Figure S1.

Generation of a stable cell line expressing fluorescently tagged PKR. (A) Schematic representation of the expression construct encoding mRuby- and FLAG-tagged PKR of human origin. The mRuby fluorescent protein (236 aa) was inserted between residues 221 and 222 of human PKR. (B) Representative micrograph showing that mRuby-PKR is a cytosolic soluble protein. Scale bar: 10 µm. (C) Western blot showing the level of expression of mRuby-PKR compared with that of endogenous PKR. The relative protein amount determined by densitometry is shown below the blots. Endogenous PKR was depleted using CRISPRi. GAPDH, loading control. (D) Western blot analysis showing endogenous PKR induction in wild-type H4 cells treated with IFNβ for 16 h. GAPDH, loading control. Metrics as in C. (E) Western blot analysis comparing the activity and kinetics of endogenous PKR and mRuby-PKR in H4 cells treated with poly I:C. (F) Violin plots showing the diameter of mRuby-PKR clusters in H4 cells stably expressing mRuby-PKR and treated with poly I:C. (G) Quantification of the number of cells with endogenous PKR and mRuby-PKR clusters upon 90 min of poly I:C treatment (N = 3 experiments, n > 800; unpaired Student’s t test, nonparametric). (H) Representative time-lapse micrographs showing the formation of mRuby-PKR clusters (yellow arrowheads) in dividing H4 mRuby-PKR cells synchronized with thymidine. Scale bar: 10 µm. (I) Quantification of the half-life of mRuby-PKR in clusters after photobleaching. (J) Quantification of the mRuby-PKR immobile fraction in clusters after photobleaching. For H and I, N = 3 experiments, n = 30. Source data are available for this figure: SourceData FS1.