Journal: Nature Communications
Article Title: Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia
doi: 10.1038/s41467-023-41925-z
Figure Lengend Snippet: a Schematic illustration of transwell assays in which LN3 T-ALL cells are cultured in the presence or absence of enriched tumor-associated myeloid cells in the same or opposite chambers to determine whether T-ALL cells require close contact with myeloid cells to survive. b Quantification of viable T-ALL cells in the chamber indicated in red 6–7 days after co-culture initiation. Results were normalized to the viability of T-ALL cells making physical contact with tumor-associated myeloid cells in the bottom chamber within each experiment. Bars depict the mean + SEM of cumulative data from n = 7 experiments, each with a distinct color-coded primary T-ALL; symbols represent the mean of 2-3 replicate wells per experiment. c Representative flow cytometry plots and ( d ) quantification ofcell surface integrin expression levels of transplanted LN3 T-ALL cells (red; CD45.2 + CD5 + ) and host T cells (blue; CD45.1 + CD5 + ) from the same leukemic spleens, quantified as median fluorescence intensity (MFI) values by flow cytometry. Isotype control stains are shaded in gray. d Results were normalized to T-cell levels within each experiment. Data are compiled from n = 6 (ITGα4, ITGβ2) or 7 (ITGα9, ITGβ1, ITGαL) independent experiments, each with a distinct color-coded primary T-ALL; symbols represent individual mice. e Quantification of ICAM-1 (left) and VCAM-1 (right) protein levels on the indicated myeloid subsets from the spleens of healthy (blue) or leukemic (red) mice. Results were normalized to healthy controls within each subset. Bars depict the mean + SEM of cumulative data from n = 3 experiments, each with a distinct color-coded primary T-ALL; symbols represent individual mice. Statistical significance was determined by ( b ) a two-sided repeated measures one-way ANOVA with the Holm-Sidak correction, ( d ) two-sided paired Student t -tests, and ( e ) two-sided unpaired Student t -tests; P -values: *<0.05, **<0.01, ***<0.001. ns, not significant. Source data are provided as a Source Data file.
Article Snippet: Cells were then stained with fluorescently labeled antibodies against anti-mouse CD45.1-PerCP/Cy5.5 (A20; 1:100), CD45.2-APC/Cy7 (104; 1:100), CD5-PE/Cy7 (53-7.3; 1:200), pIGF1R-Alexa Fluor 647 (K74-218; pY1131; BD Biosciences; 1:10), pAKT-PE or -Brilliant Violet 421 (J24-618; pS473; BD Biosciences; 1:10), pPYK2-PE (L68-1256.272; pY402; BD Biosciences; 1:10), pFAK (#3283; pY397; Cell Signaling Technology; 1:200), pFAK (#3284; pY925; Cell Signaling Technology; 1:50), pFAK (44–626 G; pY861; Invitrogen; 1:200), donkey anti-rabbit IgG-PE (Poly4064; 1:400), ILK (P83A9; 1:200), donkey anti-rabbit IgG-Alexa Fluor 488 (Jackson ImmunoResearch; 1:400) and goat anti-mouse IgG-Alexa Fluor 488 (Invitrogen; 1:400) antibodies.
Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Expressing, Fluorescence
