Fig 2: THC, but not nutrient deprivation, -induced autophagy relies on the stimulation of sphingolipid biosynthesis. (A) Upper panel: Effect of THC (4 µM, 18 h) and incubation with EBSS (18 h) on the number of U87MG cells stably transfected with control (shC) or ATG5-selective (shATG5) shRNAs as estimated by the MTT test (n = 4; mean ± s.d; **, P < 0.01 from THC-treated or EBSS-incubated U87 shC cells). Lower panel: Effect of THC (4 µM) and incubation with EBSS on the induction of autophagy (as determined by MAP1LC3B-II lipidation in the presence of E64d, 10 µM; and pepstatin A, 10 µg/ml [+inh]) of U87 cells stably transfected with control (U87 shC) or ATG5-selective (shATG5) shRNAs (n = 3, a representative experiment is shown). ATG5 mRNA levels (as determined by real-time quantitative PCR) were reduced by 85 ± 3% on U87shATG5 cells when compared with U87shC cells; (n = 4). Values in the bottom of the western blots correspond to the fold change in the MAP1LC3B-II to TUBA1A ratio relative to shC U87MG cells at the initial time point of the treatments. Nd, nondetectable. (B) Effect of THC (4 µM, 1 h, 3 h and 6 h) and incubation with EBSS (i.e., nutrient deprivation, 1, 3 and 6 h) on the induction of autophagy (as determined by MAP1LC3B-II lipidation in the presence of E64d, 10 µM; and pepstatin A, 10 µg/ml [+inh]) of U87MG cells (n = 3, a representative experiment is shown). (C) Effect of THC (4 µM; 3 h) on the mRNA levels (as determined by quantitative real-time PCR) of different enzymes involved in sphingolipid biosynthesis (CERS2; CERS5; CERS6 (ceramide synthase 2, 5 and 6), DEGS1/dihydroceramide desaturase (delta[4]-desaturase, sphingolipid 1) and SPTLC1 (serine palmitoyltransferase long chain base subunit 1) of U87MG cells (n = 5; *, P < 0.05; **, P < 0.01 from Veh-treated cells). (D) Effect of THC (4 µM), ISP-1 (5 µM) and incubation with EBSS on autophagy (18 h) (as determined by MAP1LC3B immunostaining). Note that incubation with ISP-1 prevents THC but not starvation-induced autophagy of U87MG cells. Values correspond to the percentage of cells with MAP1LC3B dots relative to the total cell number of cells ± s.d; n = 3. *, P < 0.05; **, P < 0.01 from Veh-treated cells and #, P < 0.05 from THC- and EBSS-treated cells. Bar: 20 µm.

Fig 3: Effect of Tenovin-1 on Cultured Tumor Cell Lines(A) Tenovin-1 structure.(B) MCF-7 cells (expressing wild-type p53) were treated with 10 µM tenovin-1 for the indicated times. p53 levels and p21 levels were detected using DO1 (Bartkova et al., 1993) and 118 (Fredersdorf et al., 1996) mouse monoclonal antibodies, respectively. An antibody against a-tubulin (Cat. No. T9026, Sigma) was used to monitor loading efficiency.(C) MCF-7 cells were treated with 10 µM tenovin-1 for the indicated times, and p53 and p21 mRNA levels were analyzed by Taqman-PCR as described (Saville et al., 2004). Error bars correspond to standard deviation values (n = 3).(D) Toxicity of tenovin-1 on cultured tumor cells. Tumor cell lines were treated with DMSO (control) or with 10 µM tenovin-1 for 48 hr. Cell death (necrosis and apoptosis) was measured by annexin-V/propidium iodide labeling and FACS. Values correspond to the average of two independent experiments (±SD). p53 status in each cell line is indicated (DN, coexpressing the dominant-negative form of p53).

Fig 4: Effect of vitamin D on the fibrogenic activation of myofibroblasts. a RT-qPCR result: expression of a representative profibrogenic cluster of genes. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 µg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Analysis was performed 72 h post-shock. Values are folds of induction over control cultures (treated with bleomycin vehicle: PBS); veh.: vitamin D vehicle. The results presented in the figures are means ± SEM. Significance of the analysis is indicated as ***, P < 0.001. b Detection of VIM (vimentin) and ACTA2 (a-SMA) in myofibroblasts cell extracts. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 µg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Cell extracts were obtained at 72 h post-shock. PBS is the bleomycin vehicle; veh.: vitamin D vehicle. TUBA1A (tubulin a) was used as loading control. KDa: kilodaltons. Bottom panel: densitometry analysis of bands; a.u.: arbitrary units. c Representative micrographs of indirect immunofluorescence detection of ACTA2 (a-SMA fibers) and PTK2 (focal adhesion kinase), two markers of the profibrogenic activation. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 µg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Cells were processed for immunofluorescence 72 h post-shock. d Quantification of the immunofluorescence result (% of activated fibroblasts showing clear expression of a-SMA fibers and PTK2)

Fig 5: SirT2-Related Effects of Tenovins in Mammalian Cells(A) H1299 cells were treated with 40 nM trichostatin A (TSA) (Cat. No. T8552, Sigma) to deplete nonsirtuin histone deacetylase activities. Where indicated, tenovin-1 was also added. After 16 hr, samples were analyzed using antibodies against K16 acetylated histone H4 (Cat. No. 07-329, Upstate) and total H4 (Cat. No. 07-108, Upstate).(B) H1299 cells were treated for 16 hr as indicated. Samples were analyzed with antibodies to K40 acetylated a-tubulin (Cat. No. T6793, Sigma) or total a-tubulin (Cat. No. T9026, Sigma).(C) H1299 cells were permanently transfected with a control vector (pcDNA3Neo) (lanes 1–6) or a vector for SirT2 overexpression (pcDNA3Neo-SirT2) (lanes 7–12) and treated for 16 hr with 40 nM trichostatin A in the absence or presence of 1, 3, 7, or 10 µM tenovin-6. Samples were analyzed using antibodies against K40-acetylated a-tubulin, total a -tubulin, and SirT2 (Cat. No. 2313, Cell Signaling). Note that pcDNA3-SirT2 encodes SirT2 isoform-1, which is slightly larger than isoform-2. Endogenous SirT2 isoform-1 could also be detected in lanes 1 through 6 upon longer exposure of the blots. The band below the ectopic SirT2 may correspond to SirT2 isoform-2, but this was not confirmed.