anti-fade mounting medium prolong gold  (Thermo Fisher)

Journal: bioRxiv

Article Title: YAP engages RIF1 to dampen replication stress in squamous cell carcinoma

doi: 10.1101/2024.12.20.629444

Figure Lengend Snippet: ( A ) Interaction network of YAP protein interactors identified by RIME in SCC13 cells. Oval nodes indicate proteins that were previously identified as candidate regulators of SCC13 cell proliferation and/or survival in a genome wide RNA-interference (RNAi) screen (hit: ‘Yes’), while rectangular shaped nodes indicate proteins that were not identified in the RNAi screen (hit: ‘No’). Red connecting lines (edges) show previously identified YAP interactions according to the STRING Homo sapiens database, and nodes with red borders indicate known YAP interactors (manually curated from STRING and literature search); dashed red lines show manually curated known YAP interactions that were not yet present in the STRING database. Proteins were manually grouped based on function (gene names displayed, generated in Cytoscape 3.9.1 using STRING app 1.7.1). ( B ) Gene ontology terms (‘molecular functions’ and ‘biological process’) enriched within the high-confidence YAP-interacting proteins identified by RIME. ( C ) Confocal fluorescence images (z-projections) of YAP (green) and RIF1 (red) nuclear localisation in SCC13 cells. DNA was counterstained with Hoechst dye DAPI (blue). Scale bar, 10 µm. ( D , E ) Immunoblotting analysis of YAP and IgG-control immunoprecipitates and respective inputs from SCC13 (D) whole cell extracts and (E) and nuclei-enriched fractions using the indicated antibodies. Blots from one representative experiment out of two (D) and three (E) are shown. ( F , G ) Representative confocal microscopy images (z-projections) and image quantifications of (F) YAP-RIF1 and (G) YAP-TEAD1 in-situ PLA in control (siNTC/shNTC) and YAP/TAZ knockdown (siYAP&TAZ/shYAP) cells (PLA signal, green; DNA (DAPI) blue; n >250 cells (YAP-RIF1), n >500 cells (YAP-TEAD1) per condition in n-3 independent experiments). Scale bars, 5 µm. Datapoints show fold decrease in mean nuclear PLA signal intensity in siYAP&TAZ/shYAP cells compared to siNTC/shNTC cells. Lines show means from all three experiments, error bars show SD. Exact p-values are shown, one sample two-tailed t test.

Article Snippet: Coverslips were mounted onto glass microscope slides using ProLong Gold TM anti-fade mounting medium with DAPI (Fisher Scientific, UK,) or alternatively, incubated with 2 µg/ml Hoechst 33342 (Fisher Scientific, UK) in PBS for 5 minutes, washed once with PBS, and mounted with ProLong Gold TM anti-fade glass mounting medium without DAPI (Fisher Scientific, UK).

Techniques: Genome Wide, Generated, Fluorescence, Western Blot, Control, Confocal Microscopy, In Situ, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: YAP engages RIF1 to dampen replication stress in squamous cell carcinoma

