Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USCs inhibited the proliferation of PBMNCs in MLR. USC-TA+, USC-TA−, BMSCs, and SMCs were used as stimulator cells and human PBMNCs from two healthy donors were used as responder cells. One-way MLR with single donor PBMNCs (donor A or B) as responder cells and two-way MLR with aliquot half dose of two different donors’ PBMNCs (donor A and B) as responder cells were detected in the same multi-well plate. Cultures were incubated at 37 °C for 5 days in 5% CO2 and 100% humidity, the cells were checked with inverted microscope on the 5th day before further experiment. There were different cell densities of PBMNCs be noticed in different wells for 5 days mix-culture, Scale bar =50 µm (A). The proliferation of PBMNCs was assessed with the BrdU cell proliferation colorimetric ELISA. Newly synthesized BrdU-DNA was quantified using a scanning multi-well spectrophotometer (B). *, P<0.05, post-hoc paired-comparisons. MLR, mixed lymphocyte reaction; BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells; SMC + PBMNC A, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor A; SMC + PBMNC B, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor B; SMC + PBMNC A + B, two-way mixed lymphocyte reaction as human smooth muscle cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; BMSC + PBMNC A, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor A; BMSC + PBMNC B, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor B; BMSC + PBMNC A + B, two-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; USC + PBMNC A, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor A; USC + PBMNC B, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor B; USC + PBMNC A + B, two-way mixed lymphocyte reaction as human urine derived stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Incubation, Inverted Microscopy, Enzyme-linked Immunosorbent Assay, Synthesized, Spectrophotometry, Derivative Assay, Activity Assay

Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USC showed a characteristic cytokine release profile. The supernatants from USC-TA+, USC-TA−, and BMSCs cultured alone or co-cultured with PBMNCs (direct mixed culture with PBMNCs, or indirect mixed culture with PBMNCs in transwell insert for 48 h) (A) were assessed for their relative levels of cytokines and chemokines by using the human cytokine array panel A, as described in “Methods” (B). A: human cytokine array panel template; B: PBMNCs background; C: USC-TA+ culture alone; D: USC-TA− culture alone; E: BMSCs culture alone; F: USC-TA+ direct mix culture with PBMNCs; G: USC-TA− direct mix culture with PBMNCs; H: BMSCs direct mix culture with PBMNCs; I: USC-TA+ in-direct mix culture with PBMNCs in transwell insert; J: USC-TA− in-direct mix culture with PBMNCs in transwell insert; K: BMSCs in-direct mix culture with PBMNCs in transwell insert: The immunoblot of cytokine expression levels were visualized and quantifed (C). *, P<0.05, comparing with BMSCs alone; †, P<0.05, comparing direct and indirect mixed culture with PBMNCs. BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Cell Culture, Western Blot, Expressing, Derivative Assay, Activity Assay

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet: Flow cytometry enriched antibody cocktail (22 μL) panel for analyzing rarer granulocyte and myeloid cells in mouse spleen for downstream scRNA-seq

Article Snippet: CD11b Antibody, anti-mouse, VioBlue®, REAfinityTM Clone REA592 , Miltenyi Biotec , Cat# 130-113-810; RRID: AB_2726327.

Techniques: Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet:

Article Snippet: CD11b Antibody, anti-mouse, VioBlue®, REAfinityTM Clone REA592 , Miltenyi Biotec , Cat# 130-113-810; RRID: AB_2726327.

Techniques: Recombinant, Sterility, Saline, Extraction, Blocking Assay, Software, Solvent, Sequencing, RNA Sequencing, Transferring, Aerosol, Irradiation, Inverted Microscopy, Flow Cytometry, Fluorescence

Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 6. Lyn is essential to the activation of PI 3-kinase through phosphorylation of its p110 subunit. (A; Supplementary movies M1–M4) HEK 293–TLR2 cells were transfected with a fusion protein combining GFP and the PH domain of AKT. Transfected cells were either co-transfected with LynDK (500 ng/ml) or incubated with PP2. Cells were then stimulated with Pam3 (100 ng/ml) and the kinetics of the AKT–PH construct was observed by microvideoscopy using Zeiss Axiovert inverted microscope equipped with the Metafluor imaging system. Presented here are the images corresponding to 15 min of Pam3 stimulation. Controls correspond to cells incubated with DMSO and pcDNA empty vector, or cells incubated with LY294002 (25 mM), a specific inhibitor of PI 3-kinase. (B) HEK 293–TLR2 cells were transfected with pcDNA vector (vehicle) or LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Lysates were immunoprecipitated with anti-Flag Abs and recruitment of PI 3-kinase to TLR2 was observed by Western blot with anti-p85a Abs. (C) Tyrosine phosphorylation of the p85a subunit was evaluated by Western blot in HEK 293–TLR2 transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Anti-phosphotyrosine (4G10 clone) and anti-p85a Abs were used for immuno- precipitation and Western blot. (D) HEK 293–TLR2 were transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml) and lysates were immunoprecipitated with either 4G10 or anti-p110 Abs. Phosphorylation of p110 was then revealed by Western blot with anti-p110 or 4G10 Abs. Controls correspond to cells transfected with pcDNA empty vector. (E) THP1–CD14 cells were incubated with PP2 (25mM) or DMSO, stimulated with Pam3 (100 ng/ml) and lysed. Phosphorylation of p110 catalytic subunit of PI 3-kinase was revealed by Western blot of THP1–CD14 lysates immunoprecipitated with either 4G10 or anti-p110 Abs. Controls correspond to cells treated with DMSO. These results are representative of three independent experiments.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Transfection, Incubation, Construct, Inverted Microscopy, Imaging, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 7. Lyn controls the PI 3-kinase pathway through phosphorylation of its p110 subunit after TLR2 engagement. The presence of bacteria-derived acylated lipoproteins results in the heterodimerization of TLR2 with TLR1/6 in membrane microdomains. MyD88/ IRAK are recruited to the receptor and lead to degradation of IkB and translocation of NF-kB subunits. A Rac/PI 3-kinase-dependent signaling pathway has also been described, including CD14 and Lyn that contribute to the activation cluster. After tyrosine phos- phorylation of the cytoplasmic domain of TLR2, the p85a subunit of PI 3-kinase is recruited to the receptor and phosphorylated on tyrosine. A Lyn-dependent tyrosine-phosphorylation is required to activate the PI 3-kinase catalytic subunit p110 that allows for recruitment of AKT to the inner membrane, precluding a cascade that results in transactivation of the p65 subunit of NF-kB. This leads to the nuclear translocation of a functional p50/p65 NF-kB heterodimer that results in the gene expression of pro-inflammatory cytokines.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Bacteria, Derivative Assay, Membrane, Translocation Assay, Activation Assay, Functional Assay, Gene Expression