Journal:

Article Title: Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

doi: 10.1128/JVI.76.20.10497-10502.2002

Figure Lengend Snippet: Estimation of vector gene copy number in AAV-transduced livers by quantitative PCR (3 months after vector administration). A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems. Template for PCR was as follows: 100 ng of genomic DNA extracted from several random pieces of liver and subsequently combined for each individual animal. Each sample column represents an individual animal. standards, linearized plasmid pAAV-EF1α-hF.IX (0.01, 0.1, or 1 pg) mixed with 100 ng of genomic mouse DNA (extracted from untransduced animal); NC, negative control (template, genomic DNA from untransduced animal). Bands were analyzed by densitometric scanning, and intensities were quantitated with NIH Image 6.16 software. Shown is one representative blot. Ratios of band intensities (hF.IX band/mAAT band) are for the blot shown. Gene copy number estimates are average for two experiments.

Article Snippet: A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Software

Journal:

Article Title: 14-3-3? Positively Regulates p53 and Suppresses Tumor Growth

doi: 10.1128/MCB.23.20.7096-7107.2003

Figure Lengend Snippet: 14-3-3σ interacted with p53. (A) Interaction between 14-3-3σ and p53 after adriamycin treatment. A549 cells were treated with 0.2 μg of adriamycin per ml (+) or not (−) for 24 h. Equal amounts of cell lysates were immunoblotted (I.B.) with anti-p53 antibody or anti-14-3-3σ to show the expression of p53 or 14-3-3σ after adriamycin treatment. Equal amounts of cell lysates were also immunoprecipitated (i.p.) with anti-p53 antibody, then resolved in SDS-polyacrylamide gel electrophoresis, and immunoblotted with anti-14-3-3σ antibody to observe the association between p53 and endogenous 14-3-3σ. (B) Interaction between 14-3-3σ and p53 after ionizing radiation (IR). R1B/L17 cells were transfected with the indicated Flag-tagged 14-3-3σ constructs and p53 expression vectors for 48 h and irradiated with 10 Gy. After the indicated hours of ionizing radiation, equal amounts of cell lysates were immunoprecipitated with anti-p53 antibody, and the immunoprecipitates were immunoblotted with anti-Flag antibody (M2) to observe the association between p53 and Flag-tagged 14-3-3σ. Cell lysates were also immunoblotted (I.B.) with anti-p53 antibody or anti-14-3-3σ to show the expression of p53 or 14-3-3σ. (C) Schematic representation of 14-3-3σ deletion constructs. (D) Interaction between the domains of 14-3-3σ and p53. R1B/L17 cells were transfected with the indicated Flag-tagged 14-3-3σ constructs and p53 expression vectors. Cell lysates were immunoprecipitated (i.p.) with anti-Flag antibody (M2), and the immunoprecipitates were immunoblotted (I.B.) with anti-p53 (DO-1; Santa Cruz) to observe the interaction. The same blot was stripped and immunoblotted with anti-Flag antibody (M2, Sigma) to demonstrate the amount of 14-3-3σ deletion constructs expressed and immunoprecipitated. Equal amounts of protein from the cell lysates were immunoblotted with anti-p53 (DO-1) to indicate the expression of p53. (E) p53 specifically interacts with 14-3-3σ in vitro. GST-p53 immobilized on glutathione beads was incubated with in vitro-transcribed and translated 14-3-3σ domains (amino acids 1 to 161 or 153 to 248), which were 35S labeled. Bound domains were detected by autoradiography. The bottom panel shows 10% of the in vitro-translated 35S-labeled 14-3-3σ domain inputs.

Article Snippet: Cells were then fixed with methanol/acetone (1:1v/v) at room temperature for 2 min and stained for 1 h with rabbit anti-p53 (FL293, Santa Cruz) followed by 1 h of incubation with indocarbocyanine-conjugated anti-rabbit immunoglobulin antibody (Zymed).

