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: 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

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p38 MAPK (Thr80/Tyr182) antibody Clone D3F9 , Cell Signaling Technology , Cat# 4511S; RRID:AB_2139682.

Techniques: Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Expressing, CRISPR, Software, Sequencing, Modification