Fig 2: PLD2 and mTORC2 are required to convert increases in membrane tension to decreases in actin network assembly.(A) Prediction: Knockdown of the PLD2–mTORC2-based tension circuit should result in a slower decrease of actin nucleation following an increase in membrane tension. (B) Paired static tether force measurements for stimulated cells before and after 5 min of 70 mOsm hypo-osmotic shock. Connected colored lines indicate the same cell before and after hypo-osmotic shock. All conditions significantly increase their membrane tension following hypo-osmotic shock (p < 0.05). (C) Representative images of Hem1-GFP before and 10 sec after a 70 mOsm hypo-osmotic shock. Dashed white line corresponds to cell outline. Scale bar = 10 µm. Rictor and PLD2 knockdown cells are delayed in the detachment of the WAVE2 complex from the membrane following hypo-osmotic shock. See S4 Fig for complete time series of WAVE2 complex loss following 70 mOsm hypo-osmotic shock. (D) Quantification of the dynamics of Hem1-GFP on the membrane after 70 mOsm hypo-osmotic shock. Mean ± SEM. Rictor and PLD2 knockdown cells are delayed in the detachment of the WAVE2 complex from the membrane following hypo-osmotic shock (p < 0.05). Ncells: B = 8 (Ns shRNA), 12 (Rictor shRNA), and 7 (PLD2 shRNA). D = 22 (Ns shRNA), 17 (Rictor shRNA), and 12 (PLD2 shRNA). Ntethers: B = 17 (Ns shRNA), 19 (Rictor shRNA), and 9 (PLD2 shRNA). Statistics: paired t test (B) and t test (D). Boxes in all box plots (B) extend from the 25th to 75th percentiles, with a line at the median. Whiskers extend to ×1.5 IQR (interquartile range) or the max/min data points if they fall within ×1.5 IQR.

Fig 3: Activation of the AKT pathway in a neuroendocrine tumor of unknown primary origin. a Scheme depicting sample collection and processing for the TARGET clinical trial and the studies performed for the molecular analysis of the tumor specimen TAR007. WBC White Blood Cells, WG Whole Genome, WE Whole Exome. b Whole genome sequencing from the tumor and a patient-derived sample shows extensive copy number variations, including amplifications in 3q and 5p chromosomes. c Phosphokinase arrays from tumor-derived low passage cell cultures show activation of several components of the AKT-mTOR signaling pathway compared to normal primary cells NHBE (normal human bronchial epithelium) and SAEC (small airway epithelial cells). d Phospho-receptor tyrosine kinase (RTK) arrays from TAR007 low passage-derived cell cultures show different RTK activation profiles. e Western blot confirming overexpression of PIK3CA (aka p110a) and RICTOR, as well as hyperactivation of AKT in TAR007-derived cell cultures. GAPDH was used as a loading control. Uncropped blots are available in Figure S3

Fig 4: Kaplan-Meier curve of HPV-related and -unrelated OPSCC patients according to raptor and rictor expression. a Raptor expression and cumulative overall survival in HPV-related OPSCC patients. Patients with high raptor expression tended to show worse cumulative overall survival compared with those with low or medium expression, but the difference did not reach statistical significance. b Rictor expression and cumulative overall survival in HPV-related OPSCC patients. Patients with high rictor expression had significantly worse cumulative overall survival compared with those with low or medium expression. c Raptor expression and cumulative overall survival in HPV-unrelated OPSCC patients. There was no difference in cumulative overall survival in HPV-unrelated OPSCC patients according to raptor expression. Five cases were removed from the survival estimation because of poor general condition. d Rictor expression and cumulative overall survival in HPV-unrelated OPSCC. There was no difference in cumulative overall survival in HPV-unrelated OPSCC patients according to rictor expression. Five cases were removed from the survival estimation because of poor general condition

Fig 5: RRM1 phosphorylation at S631 enhances its interaction with RRM2. (A) Computational modeling based on human RRM1 and human RRM2 structure, which were obtained from PDB database (RRM1, PDB ID: 3HNE) and (RRM2, PDB ID:3OLJ), showing that S631 is localized at interface of RRM1/RRM2 interaction. The active site (A site), specificity site (S site), catalytic site and S631 on RRM1 were indicated as blue, green, cyan and red respectively. (B) H1299 cells were co-transfected with HA-RRM2 along with empty vector (EV), WT, S631A or S631D flag-RRM1, followed by IP with anti-flag affinity beads and western blot analysis with indicated antibodies. (C-D) Co-IP analysis of RRM1/RRM2 interaction in Rictor silence (C) or Rictor KO H1299 cells (D) through IP with anti-RRM1 antibody and western blot using indicated antibodies. (E) H1299 cells were treated with 1 µM PP242 or 1 µM Rapamycin for 4 hrs, followed by analysis of RRM1/RRM2 interaction through RRM1 IP.