Journal: Molecules

Article Title: A Selective and Fast Approach for Volatile Metalorganics Assaying in Wastewater

doi: 10.3390/molecules30051111

Figure Lengend Snippet: Analysis of Hg, MeHg, Sb and Se in certified reference materials using proposed method.

Article Snippet: The reference materials, CRM-CC580 estuarine sediment and ERM-CA 011a hard drinking water were obtained from LGC Standards, Warsaw, Poland.

Techniques:

Journal: PLOS Genetics

Article Title: Incomplete paralog compensation generates selective dependency on TRA2A in cancer

doi: 10.1371/journal.pgen.1011685

Figure Lengend Snippet: (A) Distribution of gene effect scores after TRA2A knockout from the Cancer Dependency Map (Public 24Q2 dataset). Red lines mark gene effect scores of -0.5 (dotted) and -1.0 (solid). (B) Immunoblot showing depletion of TRA2A protein levels after CRISPR-Cas9 targeting in NCI-H23 cells. ACTB was used as a loading control. (C,D) Competition-based proliferation assays performed in indicated Cas9 + cell lines after TRA2A KO. SgRNA+ populations were monitored over time with a co-expressed fluorescent protein marker. Plotted is the relative sgRNA+ population normalized to the Day 3 sgRNA+ population. Bar graph summarizes normalized percentage of sgRNA+ cells remaining at Day 22 or 23 after infection, n = 3. (E) Immunoblot showing depletion or rescue of TRA2A protein levels in NCI-H23 Cas9 + cells stably expressing luciferase (control) or TRA2A cDNA resistant to sg4 (sg4-res) and infected with control or TRA2A -targeting sgRNAs. (F) Competition-based rescue assay performed in indicated TRA2A -dependent Cas9 + cells also stably expressing luciferase or TRA2A cDNA resistant to sg4 (sg4-res) and subsequently infected with control or TRA2A -targeting sgRNAs. Shown is the percentage of sgRNA+ cells at Day 28 after infection, normalized to Day 3, n = 3. (G) Immunoblot showing depletion of TRA2A protein levels after CRISPRi-mediated repression in NCI-H23 cells. (H) Competition-based proliferation assays performed in indicated Zim3-dCas9 + cell lines after TRA2A KD. SgRNA+ populations were monitored over time with a co-expressed fluorescent protein marker. Shown is the percentage of sgRNA+ cells at Day 28 after infection normalized to Day 3, n = 3. (I) Immunoblots showing protein levels of TRA2A, TRA2B, and ACTB in TRA2A -independent and -dependent cell lines. Quantification was performed by normalizing protein signal to total protein stain (see ), n = 2. (J) Summary of top 10 TRA2A -dependent cell lines and 3 tumor type-matched TRA2A -independent cell lines from DepMap. (D,F,H,I) Error bars represent standard deviation from the mean. (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, and (ns) not significant, as calculated by repeated measures two-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: Primary antibodies and dilutions used were: ACTB (Abcepta #AM1829B, 1:2,000), TRA2A (GeneTex #GTX87998, 1:1,000) and TRA2B (Bethyl #A305-011A, 1:5,000).

Techniques: Knock-Out, Western Blot, CRISPR, Control, Marker, Infection, Stable Transfection, Expressing, Luciferase, Rescue Assay, Staining, Standard Deviation, Comparison

Journal: PLOS Genetics

Article Title: Incomplete paralog compensation generates selective dependency on TRA2A in cancer

doi: 10.1371/journal.pgen.1011685

Figure Lengend Snippet: (A) Schematic representation of CRISPR screen for genetic modifiers of TRA2A dependency. (B) Scatterplot of log2 fold-change (logFC) in A549 cells between control compared to the plasmid DNA library (x-axis) and TRA2A KO compared to the plasmid DNA library (y-axis), calculated by MAGeCK. (C) (Left) Distribution of genes ranked by the log2 fold-change (logFC) between TRA2A KO and control conditions in A549, calculated by MAGeCK. The blue and red dots indicate two standard deviations from the mean log2 fold-change. (Right) Log2 fold-change between TRA2A KO and control conditions of the individual guides targeting genes representing the 10 most depleted genes in A549 cells. (D) Log2 fold-change of TRA2B sgRNAs between control or TRA2A KO conditions relative to plasmid DNA library. (E) Immunoblot confirming dual depletion of TRA2A and TRA2B protein levels after CRISPRi targeting. ACTB was used as loading control. (F) Dual-guide CRISPRi competition assays in indicated Zim3-dCas9 + cell lines, n = 3. Control condition indicates sgRNAs targeting AAVS1 and ROSA26 safe harbor loci, AARS1 KD indicates pan-essential positive control. Values indicate Day 28 percent sgRNA+ population normalized to Day 3 percent sgRNA+ population. Error bars represent standard deviation from the mean. (****) P < 0.0001, as calculated by repeated measures two-way ANOVA followed by Dunnett’s multiple comparison test. (G) Quantification of percent AnnexinV+ apoptotic cells upon negative control or TRA2A/B DKD at Day 7 post-infection as measured by flow cytometry in the indicated cell lines. Error bars represent standard deviation from the mean. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001, as calculated by two-tailed unpaired t-test. Schematic in (A) created in BioRender under License: https://BioRender.com/o10ks6p .

