Journal: Arthritis Research & Therapy

Article Title: Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

doi: 10.1186/ar4456

Figure Lengend Snippet: α(1,2)-linked fucosylated proteins are expressed in rheumatoid arthritis (RA). (A) RA synovial tissue (ST) homogenates contained more α(1,2)-linked fucosylated proteins than did osteoarthritis (OA) or normal (NL) STs (normalized to total protein concentration). Results are expressed as a ratio of the amount of fucosylated BSA/total proteins in the ST homogenates using BSA as a standard. (B) Photographs of RA ST. The far left panel shows staining with mouse anti-collagen 1. The middle panel shows staining with Ulex Europeaus Agglutinin 1 lectin (UEA-1) and goat anti-UEA-1. The right panel shows merging of the left panel and middle panel. Yellow indicates α(1,2)-linked fucosylated proteins associated with RA ST fibroblasts and the blue indicates DAPI staining of the tissue (original magnification 100×). (C) α(1,2)-linked fucosylated proteins in RA synovial fibroblast-conditioned medium were significantly higher than in OA and NL synovial fibroblast-conditioned medium. (D) α(1,2)-linked fucosylated proteins in RA synovial fibroblast cell lysates were significantly higher than in NL synovial fibroblast cultures: 2′fucosyllactose-bovine serum albumin (2′FL-BSA) was used as a standard.

Article Snippet: UEA-1, 2 μg/ml (Vector laboratories Inc, Burlingame, CA, USA) was added for 90 minutes followed by 10 μg/ml biotinylated goat anti-UEA-1 antibody (Vector laboratories Inc) for 60 minutes, then streptavidin-horseradish peroxidase (HRP) for 30 minutes.

Techniques: Protein Concentration, Staining

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: Arthritis Research & Therapy

Article Title: Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

doi: 10.1186/ar4456

Figure Lengend Snippet: α(1,2)-linked fucosylated proteins are expressed in rheumatoid arthritis (RA). (A) RA synovial tissue (ST) homogenates contained more α(1,2)-linked fucosylated proteins than did osteoarthritis (OA) or normal (NL) STs (normalized to total protein concentration). Results are expressed as a ratio of the amount of fucosylated BSA/total proteins in the ST homogenates using BSA as a standard. (B) Photographs of RA ST. The far left panel shows staining with mouse anti-collagen 1. The middle panel shows staining with Ulex Europeaus Agglutinin 1 lectin (UEA-1) and goat anti-UEA-1. The right panel shows merging of the left panel and middle panel. Yellow indicates α(1,2)-linked fucosylated proteins associated with RA ST fibroblasts and the blue indicates DAPI staining of the tissue (original magnification 100×). (C) α(1,2)-linked fucosylated proteins in RA synovial fibroblast-conditioned medium were significantly higher than in OA and NL synovial fibroblast-conditioned medium. (D) α(1,2)-linked fucosylated proteins in RA synovial fibroblast cell lysates were significantly higher than in NL synovial fibroblast cultures: 2′fucosyllactose-bovine serum albumin (2′FL-BSA) was used as a standard.

Article Snippet: To determine if α(1,2)-linked proteins were expressed on RA ST synovial fibroblasts, mouse anti-human collagen-1 (Abcam, Cambridge, MA, USA) and goat anti-UEA-1 (Vector laboratories Inc.) were used.

Techniques: Protein Concentration, Staining