Fig 2: HDAC inhibitors have anti-tumour effects on Tsc1i?EC tumour in vivo.a, b Cell death analysis of Tsc1i?EC tumour cells upon SAHA (5 µM), CI994 (25 µM), TMP269 (20 µM), Tubastatin A (Tub A, 20 µM) or DMSO treatment for 18 h. Representative results (a) and mean ± SD of MFI (b) were shown. c Western blot analysis of Cl. Casp3, p-rpS6, rpS6 and Ace-H3K9 in Tsc1i?EC tumour cells upon SAHA (5 µM), CI994, TMP269, Tub A or DMSO treatment at the indicated concentrations for 24 h. d Tsc1i?EC tumour growth upon SAHA (50 mg/kg), CI994 (100 mg/kg), Tub A (100 mg/kg) or vehicle treatment for 14 days. Tumour volumes are shown as mean ± SEM; n = 3–5 mice (6–10 tumours). e Tsc1i?EC tumour volumes at day 14. *P < 0.05, **p < 0.01; n.s., no significance. f, g Immunohistochemistry (IHC) analysis of Cl. Casp3, phospho-histone H3 at Ser10 (p-histone H3), Ki67 and LC3B (f). Scale bar, 100 µm, ×40 magnification. Graphs depict the percentage of positive staining areas in each group (g) shown as mean ± SD. *P < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: The phosphorylation of rpS6 is elevated in HL-60 macrophages and is sensitive to ERK1/2 signaling inhibition.(A and B) Western blots displaying the expression levels of rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and ß-Tubulin. The expression levels of these proteins were compared between HL-60 Mac, HL-60 PMN, A549 EC, KHYG-1 NK and H1299 EC cells, in panel A. Comparisons were also made between HL-60 cells treated with the solvent control, ERK1/2 signaling inhibitors (10 µM U0126 or 1 µM AZD6244), and mTOR inhibitor (100 nM Torin-1); in panel B. The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. In panel B, “rpS6+phos fold change” was calculated based on the mathematical expression shown. [Control] and [Inhibitor] are defined as the rpS6+phos/rpS6 ratios of the control and inhibitor (Torin-1, U0126 and AZD6244) treatment cells, respectively, Experimental replicates were repeated over different days and the data from two repeats are shown.

Fig 4: 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.

Fig 5: GSK3ß activity potentiates the phosphorylation of mTORC2 effectors in vivo under basal conditions. a The hippocampi from wildtype (n = 6) and GSK3ß S9A (n = 5) mice were isolated and lysed. Western blot was used to determine the levels of P-S235/S236-rpS6, P-S240/S244-rpS6 and rpS6. Tubulin is shown as a loading control. b WB analysis results. c Western blot was used to determine the levels of P-S473-Akt, Akt, P-Thr346-NDRG1 and NDRG1. Tubulin is shown as a loading control. d WB analysis results. Error bars indicate SEM. **p < 0.01, *p < 0.05 (unpaired t test)