pcc 01  (Addgene inc)

Journal: bioRxiv

Article Title: Chemical perturbations impacting histone acetylation govern colorectal cancer differentiation

doi: 10.1101/2024.12.06.626451

Figure Lengend Snippet: A. Immunoblot of KRT20 and GAPDH proteins in LS180 treated with MRK60 (2 µM) and indicated concentrations of JQAD1 (0.4 - 10 µM). B. Histone-enriched immunoblot of H3K27ac and H3 in HT-29 treated with MRK60 (5 µM) +/- JQAD1 (10 µM). C. Quantification of GFP+ percentage in HT29 KRT20-GFP reporter cell line treated with MRK60 (5 µM) +/- JQAD1 (10 µM). D. Ranked log 2 fold change of sgRNA distribution in GFP high (perturbation promotes differentiation) and mKate2 high (perturbation promotes stem cell) sorted cell fractions of published CRISPR-Cas9 screen targeting epigenetic regulators (78 genes 542 sgRNAs) using HT29 SOX9-mKate2/KRT20-GFP reporter cell line 16 ; select perturbations indicated in colored circles. E. Schematic of scRNA-seq experiment in patient-derived CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1 combination (left). UMAPs of scRNA-seq colored by treatment group (middle) or cell type (right). F. Proportion of cell types in CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1. G. Trajectory analysis of colonocytes in scRNA-seq experiment colored by treatment group. H. Expression of colonocyte differentiation signatures along drug response trajectory. Density plot shows histogram of cells in different treatment along pseudotime. ** P = 0.0042; *** P = 0.0002 I. Violin plot of colonocyte differentiation gene signature in scRNA-seq experiment by treatment groups. **** P = 7.0 x 10 -6 (left), **** P = 8.4 x 10 -8 (right) J. Violin plot of gene signatures associated with MRK60-induced gains in H3K27ac bound by HDAC1/2 in scRNA-seq experiment by treatment groups. **** P = 5.8 x 10 -10 (left), **** P = 2.1 x 10 -5 (right) K. Integrative heatmap of expression changes in stem cell and differentiation genes associated with H3K27ac, H3K9ac, and H4K8ac in bulk and single-cell RNA data sets with MRK60 and/or JQAD1.

Article Snippet: The pCC_01 - hU6-BsmBI-sgRNA(E + F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE plasmid (Addgene #139086; RRID:Addgene_139086) was used for CRISPR knockout.

Techniques: Western Blot, CRISPR, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Chemical perturbations impacting histone acetylation govern colorectal cancer differentiation

doi: 10.1101/2024.12.06.626451

Figure Lengend Snippet: A. Venn diagram showing genes bound by HDAC1/2 and MRK60, upregulated by MRK60, and gained H3K27ac upon MRK60 treatment. B. Schematic diagram showing CRISPR-Cas9 genetic screen in HT29 KRT20-GFP reporter cells. C. Ranked normalized Z-scored log 2 fold change (LFC) plots of sgRNA targeting 27 genes in HT29 KRT20-GFP reporter screen with respect to viability (top) and GFP/KRT20 (bottom) outputs D. mRNA and (E) protein expression of KRT20 and DAPK3 in HT115 cells engineered to suppress DAPK3 by CRISPRi knockdown treated with 2µM MRK60 for 72 hours. F. Integrative Genomic Viewer (IGV) snapshot depicting HDAC1/2 binding, H3K27ac, H3K9ac, and H4K8ac CUT&RUN peaks and ATAC-seq profiles at DAPK3 genomic locus. G. Immunoblots of Cdx2 CreERT2 ; Apc f/f ; R26 tdT adenoma mouse organoids treated with 5µM MRK60 and indicated doses of Ruxolitinib at 72 hours. H. Schematic diagram showing a proposed differentiation mechanism mediated by HDAC1/2 and H3K27ac

Article Snippet: The pCC_01 - hU6-BsmBI-sgRNA(E + F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE plasmid (Addgene #139086; RRID:Addgene_139086) was used for CRISPR knockout.

Techniques: CRISPR, Expressing, Knockdown, Binding Assay, Western Blot