Fig 2: 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.

Fig 3: 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 4: JMC and BOCD patients show aberrant differentiation of skeletal progenitors. (A–F) Masson Goldners staining of Control (A, D), JMC (B, E) and BOCD (C, F). Note the accumulation of fat and the small region of cartilage in the trabecular bone of JMC patients (magnification in B), and the invasion of chondrocytes in the trabecular bone of BOCD patients (magnification in C). Arrows point to calcified islands, asterisks indicate undermineralized trabecular bone. (G–I) Immunolocalization of ALP of Control (G), JMC (H) and BOCD (I). (J–L) Immunolocalization of AGGRECAN in Control (J), JMC (K) and BOCD (L). ALP staining demonstrates decreased bone mass in JMC and BOCD patients; AGGRECAN immunolocalization confirms the presence of cartilage in JMC and BOCD trabecular bone. (M–Q) Immunolocalization of DEPTOR in Control (M), JMC (N, P) and BOCD (O, Q). JMC, Jansen Methaphyseal Chondrodysplasia; BOCD, Blomstrand Chondrodysplasia; ALP, Alkaline phosphatase. Scale bars, 100 μm.

Fig 5: DEPTOR knockout enhances cell migration and invasion in prostate cancer cells, and Torin-1 abrogates the alterations of EMT markers upon DEPTOR depletion. a, b DEPTOR knockout promotes the migration of prostate cancer cells. DU145 (a) and 22RV1 cells (b) were transfected with the indicated sgRNA and then subjected to transwell migration assays. Representative images of migratory cells at 12–24 h are shown (left, a and b). The number of migratory cells was counted in five random fields per chamber insert (right, a and b). The mean ± SEM from three independent experiments are shown; n = 3; ***p < 0.001. c, d DEPTOR knockout promotes the invasion of prostate cancer cells. Cells were seeded into a 24-well plate chamber insert with Matrigel matrix and incubated for 12–24 h, followed by imaging (left). The invasive cells in five random fields per chamber insert were counted (right). The mean ± SEM from three independent experiments are shown; n = 3; ***p < 0.001. e, f DEPTOR knockout induces EMT in prostate cancer cells. DU145 (e) and 22RV1 (f) cells transfected with the indicated sgRNA were subjected to western blotting using the indicated antibodies. g, h Torin-1, but not rapamycin rescues the alterations of EMT markers upon DEPTOR depletion. DU145 (g) and 22RV1 (h) cells were treated with Torin-1 or rapamycin for 12 h and then subjected to 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