Fig 2: The translational downregulation of MCL-1 upon mTORC1 inhibition contributes to the enhanced induction of cell death by BCL-2/BCL-XL inhibitors(A and B) Immunoblots of Tsc2-/- MEFs (A) and 105K cells (B) treated with vehicle (DMSO), rapamycin (20 nM), or Torin1 (250 nM) in the presence or absence of Actinomycin D (200 ng/µL) for 24 h without serum.(C and D) Immunoblots of cells treated as in (A, B) but in the presence or absence of cyclohexamide (100 µM) for 8h.(E) Immunoblot of wild-type (WT) or 4E-BP1/2 DKO MEFs stably expressing empty vector (EGFP) or 4E-BP1 treated with vehicle (DMSO), rapamycin (20 nM), or Torin1 (250 nM) for 24 h without serum.(F–I) Immunoblot of Tsc2-/- MEFs (F) and 105K cells (H) expressing empty vector or HA-tagged MCL-1 lacking its 5'UTR treated with vehicle (DMSO) or rapamycin (20 nM) for 24 h without serum. *Apparent degradation product. Mean log2 fold change in absorbance (570 nM) of solubilized crystal violet dye for Tsc2-/- MEFs (G) and 105K cells (I) treated with vehicle (DMSO), rapamycin (20 nM), ABT-263 (500 nM (I) or 1 µM (G)), or the combination after 72 h in 0.5% serum is graphed as mean ± SD relative to Day 0. n = 2. **p<0.01, ***p<0.001 (two-way ANOVA).(J) Schematic summarizing findings, created with Biorender.com.

Fig 3: PRL-3-driven mTOR activation correlates with increased Akt-TSC2-Rheb signalling.(a) HCT116 cells overexpressing EGFP vector only (Vec), EGFP-tagged wild-type PRL-3 (PRL-3) or EGFP-tagged catalytic-inactive PRL-3 C104S (C104S) were cultured for 24 h in full media under normoxia (Ctrl), hypoxia (Hx), or serum-free (SF) conditions, prior to lysis and western blot analysis with the indicated antibodies. (b) HCT116 cells overexpressing Vec, PRL-3 or C104S were cultured for 1 h in full media under normoxia (Ctrl) or amino-acid starved (AA-) conditions, prior to analysis as in (a). (c) HCT116 cells stably expressing shRNA against PRL-3 (shPRL-3) or control shRNA (shCon) were cultured and analysed as in (a). (d) Cell lysates from (a) were immunoprecipitated with a configuration-specific anti-Rheb-GTP antibody and analysed by immunoblotting with the indicated antibodies. (e) Cell lysates from (b) were analysed as in (d). (f) Proposed signalling pathway for PRL-3 to mTOR substrates, 4EBP1 and p70S6K. Representative full immunoblots for the cropped images presented here are provided in the Supplementary Information.

Fig 4: E2 is verified to facilitate trained immunity in primary BMDMs from female and male mice. (A) In vitro trained immunity model for BMDMs. (B) Flow cytometry was used for testing the purity of BMDMs induced by in vitro culture. (C) The mRNA levels of TNFα and IL-6 in male/female BMDMs were detected by qPCR to determine the different intensity of trained immunity between genders. (D) The protein concentrations of TNFα and IL-6 from the supernatant from male/female BMDM cultures were detected by ELISA to determine the different intensity of trained immunity between genders. (E) E2 activated hallmarks of trained immunity, such as Akt, 4EBP1, and S6 by western blot. (F) E2 promoted M1 polarization in TI + LPS group from male and female mice. Meanwhile, E2 maintained the M2 polarization to inhibit the effect of TI (n ≥ 3/group). #p < 0.05, ##p < 0.01, and ###p < 0.001, paired Student's t-test comparing β-glucan + LPS group and LPS group. *p < 0.05, **p < 0.01, and ***p < 0.001, paired Student's t-test comparing with control group. ∧∧p < 0.01, paired Student's t-test comparing between β-glucan + LPS groups with or without E2.

Fig 5: Hypoxia and proteasomal inhibition independently cause mTORC1 suppression in MM.A A relative correlation between mRNA expression level of TSC2 and HIF1β in CD138 + bone marrow plasma cells from healthy subjects (n = 22) and newly diagnosed MM patients (n = 559). Axis units are arbitrary. Error bars represent S.E.M. B, C Shown are Kaplan–Meier plots for the overall survival (OS) and progression-free survival (PFS) of MM patients with differential expression of TSC2 (B) and HIF1β (C) from GMMG HD4 and MM5 trials. D Subcutaneous tumor growth of wt, NPRL2 KO, and TSC2 KO MM.1S cells into NCG mice (n = 4). Shown is the average relative tumor weight ± S.E.M. and the asterisk represents p < 0.05 of unpaired two-tailed student’s t-test between wt and KO. ns, not significant. E Cells were cultured in fresh media for 4 h in either normoxia or hypoxia. mTORC1 response was assessed by immunoblotting of its downstream phosphorylated and total effectors, S6K1, S6, and 4E-BP1. p97 was used to confirm equal protein loading in each lane. Shown are technical duplicates in each condition of a typical experiment of three repetitions. F, G Cells were cultured in fresh media for 16 h, then treated with IXZ [32 nM]. Following the treatment, cells were collected for immunoblotting in increasing time points as indicated. Shown are immunoblots for downstream effectors of mTORC1. Ubiquitin immunoblot was used to assess proteasomal inhibition. Shown are a typical immunoblots out of three independent experiments. nor, normoxia. hyp, hypoxia, IXZ, ixazomib.