Fig. 4
From: Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo
Molecular reconstitution confirms essentiality of ADAM10 in PDX ALL in vivo. A Schematic illustration of different ADAM10 expression variants. Upper construct: full-length ADAM10 protein, middle panel: constitutive catalytically active ADAM10 variant (ACT), lower panel: enzymatically inactive ADAM10 variant, lacking the metalloproteinase domain (ΔMP); SP: signal peptide, PRO: prodomain, DI: disintegrin domain, CR: cysteine-rich domain, TM: transmembrane domain, CP: cytoplasmic domain. B Confocal microscopy pictures of expression of recombinant ADAM10. ADAM10 was stained (Alexa Fluor (AF) 647) on fixed un-permeabilized HEK293T control (Plain), ADAM10 KO cells or ADAM10 KO cells with re-expression of the active ADAM10 construct (ADAM10 KO + ACT). DAPI was used for nuclear staining. Representative images of three independent experiments are shown. C Immunoblot of ADAM10 expression in crude cell lysates (cytosol) and membrane fraction (membr.) in HEK293T wildtype or ADAM10 KO cells with or without expression of constitutively active-ADAM10 (ACT). Syntaxin 4 and β-Actin were used as membrane protein marker and loading control, respectively. D Workflow of in vivo competitive ADAM10 reconstitution assays. E Representative flow cytometry analysis of T-Sapphire expression. T-Sapphire expression levels were compared between the ADAM10 KO PDX cells transduced with the iRFP mock vector (CTRL) and the active-ADAM10 (ACT)- or ΔMP-ADAM10 (ΔMP)-expressing cells after isolation from the animals. One representative histogram out of three individual animals per group is shown. F ADAM10 mRNA expression in parental split-Cas9-positive ALL-199 (CTRL), ADAM10 KO ALL-199 cells transduced with the iRFP mock vector (KO + CTRL) and ADAM10 KO reconstituted with the ACT or ΔMP variants was determined by qRT-PCR and normalized to GAPDH. Box indicates median, 25th and 75th percentile; whiskers indicate 25th percentile - 1.5 IQR (inter-quartile distance) and 75th percentile + 1.5 IQR. * p < 0.05 by unpaired t-test. G Distribution of subpopulations of the competitive in vivo ADAM10 reconstitution assay. Percentages of cells expressing the indicated ADAM10 variant from the injection mixture (Input) are compared to PDX cells isolated from murine BM (Output) after a similar in vivo growth period of 8–9 weeks. Violin plot with median indicated by dashed line and 25th and 75th percentile by dotted lines. Data of three animals is shown. ns not significant, * p < 0.05 by paired t-test