Journal: PLoS ONE

Article Title: An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8 + T Cells in Mice

doi: 10.1371/journal.pone.0088205

Figure Lengend Snippet: Lung, liver, spleen, kidney and bone marrow, were collected 6 and 20 weeks after NSG mice were given recombinant AAV9 vectors (5×10 9 GC of AAV9-GM-CSF, AAV9-IL-3, and AAV9-IL-15 by i.v., or 1×10 11 GC of AAV9-A2 by i.v. and intrathoracically), and DNA was isolated from the respective organs. (A) The number of AAV9 vector GC was determined by using a set of primers specific to AAV9 vector. (B) The number of GC specific for the transgenes - HLA-A2, hGM-CSF, hIL-3 and hIL-15 - was determined by using a set of primers to the respective transgene. There was a low number (10–10 3 GC; depending on the transgene) of GC detected in naïve NSG mice, as a non-specific background, and, therefore, this background GC number was subtracted from the GC number in experimental groups.

Article Snippet: Human GM-CSF, IL-3, IL-4, IL-7, and IL-15 cDNA were purchased from OriGene.

Techniques: Recombinant, Isolation, Plasmid Preparation

Journal: PLoS ONE

Article Title: An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8 + T Cells in Mice

doi: 10.1371/journal.pone.0088205

Figure Lengend Snippet: (A) Schematic representation of the strategic methodology for engrafting human CD34 + cells in AAV9-transduced NSG mice. NSG mice were inoculated with AAV9-hucytokines or AAV9-A2 and 2 weeks later, mice were irradiated to myeloablate mouse immune cells. The next day, mice were transplanted i.v. with 1×10 5 human CD34 + cells previously isolated from human fetal liver. (B) The level of human CD45 + cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34 + cells into NSG mice transduced with AAV9 encoding human IL-3, IL-4, IL-7, IL-15, or GM-CSF. (C) The level of human CD45 + cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34 + cells into NSG mice transduced with individual or combination of AAV9 encoding selected human cytokines, IL-3 (5×10 9 GC), IL-15 (5×10 9 GC), and GM-CSF (1×10 9 GC). (D) The level of human CD45 + cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34 + cells into NSG mice transduced with AAV9-A2.

Article Snippet: Human GM-CSF, IL-3, IL-4, IL-7, and IL-15 cDNA were purchased from OriGene.

Techniques: Irradiation, Isolation, Transduction

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: Overexpression of circTRAPPC6B inhibited intracellular Mtb growth while activating autophagy in Mtb‐infected macrophages. (a, b) After transfection with circTRAPPC6B overexpressing plasmids or control plasmids for 24 h, THP‐1 macrophages (a) and macaque spleen macrophage (b) were infected with Mycobacterium H37Rv at MOI = 1 for 3 or 7 days. The growth of the bacilli was evaluated by CFU count. The data were obtained from six independent experiments. Data are expressed as mean ± SEM. * P < 0.05 vs day 0 ( n = 6). (c, d) After transfection with siRNA against circTRAPPC6B (c) or plasmids overexpressing circTRAPPC6B (d) for 24 h, THP‐1 macrophages were infected with Mycobacterium H37Rv at MOI = 1 for 24 h. Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs uninfected and untransfected control. ## P < 0.01 vs H37Rv‐infected but untransfected control. (e) After transfection with circTRAPPC6B overexpressing vectors or control vectors (NC) for 24 h, THP‐1 macrophages were infected with GFP‐BCG at MOI = 10 for 24 h. THP‐1 macrophages were incubated with anti‐LC3 for 2h at room temperature and then fluorescently labelled secondary Ab for 1 h at room temperature. Representative immunofluorescence confocal image of THP‐1 macrophages showing the change of LC3B puncta and GFP‐BCG colocalisation. Scale bar, 20 μm. (f, g) Quantification assay of e. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta or colocalisation of LC3B with GFP‐BCG of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs NC ( n = 3). MOI, multiplicity of infection; CFU, colony‐forming unit; GFP, green fluorescence protein; BCG, Bacillus Calmette‐Guérin.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Over Expression, Infection, Transfection, Control, Western Blot, Expressing, Incubation, Immunofluorescence, Fluorescence

