Journal: Cell Death & Disease

Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

doi: 10.1038/s41419-020-02780-8

Figure Lengend Snippet: a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

Article Snippet: The following primary antibodies were used: ɣH2AX (Ser139) (2577, Cell Signaling Technology, Danvers, MA, USA); H2AX (ab11175, Abcam, Toronto, Canada); phospho-ATM (Ser1981) (200–301–400, Rockland Immunochemicals, Inc., Limerick, PA, USA); ATM (2873, Cell Signaling Technology); phospho-RPA32 (Ser33) (A300–246A, Bethyl Laboratories, Montgomery, TX, USA); RPA32 (ab2175, Abcam); phospho-Chk1 (Ser345) (2348, Cell Signaling Technology); phospho-Chk1 (Ser317) (12302, Cell Signaling Technology); phospho-Chk2 (Thr68) (2661, Cell Signaling Technology); BRCA1 (NB100–404, Novus Biologicals, Centennial, CO, USA); MLH1 (4256, Cell Signaling Technology); MSH2 (2017, Cell Signaling Technology); and GAPDH (5174, Cell Signaling Technology).

Techniques: Incubation, Western Blot, Transfection, Negative Control, Crystal Violet Assay, Control, Knockdown, Standard Deviation, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 1. Rescue of BRCA1 inhibition of ER- transcriptional activity by the HPV E7 and E6 and by other DNA tumor viral oncoproteins. A, domain structure of the DNA tumor virus oncoproteins studied. B, rescue of wtBRCA1 repression of ER- activity by HPV-E7 and HPV-E6 oncogenes. C, rescue of wtBRCA1 repression of ER- activity by adenovirus E1A oncogene. D, rescue of wtBRCA1 repression of ER- activity by the SV40 large T oncogene. E, wtBRCA1 only minimally represses ER- activity in human cervical cancer cell lines. For methodology, subconfluent proliferating cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid (ERE-TK-Luc)(0.25gDNAforeachplasmid)usingLipofectamineTM,asdescribedunder“MaterialsandMethods.”Thecellswerewashed,incubatedwithoutorwith17-estradiol(E2, 10 nM or 1 M) for t 24 h, and harvested for luciferase assays. Luciferase activity was expressed relative to the negative control (0 E2) (B–D) or as a percentage of the E2 positive control (E). The values are expressed as means S.E. of four replicate wells. Each experiment was performed at least twice to ensure reproducibility of the findings. Abbreviations and acronyms are as follows: CDK2, cyclin-dependent kinase 2; CR1/2, conserved region 1/2; CtBP, C-terminal binding protein; C-TERM, C-terminal domain; CXXC, one-half of a zinc coordination motif; cycA, cyclin A; ETQL, PDZ family protein-binding motif; hDLG, homolog of discs large, Drosophila; hScrib, homolog of Scribble, Drosophila; HR, host range domain; Hsc70, heat shock cognate protein 70 kDa; LXCXE, consensus retinoblastoma family protein-binding motif; MAGI-1, membrane-associated guanylate kinase inverted-1; MUPP-1, multiple PDZ domain protein; mut-SV40T, mutant SV40 large T oncogene with defective for RB-binding; NLS, nuclear localization signal; N-TERM, N-terminal domain; PCAF, p300/ CBP-associated factor; PLDLS, CtBP-binding motif; RNA pol II, RNA polymerase II; SV40T, simian virus 40 large T oncogene; TBP, TATA box-binding protein; TEF-1, transcriptional enhancer factor-1; TFIIB, transcription factor IIB; wt, wild-type.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Inhibition, Activity Assay, Virus, Transfection, Expressing, Plasmid Preparation, Luciferase, Negative Control, Positive Control, Binding Assay, Protein Binding, Membrane, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 2. Capture of C-terminally truncated BRCA1 proteins by full-length HPV-E7 and HPV-E6. A, domain structure of BRCA1 protein fragments. B, GST Western blot showing the expression of GST-E7 and GST-E6. The GST-E7 and GST-E6 fusion proteins were visualized by Western blotting using an antibody directed against GST. For reference, 100 l of GST-E7 or GST-E6 was used in the capture assays. C, expression of IVT BRCA1 proteins. The SDS-PAGE autoradiograph shows 10% of the input of each BRCA1 protein used for the GST capture assays. C and E, the arrows indicate the correct bands. D, capture of IVT full-length BRCA1 by GST-E7 and GST-E6. The binding of full-length BRCA1 (amino acids 1–1863) to beadscoatedwithGST-E7orGST-E6wasdeterminedbySDS-PAGEautoradiography.E,captureofsmallerBRCA1proteinfragmentsbyGST-E7andGST-E6.ThebindingofC-truncated BRCA1 proteins to beads coated with GST-E7 or GST-E6 was visualized by SDS-PAGE autoradiography. The abbreviations used are as follows: AD1, activation domain 1; BACH1, BRCA1-associated C-terminal helicase 1; BARD1, BRCA1-associated RING domain 1 protein; BRCT1/2, BRCA1 C-terminal domain 1/2; CtIP, CtBP-interacting protein; HDAC1/2, histone deacetylase 1/2; LMO4, LIM domain only protein 4; NES, nuclear export signal; NLS1, nuclear localization sequence 1; RHA, RNA helicase A; RbAp46/38, retinoblastoma-associated protein 46/48 kDa; TAD, transcriptional activation domain.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Capture-C, Western Blot, Expressing, SDS Page, Autoradiography, Binding Assay, Activation Assay, Histone Deacetylase Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 3. Capture of different IVT BRCA1 protein fragments by full-length GST-E7 and GST-E6. A, domain structure of BRCA1 protein and protein fragments. B, capture of different IVT BRCA1 proteins by full-length GST-E7 protein. The ability of GST-E7 to capture a set of IVT BRCA1 protein fragments spanning the length of the BRCA1 protein was determined. The input lane represents 10% of the IVT protein used in the GST capture assays. C, capture of different IVT BRCA1 proteins by full-length GST-E6 protein. Assays were performed as described in A, except that GST-E6 was used as the bait. D, capture of IVT N-terminal BRCA1 proteins by full-length GST-E7 protein. A group of N-terminal BRCA1 protein fragments was tested for capture by full-length GST-E7 protein. E, capture of IVT N-terminal BRCA1 proteins by full-length GST-E6 protein. A group of N-terminal BRCA1 protein fragments was tested for capture by full-length GST-E6 protein. F, capture of IVT BRCA1 BRCT1 by full-length GST-E7 and GST-E6 proteins. An IVT BRCA1 protein fragment containing the first BRCT1 was tested for capture by the GST-E7 and GST-E6 proteins. NES, nuclear export signal; AD1, activation domain 1.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Activation Assay

