulated Nrf2 binding activity to ARE present in Prdx6 human promoter. SRA-hLECs were cultured in the presence of DMSO (control vehicle) or with different concentrations of SFN for 1 h. Nuclear fractions were isolated and processed for gel-shift assay. A strong Nrf2/DNA complex was formed with SRA-hLECs treated with 8 μM of SFN for 1 h (B, lane 1 vs 2 vs 3). In contrast, mutant probe did not act similarly, validating that the Nrf2/DNA complex on gel-shift was specific. ( C ) Gel-shift and antibody depletion assay showed SFN amelioration of Nrf2 binding activity to ARE in the Prdx6 promoter in concentration- and time-dependent fashion. SRA-hLECs were treated with different concentrations of SFN for different time periods. Nuclear extracts containing equal amounts of proteins were incubated with radiolabeled ARE probe. A relative modulation in Nrf2/DNA complex intensity was observed, and was related to concentration and time of exposure as shown in a representative figure (C, lane 1 vs 2 and 3; Lane 2 vs 3 and lane 4 vs 5 and 6; lane 5 vs 6). In contrast, antibody depletion assay showed reduced band intensity or ablation of DNA/Nrf2 complex (C, lanes 7, 8, 9, 10, 11 and 12). Bold bases represent mutation sites; mutated base(s) as shown and underlined denote core ARE sequences in Prdx6 promoter. NS denotes nonspecific band.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: Functional Assay, Binding Assay, Gel Shift, Antibody Depletion Assay, Derivative Assay, Concentration Assay, Incubation, Mutagenesis, Activity Assay, Disruption, Cell Culture, Control, Isolation

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: In vivo DNA binding assay revealed that SFN reinforced binding activity of Nrf2 in SRA-hLECs and aging/aged primary hLECs. ( A ) Schematic representation of the regulatory region of proximal promoter of human Prdx6 gene-containing ARE binding sites showing primer location and sequences used in ChIP assay. (B) SFN induced increase in DNA binding activity of Nrf2 to Prdx6 gene promoter containing ARE site in SRA-hLECs. ChIP experiment was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis Kit. Chromatin samples prepared from SRA-hLECs treated with varying concentrations (0, 3 µM and 6 µM) of SFN for 24 h were subjected to ChIP assay with a ChIP grade antibody, anti-Nrf2 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primers designed to amplify −400 to −305 region of the human Prdx6 promoter bearing Nrf2/ARE sites as shown. Histogram shows the amplified DNA band visualized with real-time PCR analysis. DMSO (0) vs 3 µM and 6 µM SFN and 3 µM vs 6 µM SFN treatment; *p < 0.001. ( C ) SFN reinforced the enrichment of Nrf2 to its responsive element, ARE, present in Prdx6 gene promoter in aging/aged primary hLECs. ChIP assay was conducted using anti-Nrf2 antibody. Immunoprecipitated DNA fragments were purified and processed for qPCR analysis using primers indicated above and in the Methods section, but in primary hLECs of variable ages treated with different concentrations (0, 3 µM, 6 μM) of SFN. Histograms represent the concentration dependence of SFN-induced enrichment of Nrf2 at ARE sites in Prdx6 gene promoter. Open vs gray and black bars and gray vs black bar; * p < 0.001. Data revealed a significant augmentation of Nrf2 binding to ARE by SFN in all ages of LECs, but in aged cells there was a loss in Nrf2 binding to ARE. ( D ) SFN failed to activate mutant Prdx6 promoter disrupted at Nrf2/ARE site. Upper panel, diagram of 5′-regulatory region of Prdx6 promoter spanning from −918/+30 bp containing ARE site and its mutant plasmid linked to CAT reporter gene used for CAT activity. Lower panel, CAT activity of the wild-type (WT) Prdx6 promoter and its mutant (Mut) at ARE site and empty CAT vector in SRA-hLECs treated with SFN or DMSO (control). Wild-type or its mutant Prdx6 promoter construct along with pGFP-Vector were cotransfected into SRA-hLECs and CAT activity was measured. CAT activity (lower panel) was normalized to GFP readings (O.D.). Histogram represents the mean ± S.D. obtained from three independent experiments. WT vs Mut and DMSO vs SFN treated samples; *p < 0.001. ( E ) SFN reinforced Prdx6 transcription in aging/aged primary human LECs. As described above, hLECs of variable ages were transfected with the same wild-type Prdx6 promoter ARE site (upper panel). Lower panel, relative CAT activity of the wild-type promoter in SFN-treated aging/aged hLECs. All data are presented as mean ± S.D. values derived from three independent experiments. DMSO vs SFN treated samples; younger (21 y old) vs aging samples; *p < 0.001.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: In Vivo, DNA Binding Assay, Binding Assay, Activity Assay, Control, Amplification, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Concentration Assay, Mutagenesis, Plasmid Preparation, Construct, Transfection, Derivative Assay

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Prdx6 knockdown experiments revealed that SFN exerted its cytoprotective activity against UVB-induced cell injuries through Prdx6 regulation. (A) Survival experiment showing increased susceptibility of As-Prdx6 transfected cells to UVB-induced oxidative stress. SRA-hLECs were transfected with As-Prdx6 (4 μg), and the effect of As-Prdx6 was confirmed through immunoblotting with anti-Prdx6 antibody (data not shown). The transfectants were divided into different groups as shown and equal numbers of cells were cultured for assay to avoid transfection effect. Survival assay (MTS assay) showed a significant reduction in viability of As-Prdx6 (lined bar) transfectants compared to empty-vector transfectants (gray and black bars) against UVB stress. ( B ) H2-DCF-DA assay showing ROS levels after UVB stress as indicated. Result are presented as Fluorescent Unit. ( A and B ), Open vs gray bar, gray vs black bar and black vs lined bar; *p < 0.001. ( C and D ) SFN protected primary rLECs against UVB exposure. ( C) rLECs were pretreated with 2.4 µM and 4.8 μM of SFN or DMSO (vehicle control) and then exposed to UVB stress. Effects on viability were determined after 24 h by MTS assay. ( D ) Effect of SFN on lowering ROS expression. rLECs were treated with DMSO, 2.4 µM or 4.8 μM of SFN and were exposed to UVB stress as indicated. ROS expression was quantified. All histograms are presented as the mean ± S.D. values derived from two independent experiments. C and D, open vs gray bar and gray vs black bar; *p < 0.001. ( E and F ) SFN rescued primary aging hLECs from UVB stress. ( E ) SFN augmented viability of aging hLECs undergoing UVB stress. Cultured hLECs of variable ages were exposed to UVB as indicated, and effects on cell viability were determined after 24 h by MTS assay. ( F ) Effect of SFN on lowering ROS expression. hLECs were treated with SFN as in (E). ROS levels were measured with H2-DCF-DA. Histogram represents the data mean ± S.D. obtained from two independent experiments. 21 y vs 56 y and 62 y, 56 y vs 62 y, Open vs gray bar and gray vs black bar; ** p < 0.05, * p < 0.001.

Article Snippet: GST π , Catalase, and Prdx6 as well as Nrf2 gene expressions were analyzed with RT-PCR on 7300 Real Time PCR System (Applied Biosystems) using the primers designed for each molecule of rat genes (TaqMan; ratPrdx6 probe ID: Rn01759191_g1; rat catalase probe ID: Rn00560930_m1; rat GST π probe ID: Rn00561378_gH) or human genes (Universal probe library for human; Prdx6 probe ID: NM_004905.2; catalase probe ID:NM_001752.3; GST π probe ID:NM_000852.3; Nrf2 probe ID: NM_001145413 and β-actin probe ID: NM_001101.3).

