Journal: Frontiers in Endocrinology

Article Title: Seviteronel, a Novel CYP17 Lyase Inhibitor and Androgen Receptor Antagonist, Radiosensitizes AR-Positive Triple Negative Breast Cancer Cells

doi: 10.3389/fendo.2020.00035

Figure Lengend Snippet: Differential effects on AR and AR targets with enzalutamide and seviteronel treatment. AR+ TNBC cells were treated with 5 μM enzalutamide or seviteronel ± 10 nM DHT. RT-qPCR was used to assess mRNA expression of (A) AR , (B) AQP3 , and (C) SEC14L2 in MDA-MB-453 cells. (D) Protein levels of p-DNAPKcs, total DNAPKcs, and AR were measured by immunoblot in MDA-MB-453 cells. Gene expression data represent mean ± SEM for three independent experiments, and immunoblots are representative of triplicate experiments. NS, p is not significant, * p < 0.05, ** p < 0.01.

Article Snippet: Proteins were detected using antibodies for phospho-DNAPKcs (Abcam ab124918, 1:1,000), total DNAPKcs (CST 12311, 1:1,000), androgen receptor (Millipore 06-680, 1:1,000), GAPDH (CST 2118, 1:1,000), and β-Actin (8H10D10, Cell Signaling 12262S, 1:50,000).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Caveolin-1 is Associated with Tumor Progression and Confers a Multi-Modality Resistance Phenotype in Pancreatic Cancer

doi: 10.1038/srep10867

Figure Lengend Snippet: A . Quantification of the percentage of cells with mitotic catastrophe after 3 Gy radiation (left panels, *p < 0.05). Representative immunofluorescence images of presence or absence of mitotic catastrophe at 72 hours in scrambled control and siCav-1 cells (right panels). B . Quantification of average number of nuclear pH2A.X foci per cell at multiple time points (*p < 0.05). Representative immunofluorescence staining for pH2A.X foci after 3 Gy radiation at 24 hours in scrambled control siRNA and siCav-1 cells (right panels). C . Top - Quantification of average number of nuclear BRCA1 foci per cell at multiple time points (left; *p < 0.05). Representative immunofluorescence staining of BRCA1 foci after radiation at 24 hrs in scrambled control siRNA vs siCav-1 (right panels). Bottom - Quantification of average number of nuclear pDNA-PK foci per cell (left; *p < 0.05). Representative immunofluorescence staining of pDNA-PK foci after radiation at 24 hrs in scrambled control siRNA-and siCav-1 (right panels). D . Immunoblotting of scrambled control and siCav-1 treated cells with or without radiation at different time points for expression of phosphorylated BRCA1, total BRCA1, phosphorylated DNAPK, total DNAPK, and beta actin as control (top panel). Note the reductions in phospho-BRCA1 and phospho-DNA-PK at 24 hrs after radiation with Cav-1 depletion. Loss of Cav-1 also increases the degree of radiation-induced activation of apoptotic signaling at 24 hours after 3Gy (bottom panel). For nuclear foci and mitotic catastrophe experiments, at least 50 cells were counted per datapoint.

Article Snippet: Anti- cleaved caspase-9, cleaved PARP, phospho-Akt, total Akt, phospho-BRCA1, phospho-DNAPK, total BRCA1, total DNAPK, phospho-JAK2, total JAK2, phospho-STAT3, total STAT3, PIAS3, Src (Y416), Src (Y527), total Src, SOCS2, phospho-JNK, total JNK, phospho-p38, total p38, phospho-ERK1/2, total ERK1/2, alpha tubulin, phospho-H2.AX, beta actin and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Role of Atypical Protein Kinase C in Estradiol-Triggered G 1 /S Progression of MCF-7 Cells