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 4. Capture of IVT full-length E7 and E6 proteins by various GST-BRCA1 pro- tein. A, capture of full-length IVT E7 by different GST-BRCA1 proteins. The ability of a set of overlapping GST-BRCA1 proteins to capture in vitro translated full-length E7 was tested. B, capture of full-length IVT E6 proteins by different GST-BRCA1 proteins. The ability of a set of overlapping GST-BRCA1 proteins to capture in vitro translated full- length E7 was tested. C, Western blot showing the expression of different GST-BRCA1 proteins.ThedifferentGST-BRCA1proteinsutilizedinAandBwerevisualizedbyWestern blottingusinganantibodydirectedagainstGST.Thecorrectbandisindicatedbyaboxin the figure.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: In Vitro, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 5. In vivo association of BRCA1 and the E6 and E7 proteins. A, IP of endoge- nous E7 co-precipitates E7 and BRCA1 in SiHa cells. Subconfluent proliferating SiHa human cervical cancer cells were subjected to IP using an anti-E7 antibody and Western- blotted to detect E7 and BRCA1. See “Materials and Methods” for details. B, IP of endog- enous BRCA1 co-precipitates BRCA1 and E7 in SiHa cells. Subconfluent proliferating SiHa cells were subject to IP using an anti-BRCA1 antibody and Western-blotted to detect BRCA1 and E7. See “Materials and Methods” for details. C, IP of BRCA1 co-precipitates BRCA1 and exogenous FLAG-tagged E6 in 293T cells. Subconfluent proliferating 293T cells were transfected overnight with FLAG-E6 and wtBRCA1 expression vectors (10 g ofeachvectorper100-mmdish)usingLipofectamineTM,post-incubatedfor24htoallow gene expression, immunoprecipitated using an anti-BRCA1 antibody, and Western-blot- ted to detect the BRCA1 and FLAG-E6 proteins. D, anti-FLAG IP co-precipitates exoge- nous FLAG-E6 and BRCA1 in 293T cells. 293T cells were transfected as described above in C, post-incubated for 24 h to allow gene expression, immunoprecipitated using an anti- FLAG antibody, and Western-blotted to detect the FLAG-E6 and BRCA1 proteins.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: In Vivo, Western Blot, Transfection, Expressing, Gene Expression, Immunoprecipitation, Incubation