Techniques: Knockdown, Activity Assay, Transfection, Western Blot, Cell Culture, Clonogenic Cell Survival Assay, MTS Assay, Plasmid Preparation, Control, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A and B, mRNA expression levels of LEDGF/p75 ( black bars ) and Sp1 ( gray bars ) were analyzed by real time PCR. Total RNA was isolated from LECs separated from lenses of human subjects of different age groups and reverse transcribed cDNA was subjected to real time PCR analysis with specific primers as detailed in . Age group 1 (n = 4, 16–26 years); Age group 2 (n = 3, 34–42 years); Age group 3 (n = 7, 52–75 years). n; denotes number of subjects. The data represent the mean ± S.D. from three independent experiments (** p<0.001 ). C, Western analysis of LEDGF/p75 and Sp1 protein using their corresponding specific antibodies. hLECs isolated from eye lenses of 24- and 64-year-old human subjects were cultured as described in . Cellular proteins from confluent cells were extracted, and equivalent amounts were loaded onto SDS-PAGE, transferred to a PVDF membrane and processed for immunoblotting. Western analysis showed the expression levels of LEDGF/p75 ( upper panel) and Sp1 ( middle panel). Lower panel, membrane probed with β-actin antibody as loading/internal control. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Each band of blot was quantified using densitometer shown at the right . Images are representatives from three independent experiments. D and E, Sp1 upregulated expression of LEDGF/p75 protein and mRNA in hLECs in dose dependent fashion. hLECs were transfected with either pCMV-vector or increasing amounts of pCMV-Sp1 (2, 4 and 8 µg) as indicated and described in section. Total Protein and RNA were extracted after 48 h of transfection and were used for Western analysis (D) and real time PCR (E) respectively, using specific probes. D, left , Western analysis data showing the expression levels of LEDGF/p75 ( upper panel) in cells transfected with plasmid encoding Sp1 at different concentrations ( middle panel). Lower panel, membrane probed with β-actin antibody. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Right , Histogram displaying relative protein band density indicated as values ± S.D. of three independent experiments. E, Histogram showing the values (mean ± S.D.) of Sp1 concentration-dependent expression of LEDGF/p75 mRNA (black bars vs gray bars) obtained from three independent experiments (** p <0.001). F, A Sp1 inhibitor, artemisinin, reduced expression of LEDGF/p75 in LECs in dose-dependent manner. Cultured cells were treated with either increasing concentrations of artemisinin (50, 150 and 300 µM) or with vehicle control. Cell lysates were resolved onto SDS-PAGE and analyzed by Western blot for the effects of artemisinin on expression of LEDGF/p75 and Sp1 protein. Relative band density in pixels is shown below the Western blot images (* p <0.01, ** p <.001). β-actin was used as internal control. G and H, Representative immunoblots showing depletion of Sp1 using Sp1 Knockdown assay. Sp1-specific shRNA constructs were transiently (G) and stably (H) transfected as described in section. Protein lysate was prepared and Western analysis was carried out. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1 or LEDGF/p75 or β-actin. Relative band density in pixels is shown below the Western blot images (** p <.001).

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Reverse Transcription, Western Blot, Cell Culture, SDS Page, Membrane, Control, Stripping Membranes, Transfection, Plasmid Preparation, Concentration Assay, Knockdown, shRNA, Construct, Stable Transfection

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, The 5′- sequence ranging from nucleotides −315 to +35 contained three putative Sp1 binding sites as predicted by MatInspector (Genomatix). The consensus sequences for the predicted Sp1 sites (G/C boxes) are shown in bold and sites are denoted as Sp1-1, Sp1-2 and Sp1-3. Underlining is used to show the oligonucleotides employed in gel-shift and gel-shift immuno-deletion assays. The transcription start site is indicated by +1 , and letter Mlu I and Nhe I restriction sites used for preparing LEDGF/p75 -CAT constructs are shown in italic and bold. B, Nucleotide sequences alignment of the proximal promoter of mouse, rat and human LEDGF/p75 gene (NCBI, BLAST and alignment tools). Sequences highlighted in gray are highly conserved among these species, and three evolutionarily conserved Sp1 binding sites are shown in bold letters.

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Sequencing, Binding Assay, Gel Shift, Construct

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Left half, diagrams showing the 5′-deletion constructs of LEDGF/p75 promoter linked to CAT reporter gene used for transient transfections. Right half, CAT activity of the LEDGF/p75 promoter deletion constructs and empty CAT vector in hLECs. 5′-deletion mutant constructs and pGFP were cotransfected into hLECs. 48 h later, protein was extracted and CAT activity was measured. CAT activity ( right ) was normalized to GFP readings (O.D.). The data represent the mean ± S.D. from three independent experiments. B, Point mutation analysis showing Sp1 site-dependent transcriptional activity of the LEDGF/p75 gene promoter in hLECs. Left half, schematic representation of Sp1-site-directed mutants of LEDGF/p75 promoter linked to CAT. Right half, CAT activity of the wild-type (WT) and its mutant constructs (Mut-3, Mut-2, Mut-1 and Mut- 1+2+3) and empty CAT vector in hLECs. All data are presented as the mean ± S.D. derived from three independent experiments (* p <0.01, ** p <0.001).

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Construct, Transfection, Activity Assay, Plasmid Preparation, Mutagenesis, Derivative Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Representative gel-shift mobility assays showing Sp1 binding to radiolabeled oligonucleotide probes containing consensus Sp1 sites as indicated. Nuclear extracts isolated from hLECs were incubated with 32 p-labeled probes containing Sp1 binding sites (WT-probes) or their corresponding mutants (Mut probes). Nuclear extracts bound to oligos containing Sp1 sites and yielded to complex, Sp1/DNA (Cm1) (A, lanes 1, 3 and 5). No complex occurred with mutant probes (A, lanes 2, 4, and 6). The oligonucleotide probes of both wild-type and mutated sequence used in assay are shown adjacent to image. B, Gel-shift assay showing the binding of Sp1 in nuclear extract of Sp1 overexpressed with hLECs to 32 p-labeled probes with its site. Nuclear extract isolated from cells transfected with plasmid encoding Sp1 or its corresponding vector was incubated with WT-probe1 or standard control probe (sc-2502; Santa Cruz Biotech). The DNA-protein complex was resolved on a 5% acrylamide gel. A discrete Sp1 expression-dependent DNA-protein complex was observed (B; lanes 1 vs 3) in comparison to vector transfected cells (lane 1), while the mutated probe failed to generate the complex (B, lanes 2 and 4). B, Right (lanes 5 and 6) , depletion of endogenous Sp1 with its specific antibody. Nuclear extracts were incubated with either anti-Sp1 antibody (lane 6) or normal rabbit IgG (lane 5), and recovered nuclear extracts were incubated with the same probes (lanes 5 and 6). Lanes 7 and 8, standard control containing • Sp1 site (sc-2502, Santa Cruz Biotech) or its @ mutant (sc-2503) processed for gel-shift assay using the same nuclear extracts. Extreme right , Depletion assay using anti-Sp3 antibody with nuclear extract showing no change in Sp1/DNA complex (lane 10) and the complex was indistinguishable from Lane 9. Images are representatives from three independent consistent observations.

