Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: B cells were harvested from the spleen of an Exosc10COIN/LacZ mouse and fixed and prepared for 3D-STORM after 72 hrs of treatment with (1) stimulation cocktail and (2) 4OHT+stimulation cocktail. Reconstructed two color 3D STORM (super-Aresolution) image from a data set of 50,000 frames with Atto488 labeled AID, AlexaFluor647 labeled Mtr4 and DAPI labeled nucleus. Three dimensional views of the boxed region show spatial distribution of AID and Mtr4 molecules inside the nuclei of B cells isolated from (A) wild type cells and (D) Exosc10 knockout cells. Histogram of the distribution of interactions of AID and Mtr4 calculated in the B cell nucleus of (B) wild type & (E) Exosc10 knockout cells and in the corresponding cytoplasms (C) & (F), by using Matlab (2014b, MathWorks) software. (G) Comparison of the distribution of paired interaction of AID and Mtr4 in the nucleus versus cytoplasm by one way ANOVA (Tukey-Kramer test) method in Matlab (2014b, MathWorks) software. Comparison of the distribution of paired interaction of AID and Mtr4 were calculated in the cytoplasm versus nucleus of (H) wild type & (I) Exosc10 knockout B cells using a Student’s t-test in Matlab (2014b, MathWorks) software and P values are noted in the graph. (J) Comparison of the distribution of paired interaction of AID and Mtr4 in the nuclear center versus displaced from center versus cytoplasm by one way ANOVA (Tukey-Kramer test) method in Matlab (2014b, MathWorks) software. (K) Reconstructed two color 3D STORM (super-resolution) image with Alexa488 labeled AID, AlexaFluor647 labeled Mtr4 and DAPI labeled nucleus of wild type Exosc10 cells. Histogram of the distribution of interactions of AID and Mtr4 calculated in (L) nucleus center and (M) displaced from center of wild type cells, by using Matlab (2014b, MathWorks) software. All of the 3D STORM imaging was performed in three different B cells (from independent experiments) and repeated three or more times. 3D STORM super resolution image magnification is ×100. Scale bar: 1μm(K). Error bars indicate S.D. (P values: ** <0.01, *** <0.001)

Article Snippet: Alexa488 (Against-Goat) , Jackson Immuno Research , 805-547-008.

Techniques: Labeling, Isolation, Knock-Out, Software, Comparison, Imaging

Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alexa488 (Against-Goat) , Jackson Immuno Research , 805-547-008.

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Modification, Electron Microscopy, Shear, Transgenic Assay, Sequencing, Subcloning, Software

Journal: Frontiers in immunology

Article Title: Graphene quantum dots induce cascadic apoptosis via interaction with proteins associated with anti-oxidation after endocytosis by Trypanosoma brucei .

doi: 10.3389/fimmu.2022.1022050

Figure Lengend Snippet: FIGURE 3 Cytotoxicological effects graphene quantum dots (GQDs) on Trypanosoma brucei 12 and 24 h post-exposure. (A) Quantitative analysis of T. brucei intracellular reactive oxygen species (ROS) values based on the results in Supplementary Figure S7. The intracellular ROS levels increased with prolonged exposure and increased doses. (B) Alterations of the MMP (DYm) in T. brucei following treatment with GQDs. The levels of DYm decreased with increased doses and duration of exposure. A JC-10 fluorescent probe was applied for the detection using flow cytometry (Supplementary Figure S8). (C) Increased cytoplasmic Ca2+ concentration of T. brucei following treatment with GQDs for 12 and 24 h, demonstrated by flow cytometry. Changes in the intracytoplasmic Ca2+ concentration captured by confocal microscopy, which can be seen in Supplementary Figure S9. (D) Intracellular ATP was significantly decreased in T. brucei after exposure to GQDs. (E) Exposure to GQDs resulted in the developmental arrest of T. brucei. The proportions of cell cycle dispersion are shown in graphs, predicated in the flow cytometry data in Supplementary Figure S10. (F) Flow cytometry analysis of the increased DNA fragmentation in T. brucei after treatment with varying concentrations of GQDs for 12 and 24 h using TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. T. brucei parasites that were not exposed to GQDs served controls. All values are the mean ± SD of triplicates. Student’s t-test was used to obtain p- values. *p < 0.05, **p < 0.01, ***p < 0.001 (compared to the control).

Article Snippet: The annexin V-FITC Apoptosis Detection Kit (C1062L), Cell Cycle and Apoptosis Analysis Kit (C1052), Cell Counting Kit-8 (C0038), and one-step TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) Apoptosis Assay Kit (C1088) were provided by Beyotime Biotechnology (Suzhou, China).

Techniques: Cytometry, Concentration Assay, Confocal Microscopy, Dispersion, Flow Cytometry, TUNEL Assay, Staining, Control

Journal: Frontiers in Oncology

Article Title: Novel Breast-Specific Long Non-coding RNA LINC00993 Acts as a Tumor Suppressor in Triple-Negative Breast Cancer

doi: 10.3389/fonc.2019.01325

Figure Lengend Snippet: LINC00993 suppresses the growth of triple-negative breast cancer (TNBC) cells in vitro . (A) Structure of LINC00993 expression plasmid for adenovirus. (B) LINC00993 expression adenovirus infection efficiency showed by fluorescence microscope in MDA-MB-231 cells. Original magnification, ×400. Scale bars, 50 μm. (C) Expression of LINC00993 detected by qRT-PCR. MDA-MB-231 cells were infected by adenovirus for 24 h, and RNA was extracted. (D) Image of clone formation assay. (E) Number of clones were counted 2 weeks after plantation. (F) MDA-MB-231 cells were planted into 24-well plates. Twenty-four hours later, adenovirus was added to each well. Three wells of cells were digested and counted every 24 h. (G) LINC00993 expression caused apoptosis shown by TUNEL assay. Green points reflected apoptosis, and we used DAPI to stain DNA. Positive control cells were treated with DNase I, negative control cells were collected without adding TUNEL reaction buffer. Original magnification, ×100. Scale bars, 50 μm. (H) Effect of LINC00993 on cell cycle detected by flow cytometry. (I) Flow cytometry cell cycle results shown in a bar plot. (J) Invasive ability tested by Transwell assay. Twenty thousand cells were plated in each well, cells were observed after 24 h of incubation. (K) Bar plot for the number of cells that migrated across the membrane. *** P < 0.001, based on Student's t -test. Data were presented as mean ± SEM.

Article Snippet: To analyze cell apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed with One Step TUNEL Apoptosis Assay Kit (FITC, Beyotime, China) according to the manufacturer's instructions.

Techniques: In Vitro, Expressing, Plasmid Preparation, Infection, Fluorescence, Microscopy, Quantitative RT-PCR, Tube Formation Assay, Clone Assay, TUNEL Assay, Staining, Positive Control, Negative Control, Flow Cytometry, Transwell Assay, Incubation, Membrane