Figure 5

Expression of Per1 (A and C) and Bmal1 (B and D) in esiRNA transfected murine Melan-a melanocytes and B16-F10 melanoma cells after heat stimulus (39.5 °C). Melan-a or B16-F10 cells were kept during three days in constant dark and temperature (37 °C). At the beginning of the 4^th day, Melan-a cells were synchronized by two medium changes, and after further 24 h cells were transfected with esiRNA against melanopsin or EGFP (both at 10 nM) using Lipofectamine 3000 transfection kit. B16-F10 cells were transfected with esiRNA, as described above, at the beginning of the 4^th day. In both experimental scenarios, 48 hours after transfection, cells were divided into four groups: (1) control group at 37 °C in the presence of esiRNA EGFP (10 nM); (2) heat-stimulated (39.5 °C) group in the presence of esiRNA EGFP (10 nM); (3) group at 37 °C in the presence of esiRNA melanopsin (10 nM); (4) heat-stimulated (39.5 °C) group in the presence of esiRNA melanopsin (10 nM). Boxplots show the median, quartiles, maximum, and minimum expression values of each gene transcript normalized by Rpl 37a and expressed relative to the minimal value of the esiRNA EGFP group at 37 °C (N = 5–11). Total RNA was extracted immediately and 1 h after the end of the stimulus for Melan-a and B16-F10 cells, respectively. Statistical analysis was performed by One-way ANOVA followed by Tukey’s test.