Techniques: Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Transfection, Construct, Irradiation, In Vitro, Incubation, Labeling, Autoradiography

Journal:

Article Title: 14-3-3? Positively Regulates p53 and Suppresses Tumor Growth

doi: 10.1128/MCB.23.20.7096-7107.2003

Figure Lengend Snippet: 14-3-3σ increased p53 stabilization. (A) Ectopic expression of 14-3-3σ increased the stabilization of p53. H1299 cells were transfected with equal amounts of p53 expression vector and increasing amounts of the Flag-tagged 14-3-3σ expression vector. Equal amounts of protein from cell lysates were immunoblotted with anti-p53, anti-Flag, and antiactin. Actin served as a loading control. (B) Immunofluorescence studies of p53 stability. R1B/L17 cells were transfected with either plasmid pCMV expressing p19ARF, p53, or 14-3-3σ or with an empty cytomegalovirus vector to examine the immunostaining of endogenous p53 as detected by immunofluorescence. The cells that received the indicated plasmids were seeded at 2 × 103 cells per chamber slide. The cells were fixed, and p53 was immunodetected with anti-p53 antibody (FL293; Santa Cruz), followed by indocarbocyanine-conjugated anti-rabbit immunoglobulin (B, D, F, and H). DAPI staining was used to show the localization of nuclei (A, C, E, and G). (C) p53 binding activity of 14-3-3σ and p19ARF. R1B/L17 cells were transfected with the indicated expression vectors. Cell lysates were immunoprecipitated (i.p.) with anti-p53 antibody (DO-1) and immunoblotted (I.B.) with anti-Flag antibody (M2) to observe the association between p53 and 14-3-3σ or p19ARF. Immunoprecipitation with normal rabbit serum (NRB) was used as a negative control. Equal amounts of protein from cell lysates were immunoblotted with anti-p53 to indicate the expression of p53. Actin served as a loading control.

Article Snippet: Cells were then fixed with methanol/acetone (1:1v/v) at room temperature for 2 min and stained for 1 h with rabbit anti-p53 (FL293, Santa Cruz) followed by 1 h of incubation with indocarbocyanine-conjugated anti-rabbit immunoglobulin antibody (Zymed).

Techniques: Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Immunostaining, Staining, Binding Assay, Activity Assay, Immunoprecipitation, Negative Control

Journal:

Article Title: 14-3-3? Positively Regulates p53 and Suppresses Tumor Growth

doi: 10.1128/MCB.23.20.7096-7107.2003

Figure Lengend Snippet: 14-3-3σ inhibited Mdm2-mediated p53 ubiquitination. (A) 14-3-3σ stabilized p53 in the presence of Mdm2. H1299 cells were cotransfected with indicated pCMV-Mdm2, pCMV-p53, and increasing amounts of pCMV-Flag-14-3-3σ (0 μg, 0 μg, 1 μg, 4 μg, and 8 μg). Equal amounts of protein from cell lysates were immunoblotted with anti-Mdm2, anti-Flag, and antiactin. Levels of tubulin are shown as equal loading controls. (B) 14-3-3σ inhibited p53 turnover. 293T cells transiently transfected with pCMV-14-3-3σ and vector/pCMV-p53 were pulse-labeled with [35S]methionine for 30 min and chased for the indicated time. (C) 14-3-3σ accelerated Mdm2 degradation. 293T cells transiently transfected with pCMV-14-3-3σ and vector/pCMV-Mdm2 were pulse-labeled with [35S]methionine for 30 min and chased for the indicated time. Cells were harvested, and the amount of labeled Mdm2 or p53 protein immunoprecipitated at each time point was detected by phosphoimager for quantitation. p53 or Mdm2 remaining is indicated graphically. (D) 14-3-3σ inhibited Mdm2-mediated p53 ubiquitination. H1299 cells were cotransfected with equal amount of Mdm2 (3 μg), pCMV-p53 (1 μg), histidine-tagged ubiquitin-expressing vector (1 μg), and increasing amount of pCMV-Flag-14-3-3σ (0 μg, 1 μg, 4 μg, and 8 μg). The cell lysates were harvested, and the histidine-tagged ubiquitin-containing protein complexes were pulled down with His-Bind resin (Novagen). The protein complexes were then resolved by SDS-12% polyacrylamide gel and probed with anti-p53 antibody to observe the histidine-tagged ubiquitinated p53.

Article Snippet: Cells were then fixed with methanol/acetone (1:1v/v) at room temperature for 2 min and stained for 1 h with rabbit anti-p53 (FL293, Santa Cruz) followed by 1 h of incubation with indocarbocyanine-conjugated anti-rabbit immunoglobulin antibody (Zymed).

