Journal: Cell Reports

Article Title: Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M

doi: 10.1016/j.celrep.2021.108759

Figure Lengend Snippet:

Article Snippet: Click-iT RNA Alexa Fluor 594 Imaging Kit , Zymo Research , Cat#R1015.

Techniques: Recombinant, Staining, Transfection, Random Primed, DNA Labeling, Imaging, Western Blot, Generated, Software

Journal: Plant Cell Reports

Article Title: 5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase–mitotic cells in apical root meristems of Allium cepa

doi: 10.1007/s00299-020-02545-9

Figure Lengend Snippet: Analysis of DNA replication in A. cepa RAMs by EdU incorporation. A Schematic representation of copper(l)-catalyzed Alexa Fluor ® 555 azide-alkyne "click" reaction used to detect replicating DNA. B RAM cell populations labeled with EdU ( a – c ) and the same cells counterstained with DAPI ( a – c ) in the control seedlings ( a , a ), after 1 h ( b , b ) and 48 h ( c , c ) incubations with 5-AU. Scale bar = 20 μm. (C) EdU labeling of RAM cells at successive stages of interphase: G1 ( a ), early S ( b ), middle S ( c ), late S ( d ), G2 ( e ), with corresponding images of the same cells counterstained with DAPI ( a – e ). Scale bar = 10 μm. D Single molecule DNA fibers isolated from actively replicating cell nuclei after their 48 h treatment with 5-AU. Scale bar = 10 μm ≈ 20 kbp. E Mean percentage (± SD) of EdU-labeled cells (labeling index; dotted diagram) and mean (± SD) intensity of EdU labeling (Fluorescence intensity, lined diagram), calculated as a percentage of the maximum possible brightness (pixel value = 255) evaluated for selected groups of mid-S-phase cell nuclei (determined microfluorimetrically following DAPI staining). The data represent values obtained for the control and 5-AU-treated onion RAM cell nuclei at successive periods of incubation. When compared with the control, statistically significant changes in mean values (± SD) are marked by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001

Article Snippet: After washing with PBS, DNA replication in nuclei were visualized using Click-iT DNA Alexa Fluor ® 555 Imaging Kit (Thermo Fisher Scientific), according to the supplier’s instructions.

Techniques: Labeling, Control, Isolation, Fluorescence, Staining, Incubation

Journal: Plant Cell Reports

Article Title: 5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase–mitotic cells in apical root meristems of Allium cepa

doi: 10.1007/s00299-020-02545-9

Figure Lengend Snippet: Analysis of nascent transcription level in A. cepa RAM cells using EU incorporation followed by Click-iT ® RNA Alexa Fluor ® 488 imaging. A Detection of RNA synthesis in the control root tip cells ( a ) and after 72 h 5-AU treatment ( b ); corresponding images of the same cells counterstained with PI ( a and b ); note faint fluorescence signal emitted in prophase ( a , a ; arrows) and metaphase cells ( b , b ; arrows). Scale bar = 20 μm. B Changes in nascent transcription level during successive stages of mitosis in the control onion cells at prophase ( a ), metaphase ( b ), anaphase ( c ), and telophase ( d ), with corresponding images of the same cells counterstained with PI ( a – d , respectively). Scale bar = 10 μm. C Increased EU incorporation into nascent transcripts observed in elongated prophase ( a ) and the biphasic IM cells after 72 h treatment with 5-AU ( b ); corresponding images of the same cells counterstained with PI ( a , b , respectively). Scale bar = 20 μm. D Microfluorimetric evaluation of mean nascent transcription levels measured for the nucleus and nucleolus in the control and 5-AU-treated onion RAM cells. When compared with the control, statistically significant changes in mean fluorescence levels (± SD) expressed in arbitrary units [a.u.] are marked by asterisks: * indicates p < 0.05 and ** indicates 0.005

Article Snippet: After washing with PBS, DNA replication in nuclei were visualized using Click-iT DNA Alexa Fluor ® 555 Imaging Kit (Thermo Fisher Scientific), according to the supplier’s instructions.

Techniques: Imaging, Control, Fluorescence

Journal: Plant Cell Reports

Article Title: 5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase–mitotic cells in apical root meristems of Allium cepa

doi: 10.1007/s00299-020-02545-9

Figure Lengend Snippet: Nascent protein synthesis detected using Click-iT® HPG Alexa Fluor® 488 imaging in A. cepa RAM cells. (A) Immunofluorescence of proteins: control (a), 72 h treatment with 5-AU (b), negative control series performed using (50 µM) cycloheximide (c); corresponding images of the same cells counterstained with DAPI (a’–c’). Scale bars = 20 μm. (B) Microfluorimetric evaluation of protein synthesis levels (Fluorescence intensity [a.u.]) expressed in arbitrary units, calculated as mean pixel value (± SD) measured at the cytoplasm area. When compared with the control, the fluorescence intensity in 5-AU-treated is increased, with the statistical significance at p < 0.001

Article Snippet: After washing with PBS, DNA replication in nuclei were visualized using Click-iT DNA Alexa Fluor ® 555 Imaging Kit (Thermo Fisher Scientific), according to the supplier’s instructions.

Techniques: Imaging, Immunofluorescence, Control, Negative Control, Fluorescence