Fig 2: Hematoxylin and eosin histochemical staining of mouse brain with and without AAV9-cTuberin vector injection following AAV1-Cre injection.Tsc2-floxed mouse pups were either not injected (naïve) or injected ICV in both ventricles (1 × 1012 vg/kg) with an AAV1-Cre vector at P0. At 21 days, some mice were injected IV with AAV9-cTuberin (9 × 1011 vg/kg) or noninjected. At 42 days, all mice were euthanized. (A) Naïve, noninjected brain (black arrowhead indicating the choroid plexus). (B and C) Tsc2-floxed mice with AAV1-Cre at P0 and no further injection showed (B) proliferation of ependymal/subependymal cells (asterisk) and (C) subependymal nodules. (D) Little-to-no subependymal overgrowth was detected in mice receiving both the P0 AAV1-Cre ICV injection and P21 IV AAV9-cTuberin injection. Representative images are shown. Magnification bar, 100 µm. CC, corpus callosum; LV, lateral ventricle.

Fig 3: TSC1 loss predisposes cells to genotoxic stress-induced cell deathA. Fluorescent microscopy of TSC1+/+ and TSC1-/- MEFs untreated or treated with Hydroxyurea (HU, 2 mM) and Adriamycin (Adr, 0.5 µg/ml), respectively for 20 h. Hoechst33342 (membrane permeable, live-cell nuclear stain) and Propidium iodide (membrane impermeable dead cell stain) mark live and dead cells. Scale bar = 20 µm. See also Supplementary Figure S1. B. Propidium iodide exclusion flow cytometry for cell death estimation. TSC1+/+ and TSC1-/- MEFs untreated or treated with Hydroxyurea (HU, 2 mM) and Adriamycin (Adr, 0.5 µg/ml) respectively for 20h. Wherever indicated, mTORC1 Inhibitors Rapamycin (Rapa, 20 nM) or Torin1 (10 nM) were spiked 2h prior to genotoxic treatments. Data-set are a mean of duplicate samples from three independent experiments. Error bars represent standard deviation (SD) of the three repeats. One-way non-parametric ANOVA (for group comparisons) and the non-parametric Mann-Whitney U test (for pair-wise comparison) were used for statistical analysis. *P<0.05, **p<0.01, ***p<0.001, n.s.- not significant. Pair-wise significance is as indicated (TSC1+/+ vs TSC1-/-). See also Supplementary Figure S5. C. Representative western blot of WT and TSC1-/- MEFs treated as in (B). D. siRNA-mediated acute knockdown of TSC2 in wild type MEFs followed by genotoxic treatment and immunoblot detection of the indicated proteins.

Fig 4: MBH FKBP51 regulates autophagy in an inversed U-shaped manner.FKBP51 deletion is depicted in green, and FKBP51 overexpression is depicted in blue. (A) Representative blots of autophagy and mTOR markers in FKBP51MBH-KO mice. (B) Quantification of FKBP51 deletion. (C) FKBP51 deletion reduced LKB1 and AMPK binding to WIPI4 as well as (D) AMPK phosphorylation at T172. (E) TSC2-WIPI3 binding was decreased in FKBP51MBH-KO animals. (F) Quantification of mTOR substrate pp70S6K (T389). (G) LC3B-II and (H) p62 levels in the MBH. (I) Representative blots of autophagy and mTOR marker in FKBP51MBH-OE mice. (J) Quantification of viral FKBP51 overexpression. (K) FKBP51 overexpression reduced LKB1 and AMPK binding to WIPI4. (L) Quantification of AMPK phosphorylation at T172. (M) TSC2-WIPI3 binding was decreased. (N) Quantification of pp70S6K phosphorylation at T389. (O) To assess autophagic flux FKBP51MBH-OE, animals were treated with chloroquine (50 mg/kg), and LC3B-II levels were analyzed 4 hours after treatment. (P) FKBP51 overexpression blocked autophagic flux and resulted in an accumulation of p62. (Q and R) Quantification of FKBP51, p62, and BECN1, while titrating AAV-HA-FKBP51 virus into mouse neuroblastoma cells. (S) MBH FKBP51 regulates autophagy and mTOR signaling in a dose-dependent manner. All data are shown as ±SEM. Data are shown as the relative protein expression compared to control; for (A) to (N), an unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Common AR/ARv567es binding sites function as androgen-responsive enhancers. (A) Gene track view of ChIP-seq data at the FASN locus. Common AR/ARv567es binding sites (ARBS) are indicated. Data are from cells treated with dihydrotestosterone (DHT) or vehicle (ethanol, ETH) as indicated. (B) Gene track view of ChIP-seq data at the TSC2 locus. A common AR/ARv567es binding site in TSC2 exon 37 is indicated. (C) Validation of androgen-mediated recruitment of AR to FASN and TSC2 ARBSs in R1-AD1 cells by ChIP-qPCR. (D) Validation of constitutive ARv567es binding to FASN and TSC2 ARBSs in R1-D567 cells by ChIP-qPCR. (E) FASN and TSC2 ARBSs were tested for enhancer response to the synthetic androgen mibolerone (MIB) by luciferase reporter assay in R1-AD1 cells.