doi: 10.1128/mcb.24.17.7643-7653.2004

Figure Lengend Snippet: FIG. 1. Involvement of aPKC in S-phase entry and ERK-2 nuclear translocation induced by estradiol in MCF-7 cells. Quiescent MCF-7 cells were seeded on coverslips. (A) Cells were untransfected or transfected with the indicated plasmids and left unstimulated or stimulated with 10 nM estradiol. Untransfected cells were also stimulated with estradiol in the presence of 1 M GO6976 (Calbiochem). After BrdU pulsing, coverslips were analyzed for BrdU incorporation. In transfected cells, this was calculated as the percentage of BrdU-positive cells [(number of transfected BrdU-positive cells/number of transfected cells)] 100 and compared to BrdU incorporation of untransfected cells from the same coverslips. For each plasmid, data are derived from scoring of at least 200 cells. Results of more than two independent experiments have been averaged; values shown are means and standard errors of the mean. A construct expressing the Myc-tagged pSG5 empty plasmid was transfected into quiescent MCF-7 cells as a control. The cells were left unstimulated or stimulated with estradiol. BrdU was added, and after 24 h the coverslips were analyzed for DNA synthesis. A total of 62% of the Myc-tag-expressing cells entered S phase, whereas the percentage of BrdU incorporation in unstimulated cells was less than 13%. (B) Cells were transfected with the Myc-tagged ERK-2 wild type along with either GFP-antisense dominant negative PKC (GFP-PKC dn as) or GFP-sense dominant negative PKC (GFP-PKC dn s) (left panels). The middle panels show the staining of the Myc-tagged ERK-2 wild type. The results of corresponding nuclear staining (Hoechst) are shown (right panels). The micrographs represent the results of two independent experiments. (C) Quiescent MCF-7 cells were transfected with either wild-type or dominant negative Myc-tagged PKC. Expression of PKC was revealed by the anti-Myc-tagged antibody (top). In the results shown in the upper panels, the cells were left unstimulated or stimulated for 3 min with 10 nM estradiol, and lysates were immunoblotted with the antibodies against the indicated proteins. In the results shown in the lower panels, the cells were cotransfected with the HA-tagged Akt wild type. They were then left unstimulated or stimulated for 3 min with 10 nM estradiol. Lysates were immunoprecipitated with anti-HA-tagged antibody. Immunoprecipitated proteins were hybridized with the antibodies against the indicated proteins. wt, wild type; dn, dominant negative.

Article Snippet: The anti-phospho-PKC / (Thr410/Thr403), phospho-PKC / (Ser643/Ser66), phospho-PKC / II (Thr638/ Thr641), phospho-PKC (pan), and phospho-Akt (Ser473) antibodies were obtained from Cell Signaling.

Techniques: Translocation Assay, Transfection, BrdU Incorporation Assay, Plasmid Preparation, Derivative Assay, Construct, Expressing, Control, DNA Synthesis, Dominant Negative Mutation, Staining, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Role of Atypical Protein Kinase C in Estradiol-Triggered G 1 /S Progression of MCF-7 Cells

doi: 10.1128/mcb.24.17.7643-7653.2004

Figure Lengend Snippet: FIG. 2. Involvement of aPKC in the p27 nuclear localization modulated by estradiol in MCF-7 cells. Quiescent MCF-7 cells were seeded on coverslips. (A) MCF-7 cells were left untreated (top) or treated with 10 nM estradiol for 8 h (bottom). p27 localization was analyzed by fluorescence microscopy with anti-p27 MAb (left panels). The corresponding nuclear staining (Hoechst) is shown (right panels). (B) Cells were untransfected or transfected with the indicated plasmids and then left unstimulated or stimulated with 10 nM estradiol for 8 h. The p27 staining of cells was then analyzed by fluorescence microscopy. p27 staining of transfected cells was compared with that of nontransfected cells from the same coverslips. Data are derived from scoring of at least 200 cells for each coverslip, and results are expressed as the percentage of cells showing predominantly p27 nuclear staining. Results of more than three independent experiments have been averaged; the values shown are the means and standard errors of the mean. The MCF-7 cells used were also analyzed for BrdU incorporation. Incorporation was observed in 12% of total nuclei of unstimulated cells. This value shifted to 68% after cells were treated with estradiol. Overexpression of either Myc-tagged dominant negative PKC, A221–MEK-1 or Myc-His-tagged (dn) Akt inhibited the estradiol-stimulated BrdU incorporation by 68, 70, and 82%, respectively. E2, estradiol.

Article Snippet: The anti-phospho-PKC / (Thr410/Thr403), phospho-PKC / (Ser643/Ser66), phospho-PKC / II (Thr638/ Thr641), phospho-PKC (pan), and phospho-Akt (Ser473) antibodies were obtained from Cell Signaling.

