Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Selective Cannabinoid 2 Receptor Stimulation Reduces Tubular Epithelial Cell Damage after Renal Ischemia-Reperfusion Injury

doi: 10.1124/jpet.117.245522

Figure Lengend Snippet: (A) Functional activities of SMM-295 in the CB2 ACTOne assay (open circles), CB1 ACTOne assay (open triangles), and parental ACTOne cells containing only the CNG ion channel (filled squares). The functional activation of CB2 by the internal control (CP 55,940; filled diamonds) is shown. (B) Western blot analysis of signaling proteins after brief exposure (15 minutes) to SMM-295 in rat NRK-52E proximal tubule epithelial cells. Activation of prosurvival Akt/PKB and MAPK (ERK1/2 and p38) was detected using specific antibodies. β-actin was used as a loading control. ERK1/2, extracellular signal–regulated kinase 1/2.

Article Snippet: Human embryonic kidney (HEK)–cyclic nucleotide gated (CNG), HEK-CNG+CB1, and HEK-CNG+CB2 cells were obtained from Codex BioSolutions.

Techniques: Functional Assay, Activation Assay, Western Blot

Journal: Nature cancer

Article Title: Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling

doi: 10.1038/s43018-019-0018-6

Figure Lengend Snippet: a, SF295 cells were transduced with multiple guides targeting the MTF-1 gene. Following selection, genomic DNA was isolated, and the targeted region was amplified by PCR. Results from the NGS CRISPR assay are shown as percent indel formation. b, Differentially expressed genes in SF295 glioma cells following MTF-1 knockout by CRISPR/Cas9. Loss of MT1E, MT1X, and MT2A expression was observed upon MTF-1 knockout. Gene expression across three independent cell wells per cell line were measured by mRNA sequencing. Two-sided p-values for differential gene expression following MTF-1 knockout vs. parental cell line were calculated with DESeq2 and corrected for multiple hypothesis testing using the Benjamini-Hochberg method. c, Drug sensitivity of SF295 cells with and without MTF-1 knockout. MTF-1 does not alter sensitivity to control chemotherapeutic bortezomib. Mean viability across 3 independently treated wells is shown, with standard deviation indicated by error bars. d, Western immunoblot validation of SLC26A2 knockout in OVISE ovarian and A2058 melanoma cancer cell lines. The SLC26A2 protein is known to migrate across a range of molecular weights due to glycosylation. Results are representative of two independent experiments. e, OVISE cells were transduced with multiple guides targeting the SLC26A2 gene. Indel frequency at the SLC26A2 CRISPR Cas9 cut sites was assessed by NGS CRISPR assay. f, ABCB1 western blot with and without CRISPR knockout of ABCB1 in the LS1034 colon cancer cell line. Western blot was performed once. g, Percent indel formation at genomic cut site in LS1034 ABCB1 CRISPR knockout lines assessed by NGS CRISPR assay. h, Cellular viability of LS1034 CRISPR knockout lines after treatment with tepoxalin for 5 days. Mean viability across 3 independently treated wells is shown, with standard deviation indicated by error bars.

Article Snippet: Cell lines Parental cell lines were obtained from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) project 10 prior to PRISM barcoding (see https://portals.broadinstitute.org/ccle for original sources).

Techniques: Knock-Out, CRISPR, Transduction, Selection, Isolation, Amplification, Expressing, Gene Expression, Sequencing, Control, Standard Deviation, Western Blot, Biomarker Discovery, Glycoproteomics

