FANCC down-regulation leads to aberrant activation of stress-response pathways and MMP-7 overexpression in HeLa cells. (A) Immunoblot showing monoubiquitination of FANCD2 in FANCC siRNA–transfected cells. Forty-eight hours after siRNA transfection, cells were treated with mitomycin C (MMC) (500 ng/mL) or 8-methoxypsoralen (10−5M) + UVA (10 kJ/m2) (8-MOP) and lysed 24 hours later. FANCC down-regulation impacts on DNA damage–induced FANCD2 monoubiquitination. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Western blot analysis of IκBα in HeLa cells depleted of FANCC for 3 days. Actin was used as a loading control. (C) Detection of p50 and p65 subcellular localization by confocal immunofluorescence microscopy. HeLa cells treated with FANCC siRNA were subjected to immunostaining of p50 (green fluorescence) and p65 (red fluorescence) 72 hours after transfection. (D) NF-κB transcriptional activity in HeLa cells depleted for FANCC. Twenty-four hours after siRNA treatment, cells were cotransfected with the reporter pNF-κB-Luc and phRL-TK for 48 hours before the analysis of the luciferase activity. Luciferase activity in GFP-siRNA–transfected 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 < .05. (E) Immunoblot showing the level of phosphorylation of MAPK players in cells with a reduced FANCC expression compared with mock-transfected and unperturbated cells. Cells were collected 72 hours after transfection and actin was used as a loading control. Vertical lines have been inserted to indicate a repositioned gel lane. (F) MMP-7 promoter activity in mock- and siRNA FANCC–transfected HeLa cells. Twenty-four hours after siRNA treatment, cells were cotransfected with the reporter pGL3-hMMP-7 and phRL-TK, and induced luciferase activity was measured 24 hours later. Luciferase activity in GFP-siRNA–transfected 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. (G) MMP-7 mRNA steady-state level in FANCC-deprived or mock-transfected HeLa cells was determined by quantitative RT-PCR. The relative mRNA level (normalized as function of 18S rRNA content) in GFP-siRNA–transfected cells was fixed to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .01. (H) MMP-7 promoter activity in 50 μM curcumin-treated FANCC-deprived cells assessed by luciferase activity as in panel F. *P < .01. (I) MMP-7 mRNA steady-state level in FANCC-deprived HeLa cells treated with curcumin (50 μM) as assessed by quantitative RT-PCR as in panel G. *P < .01. (J) MMP-7 promoter activity in siRNA FANCC–transfected HeLa cells treated with SB203580 (p38 inhibitor, 10 μM) and PD98059 (ERK inhibitor, 30 μM) for 18 hours. *P < .05 compared with DMSO treatment.