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Gel Shift, Binding Assay, Isolation, Incubation, Labeling, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Control, Acrylamide Gel Assay, Expressing, Comparison, Depletion Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Schematic illustration of 5′-proximal promoter region of LEDGF/p75 containing Sp1 binding sites. Genomic DNA was cross-linked to immobilize bound proteins in vivo , was sheared and immunoprecipitated with anti-Sp1 or unrelated antibody rabbit IgG, and was amplified by PCR with primer specific to the region. The quantity of each input DNA was initially measured equalized by O.D. A representative gel stained with ethidium bromide is shown. As a control measure, amplification of the −2499/−2277 region (*) devoid of Sp1 elements of LEDGF/p75 promoter following immunoprecipitation was performed. B, Photographic image of the amplified DNA band visualized with ethidium bromide staining. M, molecular weight marker. Lower half, primers used for amplification of specific region containing Sp1 sites ( ** Sp1-1, *** Sp1-2, **** Sp1-3) and not related to Sp1 binding sites ( * ).

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Binding Assay, In Vivo, Immunoprecipitation, Amplification, Staining, Control, Molecular Weight, Marker

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Interrupting Sp1 activity by artemisinin interrupted LEDGF/p75 promoter activity in a concentration-dependent manner. Upper panel, a diagram of the LEDGF/p75 promoter representing three Sp1-binding sites (−170/+35) used for CAT activity. A selective Sp1 inhibitor , artemisinin, reduced the activity of LEDGF/p75 promoter in LECs in dose-dependent fashion. Artemisinin or its diluents (control) were added to culture medium of LEDGF/p75 promoter constructs or empty vector transfected cells monolayer. Cells were disrupted and CAT activities were measured as described in the section. Data are the mean of three experiments, and error bars indicate standard deviation (** p<0.001 ). B and C, Influence of Sp1 overexpression on transcriptional activity of LEDGF/p75 promoter. Plasmid encoding pCAT- LEDGF/p75 (−170/+35) or pCAT-V was cotransfected into hLECs ( B ) and Cos7 cells ( C ) with indicated amounts of pCMV-Sp1. Following CAT assay, CAT values were analyzed and represented as histograms, with Sp1 (black bar) or without Sp1 (open bar) overexpression. Empty CAT vector shows insignificant CAT activity (gray bar). Transfections were carried out as described in and level of Sp1 protein was evaluated using Western analysis (B and C, Upper panel). The data are representative of at least three independent experiments. Each value represents the mean ± S.D. (** p<0.001 ).

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Activity Assay, Concentration Assay, Binding Assay, Control, Construct, Plasmid Preparation, Transfection, Standard Deviation, Over Expression, Western Blot

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, SL2 cells, a Drosophila cell line, were transfected with indicated amounts of pPac-Sp1 (lanes 2 and 3) or pPac-V (lane 1). The expression level of Sp1 protein was examined by Western blot. Relative band density is shown below (A, gray bar vs black bar). B, Increasing Sp1 expression selectively increased LEDGF/p75 promoter activity in SL2 cells. SL2 cells were cotransfected with pPac-Sp1 or pPac-vector (pPac-V) and pCAT- LEDGF/p75 wild-type (pCAT-LED) or its mutant (pCAT-LED-Mut) reporter plasmid or pCAT vector (pCAT-V). Cells were processed to assay CAT activity as described in . Results were expressed relative to activity of the LEDGF/p75 reporter activity in the presence of pPac-V or pPac-Sp1 and are presented as histograms: pPac-Sp1 with WT promoter (gray bar), and pPac-Sp1 with mutant promoter activity (open bar). The results are mean ± S.D. of three independent experiments (** p<0.001 ). C, Sp1 directly and exclusively bound to its sites in LEDGF/p75 promoter. Nuclear extract was isolated and processed and then incubated with radio-labeled DNA probe containing Sp1 site (Probe 1 or standard control probe, • Sp1). Nuclear extract from pPac-Sp1 overexpressed cells bound strongly to probe containing wild-type Sp1 consensus sequence (lane 2), but nuclear extract from pPac-vector transfected cells showed no binding with either wild-type or mutant probe (lanes 1 and 3). Right panel, Nuclear extracts of pPac-Sp1 transfected cells incubated with standard control probe containing Sp1 site (sc-2502, lane 4) or its mutant (sc-2503, lane 5) and nuclear extract from pPac vector transfected cells incubated with standard probe (lane 6).

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Transfection, Expressing, Western Blot, Activity Assay, Plasmid Preparation, Mutagenesis, Isolation, Incubation, Labeling, Control, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Repression of LEDGF/p75 transcription by Sp1 Sumoylation. Cells were transfected or cotransfected with pCAT- LEDGF/p75 (pCAT-LED) or pCAT vector (pCAT-V) and/or with increasing amounts of a plasmid encoding Sumo1 (pEGFP-Sumo1) as indicated. Cells were disrupted at predefined times and processed for CAT assay. Data indicate CAT activity in cells overexpressing different amounts of Sumo1 (A, gray bars) and without Sumo1 (black bars). Experiments were performed three times, and data are presented as mean ± S.D. B, ChIP assay coupled with desumoylation and DNA-protein complex dissociation experiments showed that the effect of Sumo1 on the abundance of Sp1 was concentration-dependent. hLECs were transfected with either pCMV-Sp1 alone or cotransfected with pEGFP-Sumo1 or pFLAG-Senp1. ChIP assay was performed in duplicates from each sample with anti-Sp1 or anti-Sp3 antibody or control IgG. Following processing, one set of precipitated samples was submitted for PCR analysis of Sp1 responsive region of LEDGF/p75 promoter (B) as described in . In another set of experiments, DNA bound proteins were eluted with high salt solution, and Western analysis was performed on elutes to measure Sp1 prevalence by anti-Sp1-antibody (C: lane 1, pCMV-Sp1; lane 2, pEGFP-Sumo1; lane 3, pFLAG-Senp1; lane 4, pCMV-Sp1 plus pEGFP-Sumo1; lane 5, pCMV-Sp1 plus pFLAG-Senp1; p-vector). Images shown in the panel (B) are of representatives of Sp1-3 (site 3) region. Similar results were obtained with Sp1-1 (site 1) and Sp1-2 (site 2) in the LEDGF/p75 promoter when ChIP-PCR analysis was done (data not shown). Following stripping of Sp1 immunoblotted membrane, the same membrane was reprobed with Sp3 specific antibody, and no bands were observed (D).

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Transfection, Plasmid Preparation, Activity Assay, Concentration Assay, Control, Western Blot, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, hLECs were transfected with either mock, negative control siRNA or LEDGF/p75 siRNA. Following transfection, cell extracts were prepared and expression was examined by Western analysis using Anti-LEDGF/p75 antibody (A). Relative density in pixels is shown on the right . B, Control siRNA (siControl) or LEDGF/p75 siRNA (siLEDGF/p75) transfected cells were seeded in 12-well plates and submitted to UVB exposure as described in . A survival assay-MTS assay was conducted, and data shown are mean ± S.D. values of three independent experiments. ** p<0.001 compared with control siRNA. C, Sp1 overexpression in cells with siRNA LEDGF/p75 conferred resistance against UVB stress. LEDGF/p75 siRNA transfected hLECs were transiently re-transfected with pCMV-Sp1 and then exposed to UVB stress. MTS assay was performed to evaluate vulnerability. * p<0.01 compared with respective controls. D, Sp1 or LEDGF/p75 overexpression in hLECs provided cytoprotection against stress induced by UVB. Cells were cultured and exposed to different doses of UV stress as indicated. Cell viability was analyzed using MTS assay as described in . * * p<0.001 compared with respective controls. Data represent mean ± S.D. from three independent experiments.

Article Snippet: Sp1 expression in human lens epithelial cells was silenced with Sp1 shRNA Plasmid (h) (Santa Cruz Biotechnology).