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 6. Capture of BRCA1 by truncated and mutant GST-E7 and E6 proteins. A, schematic diagrams of the truncated/mutant E7 proteins used in this study. B, Western blot showing the expression of truncated and mutant GST-E7 proteins. A set of truncated and mutant GST-E7 proteins were visualized by Western blotting, using an anti-GST antibody. C,captureofIVTBRCA1-(1–302)bytruncatedandmutantGST-E7proteins.DifferentGST-E7proteinsshowninAweretestedfortheirabilitytocaptureaninvitrotranslatedN-terminal portion of BRCA1 (amino acids 1–302). D, capture of IVT BRCA1-(1–771) by truncated and mutant GST-E7 proteins. Different GST-E7 proteins from A were tested for their ability to capture an invitrotranslatedN-terminalportionofBRCA1correspondingtoaminoacids1–771.E,schematicdiagramsofthetruncated/mutantE6proteinsusedinthisstudy.F,Western blot showing the expression of truncated and mutant GST-E6 proteins. A set of truncated and mutant GST-E6 proteins were visualized by Western blotting, using an anti-GST antibody. G, capture of IVT BRCA1-(1–302) by truncated and mutant GST-E6 proteins. The various GST-E6 proteins shown in D were tested for their ability to capture IVT BRCA1-(1–302).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Mutagenesis, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 7.RescueofBRCA1repressionofER-activitybywild-typeversusmutantE7andE6.A,rescueofBRCA1repressionofER-bymutantversuswild-typeE7andE6proteins. Subconfluent proliferating MCF-7 cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid (ERE-TK-Luc) (0.25 g DNA for each plasmid) using LipofectamineTM. The cells were washed, incubated without or with 17-estradiol (E2, 10 nM) for t 24 h, and harvested for luciferase assays. Luciferase activity was expressed as a percentage of the E2-positive control; and the values are expressed as means S.E. of four replicate wells. Each experiment was performed at least twice to ensure reproducibility of the findings. B and C, dose responses for the rescue of BRCA1 repression of ER- activity by the wild-type E7 (B) and E6 (C) expression vectors. AssayswerecarriedoutinMCF-7andT47DcellsasdescribedaboveinA,exceptusingdifferentquantitiesoftheE7andE6expressionvectors.Thetotalcontentoftransfectedplasmid DNA was kept constant by the addition of the appropriate quantity of control plasmid.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Positive Control, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 8. Effect of HPV oncoproteins E7 and E6 on other functional activities of BRCA1. A, ability of E7 and E6 to rescue the BRCA1 inhibition of E-box-Luc reporter activity. Subconfluent proliferating MCF-7 or T47D cells in 24-well dishes were trans- fected overnight with the indicated expression vectors and a reporter driven by the c-Myc E-box upstream of a minimal promoter and the luciferase gene (E-box-Luc) (see Ref. 29). After transfection, the cells were washed, post-incubated for 24 h to allow gene expression, and harvested for luciferase assays. Luciferase activity was expressed as a percentage of the positive control (E-box-Luc only, no wtBRCA1, E7, or E6); and the values are expressed as means S.E. of four replicate wells. B, ability of E7 and E6 to rescue the BRCA1 repression of hTERT promoter activity. Assays were performed as described in A, except that a reporter containing the human TERT core promoter upstream of the luciferase gene (29) was utilized instead of the E-box-Luc reporter. Lucif- erase activity was expressed as a percentage of the positive control (hTERT-Luc only, no wtBRCA1, E7, or E6), and the values are expressed as means S.E. of four replicate wells.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Functional Assay, Inhibition, Activity Assay, Expressing, Luciferase, Transfection, Incubation, Gene Expression, Positive Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 9. Effects of the E6 and E7 oncoproteins on BRCA1, p53, and RB1 protein levels.SubconfluentproliferatingMCF-7orT47Dcellsweretransientlytransfectedover- night with empty pcDNA3 vector, with FLAG wild-type E6 or zinc finger defective mutant E6 (E6-(C66,136G)) (A), or with FLAG wtE7 or zinc finger defective mutant E7 (E7-(C91G)) (B) expression vectors. The cells were washed, post-incubated for 24 h to allow gene expression,andharvestedforWesternblottingtodetecttheE6andE7oncoproteins,the tumor suppressors BRCA1, p53, and RB1, and -actin (control for loading and transfer).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Incubation, Gene Expression, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 11. Effect of knockdown of BRCA1 on the ability of the E6 and E7 oncopro- teins to stimulate hTERT promoter activity. A, hTERT promoter assays. Subconfluent proliferating MCF-7 or T47D cells in 24-well dishes were preincubated for 48 h with a previously validated BRCA1 siRNA (29) or the control siRNA (50 nM); transfected over- night with wtE6, wtE7, or pcDNA3 vector; and post-incubated for 24 h to allow gene expression. Luciferase activity was expressed as a percentage of the positive control (hTERT-Luc only, no siRNA, E6, or E7). The values shown are means S.E. of four replicate wells. The data are shown on a linear scale to more easily delineate 2–3-fold changes in reporter gene activity. B, effect of siRNAs on BRCA1 protein levels. Subconfluent prolif- erating MCF-7 cells were treated with no siRNA (vehicle only), BRCA1 siRNA, or control siRNA (50 nM) for 72 h and harvested for Western blotting to detect BRCA1 or -actin (control for loading and transfer).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Knockdown, Activity Assay, Control, Transfection, Plasmid Preparation, Incubation, Gene Expression, Luciferase, Positive Control, Western Blot

