Journal: bioRxiv

Article Title: In vivo screening of tumor-hepatocyte interactions identifies Plexin B2 as a gatekeeper of liver metastasis

doi: 10.1101/2023.05.15.540681

Figure Lengend Snippet: a , Glutamine synthetase immunoreactivity in AlbCre;dCas9-SPH mice upon co-injection of SB100X and PT4-U6-sgGlul or PT4-U6-sgNT . Scale bar, 100 μm. CV, central vein. b , Glul gene expression of whole liver extracts, as assessed by qRT-PCR. Two-tailed unpaired t-test. n = 2. Log2FC normalized to Gapdh. c , Hydrodynamic tail vein injection (HTVI) of SB100X and PT4-CMV-GFP transposon. Top, endogenous GFP fluorescence. Scale bar, 100 μm. Bottom left, representative flow cytometry plot of GFP + hepatocytes (gated as CD45 - CD31 - ) 1 week after co-injection of SB100X and PT4-CMV-GFP . Bottom right, percentage of GFP + hepatocytes. AlbCre;dCas9-SPH mice (n = 5). Barplots indicate mean ± standard deviation (SD). d , sgRNA abundance in plasmid vs sorted GFP + hepatocytes. Pearson’s correlation test. e , Representative micrograph of AKPS organoids expressing sLP-mCherry. Scale bar, 50 μm. Inset shows sLP-mCherry punctae. Scale bar, 10 μm. f , Representative flow cytometry plots of mCherry niche-labeling in vivo upon AKPS injection. Gated on CD31 - CD45 - hepatocytes. g , Fluorescent micrograph of AKPS hepatic metastasis expressing sLP-mCherry 1 week post intrasplenic injection. Nuclei are labeled with DAPI. Scale bar, 100 μm. Arrowheads indicate sLP-mCherry punctae in labeled proximal hepatocytes. Scale bar, 20 μm.

Article Snippet: Transposon plasmid (PT4-CMV-GFP (Addgene #11704), PT4-U6-sgRNA-CMV-GFP) and transposase overexpression plasmid pCMV(CAT)T7-SB100 (Addgene #34879) was allowed to equilibrate at room temperature (RT), transferred to a 3 mL syringe mounted with a 27G needle, and then injected into the tail vein in a continuous motion.

Techniques: Injection, Gene Expression, Quantitative RT-PCR, Two Tailed Test, Fluorescence, Flow Cytometry, Standard Deviation, Plasmid Preparation, Expressing, Labeling, In Vivo