Journal: Nature Communications

Article Title: BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors

doi: 10.1038/s41467-019-11048-5

Figure Lengend Snippet: Upregulation of innate immune response genes in BRCA2-deficient human cells. a Venn diagram of common genes upregulated in H1299 and MDA-MB-231 human cells, upon chronic (28 days) DOX-induced BRCA2 depletion. b Gene set enrichment analysis (Gene Ontology—Biological Process database) of the 28 genes upregulated upon chronic BRCA2 inactivation in both H1299 and MDA-MB-231 cells. c , d Whole-cell extracts prepared after 4 and 28 days of DOX treatment were immunoblotted as indicated. GAPDH was used as a loading control. Phosphorylation site are indicated in red

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal antibodies raised against BRCA2 (1:1000, OP95, Calbiochem), GAPDH (1:30000, NB600-502, Novus Biologicals), α-Tubulin (1:30,000, TAT-1, Cancer Research UK Monoclonal Antibody Service); rabbit monoclonal antibody raised against ERK1/2 (1:5000, 4695, Cell Signaling), STING (1:1000, 13647, Cell Signaling), IRF3 (1:1000, AB76409, Abcam), phospho-IRF3 (1:1000, AB76493, Abcam), STAT1 (1:1000, 9175, Cell Signaling), phospho-STAT1 (1:1000, 9167, Cell Signaling); rabbit polyclonal antibodies raised against phospho-KAP1 (1:1000, A300-767A, Bethyl Laboratories), KAP1 (1:5000, A300-274A, Bethyl Laboratories), SMC1 (1:5000, A300-055A, Bethyl Laboratories).

Techniques: Control, Phospho-proteomics

Journal: Nature Communications

Article Title: BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors

doi: 10.1038/s41467-019-11048-5

Figure Lengend Snippet: Time course of innate immune response activation in BRCA2-deficient cells. a H1299 cells expressing a DOX-inducible BRCA2 shRNA were grown in the presence or absence of DOX for 28 days. Cells collected every 2 days were subjected to quantitative RT-PCR analyses using primers specific for indicated genes. mRNA levels were expressed relative to the gene encoding β-actin and to day 2 (2 −ΔΔCT ). n = 3 independent experiments, each performed in triplicates. Each dot represents a single replicate. b Whole-cell extracts prepared at indicated times after DOX addition were immunoblotted as shown. GAPDH was used as a loading control. Phosphorylation sites are indicated in red

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal antibodies raised against BRCA2 (1:1000, OP95, Calbiochem), GAPDH (1:30000, NB600-502, Novus Biologicals), α-Tubulin (1:30,000, TAT-1, Cancer Research UK Monoclonal Antibody Service); rabbit monoclonal antibody raised against ERK1/2 (1:5000, 4695, Cell Signaling), STING (1:1000, 13647, Cell Signaling), IRF3 (1:1000, AB76409, Abcam), phospho-IRF3 (1:1000, AB76493, Abcam), STAT1 (1:1000, 9175, Cell Signaling), phospho-STAT1 (1:1000, 9167, Cell Signaling); rabbit polyclonal antibodies raised against phospho-KAP1 (1:1000, A300-767A, Bethyl Laboratories), KAP1 (1:5000, A300-274A, Bethyl Laboratories), SMC1 (1:5000, A300-055A, Bethyl Laboratories).

Techniques: Activation Assay, Expressing, shRNA, Quantitative RT-PCR, Control, Phospho-proteomics

Journal: Nature Communications

Article Title: BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors

doi: 10.1038/s41467-019-11048-5

Figure Lengend Snippet: ISG induction in HR-deficient human cells and tumors and olaparib impact on this process. a Upregulation of innate immune response genes in BRCA2 -deleted ovarian tumors ( n = 4) versus tumors with median BRCA2 mRNA expression ( n = 145). b Upregulation of innate immune response genes in RAD51 -deleted ovarian tumors ( n = 11) versus tumors with median RAD51 mRNA expression ( n = 145). Dots in graphs represent individual tumors. Middle line (white), median; box limits 25 and 75 percentiles; whiskers, minimum and maximum values. c H1299 cells expressing a DOX-inducible BRCA2 shRNA were grown in the presence or absence of DOX for 4 days. Then, olaparib (1 or 10 µM) was added for 72 h, followed by quantitative RT-PCR analyses. Primers specific for the indicated genes were used. mRNA levels were expressed relative to the gene encoding β-actin and to untreated (−DOX) control cells (2 −ΔΔCT ). Error bars represent SD of n = 3 independent experiments. *, p < 0.05 (unpaired two-tailed t test). d Whole-cell extracts from cells treated as in ( c ) were immunoblotted as shown. GAPDH and SMC1 were used as loading controls. Phosphorylation sites are indicated in red. e Cells treated as in ( c ) were pulse-labeled with EdU for 30 min. Frequency of cells in G1, S, and G2/M stages of the cell cycle were determined using FACS analyses of EdU incorporation and propidium iodide staining. Error bars represent SD of n = 3 independent experiments

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal antibodies raised against BRCA2 (1:1000, OP95, Calbiochem), GAPDH (1:30000, NB600-502, Novus Biologicals), α-Tubulin (1:30,000, TAT-1, Cancer Research UK Monoclonal Antibody Service); rabbit monoclonal antibody raised against ERK1/2 (1:5000, 4695, Cell Signaling), STING (1:1000, 13647, Cell Signaling), IRF3 (1:1000, AB76409, Abcam), phospho-IRF3 (1:1000, AB76493, Abcam), STAT1 (1:1000, 9175, Cell Signaling), phospho-STAT1 (1:1000, 9167, Cell Signaling); rabbit polyclonal antibodies raised against phospho-KAP1 (1:1000, A300-767A, Bethyl Laboratories), KAP1 (1:5000, A300-274A, Bethyl Laboratories), SMC1 (1:5000, A300-055A, Bethyl Laboratories).

Techniques: Expressing, shRNA, Quantitative RT-PCR, Control, Two Tailed Test, Phospho-proteomics, Labeling, Staining

Journal: Nature Communications

Article Title: BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors

doi: 10.1038/s41467-019-11048-5

Figure Lengend Snippet: Effect of PARP inhibitor on ISG expression in BRCA1/2-deficient tumors. a Schematic representation of tumor treatment. Tumor-bearing mice were treated on indicated days with PARP inhibitor talazoparib or with solvent control, both orally administered (p.o.). Total RNA was prepared from tumors collected at the end of the treatment. b BRCA1-deficient MDA-MB-436 human cells (1 × 10 6 ) were injected into the mammary fat pad and c HCT116 ( BRCA2 +/+ or BRCA2 −/− ) human cells (3 × 10 6 ) were injected intramuscularly into the hind leg muscles of CB17/SCID mice and allowed to generate tumors which were then treated as in ( a ). mRNA levels of indicated genes were determined using quantitative RT-PCR analyses and were expressed relative to the gene encoding β-actin ( b ) or GAPDH ( c ) and to solvent-treated control tumors (2 −ΔΔCT ). Error bars represent SD of n = 3 ( b , c BRCA2 −/− ) or n = 5 ( c , BRCA2 +/+ ) tumors, for which quantitative RT-PCR reaction was performed in triplicates. NS , p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired two-tailed t test)

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal antibodies raised against BRCA2 (1:1000, OP95, Calbiochem), GAPDH (1:30000, NB600-502, Novus Biologicals), α-Tubulin (1:30,000, TAT-1, Cancer Research UK Monoclonal Antibody Service); rabbit monoclonal antibody raised against ERK1/2 (1:5000, 4695, Cell Signaling), STING (1:1000, 13647, Cell Signaling), IRF3 (1:1000, AB76409, Abcam), phospho-IRF3 (1:1000, AB76493, Abcam), STAT1 (1:1000, 9175, Cell Signaling), phospho-STAT1 (1:1000, 9167, Cell Signaling); rabbit polyclonal antibodies raised against phospho-KAP1 (1:1000, A300-767A, Bethyl Laboratories), KAP1 (1:5000, A300-274A, Bethyl Laboratories), SMC1 (1:5000, A300-055A, Bethyl Laboratories).

Techniques: Expressing, Solvent, Control, Injection, Muscles, Quantitative RT-PCR, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LSD1 modulates stress-evoked transcription of immediate early genes and emotional behavior

doi: 10.1073/pnas.1511974113

Figure Lengend Snippet: Western blot analysis showing that the additional 57-kDa SRF-related band is present only in hippocampal protein extracts and not in a nonneuronal cell line (HeLa). Total protein lysates from HeLa cells (lane1) and mouse hippocampus (lane2) were separated on SDS/PAGE and immunodecorated with anti-SRF antibody and anti-GAPDH antibody.

Article Snippet: The following antibodies were used for Western blot or immunoprecipitation experiments: anti-LSD1 (C69G12; Cell Signaling Technology); anti-SRF (D71A9; Cell Signaling Technology); anti-CoREST (07-455; Merck Millipore); anti-HDAC2 (ab7029; Abcam); anti-GAPDH (NB300-320; Novus Biological); anti-α/β tubulin (2148; Cell Signaling Technology); and anti-HA (sc-7392; Santa Cruz Biotechnology).

Techniques: Western Blot, SDS Page

Journal: Yonsei Medical Journal

Article Title: Changes in Inward Rectifier K + Channels in Hepatic Stellate Cells During Primary Culture

doi: 10.3349/ymj.2008.49.3.459

Figure Lengend Snippet: Relative inward rectifier K + channel and SUR gene expression in HSC. (A) The inward rectifier K + channel α-subunit gene expression in HSC was measured using real-time RT-PCR. The bar graphs show the relative gene expression for each inward rectifier K + channel subfamily (K ir 1.1, K ir 2.1 - K ir 2.4, K ir 3.1 - K ir 3.4, K ir 4.1 - K ir 4.2, K ir 5.1, K ir 6.1 - K ir 6.2, and K ir 7.1). (B) SUR gene expression was measured. The bar graphs show the relative gene expression for SUR1, SUR2A, and SUR2B. The HSC were used at 1 day, 1 week, 2 weeks, and 3 weeks of culture. The expression levels were normalized to GAPDH and calibrated by K ir 2.1 expression in the HSC cultured for 1 day. The data are shown as the mean ± SEM (n = 3). (C) K ir 2.1 and K ir 6.1 protein expression were measured by Western blotting. There was a band (55 kD) in the anti-K ir 2.1 membrane (Upper) as well as the anti-K ir 6.1 membrane (Lower). Both membranes were re-probed with anti-GAPDH antibody as shown. The data is representative of three independent experiments.

Article Snippet: The anti-K ir 2.1 and anti-K ir 6.1 antibodies were obtained from Alomone Labs (Jerusalem, Israel), the anti-GAPDH antibody was acquired from Novus Biologicals (Littleton, CO, USA), and Western Lightning was supplied by PerkinElmer Life Sciences (Gaithersburg, MD, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Membrane