Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-53BP1 , Novus Biologicals , Cat# NB100-904.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: (A) After transfection with indicated siRNA SMARTpools, cells were treated +/− 10 Gy IR and harvested at 1 h or 6 h post-IR, along with unirradiated controls. Acid-extracted histones and corresponding cytoplasmic fractions were analyzed by Western blot. Quantification of three independent replicates is depicted and significance determined using paired t-test following normalization to siControl UT 1h, mean +/− SEM *, p<0.05. (B) Cells were treated as in (A); cytoplasmic and nuclear fractions were prepared and analyzed by Western blot. (C) Control and ACLY-silenced cells were treated with 2 Gy IR, and 4 h later immunofluorescence imaging was performed for BRCA1, 53BP1, or γH2AX, scale bar- 200 M. Representative experiment from n=2 per cell line and condition with quantification of 10 fields/experiment, mean +/− SEM, *, p<0.05, **, p<0.01. (D) Control and ACLY-silenced cells were treated with 2 Gy IR imaged for cyclin A and BRCA1 after 4 h, scale bar-200 M. Representative experiment from n=2. Quantification of BRCA1 foci in cyclin A+ cells in 10 fields, mean +/−SEM, *, p<0.05. See also Figure S1.

Article Snippet: Antibodies used for immunofluorescence (IF): BRCA1 (1:100, Santa-Cruz Biotechnology), 53BP1 (1:300, Novus Biologicals), γH2AX S139 (1:500, Millipore or Cell Signaling).

Techniques: Transfection, Western Blot, Immunofluorescence, Imaging

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: (A) U2OS reporter cell line transfected with indicated siRNA SMARTpools was treated with Shield-1 and 4-OHT for 5 h to induce DSBs by mCherry-Lac1-Fok1. Colocalization of BRCA1 or 53BP1 with Fok1 was imaged by IF, scale, 200 M. Representative experiment from n=3 each for 53BP1 and BRCA1. Quantitation of 15–20 fields from each sample is depicted, ****, p<0.0001, n.d.- not detected (lower left panel); A.U.- Arbitrary units. Re-drawn schematic of the reporter assay, previously published (Tang et al., 2013) is depicted (lower right panel) (B) BRCA1 colocalization with Fok1 was examined in 53BP1-deficient U2OS reporter cells. (See also Figure S2B.) n.s.- not significant; A.U.- Arbitrary units. (C) AcH4 ChIP using different primer sets for endogenous loci was performed in a U2OS-TRF1-Fok1 reporter cell line that induces DSBs within telomeres (Tang et al., 2013). DSBs were induced by treatment with 4-OHT for 5 h; mean +/− SEM for 5 h, *, p<0.05, **, p<0.01, n.s.- not significant, n.d.- not detected. See Figure S3 for primer locations. (D) HR assay performed in the DR-GFP U2OS reporter line (Pierce et al., 2001). Cells were transfected with individual siRNAs for 24 hours (siACLY A or siACLY B), followed by transfection of the endonuclease Sce-1 for an additional 48 h. GFP positivity, indicating HR efficiency, was analyzed by flow cytometer, mean +/− SEM, **, p<0.01; ***, p<0.001. (E) To assay HR, cells were transduced with EV or ACLY-expressing lentivirus. Cells were transfected with siRNA SMARTpools, followed by Sce-1 as in (D). Data is representative of 2 independent experiments. Mean +/− SEM is graphed, ***, p<0.001. (F) ACLY was silenced using two independent shRNAs in HeLa cells expressing TRF2ΔB/ΔM. NHEJ was assessed by scoring end-to-end chromosomal fusions. Mean +/− SEM **, p<0.01, ***,p<0.001. See also Figure S2 and Figure S3.

Article Snippet: Antibodies used for immunofluorescence (IF): BRCA1 (1:100, Santa-Cruz Biotechnology), 53BP1 (1:300, Novus Biologicals), γH2AX S139 (1:500, Millipore or Cell Signaling).

Techniques: Transfection, Quantitation Assay, Reporter Assay, Flow Cytometry, Transduction, Expressing

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: (A) EV or myc-tagged mACLY constructs (WT or H760A) were transfected in LN229 sgACLY (clone 3.8) cells, followed by irradiation with 2 Gy IR. Cells were fixed after 4 h and co-stained for myc-tag (representing ACLY) and BRCA1. Quantification on myc-tag+ cells (or all nuclei for EV controls) was performed from 25 different fields per sample using ImageJ, mean +/−SEM, **, p<0.01, ****, p<0.0001;, n.s.-not significant. Scale bar-200 M. (B) Experiment was conducted as in (A), with analysis of 53BP1 and myc-tag. (C) Experiment was conducted as in (A), using myc-tagged mACLY constructs (WT, S455A, or S455D). (D) ACLY null LN229 cells were co-transfected with empty vector and GFP, WT mACLY and GFP, or with mACLY-NES and GFP for 48 h, and fixed 4 h after 2Gy IR treatment. IF was performed for BRCA1; GFP and BRCA1 was imaged. Quantification was performed with at least 25 fields per sample using ImageJ scoring GFP positive cells, mean +/−SEM. ***, p<0.001, n.s.- not significant. See also Figure S6.

