Additional file 4: of A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells

Figure S4. Enhancement of ABCB5-signals in keratinocytes and external melanoma cells upon docetaxel treatment is confirmed by a second anti-ABCB5 antibody. Tri-culture spheroids were generated by 3D cultivation of CCD-1137Sk cells for 3 days, followed by the combined addition of HaCaT and SK-MEL-28 cells. HaCaT and SK-MEL-28 cells were labeled with CellTrackerRed CMPTX and CellTrackerGreen CMFDA dye, respectively. After another 2 days, tri-culture spheroids were treated with 0.01 ‰ of DMSO as control (A-C) or 100 nM docetaxel in DMSO (D-F) for 48 h. Spheroids were cryosectioned into 10-μm thick slices and immunostained with mouse anti-ABCB5 antibody MA5–17026. (A and D) Overlay images of the confocal sections shown in B and E. In overlays, ABCB5 signals, melanoma cells, keratinocytes, and nuclei are depicted in red, green, yellow, and blue, respectively. Scale bars: 100 μm. (C and F) Detail images of ABCB5 signals from boxed regions in B and E. (G-H) Quantification of the relative intensity of ABCB5-positive external (G) and internal (H) SK-MEL-28 cells (percentage of total). Given is mean ± SEM (n = 4 independent experiments; * P