Fig 2: Differential expression of ABCB5, ERK, Akt, p-ERK, and p-AKT in wild-type BRAF cell, PLX-resistant melanoma cells and their parent cells by real-time PCR (a) and Western blot analysis (b). a Total RNA from melanoma cells and PLX-resistant melanoma cells were isolated. Real-time PCR was performed as described in Materials and Methods. B2M was used as an internal control. The bar graphs are expressed as fold changes of PLX resistant cell lines relative to each of their parent cell lines adjusted for B2M. Each of the experiments is a representation of two independent experiments performed in triplicate (** p < 0.01, n = 3). b Western blot analysis of ABCB5, ERK, Akt, p-ERK, p-Akt expression in wild-type BRAF cells, PLX-resistant cell lines, and their parent cell lines are shown. Anti-actin was used to show the equal loading and transfer in each lane. Targeted protein/actin showed the ratio of the scanned optical density of each protein band. ABCB5 protein level increased in A2058 PLXr and SK-MEL-28 PLXr cells versus A2058 and SK-MEL-28 cells. However, ABCB5 protein level was decreased in A375 PLXr cells versus A375 cells

Fig 3: K252a inhibition of BDNF-induced TrkB activation. Analysis of HEK-Clone2 cells incubated with BDNF (75 ng/mL), and with or without K252a (100 nM) during 1 h. (A) MCARS microspectroscopy of HEK-Clone2 after a dual treatment. HEK-Clone2 cells were incubated with BDNF (75 ng/mL) with or without K252a (100 nM), during 48 and 72 h, the figure includes bright field and fluorescence (Hoechst 33342) images. MCARS images were reconstructed from signal integration at 2850 cm-1 (CH2 symmetric stretching) and 2930 cm-1 (CH3 symmetric stretching). Scale bar, 5 µm. (B) Quantification of the CH2 signal intensity in HEK-Clone2 cells by MCARS analysis after a dual treatment for 48 and 72 h. We used HEK-Clone2 with no BDNF treatment as reference. (C) Western-blot of Akt and P-Akt (phospho-Akt) realized with 50 µg of extracted protein. Full-length blots for Akt and P-Akt are presented in Supplementary Fig. 7. *p < 0.05; **p < 0.01. For all experiments N was at least equal to 3.

Fig 4: Differential expression of ABCB5, ERK, Akt, p-ERK, and p-Akt in TMZ-treated and PLX-treated melanoma cells. a Total RNA from TMZ-treated and PLX-treated melanoma cells were isolated. Real-time PCR was performed as described in Materials and Methods. B2M was used as an internal control. The bar graphs show expressed fold changes of PLX-resistant cell lines relative to each of their parent cell lines adjusted for B2M. Each of the experiments is a representation of two independent experiments performed in triplicate (** p < 0.01, n = 3). TMZ P5: Passage 5 of TMZ-treated melanoma cells. PLX P5: Passage 5 of PLX-treated melanoma cells. b Differential expression of ABCB5, ERK, Akt, p-ERK, and p-Akt in early passages of TMZ-treated and PLX-treated cells by Western blot analysis. Targeted protein/actin showed the ratio of the scanned optical density of each protein band. TMZ P5: Passage 5 of TMZ-treated melanoma cells. PLX P5: Passage 5 of PLX-treated melanoma cells

Fig 5: PLAC1 affects the AKT process and EMT-related protein levels. (A and B) AKT and pAKT proteins were detected in PLAC1-knockdown H1299 or PC-9 cells by western blotting. (C and D) The expression levels of E-cadherin and vimentin proteins were determined by western blotting in PLAC1-knockdown H1299 and PC-9 cells. ß-actin was used as an internal control. **P<0.01. PLAC1, placenta-specific protein 1; NSCLC, non-small cell lung cancer.