Journal: Nature Immunology
Article Title: Chemosensor receptors are lipid-detecting regulators of macrophage function in cancer
doi: 10.1038/s41590-025-02191-x
Figure Lengend Snippet: a , Experimental scheme of genome-wide CRISPR–Cas9 Knockout GecKO v2 Library B screening in primary murine macrophages. b , Representative plot of backbone (LGP) or library-B-infected macrophages exposed or not to conditioned media from Pten −/− Trp53 −/− tumor cells. c , Two independent experiments were performed. Graphs show the correlations between the distribution of the guides found in the CD206 − MHCII + population (MHCII) and in the CD206 bright MHCII − population (CD206) from the two experiments. Lib1, library 1; Lib2, library 2. d , Volcano plot showing genes related to the differentially enriched sgRNA guides from CD206 − MHCII + versus CD206 bright MHCII − cells. Negative regulators of the CD206 bright MHCII − population are shown in light blue, and positive regulators are shown in red. log 2 FC ± 0.56, P < 0.005. Statistical analyses and comparisons from NGS output were performed with MAGeCK. e , Western blot analysis showing the percentage of expression of total STAT6. Two independent sgRNA guides (g1 and g2) were utilized to silence Stat6 in macrophages ( n = 2). f , g , FACS analysis of control (LGP) and Stat6 -silenced (g1 and g2) macrophages following exposure to Pten −/− Trp53 −/− conditioned media, with events gated on F4/80 + CD11b + cells: LGP n = 5, g1 n = 5, g2 n = 6 ( f ); LGP n = 4, g1 n = 4, g2 n = 4 ( g ). h , Proliferation of CD8 + T cells exposed to supernatant from Untr. and CM-tr. macrophages: bar graph shows the number of divisions. i , Scratch assay: graph and curves showing the distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages ( n = 8). Statistical analyses were performed using two-tailed unpaired Student’s t -test. Values are presented as the mean ± s.e.m. All replicates represent biological replicates. Schematic in a created using BioRender.com .
Article Snippet: The mouse GeCKO v2 Library B (Addgene) was used: this library consists of 62,804 sgRNAs constructs, with three sgRNAs targeting each of the 20,661 genes of the mouse genome.
Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Western Blot, Expressing, Control, Wound Healing Assay, Two Tailed Test
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