Fig 2: CircEIF3H promotes tumor growth and metastasis in vivo.A MDA-MB-231 cells were stably transfected with the circEIF3H-overexpressing vector or control vector and injected subcutaneously into nude mice. CircEIF3H overexpression promoted tumor growth compared with the vector group. B, C Tumor growth rate (B) and tumor weight (C) of circEIF3H-overexpressing group and vector group were presented as mean ± SD; circEIF3H-overexpressing group n = 7; vector group n = 6; **P < 0.01, ***P < 0.001. D MDA-MB-231 cells were stably transfected with the circEIF3H-sh vector or control vector and injected subcutaneously into nude mice. CircEIF3H-sh inhibited tumor growth compared with the vector group. E, F Tumor growth rate (E) and weight (F) were significantly decreased in the circEIF3H-sh group. Tumor growth rate (E) and tumor weight (F) of circEIF3H-sh group and NC group were presented as mean ± SD; n = 7; **P < 0.01, ***P < 0.001. G IHC staining analysis of the expression of HSPD1, RBM8A, and G3BP1 in tumors. Scare bar = 100 µm. H, I The macroscopic view of lung metastasis nodules and H&E-stained lung section images (Scare bar = 200 µm) after tail vein injection of MDA-MB-231 cells with circEIF3H-overexpression or circEIF3H-sh (n = 5). Statistical significance was determined by a two-sided Student’s t-test.

Fig 3: Phosphomimetic substitutions do not alter nuclear localization, UG-rich RNA binding and autoregulation of TDP-43 Immunostainings showing nuclear localization of myc-TDP-43 Wt, 12D and 12A in HeLa cells. Endogenous TDP-43 expression was silenced by siRNAs, followed by transient transfection of the indicated siRNA-resistant myc-TDP-43 constructs. After 24 h, localization of TDP-43 Wt, 12D and 12A variants was visualized by TDP-43 immunostaining (mouse anti-TDP-43 antibody, Proteintech). G3BP1 (rabbit anti-G3BP1 antibody, Proteintech) and DAPI signal is shown to visualize the cytoplasm and nuclei, respectively. In the merge (right column), DAPI is show in turquoise, TDP-43 in green, and G3BP1 in magenta. Bar, 30 µm.Electrophoretic mobility shift assay (EMSA) of TDP-43-MBP-His6 variants (Wt, 12D and 12A) in a complex with (UG)12 RNA.Representative confocal images of U2OS cells stably expressing the indicated myc-TDP-43 variants (Wt, 12D and 12A) after siRNA KD of endogenous TDP-43 and induction of myc-TDP-43 expression with doxycycline. Cells were stained with mouse monoclonal anti-myc 9E10 antibody (IMB protein production facility) and DAPI. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), DAPI is shown in turquoise) and myc-TDP-43 is shown in green. Bar, 20 µm.Western Blot showing the expression levels of myc-TDP-43 variants in stable inducible Flp-In T-Rex U2OS cell lines before and after addition of doxycycline (dox). Samples were analyzed by SDS–PAGE and Western blot using a rabbit anti-TDP-43 N-term antibody (Proteintech, upper blot), mouse anti-myc 9E10 antibody (IMB protein production core facility), and rabbit anti-Histone H3 antibody (Abcam) to detect the loading control Histone H3.Quantification of TDP-43 autoregulation after dox-induced expression of myc-TDP-43 variants in U2OS cell lines. Values represent the mean ± SD of four independent experimental replicates (n = 4) of endogenous TDP-43 expression levels normalized to Wt (-Dox) condition. *P < 0.0332 and ***P < 0.0002 by one-way ANOVA with Šídák's multiple comparisons test of TDP-43 endogenous expression levels, comparing the respective non-induced (-Dox) and induced (+Dox) lines. Source data are available online for this figure.

Fig 4: The relationship of G3BP1 protein expression status and malignant biological behavior of GC cell. (A) After transfection, the expression levels of G3BP1 protein in GC cells line was assessed by Western blotting. (B) CCK-8 assay showed that silencing G3BP1 expression can inhibit the proliferation of GC cells; (C) Transwell assay showed that silencing G3BP1 expression can inhibit the migration and invasion of GC cells. (D) Wound healing assay showed that silencing G3BP1 expression can inhibit the migration of GC cells. Compared with the G3BP1-siRNA group, *P>0.05; ***P<0.01.

Fig 5: CA16-induced autophagy promoted SG clearance. (A) Atg5-deficient RD cells (shAtg5) or control RD cells (shCtrl) were infected with CA16 at an MOI of 1 for 24 h or were mock infected. All cells were treated with 2 µg/ml poly I:C every 6 h. SGs were examined by fluorescence microscopy (G3BP1 and TIA1 serve as SG markers). Representative images of stress granules are shown. Scale bars, 10 µm. (B) and (C) Quantitation of the data in (A). (D) RD cells treated with 50 nM bafilomycin A1 or DMSO were infected with CA16 at an MOI of 1 for 24 h or were mock infected. All cells were treated with poly I:C 2 µg/ml every 6 h. SGs were examined by fluorescence microscopy (G3BP1 and TIA1 serve as SG markers). Representative images of stress granules are shown. Scale bars, 10 µm. (E) and (F) Quantitation of the data in (D). Graphs show the mean ± SEM, 6 random fields and 10 cells per field were examined for confocal microscopy. *p < 0.05; **p < 0.01; ***p < 0.001.