Journal: The Journal of Experimental Medicine

Article Title: Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

doi: 10.1084/jem.20111694

Figure Lengend Snippet: JAK2 alleles that confer resistance to enzymatic inhibitors. (A) In vitro mutagenesis screen of JAK2 R683G in Ba/F3-CRLF2 cells to identify mutations that confer resistance to BVB808. (B) Sensitivity to BVB808 was reduced in Ba/F3-CRFL2/JAK2 R683G cells harboring Y931C (GI 50 , 303 nM), G935R (462 nM), or E864K (427 nM) compared with no resistance mutation (96.4 nM; P < 0.05 for all 3 mutants). Proliferation was measured based on relative fluorescence units (RFU). Error bars represent SD. (C) Ba/F3 cells expressing ATIC-ALK (ALK+), EpoR/Jak2 V617F alone (none), or EpoR/Jak2 V617F with one of the three kinase domain mutations were transfected with either of two siRNA (#4 and #2) targeting mouse Jak2. The cells were grown in the absence of IL-3, and proliferation was measured after 24 and 48 h and normalized to cells from the same background transfected with nontargeting (nt) control siRNA. Representative immunoblot of VF/Y931C cells 24 h after transfection is shown. (D) Alignment of homologous regions in JAK2 and ABL1. Blue arrows indicate mutated codons in BCR/ABL1 reported to confer imatinib resistance in patients . Black arrows indicate codons identified by in vitro mutagenesis of BCR/ABL1 and JAK2 E864K, Y931C, and G935R mutations (below). (E) Structure of the kinase domain of JAK2 indicating E864, Y931, and G935 (right), with modeling of wild type, Y931C, and G935R (left, top to bottom). (F) Chemical structures of JAK inhibitors and the HSP90 inhibitor AUY922. (G) E864K, Y931C, and G935R were introduced into Jak2 V617F and expressed in Ba/F3-EpoR cells. Proliferation was assayed 48 h after exposure to indicated drug or vehicle in triplicate on two occasions based on RFU. *, P < 0.05 compared with no resistance mutation for that drug (GI 50 in nanomolar in parentheses). (H) Dose response curves based on RFU after 48 h of treatment with indicated drugs. Each data point was obtained in quadruplicate, and the experiment was independently repeated three times. Error bars indicate SD.

Article Snippet: After another day, we changed media to no IL-3 and added 1 mg/ml neomycin (G418; Gold Biotechnology).

Techniques: In Vitro, Mutagenesis, Fluorescence, Expressing, Transfection, Western Blot

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: AGEs facilitated the production of IL-36α from keratinocytes. a The mRNA level of IL-36α in NHKs treated with BSA or BSA-AGEs for 24 h was detected by qRT-PCR. b Secretion of IL-36α from NHKs treated with BSA or BSA-AGEs for 48 h was detected by ELISA. c Expression of IL-36α (red) in lesional samples from psoriasis patients ( n = 5), and skin samples from healthy controls ( n = 5) was detected by immunofluorescence. Representative samples were shown. Nuclei were counterstained with DAPI (blue). Isotype control was shown with a lesional sample. Bar graphs represent the mean ± SD of IL-36α fluorescence intensity. rMFI: relative mean fluorescence intensity. d Serum level of IL-36α in patients with psoriasis ( n = 30) and healthy controls ( n = 30) was detected by ELISA. e Correlation between serum levels of IL-36α and AGEs was analyzed by Spearman rank correlation test in patients with psoriasis ( n = 30). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Control, Fluorescence

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: AGE-primed keratinocytes potentiate Th17 cell response via secreting IL-36α. The frequency of RORγt + T ( a ) or IL-17A + T ( b ) cells among CD4 + T cells was detected by flow cytometry analysis on PBMCs from psoriasis patients ( n = 5) incubated for 48 h by the supernatants from NHKs, with the addition of IL-36α N, IL-36R A, or rhIL-36α into the incubation system, respectively. c The secretion of IL-17A from PBMCs of psoriasis patients ( n = 5) treated with the supernatants from NHKs for 48 h with the addition of IL-36α N, IL-36 A, or rhIL-36α into the incubation system, respectively, was detected by ELISA. SN, supernatant. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: STAT1/3 signaling pathways mediate AGE-induced expression of K17 and IL-36α in keratinocytes. Protein levels of STAT1, p-STAT1, STAT3, p-STAT3, K17, Rb, p-Rb, and p27 KIP1 in NHKs pre-transfected with STAT1 ( a ) or STAT3 ( b ) siRNA prior to BSA-AGEs stimulation for 24 h was determined by Western blot assay. The mRNA level of IL-36α in NHKs pre-transfected with STAT1 ( c ) or STAT3 ( d ) siRNA prior to BSA-AGEs stimulation for 24 h was determined by qRT-PCR. Secretion level of IL-36α from NHKs pre-transfected with STAT1 ( e ) or STAT3 ( f ) siRNA prior to BSA-AGEs stimulation for 48 h was determined by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: AGEs exert pro-proliferative and proinflammatory effects on keratinocytes via RAGE. a Protein levels of RAGE, STAT1, p-STAT1, STAT3, p-STAT3 in NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 24 h were determined by Western blot assay. b Immunofluorescence analysis of p-STAT1 and p-STAT3 in NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 24 h. c Protein levels of RAGE, K17, Rb, p-Rb, and p27 KIP1 in NHKs pre-transfected with RAGE siRNA before BSA-AGEs stimulation for 24 h was determined by Western blot assay. d The mRNA level of IL-36α in NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 24 h was determined by qRT-PCR. e Secretion level of IL-36α from NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 48 h was determined by ELISA. *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Immunofluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: Schematic representation of the role of AGEs in psoriatic inflammation. Accumulated AGEs in psoriatic epidermis bind to RAGEs on the membrane of keratinocytes and activate STAT1/3 signaling pathways. Subsequently, K17 is upregulated and thus causes nuclear export and degradation of p27 KIP1 , which leads to the cell cycle progression and ultimately proliferation of keratinocytes. Meanwhile, IL-36α is overexpressed and secreted from keratinocytes and promotes Th17 cell response, which could contribute to the formation of cutaneous immune response in psoriasis.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Membrane, Protein-Protein interactions

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