Techniques: Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, Quantitation Assay, Expressing

Journal:

Article Title: 14-3-3? Positively Regulates p53 and Suppresses Tumor Growth

doi: 10.1128/MCB.23.20.7096-7107.2003

Figure Lengend Snippet: 14-3-3σ blocked Mdm2-mediated p53 nuclear export. (A) Subcellular localization of Mdm2 was affected by overexpression of 14-3-3σ. The Rat1-akt cell lines were infected with Ad-HA-14-3-3σ or Ad-β-gal. After 48 h, cells were fixed and stained with anti-Mdm2 to detect the location of Mdm2 (A, D) or stained with anti-HA antibodies to observe the location of 14-3-3σ (B, E). Nuclei were stained with DAPI dye. (B) Activity of 14-3-3σ inhibited Mdm2-mediated p53 nuclear export. R1B/L17 cells were transfected with the indicated constructs to examine the subcellular localization of GFP-p53 as detected by green fluorescence. The cells that received the indicated plasmids were seeded at 2 × 103 in a chamber slide. The location of GFP-p53 was observed under a fluorescence microscope. A total of 300 cells were counted for the location of GFP-p53 in each condition. The percentages of nuclear-positive cells (N) and nuclear and cytoplasmic-positive cells (N+C) are presented. Data shown were from a typical experiment conducted in triplicate. Bars represent standard deviations.

Article Snippet: Cells were then fixed with methanol/acetone (1:1v/v) at room temperature for 2 min and stained for 1 h with rabbit anti-p53 (FL293, Santa Cruz) followed by 1 h of incubation with indocarbocyanine-conjugated anti-rabbit immunoglobulin antibody (Zymed).

Techniques: Over Expression, Infection, Staining, Activity Assay, Transfection, Construct, Fluorescence, Microscopy

Journal:

Article Title: 14-3-3? Positively Regulates p53 and Suppresses Tumor Growth

doi: 10.1128/MCB.23.20.7096-7107.2003

Figure Lengend Snippet: 14-3-3σ enhanced p53 transcriptional activity. (A) 14-3-3σ activated the p53 luciferase reporter gene. The BDS2-3X-luc reporter containing a p53-responsive element was transfected with the indicated p53- or 14-3-3σ-expressing vectors into R1B/L17 cells. Relative luciferase activity is shown. (B) 14-3-3σ potentiated p53 transcriptional activity in a dose-dependent manner. The BDS2-3X-luc reporter was transfected with increasing amounts of the 14-3-3σ expression vector into p53-null MEF cells. The BDS2-mutant-luc reporter with a mutated p53-responsive element was used as a negative control. Relative luciferase activity is shown. (C) Oligomerization assay. H1299 cells were transfected with the indicated plasmids. Total cell extracts were incubated with 0.01% glutaraldehyde to determine the levels of the oligomerization of p53. p53 oligomers were immunoblotted (I.B.) with anti-p53 antibodies. (D) 14-3-3σ increased the interaction between p53 molecules. H1299 cells were transfected with pCMV-His-tagged-p53 and pCMV-GFP-p53 in the presence or absence of 14-3-3σ. His-tagged p53 was bound with His-Bind beads, and His-tagged p53-associated GFP-p53 was observed with the anti-GFP antibody. (E) Transcriptional activation of p53 target gene p21. R1B/L17 cells were infected (inf) with Ad-14-3-3σ (σ) or Ad-β-gal (C) or not infected (−). After 48 h, total RNAs were prepared. Northern blot analysis was performed on total RNAs to examine the expression of the p21 gene. Signals for GAPDH are shown to indicate the integrity and quantity of the RNA.

Article Snippet: Cells were then fixed with methanol/acetone (1:1v/v) at room temperature for 2 min and stained for 1 h with rabbit anti-p53 (FL293, Santa Cruz) followed by 1 h of incubation with indocarbocyanine-conjugated anti-rabbit immunoglobulin antibody (Zymed).

Techniques: Activity Assay, Luciferase, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Negative Control, Incubation, Activation Assay, Infection, Northern Blot

Journal:

Article Title: 14-3-3? Positively Regulates p53 and Suppresses Tumor Growth

doi: 10.1128/MCB.23.20.7096-7107.2003

Figure Lengend Snippet: Model for effects of 14-3-3σ in regulating p53's activity.

Article Snippet: Cells were then fixed with methanol/acetone (1:1v/v) at room temperature for 2 min and stained for 1 h with rabbit anti-p53 (FL293, Santa Cruz) followed by 1 h of incubation with indocarbocyanine-conjugated anti-rabbit immunoglobulin antibody (Zymed).

