Figure 1.
Confocal microscopy of DAPI-stained SH-SY5Y cells treated for up to 1 h with BEO.
SH-SY5Y cells were exposed to the indicated concentration of BEO (0.005–0.02%) for 30 or 60 minutes. Apoptotic nuclear morphological changes, such as chromatin condensation and marginalization (arrows), are evident in cells exposed to BEO 0.02%, whereas no nuclear alterations are present in cells exposed to lower concentrations (0.005–0.01%). Images are representative of three independent experiments.
Figure 2.
BEO activates autophagy in SH-SY5Y cells.
(A) Concentration-dependent induction of autophagy by BEO. Representative immunoblot showing LC3I to LC3II conversion and reduced p62 levels following exposure of SH-SY5Y to increasing concentrations of BEO. Cells were incubated with medium containing either vehicle (ethanol, 0.0045–0.027%) or BEO (0.005–0.03%) for 1 h. GAPDH was used as loading control. LC3II/LC3I optical density (OD) ratio for the reported blot is shown. Histogram shows the results of densitometric analysis of p62 levels normalized on GAPDH values and expressed as percentage of vehicle from three independent experiments (mean ± s.e.m.). *P<0.05,**P<0.01, P<0.001*** vs 0.005% BEO (ANOVA followed by Tukey-Kramer multiple comparisons test). (B, C) Effect of BafA1 pretreatment on LC3II accumulation in SH-SY5Y exposed to BEO. Cells were preincubated for 2 h with BafA1 (100 nM) and then treated with BEO 0.01% (B) or 0.02% (C) for 1 h. LC3II levels were detected by western blotting. Histogram in (C) shows the results of densitometric analysis of LC3II relative to internal control and reported as mean ± s.e.m of three independent experiments. **P<0.01, ***P<0.001 vs vehicle treated cells, ## P<0.01 vs BafA1 given alone (ANOVA followed by Tukey-Kramer multiple comparisons test). (D) Effect of autophagy inhibition by BafA1 on the percentage of viable, apoptotic and necrotic cells induced by BEO. Cells were incubated with BafA1 for 2 h and cell viability was assessed by cytofluorimetric analysis of FDA/PI stained cells 1 h after the addition of 0.02% BEO. Data are the mean ± s.e.m. of four independent experiments. *P<0.05, **P<0.01, ***P.001 vs vehicle treated cells (ANOVA followed by Tukey-Kramer multiple comparisons test).
Figure 3.
Accumulation of endogenous LC3 positive structures in cells exposed to increasing concentrations of BEO.
SH-SY5Y cells were exposed to vehicle (ethanol 0.018%) or BEO (0.005–0.02%) for 30 minutes and immunostained with anti-LC3 antibody; nuclei were counterstained with DAPI. Confocal analysis of endogenous LC3 intracellular distribution shows a dose-dependent increase of LC3 puncta (autophagosomes) in cells treated with BEO 0.01% and 0.02% as compared to vehicle or BEO 0.005% treated cells. The described patter of LC3 distribution was observed in three independent experiments.
Figure 4.
Fusion of autophagosomes and lysosomes in BEO-treated cells.
(A) SH-SY5Y were treated with BEO 0.02% for 15 min, fixed and double stained with LC3 and LAMP-1 antibodies. Nuclei were counterstained with DAPI. Representative images were taken with a confocal microscopy and higher magnification views of the boxed area from the merged image are shown. White arrows indicate areas of fusion (yellow signal) between LC3 positive autophagosomes (green) and LAMP1 positive endosomes and/or lysosomes (red) indicative of a functional autophagic maturation. (B) Immunoblot showing the early conversion of LC3I into LC3II and the reduction of p62 following 15 min incubation with BEO. GAPDH was used as loading control. Images are representative of three independent experiments. (V = vehicle, 0.018% ethanol).
Figure 5.
BEO activates autophagy in MCF7 cells.
Representative immunoblot showing LC3I to LC3II conversion and reduced p62 levels following exposure of MCF7 cells to increasing concentrations of BEO. Cells were incubated with medium containing either vehicle (ethanol, 0.009–0.027%) or BEO (0.01–0.03%) for 1 h. GAPDH was used as loading control. LC3II/GAPDH optical density (OD) ratio for the reported blot is shown. Histogram shows the results of densitometric analysis of p62 levels normalized on GAPDH values and expressed as percentage of vehicle from three independent experiments (mean ± s.e.m.). ***P<0.001 vs vehicle (Student's t test).
Figure 6.
Stimulatory effects of BEO on autophagy still occur in serum-starved cells and overcome beclin-1 knockdown.