Article Snippet: Primary antibodies and dilutions used were: ACTB (Abcepta #AM1829B, 1:2,000), TRA2A (GeneTex #GTX87998, 1:1,000) and TRA2B (Bethyl #A305-011A, 1:5,000).

Techniques: CRISPR, Control, Plasmid Preparation, Western Blot, Positive Control, Standard Deviation, Comparison, Negative Control, Infection, Flow Cytometry, Two Tailed Test

Journal: PLOS Genetics

Article Title: Incomplete paralog compensation generates selective dependency on TRA2A in cancer

doi: 10.1371/journal.pgen.1011685

Figure Lengend Snippet: (A) Immunoblot and quantification of TRA2B protein expression relative to ACTB in NCI-H23 and LN319 cells stably expressing luciferase control (Luc) or overexpressing TRA2B (TRA2B-OE), n = 3. Error bars represent standard deviation from the mean. (**) P < 0.01 and (***) P < 0.001, as calculated by two-tailed unpaired t-test. (B) Competition assay performed in NCI-H23 and LN319 Cas9 + cells stably expressing luciferase control (Luc) or overexpressing TRA2B (TRA2B-OE) and subsequently infected with control or TRA2A sgRNAs, n = 3. Error bars represent standard deviation from the mean. (***) P < 0.001, (****) P < 0.0001, and (ns) not significant, as calculated by repeated measures two-way ANOVA followed by Tukey’s multiple comparison test. (C) Hierarchical clustering of splicing changes detected by MAJIQ in LN319 cells stably expressing luciferase control (Luc) or overexpressing TRA2B (TRA2B-OE) and then infected with control or TRA2A sgRNAs ( TRA2A KO). Rows represent LSVs responsive to TRA2A KO compared to control from LN319 cells expressing luciferase. Heat represents the mean PSI for a given LSV, n = 3. (D) Mean dPSI of TRA2A KO-responsive LSVs for clusters C1 and C2 in luciferase and TRA2B-OE conditions. (E) RT-PCR validation of candidate TRA2A KO-responsive splicing changes that are rescued by TRA2B overexpression in NCI-H23 and LN319 cells. (F) Proposed model of TRA2A/TRA2B compensatory mechanisms in normal cells (left), upon TRA2A loss in cells with an intact paralog buffering mechanism (middle), and upon TRA2A loss in cells with a compromised paralog buffering mechanism (right).

Article Snippet: Primary antibodies and dilutions used were: ACTB (Abcepta #AM1829B, 1:2,000), TRA2A (GeneTex #GTX87998, 1:1,000) and TRA2B (Bethyl #A305-011A, 1:5,000).

Techniques: Western Blot, Expressing, Stable Transfection, Luciferase, Control, Standard Deviation, Two Tailed Test, Competitive Binding Assay, Infection, Comparison, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Over Expression

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a Glutathione S-transferase (GST) or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were mixed with ( + ) or without (−) NSC-34 cell lysates. After the rotation at 4 °C for 5 h, the glutathione beads were washed and were subjected to 5–20% gradient gel SDS–PAGE followed by immunoblotting (IB) using indicated antibodies. In western blotting using anti-GST antibody, the large smear within the molecular weights ranging 25–46 kDa in the GF-PR lane is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to represent dimerized and/or aggregated GST-derived proteins. b NSC-34 cells overexpressing EGFP-FLAG-PR100 (EGFP-F-PR100) (green) together with HA-tagged hnRNPF, hnRNPH1, or hnRNPM were fixed and were immunostained with HA (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 10 μm. The rightmost panel shows the profile image of fluorescence intensities on the lines of EGFP-FLAG-PR100 and hnRNPs-merged image. Arrows and arrowheads indicate the localization of nucleoplasm-localizing EGFP-FLAG-PR100 and the border of nucleolus-localizing EGFP-FLAG-PR100, respectively. c Lysates of NSC-34 cells overexpressing HA-tagged hnRNPF, hnRNPH1, or hnRNPM and GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with ( + ) or without (−) 20 μg/mL RNase A. After the incubation, cell lysates were mixed with GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and were subjected to immunoblotting (IB) using indicated antibodies. d , e NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG1 (Cont.) or the FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay ( d ) and dot blotting analysis with the FLAG antibody ( e ). Reverse transcription (RT) (−) was used as negative control to monitor the PCR amplification from genomic DNA. f NSC-34 cells overexpressing EGFP or EGFP-FLAG-PR100 (green) were fixed and stained with the 500 nM Quasar 570-labeled NEAT1 Stellaris probe (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. Arrowheads and arrow correspond to paraspeckles. The rightmost panel shows the profile image of fluorescence intensities on the lines of EGFP-FLAG-PR100 and NEAT1-merged image in magnified image. Arrowhead and arrow indicate the co-localization of NEAT1 with nucleoplasm-localizing EGFP-FLAG-PR100 and the nucleolus-localizing EGFP-FLAG-PR100, respectively