Techniques: Activity Assay

Journal: Nature Communications

Article Title: HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells

doi: 10.1038/s41467-017-00609-1

Figure Lengend Snippet: Identification of the chimeric HIV/ BACH2 and HIV/ STAT5B transcripts in patients under cART. a , b Scheme of the RT-PCR strategy devised to amplify putative chimeric HIV/ STAT5B ( a ) or HIV/ BACH2 ( b ) transcripts; white and gray boxes represent non-coding and coding exons (Ex), respectively. The exon numbers and the position of the first ATG codon are indicated. The integrated provirus, the LTR, and the 5′ major viral SD site are depicted in black . The arrows represent the position of the primer pairs used for RT-PCR. The black and gray bars indicate the amplified cDNA sequence from the provirus and host genome, respectively. The dashed lines indicate the splicing events. At the bottom of each panel, representative examples of the sequences of the RT-PCR products are shown. c , d Report agarose gel electrophoresis of RT-PCR products of either HIV/ STAT5B or HIV/ BACH2 obtained from PBMC of HIV-1-infected patients using the primers indicated in a , b ; the amplicon size is ~500 bp (M molecular size markers). For each sample, RT-PCR for GAPDH was also performed as positive control of amplification (data not shown)

Article Snippet: TaqMan® Gene Expression Assays (Applied Biosystem) were used to assess gene expression of STAT5B (Hs00273500_m1, exon boundary 16-17), BACH2 (Hs00222364_m1, exon boundary 8–9), and HPRT (Hs99999909_m1, exon boundary 6–7).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Agarose Gel Electrophoresis, Infection, Positive Control

Journal: Nature Communications

Article Title: HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells

doi: 10.1038/s41467-017-00609-1

Figure Lengend Snippet: Tracking and quantification of HIV/ STAT5B and HIV/ BACH2 chimeric transcripts in different hematopoietic cell subsets obtained from HIV patients under cART. a Top panel , agarose gel electrophoresis of the RT-PCR products (using the primers indicated in Fig. ) obtained from the cDNA of the indicated cell subsets purified by FACS sorting from two HIV-1 patients (ID-96, ID-77). For each cell subsets RT-PCR for GAPDH was also performed as positive control of amplification ( bottom panels ). In both patients, a specific band of 500 bp was obtained from the cDNA extracted from the purified Treg and Tcm cell subsets. In ID_77 two different bands appeared in the agarose gel due to splice variants of the STAT5B gene. In PBMC the band is detectable only when 10-fold more cDNA was loaded in the RT-PCR. The band was not detected in all the other cell types, including Tscm, T effector memory (Tem), and others. b Top panel , agarose gel electrophoresis of the RT-PCR products obtained from the cDNA of the indicated cell subsets purified by magnetic cell isolation in four HIV-1 patients (ID-76, ID-56, ID-25, ID-63). RT-PCR for GAPDH was also performed as positive control of amplification ( bottom panel ). M: molecular size marker. c , d Histograms show the average of the relative levels of the copies of the HIV/ STAT5B ( c ) and of the HIV/ BACH2 chimeric transcript ( d ) vs. HPRT copies measured by dd-PCR in Treg and Teff cells obtained from PBMC of six and three HIV-infected patients, respectively. e , f Histograms indicate the relative levels of the copies of STAT5B ( e ) and BACH2 ( f ) vs. HPRT copies measured by dd-PCR in Treg and Teff cells obtained from PBMC of six and three HIV-infected patients, respectively. Significance was determined by Mann–Whitney tests (** p < 0.01), SEM is indicated by the error bar

Article Snippet: TaqMan® Gene Expression Assays (Applied Biosystem) were used to assess gene expression of STAT5B (Hs00273500_m1, exon boundary 16-17), BACH2 (Hs00222364_m1, exon boundary 8–9), and HPRT (Hs99999909_m1, exon boundary 6–7).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Purification, Positive Control, Amplification, Cell Isolation, Marker, Infection, MANN-WHITNEY

Journal: Nature Communications

Article Title: HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells

doi: 10.1038/s41467-017-00609-1

Figure Lengend Snippet: Phylogenies of amplified chimeric HIV/STAT5B bands. Maximum-likelihood trees constructed aligning the haplotypes identified analyzing the HIV-1 sequence from the U5 to the major SD signal among different hematopoietic cell subsets for patients ID_77 and ID_96. The hematopoietic cell subset from which the specific haplotype was identified is indicated at each branch tip of the tree. The progressive number indicates different haplotypes that have been identified within the same hematopoietic cell subset. Branch length varies according to the number of substitutions as a proportion of the length of the alignment (Distance value). Scale bar is reported below each tree

Article Snippet: TaqMan® Gene Expression Assays (Applied Biosystem) were used to assess gene expression of STAT5B (Hs00273500_m1, exon boundary 16-17), BACH2 (Hs00222364_m1, exon boundary 8–9), and HPRT (Hs99999909_m1, exon boundary 6–7).

