Fig 2: G3BP1 inhibits mTORC1 in cells without SGs, related to Figure 1(A) Insulin/aa-stimulated siG3BP1 cells. n = 6.(B) Quantitation of G3BP1 in (A). Shown are data points and mean ± SEM.(C) Quantitation of RPS6KB1-pT389 in (A). Data shown as in (B).(D) Quantitation of RPS6-pS235/236 in (A). Data shown as in (B).(E) Quantitation of G3BP1 in (G). Mean ± SEM.(F) Quantitation of RPS6KB1-pT389 in (G). Data shown as in (E).(G) Time course analysis of shG3BP1 #1 cells, insulin/aa-stimulated for up to 30 min. n = 3.(H) shG3BP1 #1 cells cultured in full medium. n = 7.(I) Quantitation of G3BP1 in (H). Shown are data points and mean ± SEM.(J) Quantitation of RPS6KB1-pT389 in (H). Data shown as in (I).(K) shG3BP1 #2 cells cultured in full medium. n = 4.(L) Quantitation of G3BP1 in (K). Shown are data points and mean ± SEM.(M) Quantitation of RPS6KB1-pT389 in (K). Data shown as in (L).(N) Serum/aa-starved shG3BP1 #1 cells. Arrow, RPS6KB1-pT389 signal. n = 8, including re-analysis of improved contrast detections for data shown in Figures 1F–1H.(O) Quantitation of G3BP1 in (N). Shown are data points and mean ± SEM. n = 8, including re-analysis of data shown in Figures 1F–1H.(P) Quantitation of RPS6KB1-pT389 in (N). Data shown as in (O). n = 8, including re-analysis of improved contrast detections for data shown in Figures 1F–1H.(Q) IF of shG3BP1 #1 cells. Cells were either serum/aa-starved and stimulated with insulin/aa for 15 min; or serum-starved and treated with arsenite for 30 min. Overlay: white, EIF3A and G3BP1 co-localization; magenta, EIF3A; green, G3BP1; inserts, magnifications of yellow square. Scale bar, 10 µm. n = 3, except shG3BP1 #1, arsenite [0 min], n = 2.(R) Quantitation of data shown in (Q). Shown are data points and mean ± SEM.(S) Immunoblot performed in parallel to IF data in (Q). Insulin/aa-stimulated shG3BP1 #1 cells. n = 3.(T) Quantitation of G3BP1 in (S). Shown are data points and mean ± SEM.(U) Quantitation of RPS6KB1-pT389 in (S). Data shown as in (T).(V) Immunoblot performed in parallel to IF data in (Q). Arsenite-exposed shG3BP1 #1 cells. n = 3.(W) Quantitation of G3BP1 in (V). Shown are data points and mean ± SEM.(X) Quantitation of RPS6KB1-pT389 in (V). Data shown as in (W).

Fig 3: Effect of rapamycin and its removal on ribosome distribution. HeLa cells were incubated with 100 nM rapamycin for 4 h and then the medium was replaced with fresh medium without the inhibitor for 2 h before the lysis. (A) Total RNA was extracted and subjected to northern blot analysis for pre-rRNA species. The membrane was hybridized with probes ITS1, ITS2, 18S and 28S as previously described. (B) Polysomal profile after rapamycin removal of HeLa cells. The polysomal and non-polysomal fractions are indicated. (C) Northern blot analysis of cytoplasmic RNA extracts from each fraction of sucrose gradient and analyzed with indicated probes (RpS6, RpS19 and ß-actin). (D) Quantification of the signals is reported as a linear plot of percentage of mRNAs in each fraction.

Fig 4: Abnormal autophagy in anterior cingular cortex of GBA-PD patients. (A) Western blot analysis of autophagy and lysosomal proteins in non-GBA-PD and GBA-PD brains (left). Compared to controls, LC3B-II levels were increased in both GBA-PD and non-GBA-PD brains, whereas SQSTM1 protein only accumulated in GBA-PD brain (right). (B) Immunohistochemistry of LC3B and SQSTM1 in control and GBA-PD brains (left). Compared to controls, pyramidal neurons from GBA-PD brains showed increased LC3B and SQSTM1 immunoreactivity (right). Scale bar: 20 µm. (C) Western blot analysis of autophagy regulators MTOR, BECN1 and AMBRA1 in non-GBA-PD and GBA-PD brains (left). GBA-PD brains showed lower protein levels of p-MTOR, p-RPS6, AMBRA1 and BECN1 (right). Data are mean (% of controls) ± SE from 3 independent experiments. Compared to controls, *, p < 0.05; **, p < 0.01, One-way ANOVA followed by Bonferroni post hoc test (A and C) or student’s t test (B).

Fig 5: CDR2L and Yo co-localize with ribosomal proteins and CDR2 co-localizes with nuclear speckle proteins in cerebellar Purkinje neurons as shown by super-resolution microscopy. (A) Upper row: Human cerebellar section stained with Yo-CSF (green) and anti-rpS6 (red); the proteins co-localize in the cytoplasm (yellow; merged image). Middle row: Human cerebellar section stained with anti-CDR2L (green) and ribosomal marker anti-rpS6 (red); the proteins co-localize in the cytoplasm (yellow; merged image). Lower row: Human cerebellar section stained with anti-CDR2 (green) and nuclear speckle marker anti-SRSF2 (red); the proteins co-localize in the nucleus. No co-localization was found with anti-Yo (magenta; merged image). (B) Upper row: Rat Purkinje neuron cultures stained with anti-Yo (CSF; green) and rpS6 (ribosomes; red); co-localization was observed in the cytoplasm (yellow; merged image). Middle row: Rat Purkinje neuron cultures stained with anti-CDR2L (green) and anti-rpS6 (red); co-localization was observed in the cytoplasm (yellow; merge image). Lower row: Rat Purkinje neurons stained with anti-CDR2 (green), nuclear speckle protein (red), and anti-Yo (magenta). CDR2 and the nuclear speckle protein co-localize in the cell nucleus (yellow; merged image), whereas Yo does not. Scale bars = 10 µm.