Article Snippet: The following antibodies were used in this study: hnRNPF/H (sc-32310, RRID:AB_2248257), Splicing factor, proline- and glutamine-rich (SFPQ) (sc-374502, RRID:AB_10989589), non-POU domain-containing octamer-binding protein (NONO) (sc-166702, RRID:AB_2152178), hnRNPQ (sc-56703, RRID:AB_2200715), hnRNPA2/B1 (sc-374053, RRID:AB_10947257), and glutathione S-transferase (GST) (sc-138, RRID:AB_627677) from Santa Cruz Biotechnology (Dallas, TX); hnRNPM (A500–011A, RRID:AB_11125542) from Bethyl Laboratories (Montgomery, TX); horseradish peroxidase (HRP)-conjugated FLAG (A8592, RRID:AB_439702) from Sigma-Aldrich (St. Louis, MO); HA (11867423001, RRID:AB_390918) and HRP-conjugated HA (12013819001, RRID:AB_390917) from Roche Diagnostics (Basel, Swiss); monoclonal TDP-43 (89789, RRID:AB_2800143) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2118, RRID:AB_561053) from Cell Signaling Technology (Danvers, MA); β-Tubulin (014–25041, RRID:AB_2650453) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan); polyclonal TDP-43 (12892–1-AP, RRID:AB_2200505) and polyclonal Matrin3 (MATR3) (12202–2-AP, RRID:AB_2281752) from Proteintech Group (Rosemont, IL); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (170–6515, RRID:AB_11125142) and HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (170–6516, RRID:AB_11125547) from Bio-Rad Laboratories (Hercules, CA).

Techniques: SDS Page, Western Blot, Derivative Assay, Staining, Fluorescence, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, RNA Immunoprecipitation, Reverse Transcription, Negative Control, Amplification, Labeling

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies. Intensities of immunodetected signals of endogenous hnRNPM were densitometrically examined with an ImageJ software. b , c NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the quantitative real-time PCR analysis of hnRNPM was performed ( b ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( c ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. d , e NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( d ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( e ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. f , g NSC-34 cells were transfected with 0.2 μg/well of the pCMV-GFP or -mouse NEAT1_2 on 6-well plate. After the transfection, NSC-34 cells were infected with adenovirus encoding LacZ or hnRNPM at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( f ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( g ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. h , i NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the cell viability was detected by WST-8 assay ( h ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( i ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test

Article Snippet: The following antibodies were used in this study: hnRNPF/H (sc-32310, RRID:AB_2248257), Splicing factor, proline- and glutamine-rich (SFPQ) (sc-374502, RRID:AB_10989589), non-POU domain-containing octamer-binding protein (NONO) (sc-166702, RRID:AB_2152178), hnRNPQ (sc-56703, RRID:AB_2200715), hnRNPA2/B1 (sc-374053, RRID:AB_10947257), and glutathione S-transferase (GST) (sc-138, RRID:AB_627677) from Santa Cruz Biotechnology (Dallas, TX); hnRNPM (A500–011A, RRID:AB_11125542) from Bethyl Laboratories (Montgomery, TX); horseradish peroxidase (HRP)-conjugated FLAG (A8592, RRID:AB_439702) from Sigma-Aldrich (St. Louis, MO); HA (11867423001, RRID:AB_390918) and HRP-conjugated HA (12013819001, RRID:AB_390917) from Roche Diagnostics (Basel, Swiss); monoclonal TDP-43 (89789, RRID:AB_2800143) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2118, RRID:AB_561053) from Cell Signaling Technology (Danvers, MA); β-Tubulin (014–25041, RRID:AB_2650453) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan); polyclonal TDP-43 (12892–1-AP, RRID:AB_2200505) and polyclonal Matrin3 (MATR3) (12202–2-AP, RRID:AB_2281752) from Proteintech Group (Rosemont, IL); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (170–6515, RRID:AB_11125142) and HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (170–6516, RRID:AB_11125547) from Bio-Rad Laboratories (Hercules, CA).

Techniques: Infection, Western Blot, Software, Real-time Polymerase Chain Reaction, Transfection