Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: Confocal time-lapse imaging showed the process of extravasation, infiltration and killing of healthy donor-derived CAR T cell in the leukaemia bone marrow chip. Vessel was formed with VE-CAD-GFP-expressing HUVECs (in green). Leukaemia blast was K562-meso-19-mCherry (in yellow). CAR T cells were stained with DiD dye (in red). The video was captured at 5 min per frame within 14 h using Nikon C2i confocal microscopy and a 20× objective.

Article Snippet: Human B-ALL cells (Reh, catalogue number CRL-8286, ATCC), CD19-expressing K562-meso-19, K562-meso-19-GFP and K562-meso-19-mCherry leukaemia cell lines provided by S. Ghassemi’s lab were cultured in RPMI medium (catalogue number 11875135, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; catalogue number A5256701, Thermo Fisher Scientific), 1% penicillin/streptomycin (catalogue number 15140163, Thermo Fisher Scientific), 100 μM l -glutamine (GlutaMAX supplement, catalogue number 35050061, Thermo Fisher Scientific) and 50 μM β-mercaptoethanol (catalogue number 21985023, Thermo Fisher Scientific) in a 37 °C incubator with 5% CO 2 .

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: Confocal time-lapse imaging showed the process of extravasation, infiltration and killing of patient-derived CAR T cell in the leukaemia bone marrow chip. Vessel was formed with VE-CAD-GFP-expressing HUVECs (in green). Leukaemia blast was K562-meso-19-mCherry (in yellow). CAR T cells were stained with DiD dye (in red). The video was captured at 5 min per frame within 14 h using Nikon C2i confocal microscopy and a 20× objective.

Article Snippet: Human B-ALL cells (Reh, catalogue number CRL-8286, ATCC), CD19-expressing K562-meso-19, K562-meso-19-GFP and K562-meso-19-mCherry leukaemia cell lines provided by S. Ghassemi’s lab were cultured in RPMI medium (catalogue number 11875135, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; catalogue number A5256701, Thermo Fisher Scientific), 1% penicillin/streptomycin (catalogue number 15140163, Thermo Fisher Scientific), 100 μM l -glutamine (GlutaMAX supplement, catalogue number 35050061, Thermo Fisher Scientific) and 50 μM β-mercaptoethanol (catalogue number 21985023, Thermo Fisher Scientific) in a 37 °C incubator with 5% CO 2 .

Techniques:

Journal: Communications Chemistry

Article Title: In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies

doi: 10.1038/s42004-025-01644-6

Figure Lengend Snippet: A – C 20 µg DSBSO crosslinked Cas9-Halo peptides, spiked into a non-crosslinked background from human HeLa peptides (1:80, w:w), were either enriched using magnetic beads from Cytiva or Cube Biotech as indicated using 20 µL bead slurry and using the exact same processing workflow. Data was searched against a database containing 20,585 proteins (Cas9 + human proteome). D – F Living K562 cells were crosslinked, and enrichment was applied using both bead types under variation of the used bead slurry volume as indicated. Data was searched against the human proteome. Black dots indicate values of individual replicates and bars indicate identified average numbers of unique XL residue pairs ( A , D ), CSMs and monolink PSMs, carrying a DSBSO modification ( B , E ) or PSMs from linear peptides without any DSBSO modification ( C , F ) at 1% FDR level, and error bars indicate their standard deviation, n = ≥2 ( A – C ) and n 3 ( D – F ). Peptides were separated using a 2 h gradient in trap-and-elute configuration, and data were recorded on an Orbitrap Eclipse instrument with detailed settings indicated in the “Methods” section.

Article Snippet: DDX39B-dTag-GFP mutant K562 (DSMZ) cells were generated as follows: The DDX39B locus was CRISPR modified with a C-terminal FKBP-GFP tag using the Alt-R system of IDT, including gRNA, electroporation, and HDR enhancers.

Techniques: Magnetic Beads, Residue, Modification, Standard Deviation

Journal: Communications Chemistry

Article Title: In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies

doi: 10.1038/s42004-025-01644-6

Figure Lengend Snippet: A – C Living K562 cells were DSBSO crosslinked and processed with or without a C18 cleanup step ahead of affinity enrichment using the Cytiva beads, but without an additional enrichment. D – F Whole K562 cells or their nuclear extracts were DSBSO crosslinked employing a streamlined workflow without C18 enrichment but including orthogonal SEC fractionation. Bars indicate identified average numbers of unique inter- and intra-protein XL residue pairs after combined analysis of four SEC fractions ( A , D ), CSMs, and monolink PSMs, carrying a DSBSO modification ( B , E ) or PSMs from linear peptides without any DSBSO modification ( C , F ) at 1% FDR level. Bars show averages, dots show values from each replicate, and error bars indicate their standard deviation, n = 3 independent replicates.

Article Snippet: DDX39B-dTag-GFP mutant K562 (DSMZ) cells were generated as follows: The DDX39B locus was CRISPR modified with a C-terminal FKBP-GFP tag using the Alt-R system of IDT, including gRNA, electroporation, and HDR enhancers.

Techniques: Fractionation, Residue, Modification, Standard Deviation

Journal: Communications Chemistry

Article Title: In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies

doi: 10.1038/s42004-025-01644-6

Figure Lengend Snippet: Venn diagrams show commonly identified unique XL sites across the acquired SEC fractions from a representative replicate each from whole K562 cells ( A ) and K562 nuclei ( B ), respectively. C – F Changes of crosslink ID numbers depending on database size. Bar plots showing either all XL unique XL sites from whole cells ( C ) and nucleus ( D ) or within those only unique XL sites found on either DDX39A or B from whole cell ( E ) or nuclear extract ( F ) samples. All fractions and n = 3 replicates for each condition were searched together at 1% FDR.

Article Snippet: DDX39B-dTag-GFP mutant K562 (DSMZ) cells were generated as follows: The DDX39B locus was CRISPR modified with a C-terminal FKBP-GFP tag using the Alt-R system of IDT, including gRNA, electroporation, and HDR enhancers.

Techniques:

Journal: Communications Chemistry

Article Title: In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies

doi: 10.1038/s42004-025-01644-6

Figure Lengend Snippet: A DDX39B-GFP expressing and wt-K562 cells were DNA stained using DAPI and inspected by fluorescence microscopy at 10x or 63x magnification as indicated. GFP signal shown in green and DAPI signal shown in blue. Crosslinks found within the nuclear extracts/ analyzed using the shotgun database, plotted on the native DDX39B ( B ) or the tagged DDX39B-FKB-GFP ( C ) rank 1 3D structure model predicted from AlphaFold3 and their respective diagnostic plots. For ambiguous crosslinks, only the shortest possible connection, when plotted to the predicted 3D structure from AlphaFold2, is shown. Shown in green are crosslinks ≤35 Å and in red links >35 Å. D Possible K-K connections with ≤35 Å from previously non-satisfied crosslinks after plotting onto the monomeric DDX39B structure, now plotted to the predicted structure of the DDX39A and B complex and allowing links to fit on either protein or across both proteins. E Histogram of measured Cα to Cα distances when plotting found crosslinks to the predicted 3D structures as indicated.

Article Snippet: DDX39B-dTag-GFP mutant K562 (DSMZ) cells were generated as follows: The DDX39B locus was CRISPR modified with a C-terminal FKBP-GFP tag using the Alt-R system of IDT, including gRNA, electroporation, and HDR enhancers.

Techniques: Expressing, Staining, Fluorescence, Microscopy, Diagnostic Assay