Techniques: Microscopy, Staining, Transfection, Derivative Assay, BrdU Incorporation Assay, Over Expression, Dominant Negative Mutation

Journal: Molecular and Cellular Biology

Article Title: Role of Atypical Protein Kinase C in Estradiol-Triggered G 1 /S Progression of MCF-7 Cells

doi: 10.1128/mcb.24.17.7643-7653.2004

Figure Lengend Snippet: FIG. 4. Estradiol-induced Ras and ERK-2 activation depends on aPKC activity in hER-expressing Cos cells. Cos cells were transfected with either the pSG5 empty plasmid or hER-expressing plasmid, alone or with a wild-type or dominant negative PKC-expressing plas- mid. Quiescent cells were left untreated or treated for 3 min with 10 nM estradiol. (A) Lysates were analyzed for expression of either PKC or hER by immunoblotting with the appropriate antibodies. (B) Ly- sates were immunoprecipitated with either control (Ctrl-Ab) or anti- ERK-2 (Anti-erk-Ab) antibodies. The kinase activity was assayed with MBP as a substrate (top). Lysates were submitted to either GST or GST–Raf-RBD. Eluted proteins were immunoblotted with anti-pan Ras antibodies (bottom). ER, hER; E2, estradiol; wt, wild type; dn, dominant negative.

Article Snippet: The anti-phospho-PKC / (Thr410/Thr403), phospho-PKC / (Ser643/Ser66), phospho-PKC / II (Thr638/ Thr641), phospho-PKC (pan), and phospho-Akt (Ser473) antibodies were obtained from Cell Signaling.

Techniques: Activation Assay, Activity Assay, Expressing, Transfection, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Immunoprecipitation, Control

Journal: Molecular and Cellular Biology

Article Title: Role of Atypical Protein Kinase C in Estradiol-Triggered G 1 /S Progression of MCF-7 Cells

doi: 10.1128/mcb.24.17.7643-7653.2004

Figure Lengend Snippet: FIG. 6. The estradiol-stimulated ER/PKC/Ras complex assembly depends on PKC activation and is associated with serine phosphorylation of aPKC. (A) Quiescent MCF-7 cells were left untreated or treated for 3 min with 10 nM estradiol in the absence or presence of the antiestrogen ICI 182,780. Cell lysates were immunoprecipitated (IP) with anti-aPKC antibody (Santa Cruz). Lysate samples were incubated in parallel with control antibodies (Ctrl-Ab). Immunoprecipitates were immunoblotted with antibodies against the indicated proteins. (B) Cos cells were cotransfected with hER (ER) and wild-type (wt) or dominant negative (dn) Myc-tagged PKC. Quiescent cells were left untreated or treated for 3 min with 10 nM estradiol (E2) in the absence or presence of the antiestrogens ICI 182,780 (ICI) or OH-tamoxifen (Tam). Lysates were immunoprecipitated with anti-ER antibody (ER), and immunoprecipitates were blotted with antibodies against the indicated proteins. (C) Cos cells were transfected with hER alone or together with the wild-type or dominant negative Myc-tagged PKC. Quiescent cells were left untreated or treated for 3 min with 10 nM estradiol. Lysates were immunoprecipitated with anti-Myc-tagged antibody, and immunoprecipitates were blotted with antibodies against the indicated proteins. (D) GST-HEG14 was incubated with the 35S-labeled proteins (circles) in the absence () or presence () of 10 nM estradiol. Eluted proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed by fluorography. WB, Western blot.

Article Snippet: The anti-phospho-PKC / (Thr410/Thr403), phospho-PKC / (Ser643/Ser66), phospho-PKC / II (Thr638/ Thr641), phospho-PKC (pan), and phospho-Akt (Ser473) antibodies were obtained from Cell Signaling.

Techniques: Activation Assay, Phospho-proteomics, Immunoprecipitation, Incubation, Control, Dominant Negative Mutation, Transfection, Labeling, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Genome Research

Article Title: Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

doi: 10.1101/gr.265736.120

Figure Lengend Snippet: CiDER reporter system identifies DNA-PKcs inhibition as a means to increase CRISPR-del efficiency. (A) The CiDER endogenous reporter relies on a series of sgRNA pairs targeting exon 1 of PLXND1 gene, whose protein product is detected by flow cytometry. (B) CiDER measurements of CRISPR-del efficiency over a range of times between sgRNA delivery and cell harvesting (mean, standard deviation). (C) Independent evaluation of CRISPR-del efficiency in sorted plexin D1–negative and D1–positive cells. (Top) Representative fluorescent activated cell sorting (FACS) plot. Plexin D1–negative (APC-) and Plexin D1–positive cells (APC+) are gated. (Bottom) The fraction of nondeleted alleles in each population quantified by qPCR (mean, standard deviation). The black bar corresponds to a nontargeting control used for normalization. Green bars correspond to sorted HeLa cells with pgRNAs targeting PLXND1 (P3). (D) CRISPR-del efficiency measured by CiDER in HeLa upon DNA-PKcs inhibition (mean, standard deviation, one-tailed paired t-test). (E, top) Representative raw flow cytometry plots for HeLa upon DNA-PKcs inhibition. Plexin D1–positive cells are gated, and numbers correspond to percentage of cells. (Bottom) Representative images of individual sorted, stained HeLa cells. (F) CiDER measurements of CRISPR-del efficiency in HeLa upon DNA-PKcs inhibition with indicated small molecules (values: mean plexin D1–positive cells; *P < 0.05, one-tailed paired t-test).

