Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) CD4 surface levels on purified primary CD4 + T cells after stimulation with phytohemagglutinin (PHA). The gray-shaded histogram represents the isotype control. ( B ) ADAM10 surface levels on the same cells after nucleofection of Cas9 complexed with non-targeting (NTC) sgRNA or the TS1 sgRNA targeting ADAM10 . ( C ) ADAM10 surface levels on primary CD4 + T cells from the same donor sorted into ADAM10 + and ADAM10 − pools. ( D ) Virus replication in the FACS-sorted pools from the same donor after infection with equal amounts (0.5 ng of p24/ml) of Nef + or Nef − HIV-1 NL4-3 . Virus replication was monitored by p24 ELISA. ( E ) Replication of Nef + or Nef − HIV-1 NL4-3 in FACS-sorted ADAM10 + and ADAM10 − pools of primary CD4 + T cells from another donor (donor B) infected as in (D). Infections with Nef − HIV-1 NL4-3 were performed in triplicate. ( F ) Mean p24 values in the supernatants of the ADAM10 + and ADAM10 − pools from donor B on day 13 postinfection (pi) with Nef − HIV-1 NL4-3 ( n = 3). ** P < 0.01 (two-tailed unpaired t test).

Article Snippet: To knock out ADAM10 in Jurkat E6.1, 293T (RRID:CVCL_0063), or primary CD4 + T cells, preassembled ribonucleotide complexes consisting of purified Cas9 (Synthego) and chemically modified synthetic sgRNA TS1 (target sequence: 5′-GATACCTCTCATATTTACAC-3′) (Synthego) were delivered via nucleofection using the Cell Line Nucleofector Kit V with the Nucleofector II device (Lonza).

Techniques: Purification, Control, Virus, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell reports

Article Title: Overcoming CD226-related immune evasion in acute myeloid leukemia with CD38 CAR-engineered NK cells

doi: 10.1016/j.celrep.2024.115122

Figure Lengend Snippet:

Article Snippet: Anti-human CD2 (TS1/2) – 151Eu , Standard Biotools , Cat# 3151003B; RRID: AB_3106938.

Techniques: Purification, Functional Assay, Virus, Recombinant, Binding Assay, Staining, Western Blot, Modification, Transfection, Fluorescence, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Cell Isolation, Plasmid Preparation, Generated, Sequencing, Software

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 1 | Identification of factors colocalizing with LFA1 intracellular clusters. a LFA1 (αLβ2) intracellular cluster (ICC) formation concomitant with lymphocyte adhesion. Adhesion of lymphocytes (BAF/LFA1) to the ICAM1-displaying surface via LFA1 results in the formation of LFA1 ICCs inside the cell. LFA1 was visualized by fusing a SNAP tag to the C-terminus of β222. b Colocalization of β2-SNAP (shown in magenta) with factors involved in the regulation of vesicular trafficking (shown in green). Colocalized spots are indicated by arrows. Colocalization of LFA1 (β2) with TGN38 (TGN, n = 15), Rab7 (n = 9) or LC3 (n = 12) was quantified using Mander’s coefficient22. c Co-trafficking of β2-SNAP and CLIP-LC3 in adhered lymphocytes. Yellow arrows and cyan arrowheads indicate the ICCs containing both β2 and LC3 (see also Supplementary Movie 1). d Trajectories of co-trafficked ICCs indicated

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques:

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 2 | Importance of ATG8 family proteins for LFA1-dependent lymphocyte adhesion. a Western blotting analysis of LC3b and GABARAP in BAF/LFA1. LC3b- KO: BAF/LFA1 with deletion of LC3b; GABARAP-KO: BAF/LFA1 with deletion of GABARAP. Two independent experiments were performed. b FACS analysis of LFA1 expression in BAF/LFA1 cells. Surface expression of LFA1 was monitored with monoclonal antibodies (Mean Fluorescence Intensity, MFI: αL [No staining, WT, LC3b-KO, GABARAP-KO] = [83.3, 22400, 22300, 26500], β2 [WT, LC3b-KO, GABARAP-KO] = [23300, 16000, 23900]). Total expression of LFA1 was monitored using the SNAP tag protein fused to β2 (MFI: SNAP [No staining, WT, LC3b-KO, GABARAP-KO] = [77.1, 33700, 30300, 39900]). c Adhesion assay. Areas of cell adhesion were monitored by interference reflection microscopy. WT: n = 45; LC3b- KO: n = 45; GABARAP-KO: n = 48. d Accumulation of LFA1 at the contact surface. Surface LFA1 was stained with a dye-conjugated non-blocking monoclonal antibody and adhered to ICAM1-coated dishes in the presence of PMA. The fluorescence intensity of LFA1 at the contact surface was measured by TIRFM. WT: n = 36; LC3b-

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques: Western Blot, Expressing, Bioprocessing, Fluorescence, Staining, Cell Adhesion Assay, Microscopy, Blocking Assay

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 3 | Outside-in signaling–induced LC3 clustering is driven by AMPK acti- vation. a Role of outside-in signaling in LC3 and LFA1 co-clustering. Merged images of β2-SNAP (green) and CLIP-LC3 (red) in BAF/LFA1 cells are shown. Inside-out stimulation was mediated by PMA and SDF1. Outside-in stimulation was mediated by the following monoclonal antibodies coated on the glass bottom dish: TS1/18, which induces low-affinity dependent outside-in signaling22, and mAb24, which induces high-affinity LFA1-dependent outside-in signaling. Both inside-out and outside-in signaling pathways are activated by ICAM1 + PMA, which induces the binding of lymphocyte LFA1 to ICAM1 in a PMA stimulation-dependent manner. ICAM1-dependent adhesion of BAF/LFA1 was already verified11. Three independent experiments were performed. Scale bar: 5 µm. b Western blotting analysis of the phosphorylation of mTOR and AMPK substrates. S6K: p70 S6 kinase, a substrate of mTOR; ACC: acetyl-CoA carboxylase, a substrate of AMPK; ULK1: Unc-51 like autophagy activating kinase 1. mTOR: n = 4; AMPK: n = 4. c Effects of rapamycin and

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques: Bioprocessing, Protein-Protein interactions, Binding Assay, Western Blot, Phospho-proteomics

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 4 | The relevance of the non-canonical autophagy in outside-in signaling- dependent LFA1–LC3 co-clustering and lymphocyte adhesion. a Colocalization analysis of LFA1 + LC3+ cluster with a tandem fusion of the evectin-2 pleckstrin- homology (PH) domain (2×PH) fused with GFP. The colocalization indices of LFA1 (β2) with LC3 or 2×PH, and that of LC3 with 2×PH were calculated (n = 13). b Effects of ATG16L1 deletion and expression of ATG16L1 mutants on lymphocyte adhesion. WT: n = 47; ATG16L1-KO: n = 57; ATG16L1-KO expressing GFP-fused ATG16L1 WT (+WT, n = 49), ΔFBD (+ΔFBD, n = 57), or ΔWD (+ΔWD, n = 51) protein. c Effects of

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques: Expressing