(A) BEO enhances serum starvation-induced autophagy in SH-SY5Y cells. Neuroblastoma cells serum-starved for 24 h (FBS 0%) were exposed to BEO (0.01–0.02%) or vehicle (V, 0.018% ethanol) for 1 h. Protein extracts were analyzed by western blotting for LC3, Beclin-1 and p62 levels. GAPDH was used as internal control. Serum starvation induced autophagy in SH-SY5Y cells as demonstrated by increased LC3II/LC3I ratio and reduced p62 levels (V FBS 0% vs V FBS 10%); further lipidation of LC3 and p62 reduction was evident in BEO-treated starved cells (FBS 0%) as compared to cells maintained in normal culture conditions (FBS 10%). Images from normal and starved cells are from the same immunoblot that has been cut to remove irrelevant lanes. (B) BEO induces autophagy through a beclin-1 independent mechanism. SH-SY5Y cells were transiently transfected with specific beclin-1 siRNA or scramble non targeting sequence 48 h before treatment with BEO (0.02%; 1 h). Effective knockdown of the protein was confirmed by western blotting. Silencing of beclin-1 did not prevent LC3I/LC3II conversion and p62 reduction induced by BEO exposure (0.02%, 1 h). Immunoblot is representative of three independent experiments (V = vehicle, 0.018% ethanol).
Figure 7.
BEO-induced autophagy is mTOR independent.
(A, B) Treatment with BEO does not affect phosphorylation status of p70S6K and ULK. (A) Representative immunoblots showing the levels of phospho p70S6K (Thr 389; p-p70S6K) and phospho ULK (Ser 757; p-ULK) following treatment with BEO 0.01% and 0.02% for 5 and 15 min. Total protein extracts were analyzed by western blotting for phospho p70S6K and pULK and subsequently for total p70S6K and total ULK. GAPDH was used as internal control. (B) Histograms show the results of densitometric analysis of autoradiographic bands from three independent experiments (mean ± s.e.m.) (V = vehicle, 0.0018% ethanol). (C) BEO enhances autophagy induced by rapamycin. Neuroblastoma cells treated with rapamycin for 48h (3 µM) were exposed to BEO 0.02% or vehicle (Veh, 0.018% ethanol) for 1h. Protein extracts were analyzed by western blotting for phospho p70S6K, p70S6K, LC3 and p62. GAPDH was used as internal control. Treatment with rapamycin significantly reduced the phosphorylation status of p70S6K and induced autophagy in SH-SY5Y cells as demonstrated by increased LC3II and reduced p62 levels; no change of p70S6K phosphorylation accompanied autophagy induced by BEO; further enhancement of LC3 lipidation and p62 reduction was evident in rapamycin-treated cells exposed to BEO 0.02% for 1 h. Immunoblots are representative of three independent experiments.
Figure 8.
d-limonene is implicated in autophagy induced by BEO.
Concentration-dependent changes of LC3 lipidation and p62 expression following treatment with (A) d-limonene, (B) linalyl acetate and the corresponding vehicles for 30 min. Tubulin or GAPDH were used as loading control. Histograms show the results of densitometric analysis from three independent experiments (mean ± s.e.m.) *P<0.05 vs vehicle (Student's t test). (C, D) Representative immunoblots showing the time-dependent LC3I to LC3II conversion following exposure of SH-SY5Y cells to (C) d-limonene (500 µM) but not (D) linalyl acetate (250 µM). (E) Effects of BafA1 pretreatment on LC3 levels in SH-SY5Y exposed to d-limonene. Cells preincubated for 2 h with BafA1 (100 nM) were treated with d-limonene (750 µM) for 1 h. LC3II levels were detected by western blotting. Image from the autoradiographic film has been cut to remove irrelevant lanes. Histograms show the result of densitometric analysis from three independent experiments (mean ± s.e.m). **P<0.01, ***P<0.001 vs vehicle; ### P<0.001 (ANOVA followed by Tukey-Kramer multiple comparisons test). (F) Linalyl acetate enhances LC3 lipidation induced by d-limonene. Cells were incubated with vehicle or exposed to d-limonene (Limo, 500 µM) and linalyl acetate (LinAc, 250 µM), given individually or in combination. After 1 h incubation protein cell extracts were analyzed by western blot for LC3 levels. GAPDH was used as internal control. Histograms show the result of densitometric analysis from three independent experiments (mean ± s.e.m). # P<0.05, ## P<0.01 (ANOVA followed by Tukey-Kramer multiple comparisons test).