FIG 4.

Validation of top genetic hits and effects of the IRAK1 inhibitor in S. flexneri infection of human THP-1 cells. (A) Correlation between pooled screen and validation data. For each hit, the log₂ fold change obtained from the genome-wide CRISPRi screening data (screen phenotype) was plotted against the fold change of cell viability of genetic hits from levels in the nontargeting control cells (validation phenotype). Host cell viability was measured by trypan blue staining. sgNC80 and sgNC135 are nontargeting controls. R is the Pearson correlation coefficient. (B) Intracellular S. flexneri ΔvirG level after infection of individual knockdown THP-1 cells, which was measured by counting bacterial CFU. (C) Schematic of positive genetic hits in the TLR1/2 signaling pathway and corresponding inhibitors. (D) Cytokine and chemokine production in infected THP-1 cells with MYD88 and IRAK1 knockdown. (E and F) Viability of THP-1 cells (E) and growth of the intracellular S. flexneri ΔvirG mutant (F) postinfection in the presence or absence of the IRAK1 inhibitor IRAK1/4-Inh at different concentrations. Data represent the means ± standard deviations (SD) (n = 3) (Student's two-tailed unpaired t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).