Journal: American Journal of Cancer Research

Article Title: BRCA1 identified as a modulator of temozolomide resistance in P53 wild-type GBM using a high-throughput shRNA-based synthetic lethality screening

doi:

Figure Lengend Snippet: BRCA1 knockdown sensitizes GSCs to TMZ induced synthetic lethality selectively in p53 wild type cells. A and B. Cell viability of GSCs upon TMZ treatment upon BRCA1 kncockdown using ShRNA/siRNA against BRCA1 incomparon to scramble control. Cell viability was determined by cell titer blue assay (after 5 days of treatment) of the GSCs after BRCA1 knockdown in presence of TMZ, n = 3/data point; mean ± SD. Right panel western blot showing knock down of BRCA1 protein.

Article Snippet: The following primary antibodies were used for protein expression detection: anti-BRCA1, anti-P53, anti-rH2AX, anti-PARP, anti-caspase 3 (Cell Signaling, Boston, MA, USA), anti-FANCD2, and anti-Rad51 (Abcam, Cambridge, MA, USA).

Techniques: shRNA, Western Blot

Journal: American Journal of Cancer Research

Article Title: BRCA1 identified as a modulator of temozolomide resistance in P53 wild-type GBM using a high-throughput shRNA-based synthetic lethality screening

doi:

Figure Lengend Snippet: p53 is necesssray to induce synthetic lethality. A. Increased cell death observed in p53 wt cells (GSC11 and GSC295) in BRCA1 knockdown cells in presence of TMZ in comparison to either TMZ alone or BRCA1 knockdown alone by annexin V staining. B. Western blot analysis showing increased protein expression of cleaved caspase 3 and cleaved PARP in p53 wild type cells GSC11 cells after BRCA1 knockdown in presence of TMZ.

Article Snippet: The following primary antibodies were used for protein expression detection: anti-BRCA1, anti-P53, anti-rH2AX, anti-PARP, anti-caspase 3 (Cell Signaling, Boston, MA, USA), anti-FANCD2, and anti-Rad51 (Abcam, Cambridge, MA, USA).

Techniques: Staining, Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: BRCA1 identified as a modulator of temozolomide resistance in P53 wild-type GBM using a high-throughput shRNA-based synthetic lethality screening

doi:

Figure Lengend Snippet: P53 knockdown abolishes apoptotic capacity. A. Western blot showing knock out of p53 in GSC11 (p53 wt) cells. B and C. Flow cytometry analysis showing knock down of BRCA1 in presence of TMZ failed to induce apoptosis when p53 is knocled out in p53 wt cells. D. Western blot showing no increase of caspase 3 cleavage an PARP clevage by knockdown of BRCA1 and TMZ when p53 is knocked out in wt cells.

