Figure 2.

CCL2 is a major determinant of the secretome. (A) RNAs were harvested from control (siC) or MITF (siMi) siRNA for 96 h and were assayed by qRT–PCR for transcripts indicated on the figure. Transcript levels are represented relative to those found in control-transfected cells as mean + SD. The results in the graph are expressed as log2 values. (B) ELISA test of CCL2 levels in the CM of control (CM-siC) or senescent (CM-siMi) 501mel melanoma cells. Data, represented as mean + SD, are significantly different; (***) P < 0.001. (C) The chemotactic effect of recombinant CCL2 (100ng/mL) with or without anti-human CCL2-neutralizing antibody (100 μg/mL) was tested on naive 501mel melanoma cells in Boyden chambers, and the number of nuclei was counted using NIH ImageJ analysis software. The average values from three experiments +SD are shown. Significantly different at P < 0.001 (***). Representative images are shown. (D) 501mel cells were left untreated (CM-C) or were exposed to fotemustine (FMT, 40 mM), temozolomide (TMZ, 900 nM) or H₂O₂ (100 μM) for 96 h. CCL2 secretion levels were analyzed by ELISA. Values are expressed as mean + SD. (E) The chemotactic effect of control (CM-siC) or senescent (CM-siMi) cells, supplemented or not with anti-human CCL2-neutralizing antibody (100 μg/mL), was assessed on naive 501mel cells. The number of nuclei was counted using NIH ImageJ analysis software. Values are expressed as mean + SD. Significantly different at P < 0.001 (***). Representative images are shown.