Journal: Scientific Reports

Article Title: Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells

doi: 10.1038/s41598-021-92754-3

Figure Lengend Snippet: Characterization of EPCs and comparison of young (passage 4, P4)/senescent (passage 8, P8) porcine EPCs. Subconfluent EPCs were fixed with 4% paraformaldehyde (PFA) and stained with the indicated antibodies against UEA-1 (green a1). EPCs were incubated with DiI-labelled ac-LDL (red) for 4 h before PFA fixation (a2). Cell nuclei were visualized by DAPI staining (blue, a3, a7 and a11). Images were then merged (a4, a8 and a12). Cells were stained with anti-CD31 and anti-VE-cadherin (a5 and a6) antibodies. Panels a9 and a10 show cells stained with anti-vWF and anti-eNOS antibodies. Images were acquired by confocal microscopy with a 40 × (oil, na 1.30) magnification objective (SP8, Leica, Germany). Scale bar: 50 μm. P4 (young group) and P8 (senescent group) cells were evaluated for doubling time using a cell counting kit-8 (CCK-8; b ), for acidic β -galactosidase activity using acidic β -galactosidase staining ( c ), and for telomere length using real-time PCR ( d ). The telomere (T) repeat copy number was normalized to the single-copy (S) gene 36B4 copy number. The T/S ratio of the control group was set as 1. n = 6 for each bar. * P < 0.05 vs. the young (P4) group. h = hours. RFU = relative fluorescence units.

Article Snippet: Fluorescein isothiocyanate (FITC)-labelled Ulex europaeus agglutinin -1 (UEA-1) lectin (GTX01512) was purchased from GeneTex (CA, USA).

Techniques: Staining, Incubation, Confocal Microscopy, Cell Counting, CCK-8 Assay, Activity Assay, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Autophagy

Article Title: LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development

doi: 10.1080/15548627.2022.2074105

Figure Lengend Snippet: The deficiency in Lamp2 does not affect global cTEC/mTEC differentiation. (A) Expression (RNAseq analysis) of LAMP family members in cTECs (green) and mTECs (red) from WT mice. Error bars represent mean ± SEM. (B) LAMP2 expression in distinct thymic cell populations in 2-week-old WT and lamp2 KO mice. Left histogram: Hematopoietic (PTPRC/CD45 + ), endothelial (PECAM1/CD31 + ), mesenchymal (PDGFRA-PDGFRB [αβ] + ) and TEC (EPCAM + ) cells (n = 3), (WT and lamp2 KO subsets in color and gray, respectively); Right histogram: cTECs (ENPEP/Ly51 + UEA-1 − ), mTEC lo (ENPEP/Ly51 − UEA-1 + CD80 lo ) and mTEC hi (ENPEP/Ly51 − UEA-1 + CD80 hi ). Bars graphs represent mean fluorescence intensity (MFI) of LAMP2 expression. Data representative of 3 independent experiments (n = 12 animals). (C) cTEC and mTEC composition in embryonic day 15.5 (E15.5) and 2-week-old WT and lamp2 KO thymus. Dot plots show a representative ENPEP/Ly51 and UEA staining and graphs represent the average cellularity of c/mTECs in the indicated timepoints from 3 independent experiments (n = 11–14 animals/group). (D) Immunofluorescence analysis of thymic sections from 2-week-old WT and lamp2 KO thymus stained for DAPI (blue), UEA-1 (red) and KRT8 (keratin 8; green). Results in A-C are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The binding of biotinylated Ulex europaeus agglutinin-1 (UEA-1) (Vector Laboratories, B-1065-2), anti-ENPEP/LY51 (clone 6C3; BD Pharmingen, 553,159), anti-LY75/CD205 (clone NLDC-145; Biolegend, 138,211), and anti-PDGFRB (clone APB5; Biolegend, 136,009) were revealed by either PE-Cy7-conjugated (eBioscience, 25–4317.82) or BV711-conjugated (Biolegend, 405,241) streptavidin.