Article Snippet: The following primary antibodies were used for protein expression detection: anti-BRCA1, anti-P53, anti-rH2AX, anti-PARP, anti-caspase 3 (Cell Signaling, Boston, MA, USA), anti-FANCD2, and anti-Rad51 (Abcam, Cambridge, MA, USA).

Techniques: Western Blot, Knock-Out, Flow Cytometry

Journal: American Journal of Cancer Research

Article Title: BRCA1 identified as a modulator of temozolomide resistance in P53 wild-type GBM using a high-throughput shRNA-based synthetic lethality screening

doi:

Figure Lengend Snippet: Increased DNA damage response (DDR) in p53 wt cells coorelates with synthetic lethality. A. Western blot analysis with the indicated antibodies of protein lysates from p53 wt and p53 mut GSCS showed increased DDR protein expression in p53 wt cells than in mut p53 cells. B and C. Immunofluorescence of p53 wt and p53 mut GSCs treated with 100 µM of TMZ and siRNA BRCA1 knockdown by for 72 hours and DNA damage was evaluated by γH2AX (red). D. western blot showing increased γH2AX protein in p53 wt cells (GSC11) thank in p53 myt (GSC23) cell lines.

Article Snippet: The following primary antibodies were used for protein expression detection: anti-BRCA1, anti-P53, anti-rH2AX, anti-PARP, anti-caspase 3 (Cell Signaling, Boston, MA, USA), anti-FANCD2, and anti-Rad51 (Abcam, Cambridge, MA, USA).

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: Molecular oncology

Article Title: Patient-derived xenografts recapitulate molecular features of human uveal melanomas

doi: 10.1016/j.molonc.2013.02.004

Figure Lengend Snippet: Determination of BAP1 mRNA (A) and protein (B) expression in patients and xenograft tumors according BAP1 status (deleterious mutations taken into account). Wilcoxon test was performed to determined significance in BAP1 expressions between primary tumors ...

Article Snippet: A specific primer/probes set (assay id: Hs01109276_g1, Life Technologies) that measures the junction of exon 16 and 17 was used to performed the relative quantification to GAPDH expression (Hs03929097_g1).

Techniques: Expressing

Journal: Environmental Health Perspectives

Article Title: Associations between Maternal Tobacco Smoke Exposure and the Cord Blood CD 4 + DNA Methylome

doi: 10.1289/EHP3398

Figure Lengend Snippet: Differentially methylated CpGs identified in MACHS that were replicated in the SMKE cord blood CD 4 + study ( n = 14 exposed; n = 16 unexposed).

Article Snippet: Chr18:3412781 , 5.6 , 8.1 , cg04431731 , 0.4 , 0.04 , TGIF1 , 0.

Techniques: Methylation

Journal: Autophagy

Article Title: Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations.

doi: 10.1080/15548627.2018.1509818

Figure Lengend Snippet: Figure 4. Impaired mitophagy in GbaL444P/WT mouse hippocampal neurons. (A) Representative micrographs of basal mitophagy in WT:mt-Keima and GbaL444P/WT:mt-Keima primary hippocampal neurons (left panel). Neurons were treated with and without the lysosome protease inhibitors leupeptin and pepstatin A (Leu/Pep, 10 μM each) to determine the amount of mitochondrial degraded by lysosomes. The ratio of the 561:458 nm-excited mito-Keima fluorescence in neuronal cell bodies was calculated to reflect mitophagy activity (right panel) . Scale bar: 10 μm; (n = 40 neurons per genotype from 4–5 culture dishes; 14–15 DIV). (B) MitoGreen and LysoTracker Red colabelling in CCCP-treated WT and GbaL444P/WT primary hippocampal neuronal cultures (left panel, scale bar: 10 μm). Neurons were treated with CCCP (10 µM) for 1 h. The cells were recovered in fresh medium, loaded with MitoTracker Green (MitoGreen; 200 nM) for 30 min followed by LysoTracker Red (LysoRed, 200 nM) for 30 min at 37°C. The number of MitoGreen and LysoRed colabeled puncta (yellow) per cell body were counted as an index of mitophagy (right panel). The boxed areas (scale bar: 2 μm) are shown in higher magnification in the right 3 micrographs for clarity. Individual neurons (40 per condition) from 4–5 culture dishes (8–10 neurons per dish) were analyzed. (C) Mitochondrial levels of autophagy receptor proteins in brain tissue from WT (n = 12) and GbaL444P/WT (n = 12) mice. Brain mitochondria were fractionated and analyzed for mitophagy proteins PINK1 and PARK2, K63- and K48-ubiquitinated species, and the autophagy receptors SQSTM1, NBR1, BNIP3L and OPTN. Protein levels were normalized to VDAC1, as the abundance of VDAC1, when normalized to GAPDH, was consistent with the increase in mitochondrial content in the mutants. Data represent mean ± SE (A, B) or mean % of WT controls (C) from 3 independent experiments. Compared to WT, * p < 0.05; **, p < 0.01, Student’s-t test.