Techniques: Transfection, Negative Control, Expressing, Western Blot, Control, Clonogenic Cell Survival Assay, MTS Assay, Over Expression, Cell Culture

Journal: Genes & Development

Article Title: Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis

doi: 10.1101/gad.267633.115

Figure Lengend Snippet: Constitutive phosphorylation of the GATA-1 V205G mutant at Ser310 and high levels of FOG-1 restore erythroid maturation. ( A ) Western blot analysis showing GATA-1 V205G Ser310 phosphorylation in NIH-3T3 cells transduced with the GATA-1 V205G mutant and starved (−) or stimulated with PDGF. The GATA-1 S310A mutant was used as nonphosphorylatable negative control. ( B ) Flow cytometry analysis of c-Kit expression in G1E cells transduced with an “empty” retroviral vector (Migr) or a vector encoding GATA-1 or one of the GATA-1 mutants, as indicated. The percentage of c-Kit-negative cells in the positive EGFP fraction is indicated. ( C ) FACS analysis of Ter119 + cells to examine erythroid differentiation in G1E-ER (G1E expressing a GATA-1 construct fused to an estrogen receptor ligand-binding domain) cells transduced as in A . G1E-ER cells, G1E-ER cells transduced with Migr (G1E-ER Migr), and G1E-ER cells overexpressing FOG-1 (G1E-ER FOG-1) were transduced with a construct expressing GATA-1 or one of the GATA-1 mutants, as indicated. The results shown are means ± SEM. ( D ) Schematic representation of the erythroid differentiation capacity of each GATA-1 mutant in the conditions used in this study.

Article Snippet: After 6 d, cells were washed in PBS, depleted of mature erythroid cells by immune-magnetic depletion against Ter119 (Miltenyi Biotec, 130-049-901), and cultured for 3 d in differentiation medium (2 mM L-glutamine, 10 U/mL penicillin and 100 μg/mL streptomycin, 4 U/mL Epo, 0.5 mg/mL iron-saturated human holotransferrin; StemPro34 plus nutrient supplement [Life Technologies]).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Transduction, Negative Control, Flow Cytometry, Expressing, Retroviral, Plasmid Preparation, Construct, Ligand Binding Assay

Journal: Genes & Development

Article Title: Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis

doi: 10.1101/gad.267633.115

Figure Lengend Snippet: Defective erythroid maturation in Gata1 S310A mice treated with PPP. ( A ) Comparison of spleens from Gata1 and Gata1 S310A mice after 20 d of PPP treatment ( left ) and BFU-E colony assays of these spleens in M3234 methylcellulose medium ( right ). n = 4. ( B ) Perl's Prussian blue staining of paraffin sections of these spleens. Bar, 200 μm. ( C , D ) Ter119 ( X -axis) and CD71 ( Y -axis) FACS analysis of the Sytox-negative living cell population of the spleen ( C ) and bone marrow ( D ) from Gata1 and Gata1 S310A mice. Numbers show the percentage of cells within a particular region.

Article Snippet: After 6 d, cells were washed in PBS, depleted of mature erythroid cells by immune-magnetic depletion against Ter119 (Miltenyi Biotec, 130-049-901), and cultured for 3 d in differentiation medium (2 mM L-glutamine, 10 U/mL penicillin and 100 μg/mL streptomycin, 4 U/mL Epo, 0.5 mg/mL iron-saturated human holotransferrin; StemPro34 plus nutrient supplement [Life Technologies]).

Techniques: Comparison, Staining

Journal: Genes & Development

Article Title: Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis

doi: 10.1101/gad.267633.115

Figure Lengend Snippet: Overexpression of E2F-2 abolishes the PPP-mediated inhibition of terminal differentiation in Gata1 S310A erythroid cells ex vivo. ( A ) Quantitative RT–PCR (qRT–PCR) studies: Primary erythroid cultures of cells derived from the bone marrow of Gata1 and Gata1 S310A mice were expanded; transduced with various shRNAs, as indicated (sh-X), on day 2; selected by puromycin treatment on day 5; and transferred to differentiation medium on day 6. Total mRNA was isolated after 3 d of differentiation, and qRT–PCR was performed. ( B ) Ter119 ( X -axis) and CD71 ( Y -axis) FACS analysis of the living cell population as described in A . Numbers show the percentage of cells within a particular region. ( C ) Schematic model of the coordination of cell proliferation and maturation during erythropoiesis: The sequestration of E2F-2 by GATA-1/pRb slows progression through the G1/S phase of the cell cycle, allowing the differentiation program to start . Phosphorylation of GATA-1 by AKT increases its affinity for FOG-1, leading to the dissociation of GATA-1 from the pRb/E2F complex. Consequently, changes to the nuclear concentration of GATA-1/FOG-1 modulate erythroid gene transcription and result in the release of E2F-2 after pRb phosphorylation and the promotion of cell division. Our data suggest that the GATA-1 S310A mutation impairs the capacity of FOG-1 to dissociate the GATA-1 S310A /pRb/E2F-2 complex, thus promoting E2F-2 sequestration and inhibiting cell proliferation. The IGF-1R pathway counteracts E2F-2 sequestration by stimulating E2F-2 expression. Numbers 1–7 correspond to references as follows: (1) , (2) , (3) , (4) , (5) , (6) , and (7) .

Article Snippet: After 6 d, cells were washed in PBS, depleted of mature erythroid cells by immune-magnetic depletion against Ter119 (Miltenyi Biotec, 130-049-901), and cultured for 3 d in differentiation medium (2 mM L-glutamine, 10 U/mL penicillin and 100 μg/mL streptomycin, 4 U/mL Epo, 0.5 mg/mL iron-saturated human holotransferrin; StemPro34 plus nutrient supplement [Life Technologies]).

Techniques: Over Expression, Inhibition, Ex Vivo, Quantitative RT-PCR, Derivative Assay, Transduction, Isolation, Phospho-proteomics, Concentration Assay, Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Mutati ons t o the Arp3 or Arp2–ARPC1 CA-binding sites cause defects in Las17 binding. A , surface representation of a homology model of ScArp2/3 complex with bound Las17–CA showing location of mutations at the Arp3 and Arp2–ARPC1 sites. Binding of Las17–CA was modeled based on both crosslinking/mass spectrometry and cryo-EM data ( , ). The C and A segments of Las17 are labeled and colored gray and red , respectively. B , schematic of the supernatant depletion assays showing the construct of Las17 used to pulldown the WT or CA-binding site mutant complexes. C , binding isotherm for LZ-Las17 281–633 generated using the supernatant depletion assay in PC buffer. Reaction was run in triplicate with the same protein stocks. Concentration on x -axis is the concentration of the LZ-Las17 281–633 dimer. Error bars represent standard deviation. Error bars for last two data points were approximately the same size as the data points, so were omitted. D and E , plot of the fraction of Arp2/3 complex bound in supernatant depletion reactions containing 0.6 μM (LZ-Las17 281–633 ) 2 . Statistical significance was measured with an ordinary one-way ANOVA with p values for comparisons to WT indicated. Data points represent technical replicates. Error bars represent standard deviation. F , diagram of the Arp2 and Arp3 CA fusion complexes, in which the CA of N-WASP is fused to the N terminus of the Arp2 or Arp3 subunit.

Article Snippet: Each supernatant was spotted on nitrocellulose membranes and blotted using anti-Arp3 antibodies (Santa Cruz; catalog no.: sc-376625, 1:1000 dilution).

