Mm af microscope

Immunofluorescence images were acquired on a TSC SP8 confocal microscope (Leica GmbH) with a 63× lens. For smFISH, a widefield Leica MM-AF microscope was used with a 100× lens. smFISH images were deconvoluted using the Regularized Inverse Filter algorithm with the DeconvolutionLab2 plugin (Sage et al., 2017 (link)) in ImageJ. The smFISH spots (each corresponding to a single RNA molecule, (Raj et al., 2008 (link))) were counted using the spot count tool in the Imaris Image Analysis Software (Bitplane), and the percentage of spots in stress granules was estimated by their overlap in fluorescent signal with FMR1. At least, 20 randomly selected cells (N) were quantified per smFISH probe. The minimum and maximum display values were adjusted for each channel for visualization purposes.
For calculating the fraction of FMR1-ADARcd-V5 in stress granules, we used FIJI’s ‘’plot profile’’ plugin. The ratio of fluorescence intensity of FMR1 in stress granules and cytoplasm was calculated for 10 cells.

Plated wild-type S2 cells were first treated with KRB (see above), and fixed and labeled for endogenous FMR1 or Sec16 as described above in the immunofluorescence section. After incubation with the secondary antibody, cells were washed three times with PBS and cells were post-fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min. Following a washing three times in PBS, cells were further incubated for 5 min in 10% formamide (17899, Thermo Fisher Scientific) in DEPC-treated water. They were then incubated overnight on a droplet containing one fluorescent smFISH probe [125 nM in 1% dextransulfate (D8906, Sigma-Aldrich), 10% formamide in DEPC-treated water at 37°C] in a moistened chamber to avoid drying. Cells were washed twice for 30 min with 10% formamide in DEPC-treated water and mounted with Prolong antifade medium (plus DAPI) on a microscope slide. The TMR-oligo(dT)30× was purchased from IDT. A widefield Leica MM-AF microscope was used with a 100× lens for imaging.