Journal: bioRxiv
Article Title: Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer
doi: 10.1101/2020.01.10.901579
Figure Lengend Snippet: A Phenotypes of patient-derived organoid lines P009T and P013T. B UMAPs of organoid single cell transcriptomes. Organoid lines and medium conditions as indicated. LGR5-ISC stem cell, enterocyte and Goblet cell signatures are visualized. C Schematic representation of SLAM-Seq workflow to infer RNA dynamics (“RNA velocity”). In short, organoids were passaged and assigned to different medium conditions. After three days, nascent RNA was metabolically labelled for 2 h using 4sU. Organoids were harvested, dissociated, and fixed. RNA in single cells was alkylated, and cells were subjected to single cell sequencing. Reads were assigned to nascent or old RNA status, depending on diagnostic T-C mutational status. D Developmental trajectories inferred from RNA metabolic labelling. Bold and thin arrows indicate high versus low directionality of RNA velocity. Colors below RNA velocity show latent time (yellow: early latent time; blue: late latent time). E Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time. F Activities of TFF3 , FABP1 , MKI67 and MMP7 in organoid single cell transcriptomes, ordered along latent time. Color code for panels D-F: Red: high activity; blue: low activity.
Article Snippet: Immunostainings were performed on the BenchMark XT immunostainer (Ventana Medical Systems), using CC1 mild buffer or Ultra CC1 buffer (Ventana Medical Systems) for 30 min at 100°C, and using antibodies rabbit anti-TFF3 (1:250, Abcam, ab108599), mouse anti-FABP1 (1:1000, Abcam, ab7366), rabbit anti-OLFM4 (1:100, Atlas Antibodies, HPA077718), mouse anti-EPCAM (1:100, ThermoScientific, MS-144-P1), rabbit anti-Ki67 (1:400, Abcam, ab16667), mouse anti-Ki67 (1:50, Dako, M7240), rabbit anti-LYZ (1:1500, Abcam, ab108508), rabbit anti-EREG (1:50, Thermo Fischer Scientific, PA5-24727), anti-PARP1, mouse anti-MUC2 (1:50, Leica, NCL-MUC-2), mouse anti-CK17 (1:10, Dako, M7046), mouse anti-MMP7 (1:100, ThermoFisherScientific, MA5-14215).
Techniques: Derivative Assay, Metabolic Labelling, Sequencing, Diagnostic Assay, Activity Assay