Extended Data Fig. 3: RIOK1 silencing inhibits the assembly of stress granules in HCC cells.
a, LC-MS (left) and MS-MS spectra to identify RIOK1 (left), G3BP1 (middle) and IGF2BP1 (right) in RIOK1 immunoprecipitates (related to Fig. 2a). b-c, Illustration depicting full-length RIOK1 (b) and IGF2BP1 (c) along with the design of truncation constructs used for mapping analyses (related to Fig. 2i and j). d, Western blotting analysis of SNU449 cells with indicated constructs. e-h, Representative confocal images of RIOK1/IGF2BP1(e) and G3BP1/ IGF2BP1(f) in indicated PLC/RNF/5 cells. The average number of SGs/SG-positive cell (g) and SG index (h) based on G3BP1 immunofluorescence were shown. n = 3 independent experiments/4 images each. i-k, Representative confocal images of indicated HCCLM3 cells after treating with As2O3 (0.5 mM) to observe the dynamics of SG assembly (i). The average number of SGs/SG-positive cell (j) and SG index (k) were shown based on G3BP1 immunofluorescence. n = 3 independent experiments, 2 biological replicates. l, Representative confocal images of indicated SNU449 cells. m, Confocal image of LLPS droplet formed in vitro incorporating RIOK1–GFP, IGF2BP1–BFP and G3BP1–mCherry (n = 3 independent experiments). n, Representative smFISH images and protein expression of indicated SNU449 cells (n = 3 independent experiments). o, Quantification of SG index in the indicated SNU449 cells subjected to glutamine deprivation or donafenib as detected by eIF4G, respectively (n = 4 images, 3 biological replicates). p, WB of lysates of SNU449 cells expressing the indicated shRNAs of G3BP2 and ‘synthetic’ constructs. q-r, Representative immunostaining (q) and quantification of SG index (r) as detected by of G3BP2, eIF4G and eIF3η in SNU449 cells expressing the indicated constructs under oxidative stress (sodium arsenate, 500 μM, 1 hr). n = 4 images, 3 biological replicates. s, Representative immunostaining of SNU449 cells transfected with indicated constructs. Error bars indicate mean ± SD. Data were analyzed by unpaired, two-tailed Student’s t-tests (g, h and o), one-way ANOVA with Tukey’s multiple comparisons test (r) and two-way ANOVA with Sidak’s multiple comparisons test (j and k). All microscopy images and blots are representative of n = 3 independent experiments.
