Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A, Schematic diagram of high throughput screen of 2123 Selleckchem bioactive compound library at 100nM alone or in combination with the IC20 of TTM (dashed red line) in A375 or WM88. B,C, Normalized Percent Inhibition (NPI) of TTM + Compound versus NPI Compound graph for indicated cells treated with Selleckchem bioactive compound library alone or in combination with IC20 of TTM. Hits (blue circles) are defined as NPI TTM + Compound ≥ 20 (dashed red line). D,E, Observed effect versus expected effect graph for indicated cells of Hits from B and C. Hits (blue circles) are defined as Bliss Index ≥ 1.5 (dashed red line). F,G, Graphical representation of Hits defined as NPI TTM + Compound ≥ 20 and Bliss Index ≥ 1.5 in indicated cells. H, Venn diagram relationship between Compound Hits. I, NPI of 5 overlapping Hit Compounds from H alone or in combination with IC20 of TTM (dashed red line). J,K, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with vehicle or indicated concentrations of drugs. L,M,N,O, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with VEH or indicated concentrations of drugs. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: High Throughput Screening Assay, Drug discovery, Inhibition

Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A,C, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells stably expressing two independent Dox-inducible shRNAs (#1 and #2) against indicated genes treated with indicated concentrations of TTM without or with Dox n=3. B,D, Scatter dot plot of TTM IC50 ± s.e.m in indicated cells cells stably expressing two independent Dox-inducible shRNAs against indicated genes treated without or with Dox. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3. E,G, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. F,H, Scatter dot plot of BH3 mimetic IC50 ± s.e.m in indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. n=3. I,J, Graphical representation of Bliss Index (observed effect versus expected effect) from A375 (E) and WM88 (G) at the indicated drug combinations. Synergistic combinations are indicated by Bliss Index values >1. Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: Stable Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Phototoxic Potential of Different DNA Intercalators for Skin Cancer Therapy: In Vitro Screening

doi: 10.3390/ijms24065602

Figure Lengend Snippet: Relative cell viability of the WM983b cell line treated for 24 or 48 h with the dyes MB and AO within a range of concentration from 1.25 µM to 320 µM. Values are presented as the mean ± combined standard uncertainty ( n = 4), and statistical analysis comparing the results to the negative control was performed with a two-way ANOVA with Bonferroni post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001. MB: methylene blue, AO: acridine orange, PC: positive control, NC: negative control, SC: solvent control.

Article Snippet: The MET1 SCC cell line (human squamous cell carcinoma) was obtained from Ximbio (London, UK), HaCaT (immortalized human keratinocytes cell line) was obtained from Addexbio (San Diego, CA, USA) and WM983b (human metastatic melanoma cell line) was obtained from Rockland (Pottstown, PA, USA).

Techniques: Concentration Assay, Negative Control, Positive Control, Solvent, Control

Journal: International Journal of Molecular Sciences

Article Title: Phototoxic Potential of Different DNA Intercalators for Skin Cancer Therapy: In Vitro Screening

doi: 10.3390/ijms24065602

Figure Lengend Snippet: Relative cell population of the WM983b cell line treated for 24 h with dye molecules ( A ) MB and ( B ) AO and submitted to irradiation. Samples not treated with irradiated light (−) were used as controls for comparison with those submitted to irradiation at different light wavelengths (640 nm for MB and 457 nm for AO). The light dose applied to the irradiated cells was 2.5 J/cm². Values are presented as means ± combined standard uncertainty ( n = 6), and statistical analysis was performed with a two-way ANOVA with Bonferroni post test, where ** p < 0.01 and *** p < 0.001, in comparison with the dark control. MB: methylene blue, AO: acridine orange, PC: positive control, NC: negative control, SC: solvent control.

Article Snippet: The MET1 SCC cell line (human squamous cell carcinoma) was obtained from Ximbio (London, UK), HaCaT (immortalized human keratinocytes cell line) was obtained from Addexbio (San Diego, CA, USA) and WM983b (human metastatic melanoma cell line) was obtained from Rockland (Pottstown, PA, USA).