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: miR‐874‐3p regulated intracellular Mtb growth and autophagy in Mtb‐infected macrophages. (a) After PBMCs were freshly isolated from blood by standard Ficoll density gradient centrifugation, miR‐874‐3p expression was analysed in human PBMCs from patients with 32 active TB and 31 HC by qRT‐PCR. U6 was used as a housekeeping gene for normalising changes in miRNA gene expression. Data are expressed as mean ± SEM. ** P < 0.01 vs HC. (b) Spearman’s correlation analysis between miR‐874‐3p and circTRAPPC6B expression in PBMCs of 32 TB patients. (c) After THP‐1 macrophages were infected with H37Rv at MOI = 1 at different time points (0, 1, 2, 3, 4, and 7 days), miR‐874‐3p expression was analysed by qRT‐PCR. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. ** P < 0.01, *** P < 0.001 vs 0 d. (d, e) After transfection with miR‐874‐3p inhibitor or negative control (miR‐NC) for 24 h, THP‐1 macrophages (d) and macaque spleen macrophage (e) were infected with Mycobacterium H37Rv at MOI = 1 for 3 or 7 days. The growth of the bacilli was evaluated by CFU count. The data were obtained from six independent experiments. Data are expressed as mean ± SEM. * P < 0.05 vs day 0 ( n = 6). (f) After transfection with miR‐874‐3p inhibitor or negative control (miR‐NC) for 24 h, THP‐1 macrophages were infected with GFP‐BCG at MOI = 10 for 24 h. Then, THP‐1 macrophages were incubated with anti‐LC3B for 2h at room temperature and then fluorescently labelled secondary Ab for 1h at room temperature. Representative immunofluorescence confocal image of THP‐1 macrophages showing the change of LC3B puncta and GFP‐BCG colocalisation. Scale bar, 20 μm. (g, h) Quantification assay of f. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta or colocalisation of LC3B with GFP‐BCG of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs miR‐NC. (i, j) After transfection with miR‐874‐3p mimics (i) or inhibitor (j) for 24 h, THP‐1 macrophages were infected with Mycobacterium H37Rv at MOI = 1 for 24 h. Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, *** P < 0.001 vs uninfected and untransfected control; ### P < 0.001 vs H37Rv‐infected but untransfected cells. qRT‐PCR, quantitative real‐time polymerase chain reaction; PBMCs, peripheral blood mononuclear cells; TB, tuberculosis; HC, health control; MOI, multiplicity of infection; CFU, colony forming unit; GFP, green fluorescence protein; BCG, Bacillus Calmette‐Guérin.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Infection, Isolation, Gradient Centrifugation, Expressing, Quantitative RT-PCR, Gene Expression, Transfection, Negative Control, Incubation, Immunofluorescence, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: circTRAPPC6B regulated autophagy in Mtb‐infected macrophages via miR‐874‐3p. After transfection with circTRAPPC6B‐overexpressing vectors (pHBAd‐cir), miR‐874‐3p mimics, or corresponding negative controls as indicated for 24 h, THP‐1 macrophages were infected with BCG at MOI = 10 or Mycobacterium H37Rv at MOI = 1 for 24 h. (a) Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in H37Rv‐infected THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001. (b) THP‐1 macrophages were incubated with anti‐LC3B for 2h at room temperature and then fluorescently labelled secondary Ab for 1h at room temperature. Representative immunofluorescence confocal image of BCG‐infected THP‐1 macrophages showing the change of LC3B puncta. (c) Quantification of b. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01. BCG, Bacillus Calmette‐Guérin; MOI, multiplicity of infection.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Infection, Transfection, Western Blot, Expressing, Incubation, Immunofluorescence