Journal: eLife

Article Title: Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage

doi: 10.7554/eLife.86764

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , SIGLEC5 (NM_003830) Human Tagged ORF Clone , Origene , RC206610 , .

Techniques: Blocking Assay, Imaging, Staining, Recombinant, Plasmid Preparation, Expressing, Luciferase, Cell Culture, Transfection

Journal: Glycobiology

Article Title: Deletion polymorphism of SIGLEC14 and its functional implications.

doi: 10.1093/glycob/cwp052

Figure Lengend Snippet: Fig. 2. The absence of Siglec-14 in some individuals and its correlation with their genotype. (A) Expression of Siglec-14 on granulocytes in multiple individuals. Peripheral blood leukocytes from five individuals were stained with an antibody recognizing only Siglec-14 (clone 40-1; solid line) or both Siglec-5 and Siglec-14 (clone 194128, R&D Systems; dotted line), or with a negative control antibody (MOPC-21; gray shaded area), and analyzed by flow cytometry. Some individuals (donors 2, 4, and 5 in this panel) lacked the expression of Siglec-14, while all individuals expressed an epitope shared by Siglec-5 and Siglec-14. (B) Schematic diagram for the genomic PCR experiment. The primer pair 5F + 5R specifically amplifies a part of the SIGLEC5 gene, and the primer pair 14F + 14R a part of the SIGLEC14 gene. Genomic PCR with the primer pair 14F + 5R yields a ∼1.6 kb product only when the SIGLEC14/5 fusion gene is present. This pair can theoretically amplify a ∼17 kb fragment from the wild-type allele as well, but failed to do so under the conditions used. (C) Expected gel electrophoresis pattern of PCR products for each genotype. (D) Genomic PCR results of the same set of individuals as in A. Donors 2, 4, and 5 are homozygotes for the null allele, as expected.

Article Snippet: Two hundred micrograms of commercially available goat anti-Siglec-5/14 antibody (AF1072, R&D Systems, Minneapolis, MN) was mixed with 1 mL of Siglec-14Fc/Affigel and incubated overnight at 4◦C.

Techniques: Expressing, Staining, Negative Control, Cytometry, Nucleic Acid Electrophoresis

Journal: Glycobiology

Article Title: Deletion polymorphism of SIGLEC14 and its functional implications.

doi: 10.1093/glycob/cwp052

Figure Lengend Snippet: Fig. 3. Influence of Siglec-14 expression on TNF-α production from THP-1 macrophages. (A) Levels of Siglec-5 and Siglec-14 expression on THP-1 cells transduced with retroviral expression vectors. THP-1 cells transduced with the empty vector (black line), those expressing wild-type (red), R119A point mutant (orange), or R362A point mutant (green) of Siglec-14, and those expressing Siglec-5 (blue), were stained with an antibody that recognizes both Siglec-5 and Siglec-14 (clone 194128, R&D Systems). Gray shaded area represents staining of the parental THP-1 cells with a negative control antibody (MOPC-21). (B) Influence of Siglec-14 expression on TNF-α production. The expression of wild-type or R119A mutant Siglec-14 significantly enhanced TNF-α production, while the expression of R362A mutant failed to show any effect. Siglec-5 expression also showed no effect. ∗∗∗P < 0.01 (Student’s t-test). (C) Involvement of DAP12 in Siglec-14-induced enhancement of TNF-α production. A specific siRNA that suppresses DAP12 expression reversed Siglec-14-induced enhancement of TNF-α production to the control level, while it did not influence baseline TNF-α production in cells expressing Siglec-14 R362A mutant or mock-infected cells. Control siRNA did not show any effect. Statistical significance of the difference between means of the amount of TNF-α produced under two different treatments of the same cell type (e.g., DAP12 siRNA versus control siRNA treatment of Siglec-14-expressing THP-1) was tested. ∗∗∗P < 0.01 (Student’s t-test). (D) Suppression of DAP12 mRNA by siRNA. RT-PCR and agarose gel electrophoresis were performed as described in Material and methods.

Article Snippet: Two hundred micrograms of commercially available goat anti-Siglec-5/14 antibody (AF1072, R&D Systems, Minneapolis, MN) was mixed with 1 mL of Siglec-14Fc/Affigel and incubated overnight at 4◦C.

Techniques: Expressing, Transduction, Retroviral, Plasmid Preparation, Mutagenesis, Staining, Negative Control, Control, Infection, Produced, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis