Journal: Science Advances

Article Title: Functional antagonism of chromatin modulators regulates epithelial-mesenchymal transition

doi: 10.1126/sciadv.abd7974

Figure Lengend Snippet: ( A ) Violin plot showing the distribution of the dependency for proliferation of all CCLE NSCLC cell lines for individual genes within the indicated families. The plot shows the phenotypic dependency calculated by the DepMap project. CRISPR (Avana) CERES score and RNAi DEMETER2 score are displayed in the left and right panels, respectively. Selected genes are labeled within their families. ANOVA, analysis of variance. ( B ) Bar plot of control and mutant A549-MGT#1 cell proliferation in parallel longitudinal analysis. Statistics: Significant by t test and Holm-Sidak post hoc test ( P < 0.05; n = 4), BRD2 , EPC1 , ARID1A , and ACVR1 KOs versus control. ( C ) Bar plot of control and mutant A549-MGT#1 ± GSK126 cell colony formation assay. Statistics: Significant by t test and Holm-Sidak post hoc test ( P < 0.05; n = 3) in DMSO group: ARID2, ARID1A, DOT1L, and ACVR1 KOs; GSK126: EPC1, ARID1A, BRD2, DOT1L, KMT2A, and ACVR1 KOs. ( D ) Left: Line plot of parallel longitudinal high-content wound healing analysis of A549-MGT#1 cells with the indicated genotypes under homeostatic conditions. Each dot represents the mean in each time point. Statistics: Two-way ANOVA and Dunnet post hoc test ( n = 4). Asterisks denote significance for the indicated comparison. Antagonistic regulators of EMT and motility in A549 cells are shown to the right. ( E ) Left: Schematic representation of three-dimensional (3D) invasion assay. Right: Migration depth of DRAQ5-stained nuclei for each time point and clone normalized to time point T = 0 hours from high-content imaging. Statistical analysis for time point 24 hours shows corrected multiple t test (* P < 0.05; *** P < 0.001; n = 4). ( F ) FACS analysis (left) and quantification (right) of MGT#1 expression in lung and brain tumor cells with the indicated genotypes.

Article Snippet: The percentage of editing was analyzed by Sanger sequencing and calculated using the ICE (inference of CRISPR edits) webtool provided by Synthego.

Techniques: CRISPR, Labeling, Control, Mutagenesis, Colony Assay, Comparison, Invasion Assay, Migration, Staining, Imaging, Expressing

Journal: bioRxiv

Article Title: Human variation impacting MCOLN2 restricts Salmonella Typhi replication by magnesium deprivation

doi: 10.1101/2022.05.08.491078

Figure Lengend Snippet: ( A ) MCOLN2 knockout leads to faster S . Typhi replication inside THP-1s. Ten replicates from three experiments, except 6 hpi is six replicates from two experiments. MCOLN2 genotype, hpi, and their interaction are all significant sources of variation (p<0.0001) in a repeated measures ANOVA. Time point p values are from Šídák’s post-hoc comparison of MCOLN2 +/+ and MCOLN2 −/– . ( B ) Workflow used to sequence mRNA from intracellular S . Typhi 16 hpi in wild-type and knock out THP-1s. ( C ) Diagram of S . Typhi’s PhoPQ-induced Mg 2+ importers. ( D ) RNA-seq of intracellular S . Typhi indicates PhoP targets are upregulated more when MCOLN2 is present. Left: Normalized enrichment score (NES) from GSEA of virulence-or cation-associated S . Typhi gene sets. Significant gene set (FDR q<0.05) indicated by asterisk. Right: The log 2 (KO/WT expression) of PhoPQ regulon genes are plotted. 16 of 19 genes have a negative fold-change (FC), indicating higher expression in WT. ( E ) PhoPQ is required for most of the increase in intracellular replication observed with MCOLN2 -/- THP-1s. Nine replicates from two experiments. ( F ) S . Typhi Δ ssaT has no effect on intracellular replication in WT THP-1s and partially accounts for the requirement of phoPQ to achieve maximal replication in MCOLN2 -/- THP-1s. Ten replicates from three experiments. P values in E & F are from Šídák’s comparison of MCOLN2 +/+ to MCOLN2 −/– following two-way ANOVAs finding significant main effects and interaction (all p<0.0001). Statistics in A, E, & F use log 2 -transformed replication ratios. Bars in A, E, & F are mean ±SEM.

Article Snippet: THP-1 knock out pools were confirmed to maintain ≥85% frameshift indels by Sanger sequencing that was analyzed with the inference of CRISPR editing (ICE) webtool v2.0 ( https://ice.synthego.com ) from Synthego( ).

Techniques: Knock-Out, Comparison, Sequencing, RNA Sequencing, Expressing, Transformation Assay