Journal: Nature cancer

Article Title: Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling

doi: 10.1038/s43018-019-0018-6

Figure Lengend Snippet: a, Drug sensitivity profiles associated with high PDE3A RNA expression (n = 489 cell lines, n = 11 compounds). PDE3A gene expression from CCLE RNAseq data (log2 RPKM; red) and cell line viability (blue) are indicated by color. Compounds where PDE3A expression was the top predictive biomarker by ATLANTIS from the PRISM 2.5 μM primary screen are shown. Cell lines are ranked by mean viability of indicated compounds with a ceiling at 1 (100%). b, Thermal shift assay performed with recombinant PDE3A. Stabilization of PDE3A protein is seen for a subset of hit compounds. DNMDP is included as established positive control for strong PDE3A binding. Full ΔTm results are shown in Supplementary Table 9. Results are representative of two independent experiments. c, HeLa cell line dose response curves with PDE3A genetic loss or pharmacologic inhibition. Knockout of PDE3A was performed by CRISPR/Cas9. Parental HeLa cell killing was rescued by co-treatment with 100 nM of the non-cytotoxic PDE3A inhibitor trequinsin. Mean viability across two independently treated wells in one experiment is shown. d, Co-immunoprecipitation of V5-tagged SLFN12. PDE3A-modulating compounds induce complex formation between PDE3A and SLFN12. Anti-V5 western blot shown using lysates from HeLa cells treated with the indicated compound at 10 μM for 8 hours. The anagrelide and DNMDP lanes were run on the same gel as all other samples and were cropped for figure purposes. Anagrelide and DNMDP results are consistent across three independent experiments. All other compounds were tested twice, with the exception of norethindrone (tested once with the expected results).

Article Snippet: Cell lines Parental cell lines were obtained from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) project 10 prior to PRISM barcoding (see https://portals.broadinstitute.org/ccle for original sources).

Techniques: RNA Expression, Gene Expression, Expressing, Biomarker Discovery, Thermal Shift Assay, Recombinant, Positive Control, Binding Assay, Inhibition, Knock-Out, CRISPR, Immunoprecipitation, Western Blot

Journal: Nature cancer

Article Title: Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling

doi: 10.1038/s43018-019-0018-6

Figure Lengend Snippet: a, Drug sensitivity profiles of tepoxalin and seven additional compounds where ABCB1 RNA expression was the top predictive genomic feature of the PRISM activity profile (n = 489 cell lines). Tepoxalin was the only compound tested where high ABCB1 expression was associated with sensitivity rather than resistance to tepoxalin. Cell line viability is depicted with ABCB1 gene expression from CCLE RNAseq log2 RPKM data. Cell lines are ranked by mean viability with a ceiling at 100%. b, ABCB1 knockout is the top hit that rescues tepoxalin activity in a genome-wide CRISPR/Cas9 gene knockout screen. LS1034-Cas9 cells (pXPR_311) were infected with the Brunello sgRNA library, selected with puromycin, and treated with 16 μM tepoxalin versus vehicle control with two replicates in independent flasks. Cells were passaged every 3–4 days over a 30-day period. Two-sided p-values are computed using MAGeCK-MLE and shown versus gene-level log fold-changes. c, ABCB1 overexpression sensitizes to tepoxalin activity in a genome-wide CRISPR/dCas9 gene activation screen. LS1034-dCas9 cells (pXPR_109) were infected with the Calabrese sgRNA library, selected with puromycin, and treated with 16 μM tepoxalin versus vehicle control with two replicates in independent flasks. Cells were passaged every 3–4 days over a 14-day period. Two-sided p-values are computed using MAGeCK-MLE and shown versus gene-level log fold-changes. d, Tepoxalin cellular competition assay following ABCB1 knockout. LS1034-Cas9 cells (pXPR_311) were stably infected with Firefly luciferase and parental LS1034 cells (without Cas9) were stably infected with Renilla luciferase. Cells were mixed in 1:1 ratio an infected with the indicated sgRNA construct against ABCB1 or an intergenic region on chromosome 2 (negative control). Following puromycin selection, cell mixtures were treated with 16 μM tepoxalin. Firefly to Renilla luminescence ratio is plotted as log fold-change over time. Mean of three technical replicates is shown and results are representative of three independent experiments.

Article Snippet: Cell lines Parental cell lines were obtained from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) project 10 prior to PRISM barcoding (see https://portals.broadinstitute.org/ccle for original sources).

Techniques: RNA Expression, Activity Assay, Expressing, Gene Expression, Knock-Out, Genome Wide, CRISPR, Gene Knockout, Infection, Control, Over Expression, Activation Assay, Competitive Binding Assay, Stable Transfection, Luciferase, Construct, Negative Control, Selection