Techniques: Binding Assay, Mass Spectrometry, Cryo-EM Sample Prep, Labeling, Construct, Mutagenesis, Generated, Depletion Assay, Concentration Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Mutatio ns in ei ther CA-binding site cause defects in Arp2/3 complex activation. A , plot of the maximum polymerization rate versus Las17 281–633 concentration for reactions containing 20 nM Arp2/3 complex (WT or Arp3 site mutant), 3 μM 15% pyrene actin, and the indicated concentrations of Las17 281–633 . B , identical to A , except the activity of the WT complex is compared with the Arp2–ARPC1 site mutants. C , identical to A , except WT and mutant complexes were titrated with GST-Las17 281–633 . The concentrations on the x -axis indicate the concentration of GST-Las17 281–633 monomers. D , identical to C , except WT and mutant complexes are titrated with GST-Las17 281–633 . E , fold increased activation of Arp2/3 complexes upon dimerization of 1.5 μM Las17 281–633 . F , Maximum polymerization rate of 20 nM of WT and mutant Arp2/3 complexes determined from time courses of pyrene actin polymerization as in ( A ) but without NPF. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values from comparison to WT calculated with a Dunnet’s test. Data points represent technical replicates of the assay. Error bars represent standard deviation. NPF, nucleation promoting factor.

Article Snippet: Each supernatant was spotted on nitrocellulose membranes and blotted using anti-Arp3 antibodies (Santa Cruz; catalog no.: sc-376625, 1:1000 dilution).

Techniques: Binding Assay, Activation Assay, Concentration Assay, Mutagenesis, Activity Assay, Comparison, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Mutations in both Arp3 and Arp2–ARPC1 binding sites cause endocytic actin internalization defects. A , widefield fluorescence microscopy images of control and mutant Saccharomyces cerevisiae strains expressing mNG-Las17 and Abp1-TagRFP-T. Scale bar represents 2 μm. Plots of the trajectories of three representative endocytic events are shown to the right , based on the position of Abp1-TagRFP-T over time. The start of each trajectory is marked with a black circle , the end with a red circle , and the color gradient ( red , green , cyan , blue , violet , and magenta ) of the line indicates the time within the trajectory. B , analysis of the maximum distance traveled by Abp1-TagRFP-T puncta from their initial position on the membrane. Punta that moved more than 0.25 μm ( gray bar ) from their initial position were considered internalized. Each data point represents a single endocytic punctum. Data were collected from at least three separate videos. Error bars represent standard deviation. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values for comparison to control strain calculated with a Dunnet’s test. C , schematic of procedure for generating average number of molecules versus time plots. Plots of the average number of mNG-Las17 or Abp1-TagRFP-T molecules versus time for the Arp3(K363D) and Arp2(V142A, Y146A) mutants are shown at the bottom of the panel. D , widefield fluorescence microscopy images of mutant S . cerevisiae strains expressing mNG-Las17 and Abp1-TagRFP-T. Scale bar represents 2 μm. To the right are kymographs of individual representative endocytic events. Scale bar for distance (d) represents 1 μm, and scale bar for time (t) represents 5 s.

Article Snippet: Each supernatant was spotted on nitrocellulose membranes and blotted using anti-Arp3 antibodies (Santa Cruz; catalog no.: sc-376625, 1:1000 dilution).

Techniques: Binding Assay, Fluorescence, Microscopy, Control, Mutagenesis, Expressing, Membrane, Standard Deviation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Mutants with defective Abp1 internalization show increased Las17 concentrations and decreased Las17 deaccumulation rates. A , plot of the average maximum number of Las17 molecules at endocytic sites for the control and mutant strains. Mutants are ordered from left to right based on their influence on endocytic internalization. Error bars represent standard deviation. Dashed horizontal black line marks the average maximum number of molecules of mNG-Las17 in the control strain. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values for comparison to control strain calculated using a Dunnet’s test. Each data point represents a single endocytic punctum. B , plot of the average maximum number of mNG-Las17 molecules at endocytic sites versus the percent internalization (from Fig. 4 B ) for control and mutant strains ( orange circles , Arp3 mutants; green filled circles , Arp2-ARPC1 site mutants). C , diagram illustrating one possible mechanism for negative feedback between Las17 and filamentous actin . Las17 bound to the membrane is connected to the actin cytoskeleton via interaction of its CA segment with Arp2/3 complex at the side of a (unbranched) filament or its V (WH2) segment with the barbed end of an actin filament. The actin network treadmills inward and pulls Las17 off the membrane. D , left , plot of the number of mNG-Las17 molecules versus time for a single endocytic event. The slope of the decrease in Las17 molecules over time ( magenta line ) is used to plot the average Las17 deaccumulation rate for control and mutant strains ( right ). Statistical significance was measured as described in A . Error bars represent standard deviation. E , plot of the fluorescence intensity of mNG-Las17 and Abp1-TagRFP-T versus time for one endocytic event in which the signal of mNG-Las17 begins to decrease before the assembly of Abp1-TagRFP-T is initiated. Dashed vertical lines indicate peak of mNG-Las17 intensity and start of Abp1-TagRFP-T intensity.

Article Snippet: Each supernatant was spotted on nitrocellulose membranes and blotted using anti-Arp3 antibodies (Santa Cruz; catalog no.: sc-376625, 1:1000 dilution).

Techniques: Control, Mutagenesis, Standard Deviation, Comparison, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Filament bundling in motility reactions with WT and mutant Arp2/3 complexes. A , images of reactions with WT or mutant Arp2/3 complexes at 60 min ± 45 s adjusted at a brightness and contrast level that shows actin filament bundles clearly. Brown circles show location of bead. Scale bar represents 20 μm. B , plot of maximum fluorescence intensity of filament bundles along the trajectory of beads in WT and mutant reactions. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values for comparison to WT control calculated using a Dunnet’s test. Note that the ARPC1(F183A, K185D) has a slightly lower but statistically significant average bundle intensity than the WT. Each data point represents the measurement of a single filament bundle around the bead. C , images of beads in reactions with CA-fusion Arp2/3 complexes or a reaction without Arp2/3 complex at three time points, with each image set at the same brightness/contrast settings ( left , scale bar represents 20 μm) or zoomed-in images of each reaction at optimal time point and brightness and contrast settings to show diffuse actin networks in reactions with the CA fusion complex ( right , scale bar represents 10 μm).

Article Snippet: Each supernatant was spotted on nitrocellulose membranes and blotted using anti-Arp3 antibodies (Santa Cruz; catalog no.: sc-376625, 1:1000 dilution).

