Figure 3
Figure 3. Overexpression of MMP-7 in FA-C cells and its involvement in the aberrant secretion of the TNF-α. (A) Immunoblot showing the level of TACE in total cell extracts. Immature and mature forms of TACE are indicated by i and m, respectively. The doublets observed for each form on Western blot reflect protein glycosylation. Equal loading of the membrane was verified by ponceau red staining and/or evaluated by the intensity of the indicated (*) aspecific band. (B) Immunoblot showing the level of MMP-7 in total cell extracts (top panel) and in supernatant (bottom panel). Equal loading of the membrane was verified by ponceau red staining and/or evaluated by the intensity of the indicated (*) aspecific band. Vertical lines have been inserted to indicate a repositioned gel lane. (C) MMP-7 promoter activity in normal, corrected, and FA cells. Cells were cotransfected with the reporter pGL3-hMMP-7 and phRL-TK, as internal control. Luciferase activity in AHH1 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. (D) MMP-7 mRNA steady-state levels as determined by quantitative RT-PCR on mRNA extracted from exponentially growing lymphoblasts and normalized to 18S rRNA content. To normalize the values among the different experiments, each time the ratio MMP-7/18S was considered equal to 1 in AHH1 cells. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (E) TNF-α release in MMP-7 inhibited FA-C cells. HSC536 and HSC536Neo cells were treated with MMP inhibitor (50 μM) or its solvent (DMSO) or left untreated. Supernatants were collected 24 hours after subculturing and cytokine release was determined by ELISA. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01. (F) TNF-α release in FA-C cells after the down-regulation of MMP-7 expression. Immunoblot shows the level of MMP-7 in supernatant of FANCC siRNA–MMP-7 down-regulated cells. A cross-reactive band (*) was used as a loading control. TNF-α accumulation in the supernatant of infected HSC536Neo cells was evaluated by ELISA. Histogram represents the mean reduction (in percentage) plus or minus SD of TNF-α accumulation observed in at least 3 independent experiments. *P < .01.

Overexpression of MMP-7 in FA-C cells and its involvement in the aberrant secretion of the TNF-α. (A) Immunoblot showing the level of TACE in total cell extracts. Immature and mature forms of TACE are indicated by i and m, respectively. The doublets observed for each form on Western blot reflect protein glycosylation. Equal loading of the membrane was verified by ponceau red staining and/or evaluated by the intensity of the indicated (*) aspecific band. (B) Immunoblot showing the level of MMP-7 in total cell extracts (top panel) and in supernatant (bottom panel). Equal loading of the membrane was verified by ponceau red staining and/or evaluated by the intensity of the indicated (*) aspecific band. Vertical lines have been inserted to indicate a repositioned gel lane. (C) MMP-7 promoter activity in normal, corrected, and FA cells. Cells were cotransfected with the reporter pGL3-hMMP-7 and phRL-TK, as internal control. Luciferase activity in AHH1 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. (D) MMP-7 mRNA steady-state levels as determined by quantitative RT-PCR on mRNA extracted from exponentially growing lymphoblasts and normalized to 18S rRNA content. To normalize the values among the different experiments, each time the ratio MMP-7/18S was considered equal to 1 in AHH1 cells. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (E) TNF-α release in MMP-7 inhibited FA-C cells. HSC536 and HSC536Neo cells were treated with MMP inhibitor (50 μM) or its solvent (DMSO) or left untreated. Supernatants were collected 24 hours after subculturing and cytokine release was determined by ELISA. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01. (F) TNF-α release in FA-C cells after the down-regulation of MMP-7 expression. Immunoblot shows the level of MMP-7 in supernatant of FANCC siRNA–MMP-7 down-regulated cells. A cross-reactive band (*) was used as a loading control. TNF-α accumulation in the supernatant of infected HSC536Neo cells was evaluated by ELISA. Histogram represents the mean reduction (in percentage) plus or minus SD of TNF-α accumulation observed in at least 3 independent experiments. *P < .01.

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