Article Snippet: Antibodies used for immunofluorescence (IF): BRCA1 (1:100, Santa-Cruz Biotechnology), 53BP1 (1:300, Novus Biologicals), γH2AX S139 (1:500, Millipore or Cell Signaling).

Techniques: Construct, Transfection, Irradiation, Staining, Plasmid Preparation

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: (A) U2OS cells were treated with olaparib at indicated doses for 72 h following ACLY silencing for 24 h. Viability was assessed using trypan-blue exclusion assay, mean +/− SEM, *, p<0.05. (B) Two independent U2OS reporter clones lacking 53BP1 (sg53BP1-clone 2A and clone 3D) were treated without (solid bars) or with (striped bars) olaparib at 30 M final concentration for 72 h following ACLY silencing for 24 h. Viability was assessed using trypan-blue exclusion assay. n.s.- not significant. (C) ACLY was silenced in HeLa cells, and cells were treated with olaparib for 24 h. Samples were collected following colcemid treatment for 1.5 h and metaphases were examined for chromosomal abnormalities. Quantification was performed manually on 70–80 total metaphases from 2 different experiments which were pooled together, mean +/− SEM; *, p<0.05, **, p<0.01. See also Figure S7.

Article Snippet: Antibodies used for immunofluorescence (IF): BRCA1 (1:100, Santa-Cruz Biotechnology), 53BP1 (1:300, Novus Biologicals), γH2AX S139 (1:500, Millipore or Cell Signaling).

Techniques: Trypan Blue Exclusion Assay, Clone Assay, Concentration Assay

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used for immunofluorescence (IF): BRCA1 (1:100, Santa-Cruz Biotechnology), 53BP1 (1:300, Novus Biologicals), γH2AX S139 (1:500, Millipore or Cell Signaling).

Techniques: Western Blot, Immunohistochemistry, Recombinant, Clone Assay, Expressing, CRISPR, shRNA, Plasmid Preparation, Construct, Software

Journal: The Journal of Neuroscience

Article Title: Memory-Related Synaptic Plasticity Is Sexually Dimorphic in Rodent Hippocampus

doi: 10.1523/JNEUROSCI.0801-18.2018

Figure Lengend Snippet: Synaptic TrkB activation depends on ERα and β1 integrin, and is required for LTP in females. A, Deconvolved image shows punctate localization of pTrkB Y515 (red) and PSD-95 (green); yellow indicates double labeling (arrow). Scale bar, 2 μm. Line graph shows that TBS, compared with LFS, caused a greater rightward skew in the density frequency distribution for pTrkB-IR colocalized with PSD-95 (p < 0.0001, F(19,342) = 10.44), and MPP substantially reduced this effect (p < 0.0001, F(19,342) = 10.66; n = 10/group). Right, The percentage of double-labeled synapses with dense pTrkB-IR (≥90 units) was elevated after TBS in vehicle-treated, but not in MPP-treated, female slices (group means normalized to the LFS group mean; p = 0.0025, F(2,29) = 7.55; post hoc tests: LFS vs TBS, *p < 0.05; TBS vs TBS + MPP, ##p < 0.01). B, In slices from male rats, TBS increased both (left) the rightward skew in the synaptic pTrkB-IR density frequency distribution (vs LFS, p < 0.0001; F(19,342) = 6.794) that was not influenced by MPP (p = 0.939; F(19,342) = 0.549, n = 10/group) and (right) the percentage of PSD-95-IR synapses associated with dense pTrkB-IR (≥90 units), also not influenced by MPP (p = 0.009, F(2,29) = 5.68; Bonferroni's post-test: **p < 0.01 vs LFS). C, TrkB blocker ANA-12 (750 nm) disrupted the stabilization of CA1 LTP in female slices (p < 0.0001, t(10) = 8.36; n = 6/group). D, S-C TBS produced a marked increase in the percentage of PSDs associated with dense pTrkB-IR in wild-type mice but not in β1 integrin cKOs (p < 0.0001, F(3,36) = 25.55; post hoc tests: LFS vs TBS for wild types, ***p < 0.0001; LFS, n = 9; TBS, n = 8; LFS vs TBS for β1 cKOs, n.s.; n = 10/group). E, Image shows pCofilin-IR colocalized with PSD-95 in female CA1 SR. Right, In females, S-C TBS increased the rightward skew in the density frequency distribution for synaptic pCofilin (relative to LFS; p < 0.0001, F(19,399) = 7.69; LFS, n = 12; TBS, n = 11). F, The selective ROCK inhibitor H1152 (100 nm, 160 min) blocked S-C LTP in female slices (p = 0.0013, t(9) = 4.57; veh,, n = 6; H1152, n = 5).