Techniques: Mutagenesis, Fluorescence, Comparison, Control

Journal: The Journal of Cell Biology

Article Title: IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

doi: 10.1083/jcb.201611050

Figure Lengend Snippet: Conditional gene silencing of IFT20 is associated with defects in termination of PDGFRα signaling. (a) Experimental setup to verify IFT20 silencing efficiency in NIH3T3 sh IFT20 cells upon treatment with Dox for given times. All cells were grown for 6 d. Asterisks indicate day of Dox addition. (b) WB analysis showing IFT20 expression in NIH3T3 sh IFT20 cells during 0–6 d of Dox treatment using the experimental setup outlined in panel a. (c–e) IFM images of growth-arrested NIH3T3 sh IFT20 cells treated without (− Dox) or with Dox (+ Dox) for 6 d, showing cellular localization of IFT20. Primary cilia (arrows) were labeled with anti–acetylated α-tubulin (Ac-tub) or anti-detyrosinated α-tubulin (Glu-tub), and the ciliary base/centrosome was labeled with anti–p150 glued (asterisks). Nuclei (nu) were visualized with DIC microscopy or DAPI staining. Anti–Giantin was used to label the Golgi complex (dashed line). (f) Percentage of ciliated NIH3T3 sh IFT20 cells after Dox treatment, as indicated. Error bars represent means ± SEM ( n = 3). (g) WB analysis of phosphorylation of PDGFRα (p-PDGFRα), AKT (p-AKT), and ERK1/2 (p-ERK1/2) upon stimulation with PDGF-AA for indicated times in growth-arrested NIH3T3 shIFT20 cells treated with or without Dox treatment for 6 d. (h) Quantification of protein phosphorylations shown in panel g. Error bars represent means ± SEM ( n = 3). (i) WB analysis of c-Cbl and Cbl-b levels in growth-arrested NIH3T3 sh IFT20 cells as well as in cells stably expressing siRNA-resistant, GFP-tagged IFT20 (NIH3T3 sh IFT20 -Res ) after Dox treatment for 6 d. (j) IFM analysis of NIH3T3 sh IFT20 -Res cells with or without Dox treatment for 6 d. Cells were stained with antibodies against GFP and IFT20, and cell nuclei were visualized with DAPI staining. Primary cilia (arrows) were labeled with anti–Glu-Tub and anti–Ac-tub and are shown as shifted overlays. Quantification of ciliated NIH3T3 sh IFT20 -Res cells; error bars represent means ± SEM ( n = 3). (k) WB analysis of phosphorylation of PDGFRα (p-PDGFRα) and AKT (p-AKT) upon stimulation with 50 ng/ml PDGF-AA for indicated times in growth-arrested NIH3T3 sh IFT20 versus NIH3T3 sh IFT20 -Res cells, both treated with Dox for 6 d. (l) Quantification of protein phosphorylations shown in panel k. Error bars represent means ± SEM ( n = 3).

Article Snippet: The following antibodies were used: R&D Systems: goat anti-PDGFRα (AF1062); Proteintech: rabbit anti–human IFT20 (13615–1-AP), rabbit anti-IFT88 (13967-1-AP); Sigma-Aldrich: mouse anti–α-tubulin (T5168), mouse anti–acetylated α-tubulin (T6793), mouse anti-FLAG (M2; F1802), rabbit anti-FLAG (F7425); BD Biosciences: mouse anti-CDK1 (610038), mouse anti-p150 Glued (610474), mouse anti-GM130 (610823); Enzo life Sciences: mouse anti-giantin (ALX-804-600-C100), mouse anti–mono- and polyubiquitin (FK2; BML-PW8810); Abcam: mouse anti–c-Cbl (Ab119954), rabbit anti–c-Cbl (Ab32027), mouse anti-GFP (Ab1218), chicken anti-GFP (Ab13970), rabbit anti–detyrosinated α-tubulin (Ab48389), rabbit anti-PDGFRα (Ab134123); Cell Signaling Technology: rabbit anti-Myc (2278), rabbit anti-GAPDH (2118), rabbit anti–Cbl-b (9498), rabbit anti-Akt (9272), rabbit anti–phosphorylated Akt (Ser473; 4060), rabbit anti-Erk1/2 (9102), rabbit anti–phosphorylated Erk1/2 (Thr202/Tyr204; 9101), rabbit anti–phosphorylated Rb (9308); Santa Cruz Biotechnology: rabbit anti–c-Cblsc-170), mouse anti–c-Cbl (sc-1651), mouse anti-GFP (sc-9996), rabbit anti-GFP (sc-8334), rabbit anti–phosphorylated PDGFRα (Tyr754; sc-12911R).

Techniques: Expressing, Labeling, Microscopy, Staining, Phospho-proteomics, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

doi: 10.1083/jcb.201611050

Figure Lengend Snippet: Conditional gene silencing of IFT20 inhibits ubiquitination and internalization of PDGFRα. (a) PDGFRα ubiquitination upon stimulation with PDGF-AA for 10 min in growth-arrested NIH3T3 sh IFT20 cells cultured with or without Dox for 6 d before ligand addition. IP with anti–PDGFRα was performed on lysates of cells not transfected (left) or transfected with a construct expressing Myc-tagged ubiquitin (right), and IPs were analyzed by WB using antibodies against mono- and polyubiquitin or Myc, respectively. The experiments were repeated three times, and results from one representative experiment are shown. (b) WB analysis of surface-biotinylated PDGFRα upon stimulation with PDGF-AA for indicated times in growth-arrested NIH3T3 sh IFT20 cells treated with or without Dox for 6 d. Cell surface proteins were labeled with biotin before (internalized) or after (surface) stimulation with PDGF-AA, followed by IP with streptavidin beads. To detect internalized PDGFRα specifically, extracellular biotin was removed after stimulation with an alkaline glutathione solution. (c) Quantification of protein-band intensity for experiment shown in panel b. Error bars represent means ± SEM ( n = 3).

Article Snippet: The following antibodies were used: R&D Systems: goat anti-PDGFRα (AF1062); Proteintech: rabbit anti–human IFT20 (13615–1-AP), rabbit anti-IFT88 (13967-1-AP); Sigma-Aldrich: mouse anti–α-tubulin (T5168), mouse anti–acetylated α-tubulin (T6793), mouse anti-FLAG (M2; F1802), rabbit anti-FLAG (F7425); BD Biosciences: mouse anti-CDK1 (610038), mouse anti-p150 Glued (610474), mouse anti-GM130 (610823); Enzo life Sciences: mouse anti-giantin (ALX-804-600-C100), mouse anti–mono- and polyubiquitin (FK2; BML-PW8810); Abcam: mouse anti–c-Cbl (Ab119954), rabbit anti–c-Cbl (Ab32027), mouse anti-GFP (Ab1218), chicken anti-GFP (Ab13970), rabbit anti–detyrosinated α-tubulin (Ab48389), rabbit anti-PDGFRα (Ab134123); Cell Signaling Technology: rabbit anti-Myc (2278), rabbit anti-GAPDH (2118), rabbit anti–Cbl-b (9498), rabbit anti-Akt (9272), rabbit anti–phosphorylated Akt (Ser473; 4060), rabbit anti-Erk1/2 (9102), rabbit anti–phosphorylated Erk1/2 (Thr202/Tyr204; 9101), rabbit anti–phosphorylated Rb (9308); Santa Cruz Biotechnology: rabbit anti–c-Cblsc-170), mouse anti–c-Cbl (sc-1651), mouse anti-GFP (sc-9996), rabbit anti-GFP (sc-8334), rabbit anti–phosphorylated PDGFRα (Tyr754; sc-12911R).

Techniques: Ubiquitin Proteomics, Cell Culture, Transfection, Construct, Expressing, Labeling

