Figure 1
Figure 1. TLR-induced overproduction of IL-1β by FANCA- and FANCC-deficient cells. (A-C) THP-1 cells expressing shRNA directed against FANCA (T-shFA), FANCC (T-shFC), or a nontargeted shRNA (T-shNT) were plated at a concentration of 106 per mL, pretreated with glyburide (50 μM) for 2 hours, and stimulated with R848 (30 μM) for 24 hours. Secreted IL-1β (A) and TNF-α (B) were measured in the conditioned media by ELISA. One representative experiment of 3 is shown. Each consisted of 3 technical replicates for each condition. (C) Western blotting of indicated proteins from whole cell extracts of the same cells was performed, and molecular weight markers are indicated. (D-E) Wild-type and Fancc−/− BMDMs were plated at a concentration of 50 000 per mL, pretreated with glyburide (50 μM) for 2 hours, and stimulated with R848 (3 μM) for 24 hours. Secreted IL-1β (D) and TNF-α (E) were measured in the culture media by ELISA. The results of 3 independent experiments, each of which consisted of 3 biological replicates for each condition, are shown. Each experiment contains pooled cells from 2 wild-type mice and 2 Fancc−/− mice. (F-G) CD14+ cells from an FA complementation group A patient and an age-matched healthy donor were isolated from peripheral blood mononuclear cells using magnetic microbeads. Cells were plated at a concentration of 50 000 per mL, pretreated with BIRB 796 (500 nM) for 6 hours, and stimulated with the indicated doses of R848 for 24 hours. Secreted IL-1β (F) and TNF-α (G) were measured in the conditioned media by ELISA. One experiment with 3 biological replicates for each condition is shown. (H-I) T-shNT and T-shFC cells were plated at a concentration of 106 per mL, pretreated with glyburide (Glyb; 50 μM) for 2 hours, and stimulated with R848 (30 μM). Total RNA was isolated at 0, 6, and 24 hours after R848 treatment, and IL-1β (H) and TNF-α (I) mRNA were measured using real-time qRT-PCR and normalized to levels of 18S rRNA. One experiment with 3 technical replicates for each condition is shown. P values were calculated using a paired Student t test.

TLR-induced overproduction of IL-1β by FANCA- and FANCC-deficient cells. (A-C) THP-1 cells expressing shRNA directed against FANCA (T-shFA), FANCC (T-shFC), or a nontargeted shRNA (T-shNT) were plated at a concentration of 106 per mL, pretreated with glyburide (50 μM) for 2 hours, and stimulated with R848 (30 μM) for 24 hours. Secreted IL-1β (A) and TNF-α (B) were measured in the conditioned media by ELISA. One representative experiment of 3 is shown. Each consisted of 3 technical replicates for each condition. (C) Western blotting of indicated proteins from whole cell extracts of the same cells was performed, and molecular weight markers are indicated. (D-E) Wild-type and Fancc−/− BMDMs were plated at a concentration of 50 000 per mL, pretreated with glyburide (50 μM) for 2 hours, and stimulated with R848 (3 μM) for 24 hours. Secreted IL-1β (D) and TNF-α (E) were measured in the culture media by ELISA. The results of 3 independent experiments, each of which consisted of 3 biological replicates for each condition, are shown. Each experiment contains pooled cells from 2 wild-type mice and 2 Fancc−/− mice. (F-G) CD14+ cells from an FA complementation group A patient and an age-matched healthy donor were isolated from peripheral blood mononuclear cells using magnetic microbeads. Cells were plated at a concentration of 50 000 per mL, pretreated with BIRB 796 (500 nM) for 6 hours, and stimulated with the indicated doses of R848 for 24 hours. Secreted IL-1β (F) and TNF-α (G) were measured in the conditioned media by ELISA. One experiment with 3 biological replicates for each condition is shown. (H-I) T-shNT and T-shFC cells were plated at a concentration of 106 per mL, pretreated with glyburide (Glyb; 50 μM) for 2 hours, and stimulated with R848 (30 μM). Total RNA was isolated at 0, 6, and 24 hours after R848 treatment, and IL-1β (H) and TNF-α (I) mRNA were measured using real-time qRT-PCR and normalized to levels of 18S rRNA. One experiment with 3 technical replicates for each condition is shown. P values were calculated using a paired Student t test.

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