Fig 2: Recruitment of UPF1 to stress granules is independent of NMD inhibition.a UPF1 and G3BP1 localization in HeLa cells expressing either GFP only, repeat RNA, or GFP-tagged DPR. Stress granules are indicated by arrowheads. Scale bar, 10 µm. Similar results were obtained from four independent experiments. b Quantification of stress granule (SG)-positive cells. n = 4 independent experiments. Data are presented as mean values ± SD. ***P < 0.001, two-sided unpaired t tests. c Quantification of stress granule formation in G3BP-WT and -DKO cells. n = 3 independent experiments. Data are presented as mean values ± SD. **P < 0.01, two-sided unpaired t tests. d Changes in UPF1 target mRNA abundance in G3BP-WT and -DKO cells. Expression levels were normalized to the GFP-only control. n = 3 independent experiments. Data are presented as mean values ± SD. *P < 0.05; **P < 0.01, two-sided ratio t tests. Source data and exact P values are provided in the Source Data file.

Fig 3: Robustness of PLSR Models, and Validation of G3BP2 as a Determinant of Cisplatin Sensitivity(A and B) The minimal number of signaling metrics required for predicting sub-G1 was calculated by iteratively removing metrics. The fraction of G3BP2-related metrics among metrics with the highest VIP scores is indicated in pie charts. (A) Metrics were eliminated sequentially from the models of cisplatin-sensitive cell lines (left panel) or cisplatin-resistant cell lines (right panel) based on the relative magnitude of their coefficients in the model, from highest to lowest VIP score. (B) Metrics were sequentially eliminated from the model of cisplatin-sensitive cell lines from lowest to highest VIP score.(C) PC1 loading scores of the dynamic signaling metrics of G3BP2 and their average are plotted for the sensitive and resistant models individually.(D) MDA-MB-231 and BT549 cells were transduced with indicated shRNAs and MTT conversion after cisplatin treatment was measured. Inset bar graphs depict MTT conversion upon treatment with 7.5 µM cisplatin. sh#1 and sh#2 refer to shG3BP2#1 and shG3BP2#2, respectively. Error bars indicate SEM of three independent experiments. The p values were calculated using two-tailed Student’s t test. ***p < 0.001.(E and F) MDA-MB-231 cells with doxycycline-inducible shRNAs targeting luciferase or G3BP2 were treated with 2 µM cisplatin. At indicated time points, cell-cycle profiles were determined by flow cytometry (E). Means and SDs of percentages of G2 cells from three independent experiments are plotted (F).(G) ?H2AX levels after 2 µM cisplatin treatment for 72 h. MDA-MB-231 cells expressing inducible shRNAs against luciferase or G3BP2 were fixed and stained with anti-?H2AX antibody and propidium iodide. ?H2AX levels and DNA content were determined by flow cytometry of two independent experiments.See also Figure S6.

Fig 4: G3BP1 and G3BP2 suppress mTORC1 in a non-redundant manner and form a heterocomplex(A) Insulin/aa-stimulated siG3BP2 cells. n = 4.(B) Quantitation of G3BP2 in (A). Shown are data points and mean ± SEM.(C) Quantitation of RPS6KB1-pT389 in (A). Data are shown as in (B).(D) Quantitation of RPS6-pS235/236 in (A). Data are shown as in (B).(E) Serum/aa-starved G3BP1 KO cells. n = 4.(F) Quantitation of G3BP2 in (E). Shown are data points and mean ± SEM.(G) Serum/aa-starved siG3BP1 cells. n = 3.(H) Quantitation of G3BP2 in (G). Shown are data points and mean ± SEM.(I) Insulin/aa-stimulated G3BP1 KO cells transfected with MYC-FLAG-G3BP1 or MYC-FLAG-G3BP2 (48 h). n = 3.(J) Quantitation of RPS6KB1-pT389 in (I). Shown are data points and mean ± SEM.(K) IP against G3BP2 or mock (rabbit IgG). n = 2.(L) BiFC. Protein+C-terminal mLumin is indicated first; protein+N-terminal mLumin is indicated second. TL, transmitted light. Scale bar, 100 µm. n = 3.(M) Quantitation of data in (L). Shown are data points and mean ± SEM.See also Figure S3.

Fig 5: G3BP1 and G3BP2 reside at lysosomes(A) Quantitation of data in (B). G3BP1, green area. Mean ± SEM.(B) Sucrose density gradient separation of serum/aa-starved MCF-7 cells. n = 3.(C) Lyso-prep with ferromagnetic nanoparticles. PNS, postnuclear supernatant. n = 3.(D) PLA of G3BP1-LAMP1 in serum/aa-starved G3BP1 KO cells. PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 10 µm. n = 3.(E) Quantitation of data in (D). Shown are data points and mean ± SEM. n = 8 technical replicates.(F) Trypsin digest of lyso-preps prepared as in (C). n = 3 except for TSC2 (n = 2).(G) IP against TSC2 (TSC2 #1) or mock (mouse IgG). n = 3.(H) PLA of G3BP1-TSC2 in serum/aa-starved G3BP1 KO cells. PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 10 µm. n = 4.(I) Quantitation of data in (H). Shown are data points and mean ± SEM. n = 8 technical replicates.(J) IP against MTOR or mock (rat IgG); insulin/aa-stimulated shG3BP1 #1 cells (15 min). n = 4.(K) Quantitation of G3BP1 in (J). Shown are data points and mean ± SEM.(L) Quantitation of TSC2 in (J). Data are shown as in (K).(M) IP against TSC2 (TSC2 #2 or #3) or mock (rabbit IgG). n = 3.(N) IP against TSC2 (TSC2 #2) or mock (rabbit IgG); insulin/aa-stimulated shG3BP1 #1 cells (15 min). n = 4.(O) Quantitation of TSC1 in (N). Shown are data points and mean ± SEM.(P) Quantitation of G3BP1 in (N). Data are shown as in (O).(Q) Quantitation of LAMP1 in (N). Data are shown as in (O).See also Figure S4.