Journal: The Journal of Cell Biology

Article Title: IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

doi: 10.1083/jcb.201611050

Figure Lengend Snippet: Cbl family E3 ubiquitin ligases are degraded in cells with reduced IFT20, leading to defects in termination of PDGFRα signaling. (a) WB analysis showing expression levels of c-Cbl and Cbl-b proteins in cycling (+ serum) and growth-arrested (− serum) NIH3T3 and RPE-1 cells. CDK1 marks cycling cells. (b) WB analysis of c-Cbl and Cbl-b in growth-arrested NIH3T3 sh IFT20 cells upon addition of Dox for 1–6 d. All cells were grown for 6 d, including 24 h of serum depletion before analysis. An unspecific band stained by the IFT20 antibody is labeled with an asterisk. (c) WB analysis of IFT20 in growth-arrested NIH3T3 and RPE-1 cells subjected to mock or siRNA(siR)-mediated silencing of c-Cbl. (d) Quantitative, real-time PCR analysis of relative Ift20 , c-Cbl , and Cbl-b mRNA transcript levels in NIH3T3 sh IFT20 upon 6 d of Dox treatment, including 24 h of serum depletion. Error bars represent means ± SEM ( n = 3). (e) WB analysis of c-Cbl and Cbl-b in cycling NIH3T3 sh IFT20 cells upon Dox treatment for 3 d in combination with lysosomal (NH 4 Cl) or proteasomal (MG-132) inhibitors for 24 h or 10 h, respectively. (f) IP of endogenous c-Cbl in NIH3T3 sh IFT20 cells with or without Dox for 3 d, followed by WB analysis with antibodies against mono- and polyubiquitin. (g) WB analysis of growth-arrested NIH3T3 shIFT20 cells stably expressing FLAG–tagged WT or RING-mutant c-Cbl (NIH3T3 sh IFT20, FLAG–c-Cbl,WT and NIH3T3 sh IFT20, FLAG–c-Cbl*RING , respectively) upon 6 d of Dox treatment. (h) FLAG IP of protein lysates from NIH3T3 sh IFT20, FLAG–c-Cbl*RING cells transiently expressing GFP-tagged c-Cbl RING mutant (GFP-c-Cbl *RING ) after treatment with Dox for 4 d. Experiments presented in panels a–c and e–h were repeated at least three times, and results from representative experiments are shown. (i) WB analysis of phosphorylation of PDGFRα (p-PDGFRα) and AKT (p-AKT) upon stimulation with 50 ng/ml PDGF-AA for indicated times in NIH3T3 cells subjected to siRNA (siR)-mediated silencing of c-Cbl, Cbl-b, or both for 72 h, including serum depletion for the last 24 h before stimulation to induce growth arrest. (j) Quantification of protein phosphorylations shown in panel i. Error bars represent means ± SEM ( n = 3).

Article Snippet: The following antibodies were used: R&D Systems: goat anti-PDGFRα (AF1062); Proteintech: rabbit anti–human IFT20 (13615–1-AP), rabbit anti-IFT88 (13967-1-AP); Sigma-Aldrich: mouse anti–α-tubulin (T5168), mouse anti–acetylated α-tubulin (T6793), mouse anti-FLAG (M2; F1802), rabbit anti-FLAG (F7425); BD Biosciences: mouse anti-CDK1 (610038), mouse anti-p150 Glued (610474), mouse anti-GM130 (610823); Enzo life Sciences: mouse anti-giantin (ALX-804-600-C100), mouse anti–mono- and polyubiquitin (FK2; BML-PW8810); Abcam: mouse anti–c-Cbl (Ab119954), rabbit anti–c-Cbl (Ab32027), mouse anti-GFP (Ab1218), chicken anti-GFP (Ab13970), rabbit anti–detyrosinated α-tubulin (Ab48389), rabbit anti-PDGFRα (Ab134123); Cell Signaling Technology: rabbit anti-Myc (2278), rabbit anti-GAPDH (2118), rabbit anti–Cbl-b (9498), rabbit anti-Akt (9272), rabbit anti–phosphorylated Akt (Ser473; 4060), rabbit anti-Erk1/2 (9102), rabbit anti–phosphorylated Erk1/2 (Thr202/Tyr204; 9101), rabbit anti–phosphorylated Rb (9308); Santa Cruz Biotechnology: rabbit anti–c-Cblsc-170), mouse anti–c-Cbl (sc-1651), mouse anti-GFP (sc-9996), rabbit anti-GFP (sc-8334), rabbit anti–phosphorylated PDGFRα (Tyr754; sc-12911R).

Techniques: Ubiquitin Proteomics, Expressing, Serum Depletion, Staining, Labeling, Real-time Polymerase Chain Reaction, Stable Transfection, Mutagenesis, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

doi: 10.1083/jcb.201611050

Figure Lengend Snippet: IFT20 interacts with Cbl-b and c-Cbl, which is targeted to the cilium in PDGF-AA–stimulated cells. (a) Validation of the IFT20-c-Cbl interaction. HEK293T cells coexpressing GFP-IFT20 with either FLAG–tagged WT c-Cbl (FLAG–c-Cbl WT ) or empty FLAG vector were subjected to FLAG IP, followed by WB analysis. (b and c) Reciprocal IPs of endogenous IFT20 or c-Cbl from HEK293T cells. (d) FLAG IP of HEK293T cell extracts expressing FLAG–c-Cbl WT or FLAG–tagged RING mutant (p.C381A) c-Cbl (FLAG–c-Cbl *RING ). (e) IFT20 IP of HEK293T cell extracts expressing FLAG–c-Cbl WT or FLAG–c-Cbl *RING , followed by WB analysis. (f) IFM of WT NIH3T3 cells coexpressing GFP-tagged c-Cbl and FLAG–tagged IFT20. The dashed line indicates the Golgi complex, the arrow shows the primary cilium, and the asterisks identify the ciliary base. The nucleus was visualized with DAPI. (g) IFM of WT NIH3T3 cells expressing GFP-tagged c-Cbl. The primary cilium was marked with anti–ARL13B (arrow). The dashed line indicates the Golgi complex, and asterisk indicates the ciliary base. The nucleus was visualized with DAPI. The outline of the cells in panels f and g is highlighted with a dashed line. (h and i) IFM of WT NIH3T3 cells expressing either GFP-tagged c-Cbl (h) or stained with anti–c-Cbl (i). The Golgi complex (dashed line) was labeled with anti–GM130, and the nucleus was visualized with DAPI. (j) Localization of c-Cbl to the primary cilium in WT NIH3T3 cells, labeled with anti–Ac-tub (arrow), in WT NIH3T3 cells before and after stimulation with PDGF-AA. The asterisk marks the ciliary base. The nucleus was visualized with DAPI. (k) Localization of c-Cbl to the primary cilium in WT NIH3T3 cells and labeled with anti–ARL13B (arrow) in IMCD3 cells before and after stimulation with PDGF-AA. The asterisk marks the ciliary base. The nucleus was visualized with DAPI. (l) Quantification of relative levels of c-Cbl in the primary cilium shown in panel k. Fluorescence was normalized to background levels. For each of the three experiments, >15 cells were quantified. Error bars represent means ± SEM ( n = 3).

Article Snippet: The following antibodies were used: R&D Systems: goat anti-PDGFRα (AF1062); Proteintech: rabbit anti–human IFT20 (13615–1-AP), rabbit anti-IFT88 (13967-1-AP); Sigma-Aldrich: mouse anti–α-tubulin (T5168), mouse anti–acetylated α-tubulin (T6793), mouse anti-FLAG (M2; F1802), rabbit anti-FLAG (F7425); BD Biosciences: mouse anti-CDK1 (610038), mouse anti-p150 Glued (610474), mouse anti-GM130 (610823); Enzo life Sciences: mouse anti-giantin (ALX-804-600-C100), mouse anti–mono- and polyubiquitin (FK2; BML-PW8810); Abcam: mouse anti–c-Cbl (Ab119954), rabbit anti–c-Cbl (Ab32027), mouse anti-GFP (Ab1218), chicken anti-GFP (Ab13970), rabbit anti–detyrosinated α-tubulin (Ab48389), rabbit anti-PDGFRα (Ab134123); Cell Signaling Technology: rabbit anti-Myc (2278), rabbit anti-GAPDH (2118), rabbit anti–Cbl-b (9498), rabbit anti-Akt (9272), rabbit anti–phosphorylated Akt (Ser473; 4060), rabbit anti-Erk1/2 (9102), rabbit anti–phosphorylated Erk1/2 (Thr202/Tyr204; 9101), rabbit anti–phosphorylated Rb (9308); Santa Cruz Biotechnology: rabbit anti–c-Cblsc-170), mouse anti–c-Cbl (sc-1651), mouse anti-GFP (sc-9996), rabbit anti-GFP (sc-8334), rabbit anti–phosphorylated PDGFRα (Tyr754; sc-12911R).

