Fig 2: DUSP KD leads to a suppression of MITF-dependent gene expression.(A) mRNA levels of MITF and its related genes were analyzed by RT–qPCR 48-h post-transfection and were referred to the expression level of siNT cells. Data are normalized to GAPDH and represent mean of three independent experiments. Statistical significance was calculated between siNT versus NT+T and siDUSP4 and siDUSP4 versus siDUSP4+ Tram. (B, C) Cells were transfected with siRNA against nontargeting control and DUSP4, DUSP6, DUSP10. (B, C) Forty-eight hours later, cell lysates were analyzed by Western blot (B), and DUSP10 mRNA levels were measured by RT–qPCR (C). Data are mean ± SEM, n = 2. (D) BRAF-mutant cell lines from different tissues were transfected with siRNA against DUSP4 and BRAF. After 48 h, cell lysates were analyzed by immunoblot. (E) MITF protein levels were assessed in the indicated BRAF-mutant cell lines by immunoblot.

Fig 3: Effect of HmHae on the levels of melanogenesis-related mRNA and proteins in melan-a cells. (A) Cells (5 × 105 cells/mL) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentration of HmHae or arbutin for 24 h. mRNA was extracted using TRIzol, and expression was analyzed by reverse-transcription polymerase chain reactions; and (B) cells (1 × 105 cells/mL) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of HmHae or arbutin for three days. Total cell lysate was extracted and assayed by Western blotting using antibodies against tyrosinase, TYRP-1, TYRP-2, and microphthalmia-associated transcription factor (MITF). Equal protein loading was confirmed using ß-actin as a standard. Abt: arbutin.

Fig 4: Western blot analysis. (A) ZnPP decreases p53, MITF, and tyrosinase expressions in normal human melanocytes in a dose-dependent manner. (B) Scanned chemiluminescent signals were analyzed with the image analysis program. Each bar represents band intensity normalized with that of ß-actin. Only increase in HO-1 expression is statistically significant according to the treatment doses of ZnPP compared to control group (p=0.043). (C) CoPP (10 µM) treatments increase expressions of p53, MITF, and tyrosinase, and ZnPP (10 µM) treatments decrease expressions in p53, tyrosinase, and MITF until 72 h after treatment. (D) Scanned chemiluminescent signals were analyzed with an image analysis program. Each intensity bar represents normalized with ß-actin. Only decreases in p53 (p=0.045) and tyrosinase (p=0.025) expressions upon ZnPP treatment are statistically significant compared with control groups. All experiments were performed at least three times independently, and representative results are shown. The data represent mean±SD. C: control, CP: CoPP, ZP: ZnPP, *Statistical significance (p<0.05).

Fig 5: The critical role of DUSP4 in MAPKi-resistant cells is strictly linked to the MITF expression levels in parental melanoma cells.(A) Schematic representation of the generation of SKMEL28- and A375-resistant cells. Parental SKMEL28 cells were treated with increasing doses of trametinib (up to 20 nM) until they acquired resistance (R+T cells). Trametinib was removed from R+T in an acute (R-T acute) or sustain (R-T sustain) manner. Parental A375 cells were treated with increasing doses of vemurafenib (up to 2.5 µM) until they acquired resistance (R+V cells). Vemurafenib was removed from R+V cells in an acute way (up to 6 d, R-V cells). Clonogenic growth of parental and resistant cells is shown. (B) Lysates of SKMEL28 and A375 parental and resistant cells were analyzed by immunoblot. (C) SKMEL28 parental and resistant cells with a sustain drug withdrawal (R-T sustain) were transfected with siRNA against DUSP4 and nontargeting as the control, and the cell growth was analyzed by measuring cell confluence over time. Cell growth values were normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siDUSP4 versus siNT condition at 108 h (D) SKMEL28 parental and resistant cells were treated as in C, and cell growth was analyzed by a colony formation assay. Representative images are shown from three independent experiments. (E) SKMEL28 parental and resistant cells were treated as in (C) and after 48 h, and cell lysates were analyzed by Western blot. Band intensities were analyzed by ImageJ software, and the P-ERK/GAPDH and MITF/GAPDH ratios are indicated in the histogram. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated against siNT. (F) A375 parental and resistant cells were transfected with siRNA against DUSP4 and nontargeting as the control, and the cell growth was analyzed by measuring cell confluence over time. Cell growth values were normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siDUSP4 versus siNT conditions at 84 h. (F, G) A375 parental and resistant cells were treated as in (F), and cell growth was analyzed by a colony formation assay. Representative images are shown from three independent experiments. (H) A375 parental and resistant cells were treated as in F, and after 48 h, cell lysates were analyzed by Western blot. (I) SKMEL28 parental and R-T sustain cells were transfected with siRNA against DUSP4 and nontargeting control in the presence or absence of trametinib (0.25 nM). Cells were plated to form colonies and analyzed 7 d later. Representative images are shown from three independent experiments. (J) SKMEL28 parental and R-T sustain cells were treated as in (I) and cell lysates were analyzed by Western blot after 48 h post-transfection. Band intensities were analyzed by ImageJ software, and the P-ERK/GAPDH and MITF/GAPDH ratios are indicated in the histograms. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated against siNT.