Figure 1.
Figure 1. CRISPR-Cas9 screen identifies Cpb1 to be required for caspase-11 expression, activation, and cell death in macrophages. (A) BMDMs were treated with intracellular LPS via CTB for 16 h, and then cytotoxicity was measured. (B) RAW264.7 transformed murine macrophage cell line (RAW) was treated with CTB/LPS or transfected LPS for 16 h and cytotoxicity was measured. (C) Expression of humanized Cas9 (hCas9) was determined in WT RAW cells that were either untreated or transduced with a lentiviral construct that stably expresses hCas (WT-hCas9), or the same cells were treated with LPS/CTB for 16 h and cytotoxicity was measured. (D) A macrophage genetic screen identified KOs that confer resistance to intracellular LPS stimulation via 16 h treatment with CTB/LPS. Each plot represents a gene (x axis). Genes are ranked on the y axis according to the significance (-LogPvalue) of enrichment, which was calculated by an accumulated hypergeometric distribution function (König et al., 2007), in the CTB/LPS-treated library compared with the nonselected control population. The top 100 hits are highlighted in the extrapolated box. Blue genes, CTB receptor genes; purple genes, complement genes; red, yellow, and green, other hits followed up on. (E–I) WT and KO cell lines were treated with LPS/CTB or transfected with LPS for 16 h and cytotoxicity was measured, (I) with or without the Cpb-inhibitor (MGTA). (J) WT, Cpb1 KO, and Cpb1 KO cells complement with either Cpb1 (Cpb1 KO + Cpb1) or caspase-11 (Cpb1 KO + Casp11) on a transgene were treated with CTB/LPS for 16 h and cytotoxicity was measured. (K) WT and KO cell lines were treated with either LPS or CTB/LPS for 16 h. Supernatants and cell lysates were collected for each sample and detection of pro-caspase-11 (pro-Casp11), cleaved caspase-11 (p30 Casp11), and β-actin was evaluated via Western blot. Indicated cell lines were treated with LPS for 2 h. Relative expression of (L) caspase-11 and caspase-1 or (M) IL-6 and TNF were determined. All relative expression was calculated via qRT-PCR, compared with GAPDH expression. All cytotoxicity was measured by release of LDH. Statistics analyzed via the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are representative of at least two (D) or three (A–C and E–M) independent experiments with three technical replicates each time.

CRISPR-Cas9 screen identifies Cpb1 to be required for caspase-11 expression, activation, and cell death in macrophages. (A) BMDMs were treated with intracellular LPS via CTB for 16 h, and then cytotoxicity was measured. (B) RAW264.7 transformed murine macrophage cell line (RAW) was treated with CTB/LPS or transfected LPS for 16 h and cytotoxicity was measured. (C) Expression of humanized Cas9 (hCas9) was determined in WT RAW cells that were either untreated or transduced with a lentiviral construct that stably expresses hCas (WT-hCas9), or the same cells were treated with LPS/CTB for 16 h and cytotoxicity was measured. (D) A macrophage genetic screen identified KOs that confer resistance to intracellular LPS stimulation via 16 h treatment with CTB/LPS. Each plot represents a gene (x axis). Genes are ranked on the y axis according to the significance (-LogPvalue) of enrichment, which was calculated by an accumulated hypergeometric distribution function (König et al., 2007), in the CTB/LPS-treated library compared with the nonselected control population. The top 100 hits are highlighted in the extrapolated box. Blue genes, CTB receptor genes; purple genes, complement genes; red, yellow, and green, other hits followed up on. (E–I) WT and KO cell lines were treated with LPS/CTB or transfected with LPS for 16 h and cytotoxicity was measured, (I) with or without the Cpb-inhibitor (MGTA). (J) WT, Cpb1 KO, and Cpb1 KO cells complement with either Cpb1 (Cpb1 KO + Cpb1) or caspase-11 (Cpb1 KO + Casp11) on a transgene were treated with CTB/LPS for 16 h and cytotoxicity was measured. (K) WT and KO cell lines were treated with either LPS or CTB/LPS for 16 h. Supernatants and cell lysates were collected for each sample and detection of pro-caspase-11 (pro-Casp11), cleaved caspase-11 (p30 Casp11), and β-actin was evaluated via Western blot. Indicated cell lines were treated with LPS for 2 h. Relative expression of (L) caspase-11 and caspase-1 or (M) IL-6 and TNF were determined. All relative expression was calculated via qRT-PCR, compared with GAPDH expression. All cytotoxicity was measured by release of LDH. Statistics analyzed via the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are representative of at least two (D) or three (A–C and E–M) independent experiments with three technical replicates each time.

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