Fig 2: Changes of MITF-M level in melanoma cells treated with vemurafenib (PLX) or trametinib (TRA). (a) Drug influence on MITF-M transcript level. qRT-PCR data, normalized to the expression of a reference gene RPS17, are presented relative to control. (b) Drug-induced changes in MITF-M and SOX10 protein levels assessed by immunoblotting. GAPDH was used as loading control. The proteins were visualized by using ChemiDoc Imaging System (Biorad). The images are cropped, which is indicated by white spaces, whereas direct comparison of controls is shown in Figure 1(a). (c) Drug influence on the percentages of MITF-positive cells shown as representative density plots along with quantification from three independent experiments. Data are presented as mean ± SD. * p < 0.05.

Fig 3: Changes in expression levels of pigmentation-associated genes following acute treatment of melanoma cells with vemurafenib (PLX) or trametinib (TRA). (a) Transcript levels of MLANA, PMEL, TYR, and DCT were assessed by qRT-PCR and expressed relative to control. They are presented as mean ± SD from three independent experiments. Statistically significant differences are indicated: * p<0.05. Comparison of expression levels of pigmentation-associated genes in melanoma cell line treated either with vemurafenib or with trametinib shown relative to the median values of all six drug-treated lines (right panels of (a)). (b) DCT protein levels assessed by immunoblotting. GAPDH was used as loading control. The proteins were visualized by using ChemiDoc Imaging System (Biorad). The images are cropped, which is indicated by white spaces. (c) Drug influence on percentages of Melan-A-positive and Ki-67-positive cells shown as representative density plots along with quantification (below). Data are presented as mean ± SD. Statistically significant differences are indicated: * p<0.05. (d) Drug-induced changes in transcript levels of pigmentation-related genes after 22 h and 44 h of treatment with either vemurafenib or trametinib. qRT-PCR data were normalized to the expression of a reference gene RPS17.

Fig 4: EGF-mediated tyrosine phosphorylation of ß4 occurs in EGFR overexpressing cell lines. (A) whole cell lysates (WCL) and ß4 immunoprecipitation (IP) samples of multiple transformed and untransformed cell lines, expressing ß4 and EGFR at different levels, treated with EGF for 5 min after 20 h of serum starvation, were analyzed by Western blot for total ß4 levels, tyrosine phosphorylation of ß4, total EGFR levels, tyrosine phosphorylation of EGFR and a-tubulin levels (loading control). (B) The protein and tyrosine abundances were quantified, normalized to the levels of A431 (at 10), and visualized in a graph. ß4 tyrosine phosphorylation (IP) is normalized to total ß4 levels (IP) on the Y-axis, and total EGFR levels (WCL) were normalized to a-tubulin levels (WCL) on the X-axis. The graph shows the mean + standard deviation (SD) of 3 independent experiments. Uncropped images of merged chemiluminescent and colorimetric blots obtained with a ChemiDoc imaging system (BioRad) are shown in Suppl. Fig. 1.

Fig 5: Immunoblot of the B4GALNT2-expressed glycosyltransferase in transfected HEK293 cells. Western blot displaying the immunoblotted band corresponding to ß4GalNAc-T2 in protein samples from transfected HEK293 cells. In the left blot, samples are dissolved in Laemmli buffer with reducing agent ß-mercaptoethanol, while Laemmli buffer without reducing agent has been used for samples of the right blot. The standards (Lane 1 and 8) specify the molecular weights (kDa). Mock-transfected and untransfected cells are negative controls (Lane 2 and 7) with the B4GALNT2 wt transfected serves as the positive control (Lane 3). The glycosyltransferase is detected in all cell preparations transfected with either of the B4GALNT2-mutated constructs, rs7224888:T > C (Lane 4), rs148441237:A > G (Lane 5) or rs61743617:C > T (Lane 6). Colorimetric and chemiluminescent images of the membrane have been merged. The lower panel displays stain-free images of total protein of each blot, ChemiDoc imaging system, Bio-Rad.