doi: 10.1101/2024.12.20.629444

Figure Lengend Snippet: ( A ) Immunoblott analysis of YAP (rabbit monoclonal anti-YAP antibody; D8H1X, Cell Signalling)) and IgG-control immunoprecipitates and respective inputs from SCC13 cell extracts using the indicated antibodies. ( B ) Immunoblot analysis of lysates from cells used in in-situ PLA experiments shown in and . To achieve the strongest YAP/TAZ depletion possible, SCC13 cells expressing inducible shNTC or shYAP were treated with doxycycline for 72 hours and were in parallel also transfected with non-targeting control or YAP/TAZ-targeting siRNA SMARTpools, respectively. Tubulin was used as loading control. ( C , D ) Representative confocal microscopy images (z-projections) of in-situ PLA (PLA signal, green; DNA (DAPI)) blue) in cells labelled with (C) no primary antibodies or (D) only one of the two primary antibodies. Scale bars, 5 µm. ( E ) Representative confocal microscopy images (z-projections) of YAP-RIF1 in-situ PLA (PLA signal, green; DNA (DAPI)) blue in miotic cells. Scale bars, 5 µm. ( F ) Confocal fluorescence image (z-projection) of YAP (green) and RIF1 (red) nuclear localisation in HARA cells. DNA was counterstained with Hoechst dye (DAPI, blue). Scale bar, 10 µm. ( G ) Immunoblot analysis of YAP and IgG-control immunoprecipitates and respective inputs from HARA cell nuclei-enriched fractions (treated with Benzonase® nuclease) using the indicated antibodies. ( H ) Agarose gel electrophoresis of DNA extracts from Benzonase®-treated and untreated HARA cells shows efficient DNA degradation upon Benzonase® treatment. Left lane: Thermo Scientific™ GeneRuler 1 kb Plus DNA Ladder (Fisher, UK).

Article Snippet: Coverslips were mounted onto glass microscope slides using ProLong Gold TM anti-fade mounting medium with DAPI (Fisher Scientific, UK,) or alternatively, incubated with 2 µg/ml Hoechst 33342 (Fisher Scientific, UK) in PBS for 5 minutes, washed once with PBS, and mounted with ProLong Gold TM anti-fade glass mounting medium without DAPI (Fisher Scientific, UK).

Techniques: Control, Western Blot, In Situ, Expressing, Transfection, Confocal Microscopy, Fluorescence, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: YAP engages RIF1 to dampen replication stress in squamous cell carcinoma

doi: 10.1101/2024.12.20.629444

Figure Lengend Snippet: ( A ) Immunoblot analysis of chromatin fractions from SCC13 cells expressing a doxycycline-induced shYAP or shNTC, using the indicated antibodies. Cells were synchronised at the G1/S boundary by double thymidine block (dTB) and released into the S phase. Samples were taken at the indicated timepoints. Histone H3 (HH3) was used as loading control. Blots from one representative experiment out of two are shown. ( B , C ) Representative confocal microscopy images of (B) z-projection and (C); orthogonal projection of YAP-RIF1 in-situ PLA in SCC13 cells (PLA signal, green; DNA (DAPI) blue). In (B) cells were labelled with a 1-hour EdU pulse prior to fixation (EdU signal, red). Arrows in (C) indicate cells completing mitosis. Scale bars, 5 µm. ( D ) Immunoblot analysis of SCC13 control cells and cells exposed to 4 mM hydroxyurea (HU) for 24 hours, using the indicated antibodies. Washout, medium removal and replacement with fresh medium. Tubulin and cyclophilin B (CBP) were used as loading controls. ( E , F ) Image quantifications of (E) YAP-RIF1 and (F) YAP-γH2AX in-situ PLA in SCC13 cells treated with 4 mM HU for the indicated times. >200 cells were measured per experimental condition in n=3 independent experiments. Superplots show nuclear PLA signal intensity per cell (grey datapoints), mean nuclear PLA signal intensity of the respective cell populations in each of n=3 experiments (black datapoints), and mean nuclear PLA signal intensity from all three experiments (lines). Error bars show SD from all three experiments. Exact p-values are shown. (E) Brown-Forsythe and Welsh ANOVA test with Dunett’s multiple comparison test, (F) unpaired t test.

Article Snippet: Coverslips were mounted onto glass microscope slides using ProLong Gold TM anti-fade mounting medium with DAPI (Fisher Scientific, UK,) or alternatively, incubated with 2 µg/ml Hoechst 33342 (Fisher Scientific, UK) in PBS for 5 minutes, washed once with PBS, and mounted with ProLong Gold TM anti-fade glass mounting medium without DAPI (Fisher Scientific, UK).

Techniques: Western Blot, Expressing, Blocking Assay, Control, Confocal Microscopy, In Situ, Comparison