Figure 6.

The PARP-1/NF-κB axis plays a key role in the deleterious effect of the secretome. (A) Western blot analysis showing a kinetic experiment of siC or siMi-transfected 501mel cell lysates probed with PARP-1, MITF, and ERK2 antibodies. (B) Immunofluorescence with PAR antibody of 501mel cells transfected for 96 h with siC or siMi or with siC and exposed to H₂O₂ (100 μM). (C) Immunofluorescent staining with p65/RELA (red) and MITF (green) of 501mel cells transfected with siC or siMi for 96 h and exposed when indicated to NF-κB (sulfasalazine) or PARP-1 (3-AB) inhibitors. (D) NF-κB luciferase promoter activity of 501mel cells transfected with MITF siRNA or exposed to fotemustine (FMT, 40 mM), temozolomide (TMZ, 900 nM), or H₂O₂ (100 μM) in the presence or absence of 3-AB (20 mM). Results are expressed as percent (%) + SD of the luciferase activity from the control condition. (E) CCL2 secretion level was analyzed by ELISA in the CM of 501mel cells transfected with siC or siMi and exposed to the PARP-1 inhibitor 3-AB (20 mM) or the NF-κB inhibitor sulfasalazine (Sulfa, 500 μM) for 96 h. Values are expressed as mean + SD, significantly different from control at P < 0.001 (***). (F) 501mel cells were transfected with siC or siMi and exposed when indicated to sulfasalazine (500 μM) or 3-AB (20 mM). Forty-eight hours after transfection and inhibitor exposure, the medium was changed by DMEM 1% serum for an additional 48 h. Drug-free CM was next used in chemotaxis experiments with naive 501mel cells. Cells that had migrated were stained 24 h later with crystal violet and counted. Values represent mean + SD of two independent experiments, significantly different from control at P < 0.001 (***). Representative images are shown.