Techniques: Biomarker Discovery, Plasmid Preparation, Expressing, Mutagenesis, Staining, Labeling, Fluorescence

Journal: The Journal of Cell Biology

Article Title: IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

doi: 10.1083/jcb.201611050

Figure Lengend Snippet: PDGFRα localizes at the plasma membrane in IFT20-depleted cells. In all experiments, cells were incubated with or without Dox for 6 d, including serum depletion for 24 h before analysis. (a) IFM of NIH3T3 sh IFT20 cells expressing GFP-tagged PDGFRα (NIH3T3 sh IFT20, GFP-PDGFRα ) stained with anti–IFT20 and anti–GM130 to label the Golgi complex (dashed line). Nuclei (Nu) were visualized with DAPI. Arrows indicate receptor localization at the plasma membrane. The outline of the cells is highlighted by a dashed line. (b) WB analysis of PDGFRα and GFP-PDGFRα in NIH3T3 sh IFT20 and NIH3T3 sh IFT20, GFP-PDGFRα with or without Dox. GAPDH was used as the loading control. (c) IFM showing localization of GFP–PDGFRα to the primary cilium (arrow) labeled with anti–Ac-tub in the absence of Dox. (d) IFM of localization of endogenous PDGFRα in NIH3T3 sh IFT20 cells with or without Dox. Nuclei (Nu) were visualized with DIC microscopy. Arrows indicate receptor localization at the plasma membrane. (e and f) Relative levels of GFP fluorescence at the cell surface in NIH3T3 sh IFT20, GFP-PDGFRα cells, corresponding to a 5-µm area starting from the cell surface into the inside of the cell (e). GFP fluorescence was normalized to background levels. For each of 3 experiment, >15 cells were quantified. Error bars represent SEM ( n = 3; f). (g) IFM analysis of GFP-PDGFRα localization in migrating NIH3T3 sh IFT20, GFP-PDGFRα cells with or without Dox. Arrows indicate localization of receptors to the leading-edge membrane of the lamellipodia (lam.) in cells treated with Dox. (h) IFM analysis of GFP-PDGFRα localization in NIH3T3 sh IFT20, GFP-PDGFRα cells subjected to siRNA-mediated silencing of both c-Cbl and Cbl-b. GM130 labels the Golgi complex. Arrows indicate localization of receptors at the plasma membrane (h) and to cellular protrusions in cells treated with Dox. Nuclei were visualized with DAPI. (i) Proposed model for the role of IFT20 in modulating PDGFRα signaling. IFT20 interacts with c-Cbl and Cbl-b to orchestrate the ubiquitination and internalization of ligand-activated PDGFRα for signaling termination. In the presence of IFT20 (left), PDGFRα is activated with proper feedback inhibition at the primary cilium, but in IFT20-depleted cells with defective ciliogenesis (right), PDGFRα mislocalizes to the plasma membrane from where the receptor is overactivated because of autoubiquitination-induced degradation of c-Cbl and Cbl-b.

Article Snippet: The following antibodies were used: R&D Systems: goat anti-PDGFRα (AF1062); Proteintech: rabbit anti–human IFT20 (13615–1-AP), rabbit anti-IFT88 (13967-1-AP); Sigma-Aldrich: mouse anti–α-tubulin (T5168), mouse anti–acetylated α-tubulin (T6793), mouse anti-FLAG (M2; F1802), rabbit anti-FLAG (F7425); BD Biosciences: mouse anti-CDK1 (610038), mouse anti-p150 Glued (610474), mouse anti-GM130 (610823); Enzo life Sciences: mouse anti-giantin (ALX-804-600-C100), mouse anti–mono- and polyubiquitin (FK2; BML-PW8810); Abcam: mouse anti–c-Cbl (Ab119954), rabbit anti–c-Cbl (Ab32027), mouse anti-GFP (Ab1218), chicken anti-GFP (Ab13970), rabbit anti–detyrosinated α-tubulin (Ab48389), rabbit anti-PDGFRα (Ab134123); Cell Signaling Technology: rabbit anti-Myc (2278), rabbit anti-GAPDH (2118), rabbit anti–Cbl-b (9498), rabbit anti-Akt (9272), rabbit anti–phosphorylated Akt (Ser473; 4060), rabbit anti-Erk1/2 (9102), rabbit anti–phosphorylated Erk1/2 (Thr202/Tyr204; 9101), rabbit anti–phosphorylated Rb (9308); Santa Cruz Biotechnology: rabbit anti–c-Cblsc-170), mouse anti–c-Cbl (sc-1651), mouse anti-GFP (sc-9996), rabbit anti-GFP (sc-8334), rabbit anti–phosphorylated PDGFRα (Tyr754; sc-12911R).

Techniques: Clinical Proteomics, Membrane, Incubation, Serum Depletion, Expressing, Staining, Control, Labeling, Microscopy, Fluorescence, Ubiquitin Proteomics, Inhibition

Journal: PLoS ONE

Article Title: Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

doi: 10.1371/journal.pone.0005755

Figure Lengend Snippet: (A) DU145 cells were electroporated with cDNA expression constructs of WOX1, dn-WOX1 (DN), and/or p53. 24 hr later, the cells were exposed to C1q for 8 hr. In appropriate controls, cells were electroporated with medium (Sham) or without electroporation (Cont). No DNA fragmentation was shown in these controls. C1q increased the DNA fragmentation in cells expressing WOX1, but not p53. C1q suppressed p53/WOX1-increased DNA fragmentation. dn-WOX1 inhibited cell death caused by p53. (B) The intensity of DNA fragmentation was quantified by Photoshop, and averaged results shown in the bar graph were from two experiments. The “Sham” control (without C1q treatment) is regarded as 0%.

Article Snippet: Additional specific antibodies used in this study were against the following proteins: 1) p53, JNK1, p-JNK1 (phosphorylation at Thr183 and Tyr185), p-ERK (Tyr204 phosphorylation) from Santa Cruz Biotechnologies, 2) p-STAT3 (Tyr705 phosphorylation) and STAT3 from New England BioLab, 3) ERK from BD Transduction Laboratory, and 4) C1q from Quidel.

Techniques: Expressing, Construct, Electroporation, Control

Journal: PLoS ONE

Article Title: Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

doi: 10.1371/journal.pone.0005755

Figure Lengend Snippet: DU145 cells were cultured overnight in each indicated serum with depletion of a specific complement protein. p53 localization in the cells was determined by immunofluorescence microscopy. (A) In the absence of complement C6 (ΔC6 serum), p53 was mainly present in the cytoplasm. High molecular size HA (50 µg/ml) induced nuclear accumulation of p53 during treatment for 1 hr. (B) In the absence of serum C9 (ΔC9 serum), p53 was mostly localized in the nuclei, and HA (50 µg/ml) induced nuclear export in 1 hr. (C) To determine nuclear localization of p53, approximately 200 cells were counted. Shown in the bar graph is an average of results form two experiments. (D) In negative controls, cells were stained with Texas Red-conjugated secondary antibody only. SF, serum free; NHS, normal human serum.

Article Snippet: Additional specific antibodies used in this study were against the following proteins: 1) p53, JNK1, p-JNK1 (phosphorylation at Thr183 and Tyr185), p-ERK (Tyr204 phosphorylation) from Santa Cruz Biotechnologies, 2) p-STAT3 (Tyr705 phosphorylation) and STAT3 from New England BioLab, 3) ERK from BD Transduction Laboratory, and 4) C1q from Quidel.

Techniques: Cell Culture, Serum Depletion, Immunofluorescence, Microscopy, Staining