Fig 2: qPCR for mTOR, DEPTOR, Rictor and Raptor was carried out in triplicate. Error bars ± SEM. mTOR and raptor showed a significant increase in expression compared to controls (A, P≤0.0001 for both groups). Rictor showed a significant increase in patients aged 20-29 years (P=0.0004) (B). Expression of Rictor, Raptor and DEPTOR in CTCs using ImageStreamx Mark II flow cytometry. Channel 1: Brightfield of CTCs; Channel 2: Staining of CTCs with the nuclear marker DRAQ5 (red); Channel 3: Expression of mTOR components (green); and Channel 4: Merged images from channels 2 and 3 (C). ***P<0.001.

Fig 3: Relative DEPTOR expression was measured by qPCR in MDAH-2774 cells treated with Rap (A, 20 and 100 nM), Eve (B, 20 and 100 nM), Def (C, 100 and 1,000 nM), Tem (D, 10 nM and 100 nM), Res (E, 25 and 50 µM), BEZ (F, 10 and 100 nM) and carrier (DMSO) only control for 48 and 72 h. cDNA was synthesised from extracted RNA from 3 biological replicates for each condition *P<0.05, **P<0.01, ***P<0.001.

Fig 4: DEPTOR knockout promotes cell proliferation and clonogenic survival and the activation of S6K1 and AKT in prostate cancer cells. a DEPTOR knockout promotes the proliferation of prostate cancer cells. DEPTOR-knockout DU145 and 22RV1 cells were generated via CRISPR/Cas9 technology. Cells were seeded in 96-well plates in triplicate and then subjected to an ATPlite-based cell proliferation assay. Cell proliferation is expressed as the fold change compared with that at day 1. The mean ± SEM are shown from three independent experiments, n = 3; **p < 0.01; ***p < 0.001. b DEPTOR knockout promotes clonogenic survival of prostate cancer cells. Cells were seeded in 60-mm dishes at 500 cells per dish in triplicate and were incubated for 7–14 days, followed by staining (left) and colony counting (right). The mean ± SEM are shown from three independent experiments; n = 3; ***p < 0.001. c DEPTOR knockout causes increased phosphorylation of the mTORC1 and mTORC2 downstream effectors, S6K1 and AKT, respectively. Cells were harvested for western blotting using the indicated antibodies. The band density was quantified and expressed as the relative gray value (compared with the control), by arbitrarily setting the control value as 1

Fig 5: Expression pattern of ASS1 and mTOR-associated molecules in clinical specimens derived from human endometrial cancer tissues.(a) Representative images of immunohistochemistry of ASS1 using endometrioid carcinoma tissues with high (score 3), intermediate (score 2), low (score 1), or null (score 0) signal intensity, which were used for calculating H-scores. Scale bars, 200 μm. (b) Representative image of immunohistochemistry for ASS1, in which the representative boxed areas of the tumor center, tumor invasive front and normal uterine tissue are enlarged. Black scale bar, 5 mm. White scale bars, 500 μm. (c–e) ASS1, DEPTOR protein expression levels or ribosomal protein S6 phosphorylation (pS6) were statistically analyzed by comparing H-scores calculated from four fields at 200x magnification in the tumor invasive front or tumor center from 74 (ASS1) or 74 (DEPTOR and pS6) cases, respectively. Scale bars, 500 μm. ***p < 0.001, Welch’s t-test.