CRISPR/Cas9 gene editing of the MyD88 gene locus with mEGFP and p38 signaling in EL4-MyD88-GFP cells. (A–D) Workflow and validation for CRISPR/Cas9 editing of the MyD88 gene locus with mEGFP. (A) Schematic of the MyD88 gene locus and HDR template designed to insert a mEGFP open reading frame immediately upstream of the stop codon. (B) FACS results of WT and CRISPR/Cas9-engineered MyD88-GFP EL4 cells. Overlaid box indicates a sorting gate to select EL4-MyD88-GFP cells. (C) PCR screening of CRISPR/Cas9 editing to select homozygous edited cell clones. Schematic shows the primer design for PCR amplification of genomic DNA to detect WT, heterozygous, and homozygous edited EL4 cells. Homozygous MyD88-GFP edited cell clones are labeled in red. Three homozygous clones were retained for Western blot analysis. See Fig. S6 for uncropped blots. (D) Western blot analysis of three MyD88-GFP clones. Blots were probed with anti-MyD88, then stripped and reprobed with anti-GFP. (E) To assess the level of MAPK pathway activation, EL4 cells were fixed (60 min after SLB contact) and stained for MyD88-GFP (green) and phospho-p38 (magenta); DAPI staining of nuclei (blue). Cells were imaged with confocal microscopy. Schematic shows the position of the confocal micrograph slice. Only cells in contact with SLB functionalized with IL1β had increased phospho-p38 nuclear staining intensity. Scale bar, 5 mm. (F) Reconstructed axial view of cells shown in E showing the localization of MyD88 to the cell–SLB contact zone and phospho-p38 staining under IL1β stimulation. Scale bar, 1 mm. (G) Quantification of phospho-p38 staining intensity. Mean ± SD from n = 12 (with IL1β) and 20 cells (without IL1β). fluo, fluorescence; FSA-A, forward scatter area; MW, molecular weight.