Article Snippet: Primary antisera cocktails included rabbit antisera to pTrkB Y515 ( Wang et al., 2016a ; 1:500; catalog #NB100-92656, Novus Biologicals; RRID: AB_1218205 ), the activated conformation of β1 integrin ( Wang et al., 2016a ; 1:400; catalog #MAB2259Z, EMD Millipore; RRID: AB_94616 ), pFAK Y397 ( Bock and Herz, 2003 ; 1:500; catalog #44-624G, ThermoFisher Scientific; RRID: AB_2533701 ), pCofilin Ser3 ( Lauterborn et al., 2017 ; 1:500; catalog #ab12866, Abcam; RRID: AB_299488 ), pERK1/2 Thr202/Tyr204 ( Seese et al., 2014 ; 1:500; catalog #4370, Cell Signaling Technology; RRID: AB_2315112 ), phosphorylated (p) Src Tyr418 ( Chen et al., 2010 ; 1:500; catalog #44-660G, Thermo Fisher Scientific; RRID: AB_1500523 ), ERα (1:700; catalog #sc-542, Santa Cruz Biotechnology; RRID: AB_631470 ), or GPER1 (1:1000; catalog #ab39742, Abcam; RRID: AB_1141090 ) in combination with mouse anti-postsynaptic density-95 [PSD-95; 1:1000; catalog #MA1-045, Thermo Fisher Scientific (RRID: AB_325399 ); or catalog #ab12093, Abcam (RRID: AB_298846 )], or mouse anti-ERβ (1:700; catalog #sc-390243, Santa Cruz Biotechnology; RRID: AB_2728765 ) in combination with goat anti-PSD-95 (1:1000; catalog #ab12093, Abcam; RRID: AB_298846 ).

Techniques: Activation Assay, Labeling, Produced

Journal: Frontiers in Oncology

Article Title: Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

doi: 10.3389/fonc.2021.612354

Figure Lengend Snippet: HSP90i by NW457 leads to downregulation of DNA damage response factors, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in human glioblastoma cells. (A) Transcriptomic profiling of regulators of the DNA damage response (DDR) in LN229 and T98G cells. mRNA expression levels were determined by qRT-PCR, normalized to a matrix of 3 reference genes (18S rRNA, δ-amino-laevulinate-synthase, and β2-microglobulin), and calibrated to the results of untransformed human astrocytes. For both cell lines, three replicates were analyzed and are displayed as x-fold log2-values. (B) Time course analysis of DDR regulator protein expression in LN229 and T98G cells upon HSP90i by 10 nM NW457. Arrowheads indicate the bands that were used for quantification. Protein levels were normalized to a matrix comprising vinculin and α-tubulin and are depicted as x-fold log2-values compared to the 0 h controls. (C) Immunofluorescence microscopy of γH2AX and 53BP1 DNA damage repair foci in LN229 and T98G cells upon irradiation at 2 Gy ± HSP90i by NW457. Cells were treated with NW457 (10 nM) or DMSO for 24 h, irradiated, and fixed at the indicated times. Cells were stained for γH2AX, 53BP1, and DNA and subjected to deconvolution immunofluorescence microscopy. Scale bar depicts 10 µm. (D) Quantification of DNA damage repair kinetics from (C) . γH2AX and 53BP1 double-positive foci in at least 20 randomly picked nuclei were counted by hand. Individual data points with superimposed means ± 95% confidence intervals are displayed, and overall curve comparison was performed by two-way ANOVA. (E) Clonogenic survival of LN229 and T98G cells upon irradiation at 0–8 Gy ± HSP90i by NW457. Cells were pre-treated with 10 nM NW457 or DMSO for 24 h followed by irradiation at the indicated doses, and colony formation was allowed for 13 d ± continuous NW457 treatment. Individual data points of 4 independent experiments are shown, linear-quadratic regression lines are superimposed, and overall curve comparison was performed by two-way ANOVA.

Article Snippet: Cells were stained with monoclonal mouse anti-γH2AX (Merck Millipore) and polyclonal rabbit anti-53BP1 (Bio-Techne, Wiesbaden, Germany) antibodies diluted in 3% isotonic bovine serum albumin and 0.1% Triton X-100 for 2 h at room temperature.

Techniques: Irradiation, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Comparison