Techniques: Irradiation, Comparison, Control, Positive Control, Negative Control, Solvent

Journal: Cell

Article Title: Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

doi: 10.1016/j.cell.2022.04.014

Figure Lengend Snippet: Figure 6. Neo-interaction of BRAFV600E with KEAP1 and its collateral vulnerability (A and B) BRAFV600E stabilizes endogenous NRF2. Immunoblot (A) and densitometry analysis (B) showing NRF2 levels upon cycloheximide (CHX) chase in HEK293T cells overexpressing BRAF WT or V600E. (C) BRAFV600E activates NRF2 transcriptional activity. HEK293T cells were cotransfected with the NRF2-ARE luciferase reporter and either WT or V600E BRAF. Relative luciferase activity was measured, normalized to internal Renilla luciferase control. The data are presented as mean ± SD of three replicates from a repre- sentative experiment. ***p < 0.001. (D) BRAFV600E increases NRF2 and its target gene NQO1 protein levels in HEK293T cells transfected with GST-BRAFV600E versus WT. (E) Effect of BRAFV600E on NRF2 mRNA levels in HEK293T cells transfected with flag-NRF2 and GST-BRAF WT or V600E. nsp > 0.05. (F) KEAP1-dependency study of BRAFV600E-induced increase of NRF2 protein levels. Melanoma cells, CHL-1 (left) and HMCB (right), were transfected with KEAP1-targeting siRNA and BRAF WT or V600E plasmids as indicated. NRF2 protein expression was evaluated using western blot as indicated. (G and H) Representative blots (G) and densitometry analysis (H) of the correlation of NRF2 and its target gene NQO1 protein levels with BRAF genetic status in six melanoma cell lines with WT or V600E BRAF. The data are presented as mean ± SD from the densitometry analysis of three representative experi- ments. **p < 0.01. (I) Violin plot of the correlation between NQO1 mRNA levels and BRAF genetic status in 967 cell lines from CCLE dataset. The lines indicate mean ± SD. ***p < 0.001. (J and K) Effect of BRAF inhibitor, vemurafenib (J), or MEK1 inhibitor, selumetinib (K), on NRF2 and NQO1 protein levels in WM3482 melanoma cell line with BRAFV600E mutation. (L) Competitive binding between BRAFV600E and NRF2 to KEAP1. GST pulldown of GST-KEAP1 complex from lysate of HEK293T cells cotransfected with flag- NRF2 and with increasing amounts of flag-BRAFV600E. (M) Violin plot showing the CERES dependency scores for CRISPR knockout of KEAP1 in 342 cancer cell lines from CCLE dataset. The lines indicate mean ± SD. ***p < 0.001. (N) Parallel chemogenomic screening in a pair of isogenic MCF10A cell lines. Data were presented as percentage of inhibition in parental MCF10A cells with BRAF WT versus its V600E knock in counterpart. (O) Chemical structure of deoxynyboquinone (DNQ). (P) AUC analysis of DNQ-induced dose-dependent growth inhibition of twelve cell lines with BRAF WT or V600E. WT: CHL-1, HMCB, MCF10A, MeWO, WM3311, and RKO+/-/-; V600E: A2058, A375, MCF10ABRAF-V600E, WM3482, SK-MEL-5, and RKO. Each dot represents one cell line, and the data are presented as mean ± SD. *p < 0.05. (Q) Representative DNQ-induced dose-dependent growth inhibition of CHL-1 and WM3482 cell lines. The experiments were repeated independently three times. The data are presented as mean ± SEM from triplicates from a representative experiment. (R) Sequential combination effect of DNQ and vemurafenib in growth inhibition of WM3482 cells carrying BRAFV600E mutation. DNQ-induced dose-dependent growth inhibition was tested in three conditions: (1) DNQ alone, (2) pretreatment with 100 nM vemurafenib for 24 h followed by DNQ for 3 days (vemurafenib

