PMC

Inhibition of AA(−)-mediated autophagy leads to escape from autophagy-associated cell death. A, HL-60 cells were cultured in AA(−) or AA(+) with or without 3-MA (1 and 3 mm) for 30 min. LC3 protein was detected by Western blot analysis. The results are representative of three independent experiments. B, after a 2-h treatment with 3-MA in AA(−) or AA(+), cell death was assessed by DAPI staining and at least 100 cells were counted. Values are the mean ± S.D. from three independent experiments. *, p < 0.005.

Effects of S1P on ceramide-induced autophagy. A, HL-60 cells were treated with 10 μm C₂-ceramide (C₂-Cer) for 1 h in AA(+) medium. Autophagosome formation was detected with LC-3 staining. Scale bar, 10 μm. B, cells were treated with the combination of C₂-Cer and S1P at various concentration for 30 min in AA(+) medium. After treatment with C₂-Cer and S1P at the indicated concentrations, autophagy was assessed by MDC incorporation under fluorescence microscopy and measured using MacSCOPE. Values are the mean ± S.D. from three independent experiments. *, p < 0.005; **, p < 0.05.

Effects of S1P on autophagy-associated cell death following ceramide treatment. A, HL-60 cells were incubated in AA(+) or AA(−) with or without 20 μm S1P for 2 h. B, cells were incubated with the indicated concentrations of S1P (0, 5, 10, and 20 μm) and 10 μm C₂-ceramide (C₂-Cer) for 24 h in AA(+) medium. C, CHO (white column) and CHO/S1P₃ (gray column) cells were incubated in AA(+) or AA(−) with or without 20 μm S1P for 12 h. D, cells were incubated with various combinations of S1P (0, 10, and 20 μm) and C₂-Cer (0, 25, and 50 μm) for 6 h. Cell death was assessed by DAPI staining. Values are the mean ± S.D. from three independent experiments. *, p < 0.005; **, p < 0.05.

S1P₃ receptor is essential for S1P-mediated counteraction of autophagy. A, expression of S1P receptors (S1P_1–5) was detected by RT-PCR (upper panel) and real-time PCR (under graph) in HL-60 cells. Total RNA was extracted from cells and converted to cDNA. The real-time PCR was performed for both the target gene and GAPDH. Expression was calculated after normalizing against GAPDH in each sample and is presented as relative mRNA expression. B, cells were incubated in either AA(+) or AA(−), either alone or supplemented with 20 μm S1P in the presence or absence of CAY10444 for 30 min. LC3 protein was detected by immunoblotting, and LC3-II/LC3-I levels were quantified with ImageJ 1.43. Values are the mean ± S.D. from three independent experiments. *, p < 0.005. **, p < 0.05.

Effects of S1P on AA(−)-induced autophagy. A, HL-60 cells were incubated in AA(−) medium with 20 μm S1P for 1 h. Autophagosome formation was detected by immunostaining of LC-3. Scale bar, 10 μm. B, cells were incubated in AA(+) medium or AA(−) medium with or without 20 μm S1P for 1 h. LC3 protein was detected by Western blot analysis. C, after treatment with AA(+) or AA(−) together with or without S1P for 1 h, the cells were further incubated with 50 μm MDC for 10 min and immediately analyzed by fluorescence microscopy. The density of MDC was assessed using MacSCOPE. Scale bar, 10 μm. D, cells were incubated with AA(−) medium for the indicated times in the absence or presence of 20 μm S1P, and then the lipids were extracted. Ceramide content was measured using the diacylglycerol kinase method. Values are the mean ± S.D. from three independent experiments. *, p < 0.005; ns, no significance.

Involvement of acid sphingomyelinase (SMase) in autophagy-mediated generation of ceramide. A, HL-60 cells were cultured in AA(−) or AA(+) for 1 h after pre-treatment with 100 μm fumonisin B1 (FB1) or 100 nm myriosin (Myr) for 1 h. Autophagy was measured by MDC incorporation. B–D, cells were cultured in AA(−) or AA(+) for the indicated times, and then acid SMase (B), neutral SMase (C), and SMS (D) activities were measured using C₆-NBD-sphingomyelin and C₆-NBD-ceramide as the substrates as described under “Experimental Procedures.” Values are the mean ± S.D. from three independent experiments. *, p < 0.005. E and F, HL-60 cells were transfected with 40 nm of two species of siRNAs for acid sphingomyelinase (SMPD1-a and SMPD1-b) and scrambled sequence siRNA using MultiFectam. Efficacy of siRNAs was checked by immunoblotting (E). Seventy-two hours after transfection, cells were incubated in AA(+) medium or AA(−) medium for 1 h. MDC incorporation was examined by fluorescence microscopy, and the density of MDC was calculated from at least 200 cells (F). Values are the mean ± S.D. from three independent experiments. Scale bar, 10 μm. *, p < 0.005.

