Journal: Nucleic Acids Research

Article Title: Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition

doi: 10.1093/nar/gkw1284

Figure Lengend Snippet: XRN1 regulates ex-miRNA decay in recipient cells ( A ) Relative quantification analysis of ex-miRNA-223-3p in A549 cells. siXRN1-transfected A549 cells co-cultured with PMN overnight were harvested at the indicated periods of time post PMN removal (Time Washed, T.W.). Results are representative of three biological replicates. In the right panel, immunoblot analysis of XRN1 expression. b-ACTIN served as an equal loading control. ( B ) Immunoblot analysis of FOXO1 and EMT marker expression levels. β-ACTIN served as an equal loading control. ( C ) In vitro invasion assay of siXRN1-transfected A549 cells. A549 cells co-cultured with SPN of PMN, produced in serum-free medium, were seeded in the upper part of transwells. The number of cells attached to the bottom of a Matrigel-coated membrane after 16 h was quantified after crystal violet staining. Data represent the quantification of five biological replicates, ‘centre values’ as mean and error bars as s.d. * for P < 0.05, ** for P < 0.01.

Article Snippet: After 1 h of blocking with 5% bovine serum albumin or non-fat milk prepared in TBS-Tween buffer, the blots were incubated overnight at 4°C with antibodies against FOXO1 (clone C29H4, 1:1000, Cell Signaling Technologies, ref #2880), XRN1 (1:1000, Bethyl Laboratories, ref A-300-443A), E-CADHERIN (clone 4A2C7, 1:500, Invitrogen, ref 33–4000), SNAIL (clone C15D3, 1:1000, Cell Signaling Technologies, ref #3879), SLUG (clone A-7, 1:400, Santa Cruz Biotechnology, ref sc-166476), VIMENTIN (clone SP20, prediluted, 1:100, Abcam, ab27608) and b-ACTIN (clone AC-40, 1:10 000, Sidma-Aldrich, ref A3853) used as loading control.

Techniques: Quantitative Proteomics, Transfection, Cell Culture, Western Blot, Expressing, Control, Marker, In Vitro, Invasion Assay, Produced, Membrane, Staining

Journal: bioRxiv

Article Title: Cytoplasmic accumulation of FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and defects in inhibitory synapses

doi: 10.1101/2020.06.09.141556

Figure Lengend Snippet: ( a-b ) Representative western blot images ( a ) and respective quantifications ( b ) of cytoplasmic (left) or synaptosome (right) extracts from Fus +/+ (+/+) or Fus ΔNLS/+ (Δ/+) mice (4 months of age, 3 individual mice presented, 6 mice in total analyzed per genotype) using two antibodies recognizing the N-terminal part of the FUS protein (FUS N-ter1 and FUS N-ter2), the C-terminal part of FUS (encoding the NLS, FUS C-ter) or synaptophysin protein to show enrichment in synaptic proteins in the synaptosome fraction. Please note that the 3 FUS western blots were run on independent gels, to avoid stripping and reprobing on the same membrane for the same protein. Each of these gels were controlled for equal loading using StainFree markers, that are provided in Figure S5 with uncropped western blot images. (c) mRNA levels of the indicated genes in RNAs extracted from cytoplasmic (Cyto.) or synaptosome (Synap.) extracts from Fus +/+ (+/+) or Fus ΔNLS/+ (Δ/+) frontal cortex from 4 months old mice as assessed using RT-qPCR. All quantifications are presented relative to the +/+ cytoplasmic RNA levels set to 1. *, p<0.05 vs corresponding wild type fraction; ***, p<0.001 vs corresponding wild type fraction; #, p<0.05 vs corresponding cytoplasmic fraction of the same genotype; ###, p<0.001 vs corresponding cytoplasmic fraction of the same genotype; Two-way repeated measures ANOVA with Tukey post hoc test.

Article Snippet: Membranes were saturated with 10% non-fat milk in PBS and then probed with the following primary antibodies: Anti-Synaptophysin (Abcam, ab14692, 1:1000), Anti-FUS N-ter1 (ProteinTech, 11570, 1:1000), Anti-FUS N-ter2 (Bethyl, A-300-291A, 1/2000) and Anti-FUS C-ter (Bethyl, A300-294A, 1:2000) all diluted in 3% non-fat milk in PBS.

Techniques: Western Blot, Stripping Membranes, Membrane, Quantitative RT-PCR

Journal: Nucleic acids research

Article Title: Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.

doi: 10.1093/nar/gkaa859

Figure Lengend Snippet: Figure 1. The UV-FLIM-FRET system and the experimental outline for analysing interaction between mEGFP- and mCherry-tagged proteins before and after UV laser-induced microirradiation by three independent methods. (A) Lifetime of Atto488 at increasing excitation power until detector pile- up artefacts became apparent as a drop in measured lifetimes. (B) Live U2OS cells transiently co-transfected with mEGFP-tagged protein (donor) and either with mCherry-empty vector or with mCherry-tagged interaction partner (acceptor) and imaged before and after inducing DNA damage. Sites of laser damage are visible by increased photon count numbers caused by rapid protein recruitment to the site of laser microirradiation (indicated with an orange box within the nucleus). (C) Fluorescence lifetime decay curves plotted as a pixel-by-pixel heatmap of measured mEGFP lifetimes scaled from red (3 ns lifetime) to light blue (1.5 ns lifetime) to visualize changes in the lifetime of the donor in the presence of an acceptor directly at sites of UV laser microirradiation (orange boxes) or in the rest of the nucleus. (D) Phasors representing background fluorescence (marked with a green circle on the phasor plot), donor-only lifetime (marked with a red circle on the phasor plot, corresponding regions where no FRET occurs are highlighted in red on the FLIM image of the cell) and phasors representing shortened donor lifetime in the presence of an interacting acceptor (marked with a yellow circle on the phasor plot, corresponding regions of FRET on the FLIM map highlighted in yellow) are separated along a FRET trajectory (marked with a yellow line on the phasor plot). (E) Experimental outline for FLIM, immunofluorescence, acceptor photobleaching and LacO tethering. (F) mEGFP-ALC transfected cells were fixed after damage and damage sites identified by the accumulation of mEGFP-ALC1 and co-localization with H2AX, PAR and cyclobutane pyrimidine dimers (CPDs) detected by immunofluorescence staining. (G) Acceptor photobleaching on cells fixed after UV laser damage. Damage sites were identified by H2AX immunofluorescence staining and by increased fluorescence intensity due to protein accumulation at the damage site (marked by an orange square). Acceptor was bleached in a region marked with a red square and the intensity of mEGFP measured across a region marked with a purple square before and after bleaching the acceptor. (H) Accumulation of mCherry-tagged prey at the LacO site (white arrow) in live U2OS-2B2 cells was observed in a fluorescence three-hybrid assay when the prey was able to interact with the mEGFP-tagged bait tethered at LacO with a LacI-GFP nanobody. All scale bars indicate 10 m.

Article Snippet: Rabbit anti- H2AX (1:600; Bethyl A-300-081A), mouse anti- H2AX (1:500; Millipore JBW301), mouse anti-CPD (1:500; Cosmo Bio NMDND001) and rabbit anti-PAR polyclonal (1:1000; Trevigen 4336-BPC-100) were used for immunofluorescence.

Techniques: Transfection, Plasmid Preparation, Fluorescence, Immunofluorescence, Staining, Hybrid Assay