Fig. 1.

MITF expression modulates immune system response through a soluble, secreted factor ITGBL1 via RUNX2. a Melanoma cells were transfected with siRNA control or 2 different siRNA directed against MITF. Forty-eight hours later, activated PBMCs were added to cells and acquisition using Incucyte was performed. Quantification of melanoma cells death is displayed for each condition. b Activated PBMCs were incubated for 48 h in conditioned media from siCtl or siMITF melanoma cells and subsequently incubated with naïve 501Mel melanoma cells. Quantification of melanoma cell death after incubation with PBMCs is shown. All graphs represent mean+/−SD of 3 independent experiments. c 501Mel were transfected with two different siRNA for MITF (A) for 48 h. Protein lysates were separated by SDS page and blotted for MITF and ITGBL1 expression. HSP90 was used as a loading control. d Resting or activated PBMCs were incubated for 48 h in presence or absence of recombinant ITGBL1 (5 ng/ml). PBMCs were subsequently added to 501Mel melanoma cells and cell death was analyzed with Incucyte. Quantification of melanoma cell death is displayed as the mean+/−SD of 3 independent experiments. e WM3912 melanoma cells were transfected with control or MITF siRNA or infected with control or MITF adenoviruses. Proteins were probed for MITF and RUNX2 proteins expression. ERK2 was used as loading control. f 501Mel cells were infected with 2 different RUNX2 shRNA (sh#1, sh#2) encoding lentiviruses or its control shRNA (shScr). Proteins were probed for MITF and RUNX2 expression. ERK2 was used as a loading control. g 501Mel cell death was monitored with Incucyte experiments in presence of resting or activated PBMCs. Quantification of melanoma cell death is displayed as the mean+/−SD of 3 independent experiments. h WM9 melanoma cells were treated with 5 μM of VitD3 for 24 h, then RUNX2 and MITF proteins analyzed by western blot. ERK2 was used as loading control. i WM9 melanoma pretreated with 5 μM VitD3 were incubated with resting or activated PBMCs. Melanoma cell death was quantified using Incucyte. Results show as mean+/−SD of one representative experiment done in triplicate