Fig 2: Lipid substrate accumulation mediates mTOR hyperactivity in GD iPSC NPCs. (A) Representative immunofluorescence images of control and GD2a NPCs co-labeled for p-RPS6 (green) and DAPI (blue). GD NPCs were either untreated or treated with 5 µM of the substrate reduction compound (SRC) GZ-161 for 72 h. (B) Quantitation of p-RPS6 fluorescence signal intensity in control and GD2a NPCs with and without GZ-161 treatment. Data from >100 cells per group, assayed in at least four different fields per experiment, in three experiments. Bar graph represents fold change in the mean p-RPS6 fluorescence intensity relative to control. (C) Representative western blot showing p-mTOR (left) and p-RPS6 (right) levels in control, GD2a and GD2a NPCs treated with the SRC. Also shown is ß-actin loading control. Bar graphs represent fold p-mTOR and p-RPS6 in GD NPCs (data from GD2a and GD2b combined) relative to control, n=3-5 per group. Data are mean±s.e.m.*P<0.05 and **P<0.005 (one-way ANOVA between indicated groups). Scale bar: 50 µm (magnification 20×).

Fig 3: Raloxifene delays resumption of protein translation post-hypoxia in correlation with increased eIF2a phosphorylation and decreased mTOR signaling.A and B Puromycin incorporation assay. U251 cells were treated with 40 µM raloxifene or DMSO vehicle control for 1 h prior to 2 h hypoxic A or non-hypoxic B incubation. At various times post-hypoxic or normoxic incubation cells were treated with 10 µg/mL puromycin for 10 min and puromycin incorporation into nascent proteins was detected by anti-puromycin blot. Blots were normalized to total lane protein, and represented as ratios with DMSO time 0 normalized to 1 (bottom quantification panels). Data is presented as the mean of triplicates ± SEM, unpaired t-test *p < 0.05; **p < 0.01. C and D U251 cells were treated as in A but without addition of puromycin. Cell lysates were probed for eIF2a, phospho-eIF2a, GADD34, rpS6, and phospho-rpS6 at various times post-hypoxic C or normoxic D incubation. All blots were normalized to total lane protein with DMSO time 0 set to 1 (bottom quantification panels). The level of eIF2a phosphorylation is presented as the ratio of p-eIF2a to total eIF2a (bottom quantification panels). The level of rpS6 phosphorylation is similarly presented. Data is presented as the mean of triplicates ± SEM, unpaired t-test, and false discovery rate of 1%. *p < 0.05; **p < 0.01.

Fig 4: Validation of individual primordial follicle oocyte RNA-seq results by qRT-PCR and immunochemistry. (A) qRT-PCR analysis of PI3K/PTEN/Akt pathway genes shows no significant change in the expression of Akt-1 (nvehicle = 5 and nchemo = 6; p = 0.93), rPS6 (nvehicle = 5 and nchemo = 6; p = 0.32) or Foxo3a (nvehicle = 5 and nchemo = 6; p = 0.12) in the cyclophosphamide-treated group (chemo) as compared to the vehicle. The ‘n’ indicates the number of single primordial follicles analyzed, data are presented as mean of the ??Ct ± S.E.M. (B) There is no change in the percentage of primordial follicles stained for (phospho)-Akt (7.29 ± 0.22, n = 8 vs. 11.59 ± 0.8, n = 6; p = 0.52), p-Foxo3a (12.03 ± 1.48, n = 9 vs. 18.85 ± 1.43, n = 8 ; p = 0.63), and p-rpS6 (6.06 ± 0.14, n = 11 vs.12.55 ± 0.94, n = 7; p = 0.4) after cyclophosphamide treatment. The ‘n’ indicates the number of xenografted ovarian cortex tissues, data are presented as mean ± S.E.M.

Fig 5: The S. pombe homologues of human FLCN (bhd1) and Rab7A (ypt71) function in the same genetic pathway.(a) Cells from the indicated genotypes were grown to log phase in minimal media (EMM) supplemented with amino acids at regular (225 mg l-1) concentration, and then transferred to either EMM supplemented with amino acids at low (45 mg l-1) or high (1,125 mg l-1) concentration. After 90 min, samples were taken and subjected to western blot analysis using antibodies against the phosphorylated form of Rps6 (pRps6) and S6K1 (pS6K1), which are direct substrates of TORC1 in the fission yeast and humans. Actin was used as a loading control. (b) Wild type (WT) and mutant strains were grown at 32 °C in liquid minimal media (EMM). Once in log phase, cells were spotted in a 10-fold concentration gradient on rich (YEA) or minimal (EMM) media supplemented with amino acids (aa) at regular (225 mg l-1) or low (45 mg l-1) concentration. Rapamycin (Rap, 200 ng ml-1) was added to EMM media supplemented with regular amino acids. The grey triangles represent the concentration gradient of cells spotted on each plate. Strain genotype is indicated on the left column. (c) For each indicated genotype, an equal number of cells of opposite mating types were crossed on EMM minus nitrogen plates. The mating efficiency was determined by calculating the percentage (%) of zygotes formed for each cross out of total number of cells. At least 400 cells were counted for each measurement.