Journal: iScience

Article Title: Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

doi: 10.1016/j.isci.2023.107805

Figure Lengend Snippet: Sleeping Beauty genetic screen strategy and melanoma BRAF/MEK inhibition screen results (A) Schematic of the genetic screens. (B) Circos plot of insertion sites in unselected samples (left) and BRAF/MEKi selected samples (right). Each ring displays data from the screen for a given cell line. (C) Boxplots showing the number of unique genes mutated per 10cm plate for a given cell line screen. The right Y axis is for UACC257 data. The edges of the boxes denote the 1st and 3rd quartiles, and the line denotes the 2nd quartile. (D) Line plot showing the number of true positive drivers detected when using a subset of n samples. The X axis denotes n. True positive drivers are those that passed filtering when using the entire dataset. The error bars denote the 95% confidence interval. (E) Heatmap of the -log(q-value) for select candidates recovered in our genetic screens. The q value is the multi-hypothesis corrected p value generated by performing a chi-squared test based on the observed number of insertions in a gene and the expected number of insertions in a gene. The expected number of insertions is derived from the number of TA sites in a gene and the total number of insertions in the dataset.

Article Snippet: We performed cell-based SB mutagenesis in five BRAF V600 E/K melanoma cell lines against encorafenib (ENCOR) and binitmetinib, which are inhibitors of BRAF and MEK, respectively., , , , , , , Compared to VEM, ENCOR binds more strongly to BRAF V600 E/K , which translated to improved overall survival compared to VEM in a clinical trial.

Techniques: Inhibition, Generated, Derivative Assay

Journal: iScience

Article Title: Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

doi: 10.1016/j.isci.2023.107805

Figure Lengend Snippet:

Article Snippet: We performed cell-based SB mutagenesis in five BRAF V600 E/K melanoma cell lines against encorafenib (ENCOR) and binitmetinib, which are inhibitors of BRAF and MEK, respectively., , , , , , , Compared to VEM, ENCOR binds more strongly to BRAF V600 E/K , which translated to improved overall survival compared to VEM in a clinical trial.

Techniques: Recombinant, Reverse Transcription, Transfection, RNA Extraction, Plasmid Preparation, Software

Journal: Oncotarget

Article Title: Parthenolide induces MITF-M downregulation and senescence in patient-derived MITF-M high melanoma cell populations

doi: 10.18632/oncotarget.7030

Figure Lengend Snippet: A. Western blot analysis of BRAF mutation status in patient-derived melanoma cell populations (DMBCs). An antibody recognizing BRAF V600E but not wild type BRAF was employed. A375, a melanoma cell line harboring BRAF V600E mutation was used as a positive control. B. Doubling time of melanoma cell populations assessed as metabolic activity of acid phosphatase. (n = 3) C. qRT-PCR analysis comparing basal levels of MITF-M and MITF . D. Western blot analysis comparing basal levels of MITF. A doublet of M isoform is indicated by arrows (top). MITF (total) and MITF-M protein levels were quantified relatively to their levels in A375 cells (bottom) (n = 3). E. qRT-PCR analysis comparing basal transcript levels of tyrosinase ( TYR ) and MLANA . Relative mRNA quantity of MITF and MITF-M (panel C), TYR and MLANA (panel E) is represented after normalization to RPS17 and the level in melanocytes (NHEM). As in DMBC11 and DMBC12 cells the expression of TYR and MLANA was several hundred fold lower than in NHEM, it is displayed as zero. DMBC, patient-derived melanoma populations obtained in Department of Molecular Biology of Cancer.

Article Snippet: BRAF V600E antibodies recognizing V600E-mutated, but not wild-type BRAF were from Biomol.

Techniques: Western Blot, Mutagenesis, Derivative Assay, Positive Control, Activity Assay, Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: Reactivation of ERK and Akt confers resistance of mutant BRAF colon cancer cells to the HSP90 inhibitor AUY922

doi: 10.18632/oncotarget.10414

Figure Lengend Snippet: ( A ) Whole cell lysates from Lim1215, Caco-2, RKO and WiDr treated with AUY922 (400 nM) for indicated time points were subjected to Western blot analysis. Data are representative, n = 3. ( B ) RKO (left) and WiDr (right) cells transfected with the control or BRAF V600E siRNA were treated with AUY922 (400nM) for 48 hours. Whole cell lysates were subjected to Western blot analysis. Data are representative, n = 3. ( C ) RKO (left) and WiDr (right) cells transfected with the control or BRAF V600E siRNA were treated with AUY922 (400 nM) for 48 hours. Apoptosis was measured by PI and Annexin V staining. Data are mean ± SE, n = 3. * P < 0.05, Student's t -test. ( D ) Whole cell lysates from RKO and WiDr cells were treated with AUY922 (400 nM), and/or PLX4720 (3 μM) for 48 hours and subjected to Western blot analysis. Data are representative, n = 3. ( E ) RKO and WiDr cells were treated with AUY922 (400 nM), and/or PLX4720 (3 μM) for 48 hours. Apoptosis was measured by PI and Annexin V staining. Data are mean ± SE, n = 3. ** P < 0.01 Student's t -test.

Article Snippet: siRNAs against CDC37, Akt, and BRAF V600E were synthesized by GenePharma (Shanghai GenePharma Co, Ltd, Shanghai, China) with the following target sequences: ACACAAGACCUUCGUGGAA (CDC37 siRNA1), CGGCAGUUCUUCACUAAGA (CDC37 siRNA2), GCUCCUUCAUUGGGUACAATT (Akt siRNA1), GCGGAAGGAAGUCAUCAUUTT (Akt siRNA2), GCUACAGAGAAAUCUCGAUTT (BRAF V600E siRNA) [ ].

Techniques: Western Blot, Transfection, Staining