Alexa fluor 350

Fusion competent ICAM-1+ myoblasts were fluorescently labeled using CellTracker™ Green CMFDA (Life Technologies; 2.5 µM); whereas, fusion competent EV myoblasts were not fluorescently labeled. Cells were collected using StemPro® Accutase® (Invitrogen), suspended in differentiation medium (200 cells/µl), and ICAM-1+ and EV myoblasts were mixed in equal number in polypropylene tubes. Tubes were placed in a shaking water bath for 2 h and aliquots of cells were immobilized to slides using a cytospin centrifuge^{4 (link)}. Cells were fixed in 4% formaldehyde, stained with WGA (Alexa Fluor® 350; Thermo Scientific), and mounted with Fluoromount-G^TM (SouthernBiotech). Images in six fields of view were captured using a 10X objective on an epifluorescence microscope.
The number of aggregates and the number of myoblasts within an aggregate were quantified using a macro function written for Image Pro 7 (Media Cybernetics). The number of ICAM-1+ cells within an aggregate was expressed as a percentage of the total number of WGA +cells within the aggregate. The number of EV cells was calculated by subtracting the total number of WGA+ cells from the number of ICAM-1+ cells within an aggregate. On average, ~2,000 myoblasts per slide were counted in 6 independent experiments.

Nuclei in the dissected Malpighian tubules were counterstained by 1 μg/ml 4′,6-diamino-2-phenylindole (DAPI) in 20 μl PBS, 12 min in dark. F-actin was stained by 1 unit Texas Red™-X Phalloidin (ThermoFisher) in 20 μl PBS for 20 min. A-Mannopyranosyl and a-glucopyranosyl residues as membrane markers were stained by Concanavalin A, Alexa Fluor™ 594 Conjugate (ThermoFisher) in 20 μl PBS for 1 hour. We used 1 μl mouse monoclonal anti-3-nitrotyrosine [39B6] (Abcam) in 20 μl PBS for 1 hour and stained 4-hydroxynonenal conjugate by 1 μl mouse monoclonal anti-4-hydroxynonenal antibody (Abcam) in 20 μl PBS for 1 hour. Primary mouse antibodies were visualized by 1 μl F(ab′) 2-Goat Anti-Mouse IgG (H + L) Cross Adsorbed Secondary Antibody conjugated with Alexa Fluor 488 (ThermoFisher) in 20 μl PBS for 1 hour and 1 μl Goat Anti-Mouse IgG (H + L) Cross Adsorbed Secondary Antibody, Alexa Fluor 350, in 20 μl PBS for 1 hour. Mitochondria were visualized by the mitochondrially targeted EYFP (mito-GFP) following appropriate crosses [17 (link)].

Cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 for 6 min, blocked with 1% BSA for 30 min, incubated overnight at 4 °C with primary antibody, and finally incubated with Alexa Fluor^® 488 (#A-11055, Thermo Fisher Scientific, USA), Alexa Fluor^® 555 (#A-31570, Thermo Fisher Scientific, USA), Alexa Fluor^® 350 (#A10039, Thermo Fisher Scientific, USA) for 1 hour at room temperature. Each step was followed by two 5 min washes in PBS. For nuclear stain, prepared specimens were counterstained with DAPI (1‰) for 2 min. Fluorescent images were obtained with a confocal microscope (Olympus, Japan).

HCT116 and A431 cells were incubated with 500 nM Onalespib or complete media (control) for 24 h, followed by irradiation with 2 Gy. Live cell analysis via flow cytometry was performed 48 h, 72 h as well as 96 h after radiation. Here, cells were washed in PBS and incubated in Annexin V binding buffer (140 mM NaCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES) for 5 minutes. 5 µL of Annexin V (APC conjugated; PARTEC; 05–7–490 A or Alexa Fluor 350 conjugated; ThermoScientific; A23202) were then added to each sample at least 15 minutes before analysis. 30 seconds before a sample was run, 0.5 µg/mL DAPI (Thermo Scientific; 62248) was added. Analysis was performed on a LSR II SORP flow cytometer (BD).

Cells transfected for 24 hr with hRluc-GFP-GC-rich or Rluc-GFP-AU-rich plasmids were fixed with 4% paraformaldehyde for 20 min at RT and permeabilized in 70% ethanol overnight at 4°C. The sets of transcript-specific probes (Supplementary file 2) and the secondary Cy3 FLAP probe were designed, purchased and hybridized as previously described (Tsanov et al., 2016 (link)). Of note, none of the AU-rich probes and only 7 out of the 24 GC-rich probes hybridize to the GFP-LSM14A transcripts, resulting in either no signal or a faint signal which was easy to discriminate from the Rluc-GFP mRNA signal. Cells were further processed for immunostaining using rabbit polyclonal anti-DDX6 (1:2000; Novus Biological) and goat anti-rabbit Alexa Fluor 350 (1:300, Thermofisher) antibodies. Epifluorescence microscopy was performed on an inverted Zeiss Z1 microscope equipped with a motorized stage using a 63 × 1.32 oil immersion objective. Images were processed with Icy software.

Cells cultured on 24-well chamber slides were fixed in 4% paraformaldehyde for 15 min, treated with 0.5% TritonX-100 in PBS for 10 min and incubated with 1% BSA for 30 min at RT. For detection of specific protein, cells were incubated with primary antibodies overnight at 4 °C and followed with secondary antibodies for 1.5 h at RT. All secondary antibodies (Alexa Fluor® 488, Alexa Fluor®555, Alexa Fluor®350) were purchased from Thermo Fisher Scientific Inc. (Thermo Fisher Scientific, CA, USA). The nucleus was detected with DAPI (Sigma-Aldrich, St. Louis, MO, USA) staining. Images were acquired with laser scanning confocal microscope (Olympus, Tokyo, Japan).