Fig 2: ?Sin1 expression suppresses Akt activation but not p70S6K activation in DLD1 cellsA. Following 72 hours doxycycline (Dox) induction of Sin1 constructs, or 30 minute incubation with 1µM PP242 or 100nM rapamycin (Rapa), cell lysates were probed with the indicated antibodies. B. Quantification from 3 independent experiments indicates that Sin1?1-192 but not Sin1-FL significantly inhibits phosphorylation of the mTORC2 target Akt on S473 but not the mTORC1 targeted p70S6K on T389. Conversely rapamycin selectively inhibits T389 phosphorylation while PP242 inhibits both. C. and D. Cells were serum starved (0.5% Serum) overnight (O/N)prior to stimulation with 10% Serum for the times indicated. Phosphorylation of Akt on pT308 and pS473 were assessed relative to total Akt. GAPDH indicates protein loading. Quantification represents mean +/- S.D (n = 3). Statistical significance was assessed by 1-way (B) or 2-way (D) ANOVA and Bonferroni post hoc tests; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: Truncated Sin1 displaces endogenous Sin1 from mTORC2 in DLD1 colon cancer cellsA. Schematic indicating the domain structure of Sin1 and the constructs used to displace endogenous Sin1 from mTORC2. B. Expression of myc tagged Sin1 constructs can be detected only after induction with Doxycycline (Dox). Cells were treated with 100nM of doxycycline (+) for 72 hours and expressed proteins were detected by immunoblot of whole cell lysates with anti-myc (9E10) antibodies. C. and D. Sin1 constructs incorporate into mTORC2 and displace endogenous Sin1. Constructs were induced for 72 hours prior to immune precipitation. (C) mTORC2 subunits, mTOR and Rictor, only appear in myc immunoprecipitates after induction with doxycycline (Left panels); myc-?Sin1 cannot be directly detected in precipitates due to secondary antibody cross reaction with precipitating IgG. Right panels indicate unchanging expression levels of Rictor and mTOR in immune precipitation input lysates, which is further quantified from 3 independent experiments E. Endogenous Sin1 and Rictor immunoprecipitates demonstrate displacement of endogenous Sin1 from mTORC2. Following induction, band shifted myc-tagged FL Sin1 can be detected in Sin1 and Rictor precipitates (Left panels). Truncated ?Sin1 can be detected in Rictor, but not Sin1, immunoprecipitates as the Sin1 antibody epitope is deleted from ?Sin1. F. Quantification of Sin1 levels detected in Rictor immunoprecipitates indicates the level of endogenous mTORC2 disruption following Sin1 construct induction (data are mean +/- S.D; n = 3). Myc-?Sin1 displaces >80% of endogenous Sin1 while levels of myc-FL Sin1 associated with Rictor are comparable with endogenous Sin1 levels.

Fig 4: ?Sin1 expression suppresses DLD1 subcutaneous tumour growth in NOD/SCID miceMice were inoculated with 106 DLD1 cells on both flanks and assigned randomly to control or doxycycline (Dox) groups. Tumour burden A. and incidence B. per inoculation were assessed 3 times weekly. Data are mean +/- S.E.M. Statistical significance was assessed by ANOVA and Bonferroni post hoc test; **p < 0.01; ***p < 0.001. C. Examples of individual tumour growth rates; curves indicate exponential growth curve best fit to estimate growth rates. D. Growth rate of tumours does not differ significantly between control and doxycycline cohorts. Data are mean +/- STD E. Mice were inoculated on a single flank with DLD1 cells. Once tumours had reached a volume of 50mm3 mice were randomised into vehicle and Dox treated groups. Times indicated are post-1st treatment and statistical analysis was conducted as for A. This data is derived from a single cohort of animals. F. Growth rates for individual tumours from (E) are illustrated (student's t-test; *p < 0.05.