Techniques: Amplification, Construct, Sequencing

Journal: Nature Communications

Article Title: HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells

doi: 10.1038/s41467-017-00609-1

Figure Lengend Snippet: Effects of LV-mediated expression of STAT5B and BACH2 in in vitro-induced Treg cells. a – c Schematic structure of the LVs used to coordinately express the orange fluorescence protein (as marker transgene) and STAT5B ( a ) or BACH2 ( b ) or the control vector-expressing GFP and dNGFR c . Transgene transcript is indicated by arrows . d Scheme of the experimental strategy. CD4 + naïve T cells from PBMC or cord blood mononuclear cells of HDs were activated by anti-CD3/CD28 beads for 24 h, transduced with the indicated LVs, and cultured in presence of IL-2 and TGF-β for 2 weeks; e Relative percentage of GFP + or Orange + cells (for bidirectional SIN LVs encoding for BACH2 or STAT5B) of naïve CD4 + T cells transduced with the indicated vector and analyzed 5 days after transduction, N = 5; f Percentage of CD25 + FOXP3 + cells in CD4 + T cells transduced with the indicated vector and untransduced cells after 2 weeks of culture, N = 5; g Inhibition of proliferation (analyzed by flow cytometry for e-Fluor dilution and indicated as % suppression vs. responder alone) of anti-CD3/CD28 activated allogeneic Responder cells (R) cultured in presence of different doses (Ratio R:S were 1:1 and 1:0.25) of CD4 + CD25 + suppressive cells (S) purified from cells transduced or not with the indicated LVs, N = 5; h Proliferation rate (measured by [3 H]-thymidine incorporation) of purified CD25 + cells from T cells transduced or not with the indicated vector and activated with anti-CD3 CD28 in the absence ( black bar ) or presence ( orange bar ) of IL-2. Fold increase vs. unstimulated control cells ( N = 3) is shown, N = 5. Data are represented as Mean ± SEM. Significance was determined by one-way ANOVA with Bonferroni correction (* p < 0.05; ** p < 0.01).

Article Snippet: TaqMan® Gene Expression Assays (Applied Biosystem) were used to assess gene expression of STAT5B (Hs00273500_m1, exon boundary 16-17), BACH2 (Hs00222364_m1, exon boundary 8–9), and HPRT (Hs99999909_m1, exon boundary 6–7).

Techniques: Expressing, In Vitro, Fluorescence, Marker, Control, Plasmid Preparation, Transduction, Cell Culture, Inhibition, Flow Cytometry, Purification

Journal: Nature Communications

Article Title: HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells

doi: 10.1038/s41467-017-00609-1

Figure Lengend Snippet: Effects of the forced expression of STAT5B and BACH2 in Treg cells. a Experimental strategy. Treg cells were purified from PBMC of HDs, were activated by anti-CD3/CD28 Treg expander beads for 24 h, transduced with the indicated LVs, and cultured for 10 days in presence of Treg expander beads and IL-2 (100 U/ml). b Inhibition of proliferation (analyzed by flow cytometry for e-Fluor dilution and indicated as % Suppression vs. responder alone) of anti-CD3/CD28 activated allogeneic Responder (R) cells cultured in presence of different doses (Ratio R:S were 1:1, 1:05, and 1:0.25) of Suppressive cells (S). c , d Percentage of Orange + Treg cells expressing STAT5B ( c ) or BACH2 ( d ) measured over time and cultured alone (indicated as STAT5B and BACH2) or in presence of Treg cells transduced with the control LV ( c ) (Ratio GFP-expressing and STAT5B-expressing cells is 1:1, 1:05, and 1:025, the same for GFP-expressing and BACH2-expressing cells). Significance was determined by performing two-way ANOVA with Bonferroni correction on Log ODD transformed % values (* p < 0.05; ** p < 0.01). Data are represented as Mean ± SEM, and for all panels N = 4

Article Snippet: TaqMan® Gene Expression Assays (Applied Biosystem) were used to assess gene expression of STAT5B (Hs00273500_m1, exon boundary 16-17), BACH2 (Hs00222364_m1, exon boundary 8–9), and HPRT (Hs99999909_m1, exon boundary 6–7).

Techniques: Expressing, Purification, Transduction, Cell Culture, Inhibition, Flow Cytometry, Control, Transformation Assay

Journal: Cell reports

Article Title: TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction

doi: 10.1016/j.celrep.2020.108379

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-Myc tag , Cell Signaling , Cat# 2276S; RRID:AB_331783.

Techniques: Plasmid Preparation, Recombinant, Transfection, Blocking Assay, Purification, Gel Extraction, DNA Purification, shRNA, Software