Figure 5

Targeted validation of a set of host factors promoting or inhibiting HBD2 expression. A set of four host factors selected among those validated by counter-screening, as promoting or inhibiting HBD2 expression, was subjected to further validation by investigating their impact on HBD2 transcription in wild-type TC7 cells challenged with E. coli. Wild-type cells were silenced for 48 h using siRNA against (a) the MYD88, SUDS3 and ZNF3 promoting genes, or (b) the GATA6 inhibiting gene, and then challenged for 3 h with E. coli K12, at a MOI of 10 bacteria per cell. HBD2 transcription was finally measured by qRT-PCR. Values are presented on a logarithmic scale as the ratio of gene transcription in challenged cells compared with non-challenged cells, following scrambled siRNA (SC, white bars) or target siRNA (black bars) transfection. Data are presented as the mean ± s.d. (n = 3 biological replicates). *P < 0.05 evaluated by two-tailed Mann–Whitney U test. (c) ELISA dosage of the HBD2 peptide in supernatants of cells silenced for 48 h using scrambled siRNA (SC), siRNA against the MYD88, SUDS3 and ZNF3 promoting genes, or siRNA against the GATA6 inhibiting gene, and then challenged for 3 h with E. coli K12, at a MOI of 10 bacteria per cell. The bacterial challenge was stopped by an antibiotic treatment and supernatants were collected 24 h after the beginning of the challenge. Values are presented on a logarithmic scale in picogram of peptide per milliliter. Data are presented as the mean ± s.d. (n = 3 biological replicates). *P < 0.05 evaluated by two-tailed Mann–Whitney U test.