Article Snippet: The membranes were blocked in 5% BSA in TBS- 0.1% tween and incubated within the indicated primary antibodies: total DNA-PKcs Y393 (Abcam ab32566), pS2056 DNA-PKcs (Abcam ab124918), α-tubulin (Sigma-Aldrich T6199).

Techniques: Cider Assay, Inhibition, CRISPR, Flow Cytometry, Cell Harvesting, Standard Deviation, FACS, One-tailed Test, Staining

Journal: Genome Research

Article Title: Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

doi: 10.1101/gr.265736.120

Figure Lengend Snippet: DNA-PKcs inhibition boosts CRISPR-del independent of cell, readout, or sgRNA delivery method. (A) CRISPR-del efficiency of CiDER in HeLa upon DNA ligase 4 inhibition (mean). (B) CiDER measurement of CRISPR-del efficiency in HCT116 and HEK293T cell lines upon DNA-PKcs inhibition (mean, standard deviation, one-tailed paired t-test). (C) CRISPR-del efficiency at MALAT1-enhancer upon DNA-PKcs inhibition. (Left) The fraction of WT allele quantified by qPCR (mean, standard deviation, one-tailed paired t-test). (Center) The pgRNAs, PCR primers, and a representative agarose gel from the genomic PCR of the region. (Right) Quantification of the KO band from the gel. (D) CRISPR-del efficiency of CiDER in HeLa as a result of initiating DNA-PKcs inhibition at 24 h after lentiviral infection (MOI = 0.3).

Article Snippet: The membranes were blocked in 5% BSA in TBS- 0.1% tween and incubated within the indicated primary antibodies: total DNA-PKcs Y393 (Abcam ab32566), pS2056 DNA-PKcs (Abcam ab124918), α-tubulin (Sigma-Aldrich T6199).

Techniques: Inhibition, CRISPR, Cider Assay, Standard Deviation, One-tailed Test, Agarose Gel Electrophoresis, Infection

Journal: Genome Research

Article Title: Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

doi: 10.1101/gr.265736.120

Figure Lengend Snippet: Applying DNA-PKcs inhibition to pooled CRISPR-del screens. (A) Testing the functional benefit of DNA-PKcs inhibition. (Left) Experimental setup, with pgRNAs targeting the transcription start site (TSS) of the essential gene RPS5. A positive control sgRNA pair (P+) target the gene open reading frame and thus cause loss of function independent of deletion. (Right) Viability assays with pgRNAs targeting the nonessential AAVS1 locus. (Bottom) Viability assays after RPS5 TSS deletion (mean, standard deviation, one-tailed paired t-test). (B) Design of a typical pooled high-throughput screen to identify essential genes. (C) Composition of pgRNA library targeting both essential genes’ TSS and nonessential neutral loci. (D) Log2 fold-change (LFC) in abundance of indicated pgRNAs compared with day 0. Significance calculated by two-tailed t-test. (E) Hits reported by MAGeCK at two timepoints. Negative log10 false-discovery rate (−log10FDR) for treated and untreated samples are indicated in the y- and x-axis, respectively. Each point represents a target. A hit is called a true positive (TP) if it is targeting an essential gene and has an FDR <0.25. Points above the diagonal indicate hits with a lower FDR (higher −log10FDR) in the treated sample. Numbers in plot reflect TPs in each combination of treated/untreated cells. (F) pgRNAs targeting ERCC1 TSS: read coverage for untreated and treated samples at 1 wk. (G) Fold-change variation for individual ERCC1 pgRNAs across timepoints: x-axis, timepoint (0, 1, and 3 wk); y-axis, LFC in abundance. Orange lines represent all pgRNAs targeting essential genes.

Article Snippet: The membranes were blocked in 5% BSA in TBS- 0.1% tween and incubated within the indicated primary antibodies: total DNA-PKcs Y393 (Abcam ab32566), pS2056 DNA-PKcs (Abcam ab124918), α-tubulin (Sigma-Aldrich T6199).

Techniques: Inhibition, CRISPR, Functional Assay, Positive Control, Standard Deviation, One-tailed Test, High Throughput Screening Assay, Two Tailed Test