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER CHL-1 cell line ATCC Cat# CRL-9446; RRID:CVCL_1122 WM3311 cell line Rockland Immunochemicals Inc. Cat# WM3311-01-0001 WM3482 cell line Rockland Immunochemicals Inc. Cat# WM3482-01-0001 A2058 cell line ATCC Cat# CRL-11147; RRID:CVCL_1059 SK-MEL-5 cell line ATCC Cat# HTB-70; RRID:CVCL_0527 LOXIMVI cell line Millipore Cat# SCC201; RRID:CVCL_1381 RPMI-7951 cell line ATCC Cat# HTB-66; RRID:CVCL_1666 SK-MEL-28 cell line ATCC Cat# HTB-72; RRID:CVCL_0526 UACC257 (U257) cell line NCI-DTP Cat# UACC-257; RRID:CVCL_1779 WM88 cell line Rockland Immunochemicals Inc. Cat# WM88-01-0001; RRID:CVCL_6805 CJM cell line Gift from Dr. Stuart Scheiber; PMID: 28678785 RRID: CVCL_U797 Parental MCF10A cell line Horizon Discovery Cat# HD PAR-003; RRID:CVCL_0598 Isogenic MCF10A BRAF V600E cell line Horizon Discovery Cat# HD 101-012 Parental RKO cells (+/V600E/V600E) cell line Horizon Discovery Cat# HD PAR-007; RRID:CVCL_0504 Isogenic RKO BRAF (+/-/-)cell line Horizon Discovery Cat# HD 106-003 MCF7 cell line ATCC Cat# HTB-22; RRID:CVCL_0031 Isogenic MCF7 AKT1 WT cell line Gift from Dr. Josh Lauring; PMID: 23888070 N/A Isogenic MCF7 AKT1 E17K cell line Gift from Dr. Josh Lauring; PMID: 23888070 N/A NCI-H1299 cell line ATCC Cat# CRL-5803; RRID:CVCL_0060 HCT116 cell line ATCC Cat# CCL-247; RRID: CVCL_0291 C4-2 cell line ATCC Cat# CRL-3314; RRID:CVCL_4782 Oligonucleotides qPCR primers for NRF2: Forward: GCGAC GGAAAGAGTATGAGC; Reverse: GTTGGC AGATCCACTGGTTT IDT N/A qPCR primers for GAPDH: Forward: ATGT TCGTCATGGGTGTGAA; Reverse: AGTT GTCATGGATGACCTTGG IDT N/A ON-TARGETplus Human KEAP1 siRNA Horizon Discovery Cat# is L-012453-00-0010 Recombinant DNA OncoPPi v2 library This paper N/A OncoMut library This paper N/A TCF/LEF transcriptional luciferase reporter plasmid BPS Bioscience Cat# 60500 AP-1 transcriptional luciferase reporter plasmid Qiagen Cat# CCS-011L NRF2-ARE luciferase reporter plasmid BPS Bioscience Cat# 60514 pET15b-KEAP1-KELCH plasmid Gift from Dr. Donna Zhang; PMID: 15475350 N/A Software and algorithms Graphpad Graphpad; v7 https://www.graphpad.com/ scientific-software/prism/ Cytoscape Cytoscape; v3.9.0 https://cytoscape.org Matlab Mathworks https://www.mathworks.com/products/ get-matlab.html CARINA This paper N/A Python Python https://www.python.org Schrödinger software Schrödinger https://www.schrodinger.com ll e3 Cell 185, 1974–1985.e1–e7, May 26, 2022 Resource ll

Techniques: Western Blot, Activity Assay, Luciferase, Control, Transfection, Expressing, Mutagenesis, Binding Assay, CRISPR, Knock-Out, Inhibition, Knock-In

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: Performance of candidates (A) Schematic of potential mechanisms by which candidates drive drug resistance. (B) Representative western blot with indicated antibodies in a panel of melanoma cell lines engineered to overexpress the screen candidates, empty vector (EM), VAV1, PDGFRB, or CSF1R. (C) Dose-response of modified cell lines against ENCOR and COBI. Top row shows ENCOR data and bottom row shows COBI data. Each data point represents three replicate measurements. Viability is shown after three days of drug treatment for A375 and 451Lu and four days for SKMEL28 and PDX10. The error bars denote the 95% confidence interval. (D) Alternate visualization of data in (C) showing viability of indicated cell lines relative to VEH for 21 nM of ENCOR or COBI. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval. (E) Viability of indicated cell lines relative to day zero after three days for A375 and 451Lu and four days for SKMEL28 and PDX10. Conditions included either treatment with VEH or ENCOR. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval. (F) Representative western blot for indicated antibodies in A375 modified with enforced expression of empty vector (EM), CSF1R L301S, Y969 (CSF1R∗), and PDGFRB N666K (PDGFRB∗). Cells were treated either VEH or 300nm of ENCOR for 6 h. Right panel shows quantification of three technical replicate blots. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval. (G) Dose-response curves of A375 modified with EM, CSF1R∗, and PDGFRB∗ against ENCOR. Right panel is alternate visualization that displays data from dose-response curves for 21 nM of ENCOR. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval.

Article Snippet: Human: 451Lu , Rockland Inc. , Catalog #: 451Lu-01-0001.

Techniques: Western Blot, Plasmid Preparation, Modification, Two Tailed Test, Expressing

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: Human: 451Lu , Rockland Inc. , Catalog #: 451Lu-01-0001.

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

Journal: eLife

Article Title: A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx

doi: 10.7554/eLife.72849

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse CXCL3 ELISA Kit , Rockland , Cat#:KOA0825 , .

Techniques: Flow Cytometry, cDNA Library Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Staining, Blocking Assay, In Vivo, Control