Article Snippet: Primary Antibodies used for western blot and immunohistochemistry include: human total OXPHOS respiratory chain proteins complexes (Abcam, ab110411), MT-CO1/mitochondrial DNA encoded cytochrome c oxidase I (Abcam, ab14705), MFN1 (Santa Cruz Biotechnology, sc50330), MFN2 (Abcam, ab56889), OPA1 (BD Bioscience, BP612606), FIS1 (Proteintech, 10586–1-AP), DNM1L/DRP1 (Santa Cruz Biotechnology, sc32898), VDAC1 (Abcam, ab15895), TOMM20 (Proteintech, 11802–1-AP), TIMM23 (Proteintech, 11123-AP), CYCS/cytochrome C (Abcam, ab13575), PPIF/cyclophilin F (Abcam, ab110324), SOD2 (Abcam, ab13533), PPARGC1A/PGC1-α (Abcam, ab54481), NRF1 (Abcam, ab175932), TFAM (Abcam, ab119684), ACTB/ β-actin (Sigma, A5441), GBA (Abnova, H00002629-M01), GBA (Sigma, G4171), SNCA (Sigma, S3062), BECN1/Beclin1 (Sigma, B6186), AMBRA1 (Novus, 26190002), MTOR (Cell Signaling Technology, 2972S), p-MTOR (Cell Signaling Technology, 2971S), RPS6 (Cell Signaling Technology, 2317S), p-RPS6 (Cell Signaling Technology, 2211S), PRKN/ PARK2/parkin (Cell Signaling Technology, 2132S), PINK1 (Cell Signaling Technology, 6946S), NBR1 (Proteintech, 16004–1-AP), BNIP3L (Proteintech, 12986–1-AP), OPTN/ Optineurin (Proteintech, 10837–1-AP), CALCOCO2/NDP52 (Cell Signaling Technology, 9036S), ubiquitin (Millipore, mab1510), K48 ubiquitin (Millipore, 051307), K63 ubiquitin (Millipore, 051308), GAPDH (Novus, NB300112), LC3B (Novus, NB600-1384), SQSTM1/p62 (MBL, PM045 and ARP, 03-GP62-C), CTSD/cathepsin D (Abcam, ab75852), LAMP1 (Abcam, ab24170), 8-OHdG (Abcam, ab62623), EEA1 (Cell Signaling Technology, #2411), MYC (Abcam, ab9132), and CANX/calnexin (Sigma, MAB3126).

Techniques: Fluorescence, Activity Assay

Journal: Autophagy

Article Title: Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations.