Techniques: Expressing, Fluorescence, Staining, Immunofluorescence

Journal: Autophagy

Article Title: LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development

doi: 10.1080/15548627.2022.2074105

Figure Lengend Snippet: Defective autophagy and MHC II processing in lamp2 KO cTECs. (A-B) Autophagic flux in cTECs from 12 days old WT and lamp2 KO mice was analyzed by flow cytometry. (A) Histograms show representative Cyto-ID analysis in cTEC and mTEC from WT and lamp2 KO. Graphs represent the MFI of Cyto-ID in the indicated subsets. (B) Scheme (top left) represents autophagic flux using RFP-GFP-LC3 mice. Differential detection of GFP and RFP allows the distinction between AP (GFP + RFP + ) and AL (RFP + ). Dot plot (bottom left) represents GFP and RFP expression in cTECs of WT and lamp2 KO RFP-GFP-LC3 mice. Graphs (top right) represent the average frequency of RFP + GFP hi (yellow) and RFP + GFP low (red) cells in WT and lamp2 KO RFP-GFP-LC3 cTECs. Graphs (bottom right) represent the MFI of GFP and RFP expression in WT (gray) and lamp2 KO (blue) RFP-GFP-LC3. (C) WT and lamp2 KO RFP-GFP-LC3 cTECs (EPCAM + ENPEP/LY51 + ) were analyzed by imaging flow cytometry. Representative images are shown. Graph show the distribution of the number of RFP puncta in WT (n = 1345 cells) and lamp2 KO (n = 1422 cells) RFP-GFP-LC3 cTECs. Data in A-C include an average of 2–3 independent experiments (n = 4–6 WT and n = 4–6 lamp2 KO. (D) Flow cytometry analysis of 15G4 staining on cell surface of cTECs (ENPEP/LY51 + UEA-1 − ) and mTECs (ENPEP/LY51 − UEA-1 + ) from 12 days old WT and lamp2 KO mice. Histograms show representative staining and graphs represent the MFI of 15G4 staining in the indicated subsets. Data include an average of 4 independent experiments (n = 6 WT and 7 lamp2 KO). (E) Histograms show representative 15G4 staining on the cell surface of WT and lamp2 KO c/mTECs from lamp2 WT/KO heterozygous mice. Graphs represent the MFI found in indicated subsets in control (gray) and mutant (blue) cells (n = 8 from 4 experiments). (F) Postnatal day 3–5 WT thymus were treated overnight with chloroquine (50 μM) or with Baf-A1 (0.5 μM) for 5 h. Histograms show the representative staining with 15G4 staining in control (light gray) and chloroquine-treated (black) or Baf-A1-treated (black) in WT cTECs. Graphs represent the MFI in control (gray) and treated (dark gray/ black) cTEC (n = 4–6 animals from 3–4 independent experiments). Results in A-F are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The binding of biotinylated Ulex europaeus agglutinin-1 (UEA-1) (Vector Laboratories, B-1065-2), anti-ENPEP/LY51 (clone 6C3; BD Pharmingen, 553,159), anti-LY75/CD205 (clone NLDC-145; Biolegend, 138,211), and anti-PDGFRB (clone APB5; Biolegend, 136,009) were revealed by either PE-Cy7-conjugated (eBioscience, 25–4317.82) or BV711-conjugated (Biolegend, 405,241) streptavidin.

Techniques: Flow Cytometry, Expressing, Imaging, Staining, Mutagenesis

Journal: Journal for Immunotherapy of Cancer

Article Title: Nanoemulsion adjuvantation strategy of tumor-associated antigen therapy rephrases mucosal and immunotherapeutic signatures following intranasal vaccination

doi: 10.1136/jitc-2020-001022

Figure Lengend Snippet: Tailoring emulsified particles to monodisperse nanosized distribution rephrases mucosal signatures following intranasal vaccination. (A) Schematic illustration of the monodisperse nanoemulsion enables the antigens to pass through the mucosal epithelium and facilitate the transportation of antigens into lymphoid tissues. (B) Microarray analysis of transcription profiles induced by emulsified particles 20 hours after administration. Genes with a fold change ≥1.5 and p<0.05 compared with the PBS control. (C) Membranous (M) cell emergence and natural killer (NK) cell trafficking in nasal mucosa. Nasal mucosal tissues were harvested and phenotyped by immunohistochemical (IHC) staining. The brown signal around the blue nucleus indicates UEA-1+ and CD335+ cells, respectively (magnification, 400×). APCs, antigen-presenting cells. PBS, phosphate-buffered saline.

Article Snippet: The primary antibodies, anti-CD3, anti-CD4, anti-CD8, anti-CD335 NKp46 (BioLegend) and anti-UEA-1 (MBL, IL, USA), were applied, and the sections were then incubated at room temperature for 1 hour.

Techniques: Microarray, Immunohistochemical staining, Immunohistochemistry