Journal: Life Science Alliance

Article Title: DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF

doi: 10.26508/lsa.202101235

Figure Lengend Snippet: (A) SKMEL28 cells were transfected with siRNA against DUSP4 or nontargeting control with or without trametinib (Tram) at 0.25 nM. After 48 h, cells were collected, and RNA-seq experiments were performed. Volcano plots display differentially expressed genes from the RNA-seq analysis between siDUSP4 and siNT or between siDUSP4+ Tram and siDUSP4 (right panel). The vertical axis (y-axis) corresponds to the mean expression value of log 10 (q-value), and the horizontal axis (x-axis) displays the log 2 fold change value. Positive x-values represent up-regulated genes, and negative x-values represent down-regulated genes. MITF target genes are labeled in red . (B) Cells were treated as in (A), and 48 h later, lysates were analyzed by immunoblot. (C) Transcription factor activities were inferred based on the RNA-seq data using the msviper (Virtual Inference of Protein-Activity by Enriched Regulon analysis) algorithm. The activity of transcription factors was analyzed comparing y-axis: NES for siDUSP4 versus siDUSP4+ Tram or x-axis: NES for siNT versus siDUSP4. Positive values in both axes represent increased activity, whereas negative values represent decreased activity. (D) Cells were transfected with siRNA against DUSP4, MITF, and nontargeting as the control. 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 siNT versus siDUSP4 and siMITF conditions at 108 h. (E) Lysates from transfected cells were also analyzed by immunoblotting. (F) BRAF V600E cells from melanoma (SKMEL28 and COLO829), colorectal cancer (LS411N), and glioblastoma (DBTRG-05MG) were transfected with siRNA against BRAF and DUSP4. 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 siNT versus siDUSP4 and siBRAF conditions at 120 h.

Article Snippet: Reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher Scientific) and Opti-MEM (#51985026; Thermo Fisher Scientific) was performed according to the siTOOLS Biotech protocol using 3 nM final concentration of siPOOL nontargeting control and siPOOL against DUSP4, DUSP6, DUSP10, MITF, BRAF (siTOOLs Biotech).

Techniques: Transfection, Control, RNA Sequencing, Expressing, Labeling, Western Blot, Activity Assay

Journal: Life Science Alliance

Article Title: DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF

doi: 10.26508/lsa.202101235

Figure Lengend Snippet: (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.

Article Snippet: Reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher Scientific) and Opti-MEM (#51985026; Thermo Fisher Scientific) was performed according to the siTOOLS Biotech protocol using 3 nM final concentration of siPOOL nontargeting control and siPOOL against DUSP4, DUSP6, DUSP10, MITF, BRAF (siTOOLs Biotech).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Control, Western Blot, Mutagenesis

Journal: Life Science Alliance

Article Title: DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF

doi: 10.26508/lsa.202101235

Figure Lengend Snippet: (A) BRAF V600E cells labeled in purple (SKMEL28, A375) and NRAS Q61K/R cells labeled in green (SKMEL30, SKMEL2, respectively) were transfected with siRNA against DUSP4, MITF, and nontargeting control. After 48 h, cell lysates were analyzed by Western blot. The upper cartoons classify melanoma cells according on their MITF expression levels. High levels of MITF are shown in brown, whereas low levels of MITF are labeled in pink. 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 for each different cell line. (B) Cells were treated as in (A), and cell growth was analyzed by measuring cell confluence over time. Graphs show cell growth curves normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siNT versus siDUSP4 condition at 102 h. (C) Patient-derived cell lines containing NRAS Q61L/K mutation (WM1366 and WM3623, respectively) were transfected as in (A). After 48 h, cell lysates were analyzed by Western blot. (D) WM1366 and WM3623 cells were treated as in (A), and cell growth was analyzed by measuring cell confluence over time. Graphs show cell growth curves normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siNT versus siDUSP4 condition at 120 h. (E) Co-expression analysis of DUSP4 and MITF in human melanoma. Bivariate and rank correlation analysis of combined gene expression data (n = 124) from four separate studies available through cBioportal . Source data are available for this figure.

Article Snippet: Reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher Scientific) and Opti-MEM (#51985026; Thermo Fisher Scientific) was performed according to the siTOOLS Biotech protocol using 3 nM final concentration of siPOOL nontargeting control and siPOOL against DUSP4, DUSP6, DUSP10, MITF, BRAF (siTOOLs Biotech).

Techniques: Labeling, Transfection, Control, Western Blot, Expressing, Software, Derivative Assay, Mutagenesis, Gene Expression