Figure 8. CRISPR/Cas9-mediated genome editing reveals impaired TLR3- and TLR4-induced cytokine production in LYST-mutant human cells. (A) Schematic representation of the human LYST locus. The enlargement shows the genomic region from exon 49 to 50 of human LYST. Red, coding sequence; gray, predicted WD40 repeats; green, target sequences for Cas9n-mediated double nicking in exon 49. CDS, coding sequence. (B) Strategy for Cas9n-mediated double nicking (red arrowheads) in exon 49 of human LYST. Target sequences (blue), protospacer-adjacent motifs (PAM; red), and the distance of Cas9n-induced nicks are indicated. (C) Sequence analysis shows indel mutations for all three alleles of LYST in THP-1 clone 8.3 and clone 12.1. Red dashes indicate deleted bases, red letters show inserted bases, and numerals indicate total number of deleted/inserted base pairs. (D) WT and LYST-mutant human THP-1 cells were differentiated into macrophage-like cells. After fixation and permeabilization, cells were stained with anti–human Lamp1 (Sigma-Aldrich) and DAPI. Representative individual and overlay fluorescence images were obtained on a microscope (IX81; Olympus). Subsequent image analysis and pseudocolor processing was performed by using iVision software (BioVision Technologies). Bars, 10 µm. BF, bright field. (E) Differentiated CD14+ WT and LYST-mutant THP-1 cells were stimulated for 4 h with the indicated reagents in the presence of brefeldin A. Flow cytometric analysis shows intracellular TNF expression on CD14+ cells. Shaded, WT; red lines, LYST-mutant clone 8.3; blue lines, LYST-mutant clone 12.1. (F) Differentiated CD14+ WT and LYST-mutant THP-1 cells (clones 8.3 and 12.1) were stimulated for 2 h with 20 ng/ml LPS, 10 µg/ml poly(I:C), or left untreated. IFNB1 mRNA levels were determined by quantitative real-time PCR of duplicate measurements. The expression level of nonstimulated WT THP-1 cells was set as 1. (G) Differentiated CD14+ WT and LYST-mutant THP-1 cells (clones 8.3 and 12.1) were stimulated for 2 h with PMA/ionomycin or left untreated. IL8 mRNA levels were determined by quantitative real-time PCR of duplicate measurements. (F and G) Data are mean ± SD. All data are representative for at least two independent experiments. iono, ionomycin; rel., relative.
Figure 8.

CRISPR/Cas9-mediated genome editing reveals impaired TLR3- and TLR4-induced cytokine production in LYST-mutant human cells. (A) Schematic representation of the human LYST locus. The enlargement shows the genomic region from exon 49 to 50 of human LYST. Red, coding sequence; gray, predicted WD40 repeats; green, target sequences for Cas9n-mediated double nicking in exon 49. CDS, coding sequence. (B) Strategy for Cas9n-mediated double nicking (red arrowheads) in exon 49 of human LYST. Target sequences (blue), protospacer-adjacent motifs (PAM; red), and the distance of Cas9n-induced nicks are indicated. (C) Sequence analysis shows indel mutations for all three alleles of LYST in THP-1 clone 8.3 and clone 12.1. Red dashes indicate deleted bases, red letters show inserted bases, and numerals indicate total number of deleted/inserted base pairs. (D) WT and LYST-mutant human THP-1 cells were differentiated into macrophage-like cells. After fixation and permeabilization, cells were stained with anti–human Lamp1 (Sigma-Aldrich) and DAPI. Representative individual and overlay fluorescence images were obtained on a microscope (IX81; Olympus). Subsequent image analysis and pseudocolor processing was performed by using iVision software (BioVision Technologies). Bars, 10 µm. BF, bright field. (E) Differentiated CD14+ WT and LYST-mutant THP-1 cells were stimulated for 4 h with the indicated reagents in the presence of brefeldin A. Flow cytometric analysis shows intracellular TNF expression on CD14+ cells. Shaded, WT; red lines, LYST-mutant clone 8.3; blue lines, LYST-mutant clone 12.1. (F) Differentiated CD14+ WT and LYST-mutant THP-1 cells (clones 8.3 and 12.1) were stimulated for 2 h with 20 ng/ml LPS, 10 µg/ml poly(I:C), or left untreated. IFNB1 mRNA levels were determined by quantitative real-time PCR of duplicate measurements. The expression level of nonstimulated WT THP-1 cells was set as 1. (G) Differentiated CD14+ WT and LYST-mutant THP-1 cells (clones 8.3 and 12.1) were stimulated for 2 h with PMA/ionomycin or left untreated. IL8 mRNA levels were determined by quantitative real-time PCR of duplicate measurements. (F and G) Data are mean ± SD. All data are representative for at least two independent experiments. iono, ionomycin; rel., relative.

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