doi: 10.1080/15548627.2018.1509818

Figure Lengend Snippet: Figure 6. Dual effects of heterozygous GBA mutation on mitophagy. (A) GBA protein levels of GBA knockout (KO), MYC-WT GBA (WT) and MYC-L444P (LP) mutant GBA-overexpressing SH-SY5Y cell lines (left panel). The overexpression of MYC-WT and MYC-GBAL444P, and the deletion of endogenous GBA were validated by western blotting using anti-MYC and anti-GBA antibodies (middle panel), and by measuring GBA enzyme activity from lysosomal-enriched fractions (right panel). Compared to Mock controls (C), **, p < 0.01, One-way ANOVA, Bonferroni post hoc test; (B) MitoTracker Red, MitoTracker Green, and MitoSOX Red labeling in control, KO, WT and L444P SH-SY5Y cells. All 4 cell lines were loaded with MitoTracker Red (MitoRed, 200 µM, 20 min) and MitoTracker Green (MitoGreen, 200 µM, 20 min), or MitoSOX Red (3 μM, 10 min) prior to live cell imaging. Scale bar: 10 μm; (C-D) Quantification of MitoTracker fluorescence intensity in KO- (C) and L444P- (D) SH-SY5Y cells. MitoGreen and MitoRed fluorescence estimates the amount of total mitochondria and functional mitochondria, respectively. The ratio of MitoRed to MitoGreen fluorescence intensity was calculated to estimate the levels of functional mitochondria (n = 60–80 cells per condition from 3 independent experiments). (E) Quantification of MitoSOX Red fluorescence intensity in control, KO, WT and L444P SH-SY5Y cells (n = 60–80 cells per condition from 3 independent experiments). Both L444P and KO cells exhibited an increase in MitoSOX Red intensity, indicative of mitochondrial oxidative stress. (F) Western blot analysis of mitochondrial fraction for autophagy receptors in control, KO, WT and L444P cells. Compared to controls, L444P cells showed lower mitochondrial levels of SQSTM1, NBR1, and BNIP3L. (G) Impaired autophagy in KO and L444P cells. Control, KO, WT and L444P cells were treated with (PI+) and without (PI−) lysosome inhibitors leupeptin and pepstatin A (10 μM each). Flux of autophagy including lysosomal degradation was denoted as ‘PI+-PI−’. (H) Western blot analysis of autophagy-activating proteins AMBRA1 and BECN1 in all 4 cell lines. Data represent mean % of mock controls from 3 independent experiments. Compared to controls, L444P cells showed a reduction in protein levels of BECN1 and AMBRA1. *, p < 0.05; **, p < 0.01, One-way ANOVA, Bonferroni post hoc test.

Article Snippet: Primary Antibodies used for western blot and immunohistochemistry include: human total OXPHOS respiratory chain proteins complexes (Abcam, ab110411), MT-CO1/mitochondrial DNA encoded cytochrome c oxidase I (Abcam, ab14705), MFN1 (Santa Cruz Biotechnology, sc50330), MFN2 (Abcam, ab56889), OPA1 (BD Bioscience, BP612606), FIS1 (Proteintech, 10586–1-AP), DNM1L/DRP1 (Santa Cruz Biotechnology, sc32898), VDAC1 (Abcam, ab15895), TOMM20 (Proteintech, 11802–1-AP), TIMM23 (Proteintech, 11123-AP), CYCS/cytochrome C (Abcam, ab13575), PPIF/cyclophilin F (Abcam, ab110324), SOD2 (Abcam, ab13533), PPARGC1A/PGC1-α (Abcam, ab54481), NRF1 (Abcam, ab175932), TFAM (Abcam, ab119684), ACTB/ β-actin (Sigma, A5441), GBA (Abnova, H00002629-M01), GBA (Sigma, G4171), SNCA (Sigma, S3062), BECN1/Beclin1 (Sigma, B6186), AMBRA1 (Novus, 26190002), MTOR (Cell Signaling Technology, 2972S), p-MTOR (Cell Signaling Technology, 2971S), RPS6 (Cell Signaling Technology, 2317S), p-RPS6 (Cell Signaling Technology, 2211S), PRKN/ PARK2/parkin (Cell Signaling Technology, 2132S), PINK1 (Cell Signaling Technology, 6946S), NBR1 (Proteintech, 16004–1-AP), BNIP3L (Proteintech, 12986–1-AP), OPTN/ Optineurin (Proteintech, 10837–1-AP), CALCOCO2/NDP52 (Cell Signaling Technology, 9036S), ubiquitin (Millipore, mab1510), K48 ubiquitin (Millipore, 051307), K63 ubiquitin (Millipore, 051308), GAPDH (Novus, NB300112), LC3B (Novus, NB600-1384), SQSTM1/p62 (MBL, PM045 and ARP, 03-GP62-C), CTSD/cathepsin D (Abcam, ab75852), LAMP1 (Abcam, ab24170), 8-OHdG (Abcam, ab62623), EEA1 (Cell Signaling Technology, #2411), MYC (Abcam, ab9132), and CANX/calnexin (Sigma, MAB3126).

Techniques: Mutagenesis, Knock-Out, Over Expression, Western Blot, Activity Assay, Labeling, Control, Live Cell Imaging, Fluorescence, Functional Assay