Analysis of TNF-α mRNA expression and level and protein expression and secretion. (A) TNF-α promoter activity in FA and corrected lymphoblasts. Cells were cotransfected with the reporter pGL3-hTNF-α-LucF and phRL-TK, used as internal control. Luciferase activity in HSC536Corr cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .01. (B) Steady-state level of TNF-α mRNA in FA and corrected lymphoblasts assessed by semiquantitative RT-PCR. The histogram represents the relative level of TNF-α mRNA in FA-C–deficient compared with FANCC-corrected cells. The level in HSC536Corr was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (C) Immunoblot showing the level of TNF-α in total cell extracts prepared from HSC536 cell line either untreated (Unt) or treated for 18 hours with brefeldin A (Bref A) or dexamethasone (Dexa). A cross-reactive band (marked with asterisk) was used as a loading control. (D) Intracellular TNF-α accumulation in FA and corrected lymphoblasts. Cells were treated with dexamethasone (10 μg/mL) for 3 hours. After this incubation, cells were collected by centrifugation, washed, and resuspended in fresh medium in presence of brefeldin A (1 μg/mL) for 24 hours. Proteins were extracted at the indicated time points. TNF-α content was measured by ELISA. Each point represents the mean (± SD) of at least 3 independent experiments. (E) Kinetics of TNF-α accumulation in HSC93, HSC536Corr, HSC536, and HSC536Neo cell lines. Supernatants were collected 3, 6, and 9 hours after cell subculturing in fresh medium and cytokine release was determined by ELISA. Each point represents the mean (± SD) of at least 3 independent experiments. *P < .01.