Journal: Cell

Article Title: The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity

doi: 10.1016/j.cell.2016.11.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: IFNγ concentrations were determined using Mouse IFN-gamma DuoSet ELISA (R&D Systems, #DY485-05) according to a manufacturer’s protocol.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Recombinant, cDNA Synthesis, Selection, Transgenic Assay, Plasmid Preparation, CRISPR, Sequencing, Software

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: Journal of biotechnology

Article Title: Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

doi: 10.1016/j.jbiotec.2016.03.027

Figure Lengend Snippet: Fig. 1: Critical features for CAS9/gRNA expression affecting the genome editing efficiency. Implementing CRISPR/Cas9 in a given organism is reliant on a set of interdependent components. These features include the DNA sequence of CAS9, the type and position of the NLS for the nuclear import of Cas9, different gRNA sequences and target loci, the promoter for the expression of the gRNAs, the introduction of processing elements for RNA maturation (ribozymes) as well as transcriptional termination. Elements on the plasmid are not drawn to scale. Cas9 (blue) shown on the right side includes the gRNA (red) and the target DNA for cleavage (yellow). The illustration of the Cas9/gRNA complex was taken from the RCSB PDB (see acknowledgements).

Article Snippet: The Homo sapiens codon optimized CAS9 (HsCAS9) and the Streptococcus pyogenes CAS9 (SpCAS9) were amplified from the template vectors p414-TEF1p-Cas9-CYC1t and pMJ806 (both obtained from Addgene, Cambridge, MA, USA) using primer pairs spCas9_fw/spCas9-SV40_rv and hsCas9_fw/ hsCas9-SV40_rv respectively.

Techniques: Expressing, CRISPR, Sequencing, Plasmid Preparation

Journal: Journal of biotechnology

Article Title: Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

doi: 10.1016/j.jbiotec.2016.03.027

Figure Lengend Snippet: Fig. 2: High efficiency implementation of CAS9 and gRNA expression in P. pastoris.

Article Snippet: The Homo sapiens codon optimized CAS9 (HsCAS9) and the Streptococcus pyogenes CAS9 (SpCAS9) were amplified from the template vectors p414-TEF1p-Cas9-CYC1t and pMJ806 (both obtained from Addgene, Cambridge, MA, USA) using primer pairs spCas9_fw/spCas9-SV40_rv and hsCas9_fw/ hsCas9-SV40_rv respectively.

Techniques: Expressing

Journal: Journal of biotechnology

Article Title: Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

doi: 10.1016/j.jbiotec.2016.03.027

Figure Lengend Snippet: Fig. 4: The CRISPR/Cas9 system allows high efficiency targeting of various genes (A) and is suitable for multiplexing (B, C) in P. pastoris.

Article Snippet: The Homo sapiens codon optimized CAS9 (HsCAS9) and the Streptococcus pyogenes CAS9 (SpCAS9) were amplified from the template vectors p414-TEF1p-Cas9-CYC1t and pMJ806 (both obtained from Addgene, Cambridge, MA, USA) using primer pairs spCas9_fw/spCas9-SV40_rv and hsCas9_fw/ hsCas9-SV40_rv respectively.

Techniques: CRISPR, Multiplexing

Journal: International journal of oncology

Article Title: Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

doi: 10.3892/ijo.2016.3822

Figure Lengend Snippet: Figure 4. Detection of genome editing at RPN2 gene. (A) Cloning target RPN2 sequence using the Guide-it CRISPR/Cas9 system. A red line under AGG indicates protospacer-adjacent motif. (B) Fluorescence analysis shows that transfection with RPN2-Cas9-GFP plasmid increased GFP-expressing cells in MKN74 and KATO III cell lines. Fluorescence cell imaging were visualized 96 h following transfection. (C) Indel frequency was measured using the T7E1 assay and the percentages are shown at the bottom of each lane. (D) Protein extracted from fluorescence positive cells was analyzed for RPN2 and GAPDH by western blot assay. (E) Sanger sequencing of PCR products around gRNA binding site (red letters) and protospacer adjacent motif (PAM) site (blue letters). Wild-type reference sequences are given on the top.

Article Snippet: Anti-RPN2 Ab was from Aviva Systems and anti-GAPDH Ab was from IMGENEX.

Techniques: Cloning, Sequencing, CRISPR, Fluorescence, Transfection, Plasmid Preparation, Expressing, Imaging, Western Blot, Binding Assay

Journal: International journal of oncology

Article Title: Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

doi: 10.3892/ijo.2016.3822

Figure Lengend Snippet: Figure 6. Relationship between RPN2 and p53 in gastric adenocarcinoma. (A) p53 protein stained in brown was detected in the nucleus of primary gastric adenocarcinoma lesions. (B) Evidence for knockdown of RPN2 caused by transfection with siRNA. MKN45 cells were transfected with scrambled siRNA (control); GAPDH was used as loading control. RPN2 siRNA followed by western blot assays using anti-RPN2 antibodies. (C) The knockout of RPN2 expression in MKN45 cell line did not reduce the ability of invasion. Data are expressed as mean values ± SD from triplicate experiments.

Article Snippet: Anti-RPN2 Ab was from Aviva Systems and anti-GAPDH Ab was from IMGENEX.

Techniques: Staining, Knockdown, Transfection, Control, Western Blot, Knock-Out, Expressing

Journal: Cell research

Article Title: Genome editing with CRISPR/Cas9 in postnatal mice corrects PRKAG2 cardiac syndrome.

doi: 10.1038/cr.2016.101

Figure Lengend Snippet: Figure 3 CRISPR/Cas9 system specifically targets the H530R mutation site of PRKAG2 in human and mouse genome. (A) Schematic of candidate sgRNA target sites in human and mouse PRKAG2 gene. Lowercase characters denote the intronic region and uppercase characters denote the exonic region. PAM motifs (NGG) are highlighted in green. (B) Screening of sgRNAs by T7E1 assay in human WT and H530R knock-in ES cells. The selected sgRNA-m3 is highlighted in red. Blue ar- rows denote the digested DNA bands. (C) Frequency of indels by sgRNA-m3 at on-target (h-H530R) and off-target (h-OT1- h-OT10 and h-WT) sites in human WT and H530R ES cells. Data represent mean ± SD (n = 5). The most prevalent on-tar- get indels of PRKAG2 H530R by sgRNA-m3 are shown in the lower panel. Line denotes the sgRNA sequence, and arrow denotes the Cas9 cleavage site. (D) Frequency of indels at on-target (m-H530R) and off-target (m-OT1-m-OT10 and m-WT) sites in heart genomic DNA from the +/H530R mouse injected with AAV9-Cas9/sgRNA-m3 at P4 or P42. Data represent mean ± SD (n = 6). Most prevalent on-target indels of PRKAG2 H530R in +/H530R mouse heart genomic DNA by sgRNA-m3 injected at P4 are shown in the lower panel. (E) qPCR analysis of PRKAG2 expression in the heart from mice as indicated. Data represent mean ± SD (n = 5). Unpaired two-tailed Student’s t-test was performed for single comparison (**P < 0.01, ***P < 0.001). (F) Protein level analysis of AMPK subunits by immunoblots of heart tissues from mice as indicated.

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit monoclonal antibodies against AMPKα (#2603S), AMPKβ2 (#4148S), AMPKγ2 (#2536S), and phospho-AMPKα (T172) (#2535S) were purchased from Cell Signaling Technology (Dan- vers, MA).

Techniques: CRISPR, Mutagenesis, Knock-In, Sequencing, Injection, Expressing, Two Tailed Test, Comparison, Western Blot