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: Journal of Nanobiotechnology

Article Title: Apoptotic vesicles from macrophages exacerbate periodontal bone resorption in periodontitis via delivering miR-143-3p targeting Igfbp5

doi: 10.1186/s12951-024-02934-2

Figure Lengend Snippet: MiR-143-3p inhibited the osteogenic differentiation of BMSCs through targeting Igfbp5 . ( A ) Prediction of miR-143-5p target genes by databases. ( B ) Gene sequences for the dual-luciferase reporter assay. ( C ) Fluorescence intensity of the dual-luciferase reporter assay. ( D ) IGFBP5 expression levels in BMSCs. Effects of mimic and inhibitor on IGFBP5 mRNA ( E ) and protein ( F ) levels. ( G ) Immunofluorescence images of IGFBP5 in periodontal tissues of mice with periodontitis. Scale bar: 200 μm. ( H ) Relative fluorescence intensity of IGFBP5 in bone tissue. Effects of IGFBP5 and its inhibitor on mRNA levels of BMSC mineralization markers ( I ), ALP activity ( J ), ARS and ALP staining ( K ), and protein levels of mineralization markers ( L ). n = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Recombined IGFBP5 (HY-P70408), NBI-31,772 (HY-110135) and Z-VAD(OMe)-FMK (HY-16658) were purchased from MedChemExpress.

Techniques: Luciferase, Reporter Assay, Fluorescence, Expressing, Immunofluorescence, Activity Assay, Staining

Journal: Journal of Nanobiotechnology

Article Title: Apoptotic vesicles from macrophages exacerbate periodontal bone resorption in periodontitis via delivering miR-143-3p targeting Igfbp5

doi: 10.1186/s12951-024-02934-2

Figure Lengend Snippet: Schematic representation of macrophage-derived ApoVs inhibiting BMSC differentiation and exacerbate periodontal resorption via miR-143-3p/Igfbp5 signaling axis

Article Snippet: Recombined IGFBP5 (HY-P70408), NBI-31,772 (HY-110135) and Z-VAD(OMe)-FMK (HY-16658) were purchased from MedChemExpress.

Techniques: Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

doi: 10.1186/s12964-024-01636-4

Figure Lengend Snippet: RNF128 interacts with IL-3 receptor IL-3Rα. ( A ) RNF128 expression levels significantly elevated upon IL-3 treatment. THP-1 cells were treated with IL-3 for 1 or 2 h, and the expression of RNF128 was evaluated using qPCR. ( B ) RNF128 interacts with IL-3Rα, not IL-3Rβ. 293T cells were transfected with HA-RNF128, along with Flag-tagged IL-3Rα and IL-3Rβ, and lysed after 24 h. The lysates subjected to immunoprecipitation using an anti-Flag antibody, followed by immunoblotting with the indicated antibodies. ( C ) The reverse interaction was confirmed by immunoprecipitation using an anti-Myc antibody in cells co-expressing Myc-RNF128 and Flag-IL-3Rα. ( D , E ) The N-terminal domain of RNF128 is primarily responsible for binding to IL-3Rα. 293T cells were transfected with Flag-IL-3Rα and HA-tagged truncated form of RNF128, and the immunoprecipitation analysis was conducted using an anti-Flag antibody. ( F ) Analysis of the colocalization between RNF128 and IL-3Rα. HeLa cells co-expressing RNF128 and IL-3Rα were subjected to immunostaining, with RNF128 detected using FITC and IL-3Rα using TRITC. Fluorescence signals were observed using confocal laser scanning microscopy. Scale bars: 10 μm. The data are presented as the mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Recombinant human IL-3 (Pepro Tech, 200-03), mouse Il-3 (MCE, HY-P7062), mouse GM-CSF (Pepro Tech, 213–13) and LPS (Sigma-Aldrich, L2880) were purchased from the respective suppliers.

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Immunostaining, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

doi: 10.1186/s12964-024-01636-4

Figure Lengend Snippet: RNF128 negatively regulates IL-3-triggered signaling. ( A , B ) Effects of Rnf128 on GM-CSF-induced phosphorylation of Stat5 and transcription of downstream genes. Wild-type and Rnf128 -deficient BMDMs were treated with GM-CSF (10 ng/ml) or not for the indicated times before immunoblotting analysis and qPCR analysis. ( C , D ) Effects of Rnf128-deficient on Il-3-induced phosphorylation of Stat5. Wild-type and Rnf128 -deficient BMDMs were either untreated or treated with Il-3 (25 ng/mL) for the specified durations. Subsequently, immunoblotting analysis was conducted to detect p-Stat5 levels, with quantification illustrating the ratio of p-Stat5 to Stat5 ( D ). ( E ) Effects of Rnf128-deficient on Il-3-induced Id1 , Pim1 and Cd69 genes. ( F , G ) Effects of RNF128 knockdown on IL-3-induced phosphorylation of Stat5. RNF128 knockdown and control TF-1 cells were subjected to overnight starvation, followed by stimulation with IL-3 (20 ng/mL) for the indicated durations. Subsequently, immunoblotting analysis was conducted for p-STAT5, and the quantification of p-STAT5/STAT5 ratios is presented ( G ). ( H ) Effects of RNF128 knockdown on the transcription of the ID1, PIM1 AND CD69 genes induced by IL-3. * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical analysis was performed using two-way ANOVA with Dunnett’s post-hoc test

Article Snippet: Recombinant human IL-3 (Pepro Tech, 200-03), mouse Il-3 (MCE, HY-P7062), mouse GM-CSF (Pepro Tech, 213–13) and LPS (Sigma-Aldrich, L2880) were purchased from the respective suppliers.

Techniques: Phospho-proteomics, Western Blot, Knockdown, Control

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

doi: 10.1186/s12964-024-01636-4

Figure Lengend Snippet: The schematic diagram illustrates the role of RNF128 in the IL3/STAT5 signaling pathway. The IL-3 signaling pathway relies on the IL-3 receptor complex, comprised of IL-3Rα, responsible for IL-3 binding specificity, and the common beta chain IL-3Rβ, shared with GM-CSF and IL-5 receptors. Upon IL-3 binding to its specific receptor, IL-3Rα initiates a cascade of downstream signaling events, involving the activation of JAK kinases, particularly JAK2. This activation subsequently leads to STAT5 phosphorylation, facilitating the transcription of genes like ID1, PIM1, and CD69. Notably, RNF128 demonstrates specific binding to IL-3 receptor alpha chain IL-3Rα, distinct from the common beta chain IL-3Rβ. The functional role of RNF128 is underscored as it promotes K27-linked polyubiquitination of IL-3Rα, culminating in its degradation through the lysosomal pathway. Consequently, RNF128 plays a crucial role in tempering excessive inflammatory responses

Article Snippet: Recombinant human IL-3 (Pepro Tech, 200-03), mouse Il-3 (MCE, HY-P7062), mouse GM-CSF (Pepro Tech, 213–13) and LPS (Sigma-Aldrich, L2880) were purchased from the respective suppliers.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Functional Assay