Fig 2: NC Inhibits SIN1 Expression(A) The expression of SIN1 was measured by western blotting in MG63 cells and U2OS cells after NC treatment. (B) Quantitative results are illustrated for (A). *p < 0.01 compared with control (DMSO treatment group).

Fig 3: SIN1 mediates AURKA functions in regulating PTC progression. A The protein expression levels of SIN1 and P-AKT were examined by western blotting in OE AURKA-transfected KTC cells and siSIN1/OE AURKA-transfected KTC cells. Mg132 (20 μM) was added 6 h prior to cell harvest for WESTERN BLOT. B KTC cells were stably transduced with AURKA or siSIN1 were cultured for 14 days prior to crystal violet staining. C Transwell migration assays were carried out using KTC cells stably overexpressing AURKA and siSIN1-transfected KTC cells or the corresponding negative control (NC) cells. D The ratio of EdU-labelled cells was observed in AURKA-overexpressing KTC cells and siSIN1-transfected KTC cells compared with the control group. E, F Representative images of subcutaneous xenografts in nude mice derived from OE AURKA-transfected KTC cells or PCDH-transfected KTC cells or shSIN1/OE AURKA-transfected KTC cells. G The expression levels of AURKA, Ki67, P-AKT and SIN1 in xenografts of each group were assessed by immunohistochemical staining. The scale bar is 50 μm. Data are shown as the mean ± SD of three replicates (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig 4: Downregulation of SIN1 Inhibits Osteosarcoma Cell Invasion(A and B) Left: cell migration and invasion were detected by a Transwell chamber assay with or without Matrigel in MG63 cells (A) and U2OS cells (B) after SIN1 siRNA transfection and NC treatment. Right: quantitative results are illustrated for the left image. siRNA Ctr, siRNA control; MG63, 1.5 μM NC; U2OS, 4 μM NC; siRNA, SIN1 siRNA; Both, SIN1 siRNA+NC. (C) Top: the expression of SIN1 was detected by western blotting in osteosarcoma cells with SIN1 siRNA transfection and NC treatment. Bottom: quantitative results are illustrated for the top image. *p < 0.01 compared with control; #p < 0.05 compared with NC treatment alone or SIN1 siRNA transfection alone.

Fig 5: mTORC2 induces cGAS chromatin localization.a. Immunoblot analysis of whole-cell lysates (WCLs) from HCT116 cells treated with PI3K-mTOR pathway inhibitors (2 µM, 12 hours). b. Automated quantification of cGAS intensities in individual cell nuclei and cytosol from (B) using CellProfiler. Nuclear/cytosolic ratios are plotted as violin plots with mean for each condition. n = 50 from 3 independent experiments. Unpaired two-tailed t-test. c. Immunoblot analysis of whole cell lysates from HCT116 cells lentivirally infected with scrambled (sh-Src) or shRNAs targeting mTOR components. d. Immunofluorescence analysis and quantification cGAS subcellular distribution in HCT116 cells lentivirally infected with the scramble control or gene-targeted shRNAs. Nuclear/cytosolic ratios are plotted as violin plots with means for each condition. n = 50 from 3 independent experiments. Unpaired two-tailed t-test. e. Co-immunoprecipitation and immunoblot analysis of cGAS and mTORC2 component interaction in HCT116 cells. f. Co-immunoprecipitation of Myc-tagged cGAS mutants from HCT116 cells transfected with the indicated constructs. g. eBRET2 signal reporting the interaction between cGAS and SIN1 (mTORC2 component) in HCT116 cells expressing a GFP2-cGAS and Rluc8-SIN1 biosensor. Bars represent the means ± s.e.m. from three independent experiments. Unpaired two-tailed t-test. h. Schematic of the workflow establishing an in vitro cGAS phosphorylation system using purified mTORC2 complexes and AKT1 as a control substrate. i. Mass spectrometry analysis of in vitro cGAS phosphorylation by purified mTORC1 or mTORC2 complexes from starved (48 h) or insulin-stimulated (30 min with 100 nM insulin) HCT293T cells. Experiments were repeated three times (a, c, e, f, h) with similar results. Numerical source data and unprocessed blots are available as source data. Source data