S1P-S1P₃ receptor signaling accelerates mTOR pathway interference with ceramide-dependent autophagy. A, CHO and CHO/S1P₃ cells were cultured in AA(−) or serum-free medium (AA(+)) containing the indicated concentrations of S1P for 30 min. Each protein was detected by Western blot analysis. B and C, CHO and CHO/S1P₃ cells were cultured in AA(−) with or without 10 μm S1P for 30 min (B) or treated with 50 μm C₂-ceramide (C₂-Cer) in the presence or absence of 10 μm S1P for 30 min (C). The indicated proteins were detected by immunoblotting. D and E, CHO (D) and CHO/S1P₃ (E) cells were transfected with RFP-GFP-LC3 using the TransIT-LT1 (Mirus). After 36 h, cells were cultured in serum-free media for 3 h, and treated with each condition for 60 min. Images were obtained by fluorescent microscopy. Scale bars, 10 μm. F, CHO cell lines were cultured in AA(−) or serum-free medium (AA(+)) containing 50 μm C₂-Cer with or without 10 μm S1P for 30 min. Then, MDC incorporation was examined by fluorescence microscopy, and the density of MDC was calculated. Images were obtained by fluorescent microscopy, and cells having at least five dots were counted as autophagic cells. Values are the mean ± S.D. from three independent experiments. **, p < 0.05.

Involvement of CAPPs on mTOR dephosphorylation and ceramide-dependent autophagy. A, CHO/S1P₃ cells were treated with AA(+), AA(−), or 10 μm C₂-ceramide (C₂-Cer) for 30 or 60 min, and then cells were harvested. PP1/PP2A serine/threonine protein phosphatase activity was determined as described under “Experimental Procedures.” The specific activity expressed as arbitrary unit/min/0.3 μg of protein. B and C, CHO/S1P₃ cells were incubated with or without OA in serum-free media for 3 h. Cells were treated with C₂-Cer (B) or AA(−) (C) for 1 h. Phosphor-mTOR and mTOR was detected by Western blot analysis. D and E, RFP-GFP-LC3 was transfected in CHO/S1P₃ cells. After 36 h, cells were treated with C₂-Cer (D) or AA(−) (E) in the presence or absence of 100 nm OA. Images were obtained by fluorescent microscopy. Scale bars, 10 μm. F and G, cells were treated with 50 μm C₂-Cer (F) or AA(−) (C) with or without 100 nm OA for 12 h. Cell death was assessed by DAPI staining and at least 100 cells were counted. Values are the mean ± S.D. from three independent experiments. *, p < 0.005. **, p < 0.05.

Effect of S1P on the activation of mTOR pathway- and rapamycin-induced autophagy. A, HL-60 cells were incubated in AA(+) or AA(−) with or without 20 μm S1P for 30 min. Western blot analysis was performed using the anti-phospho-mTOR (Ser-2448) antibody or anti-mTOR antibody after immunoprecipitation using the anti-mTOR antibody. Phospho-mTOR/mTOR levels were quantified with ImageJ 1.43. Values are the mean ± S.D. from three independent experiments. **, p < 0.05. B, cells were treated with various concentrations of S1P for 30 min (upper panel) or with 20 μm S1P for the indicated times (lower panel). In vitro kinase activity in immunoprecipitated mTOR was measured. C and D, cells were incubated with AA(−) medium for 1 h in the presence or absence of 20 μm S1P with or without pretreatment with 50 nm rapamycin for 24 h. LC3 protein was determined by Western blot analysis (C), and autophagy was detected by MDC incorporation and measured using MacSCOPE (D). Scale bar, 10 μm. Values are the mean ± S.D. from three independent experiments. *, p < 0.005. E, cells were incubated in AA(−) medium for the indicated times in the presence of 20 μm S1P with or without 50 nm rapamycin pretreatment for 24 h. Western blot analysis was performed using the anti-phospho-p70 S6 kinase (P-p70 S6K), anti-p70 S6K, anti-phospho-4E-BP1, and anti-4E-BP1 antibodies. Results are representative of three independent experiments.

Autophagy and ceramide accumulation induced by amino acid deprivation (AA(−)) in human leukemia HL-60 cells. A, HL-60 cells were incubated in medium containing amino acids (AA(+)) or medium without amino acids (AA(−)). For ceramide treatment, cells were incubated with 10 μm C₂-ceramide (C₂-Cer) for 1 h in AA(+) medium. Morphological changes were detected by electron microscopy. Arrowheads indicate autophagosome formation. B, cells were treated with AA(+), AA(−), and C₂-Cer for 1 h. Upper panel, LC3 protein was detected by immunoblotting. Right panel, autophagosome formation was detected with immunocytochemistry using the anti-LC3 antibody. Scale bar, 10 μm. C, after treatment of cells with AA(+), AA(−), or C₂-Cer, cells were further incubated with 50 μm MDC for 10 min and immediately analyzed by fluorescence microscopy. The density of MDC was assessed by the program MacSCOPE. Scale bar, 10 μm. *, p < 0.005 versus AA(+). D, cells were treated with AA(−) medium for the indicated time periods. Then autophagy was measured by MDC incorporation. E, ceramide content was determined by the diacylglycerol kinase assay after lipid extraction by the Bligh and Dyer () method as described under “Experimental Procedures.” Values are the mean ± S.D. from three independent experiments. *, p < 0.005 versus 0 h; **, p < 0.05 versus 0 h.