Fig 2: Knockdown effect of the target siRNA-FA-PEG-COL nanoparticles on cell motility and invasion in vitro.(A, B) S2-013 cells were incubated with scrambled control siRNA-FA-PEG-COL nanoparticles (Scr), CCDC88A siRNA-FA-PEG-COL nanoparticles (siCCDC88A), or WASF2 siRNA-FA-PEG-COL nanoparticles (siWASF2). Motility (A) and two-chamber invasion (B) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns, mean; bars, SD. *p < 0.05 compared to cells incubated with scrambled control siRNA-FA-PEG-COL nanoparticles (Student’s t-test). (C) S2-013 cells were incubated with scrambled control siRNA-FA-PEG-COL nanoparticles (Scr) and the target siRNA-FA-PEG-COL nanoparticles against mRNAs for ARHGEF4 (siARHGEF4), LAMTOR2 (siLAMTOR2), mTOR (simTOR), and NUP85 (siNUP85). Two-chamber invasion assay was performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns, mean; bars, standard deviation (SD). *p < 0.05 compared to cells incubated with scrambled control siRNA-FA-PEG-COL nanoparticles (Student’s t-test).

Fig 3: Disease progression in the orthotopic mouse model of PDAC.(A) Representative S2-013-derived PDAC tumor tissues in S2-013 tumor-bearing mice treated with scrambled control siRNA-FA-PEG-COL nanoparticles (Scr) and target siRNA-FA-PEG-COL nanoparticles against mRNAs for LAMTOR2 (siLAMTOR2), mTOR (simTOR), and NUP85 (siNUP85). Arrow, primary tumor; arrowheads, dissemination nodules in the abdominal cavity. (B) Hematoxylin and eosin staining of representative sections of S2-013-derived PDAC tumor tissues in mice treated with scrambled control siRNA-FA-PEG-COL nanoparticles or target siRNA-FA-PEG-COL nanoparticles against mRNAs for LAMTOR2, mTOR, and NUP85.

Fig 4: RAG-GTPases and LAMTOR proteins are required for lysosomal leucine storage. (A–C) Percent 3H-leucine released from wt and RagA/B-KO MEFs or wt and Lamtor1 or Lamtor2-KO ESCs in nutrient-replete or leucine-free medium in the presence or absence of LLOMe in the chase. Western blots show loss of expression of RAGA, LAMTOR1, and LAMTOR2 in KO cells. (D) Distribution of 3H-leucine in wt and different KO MEFs or Torin1-treated MEFs entering protein synthesis (gray bars), released (black bars), or retained in cells as free amino acid (light gray bars). (E and F) Percent 3H-leucine released from control or RagA- or RagB- overexpressing MCF7 cells in leucine-free (E) or nutrient-replete (F) medium in the presence or absence of LLOMe in the chase. Asterisks in (F) show statistical significance compared to control; ns shows lack of significance between control and LLOMe. (G) Expression levels of RagA and RagB proteins in MCF7 cells compared to MCF10A cells. See also SI Appendix, Fig. S5B. Individual data points in (A–F) represent means from at least three independent biological replicates performed in triplicate. Bars show means from all biological replicates, and error bars show SD. (H) Model: essential amino acids, including leucine, transit the lysosome and are utilized in protein synthesis. Excess amino acid is released into the medium (dashed arrow). Under starvation conditions, leucine is retained in lysosomes in a RAGA/B- and LAMTOR1/2-dependent manner and utilized for protein synthesis. The inhibition of protein synthesis releases lysosomal stores.

Fig 5: LAMTOR2 regulates lysosomal positioning in dendrites of cultured hippocampal neurons, while mTORC1 inhibition has no effect. See also Supplementary Figure 2. (A,B) Effects of LAMTOR2 KD on dendritic lysosomal positioning. (A) Images of cultured hippocampal neurons stained for LAMP2 (red). Neurons were infected with an shRNA AAV directed against LAMTOR2 (shLT2) or a scrambled shRNA control (shSc) with GFP co-expression before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (B) Quantification of lysosome distribution along the dendrites, as shown in (A). Results are Means ± SEM of 8–11 neurons from 3 independent experiments, *p < 0.05, ***p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test. (C,D) Effects of mTORC1 inhibition on dendritic lysosomal positioning. (C) Images of cultured hippocampal neurons stained for LAMP2 (Red) and GFP. Neurons were treated with vehicle control or with Torin 1 (250 nM) for 4 h before being processed for immunofluorescence assay and imaging. Scale bar: 20 μm. (D) Quantification of lysosome distribution along the dendrites, as shown in (C). Data are represented as Means ± SEM. N = 6–12 neurons from 3 independent experiments, two-way ANOVA with Sidak’s post-test.