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. 2010 Aug 27;18(2):350–361. doi: 10.1038/cdd.2010.104

Figure 7.

Figure 7

SPP1 depletion triggers autophagy and activates Akt by intracellular S1P independently of its receptors. (a) MCF7 cells transfected with pEGFP-LC3 were treated with 100 nM S1P or dihydroS1P (DHS1P) for 24 h, and autophagy was quantified by confocal microscopy. Data are means±S.D. of three independent experiments. (b) Cells were treated with 100 nM S1P or DHS1P for 10 min. (c) Cells were pretreated without or with 10 μM VPC23019 or 1 μM JTE-013 for 30 min, and then stimulated with 100 nM S1P for 10 min. (b, c) Equal amounts of lysates were analyzed by immunoblotting with anti-phospho ERK1/2. Blots were re-probed for ERK to insure equal loading and transfer. (d) Cells transfected with siControl or siSPP1 were transfected 24 h later with EGFP-LC3, cultured in the absence or presence of 10 μM VPC23019 or 1 μM JTE-013, and autophagy quantified by confocal microscopy. Values reported are means±S.D. of three independent experiments. (e) MCF7 cells transfected with siControl or siSPP1 or naïve MCF7 cells were treated with vehicle, S1P (100 nM), or EGF (10 ng/ml) for 10 min. Proteins were immunoblotted with anti-phospho-Akt (Ser473) and re-probed with anti-Akt antibody. (f) MCF7 cells were treated with 10 μM S1P or dihydroS1P (DHS1P) for 2 h and sphingolipids analyzed by LC-ESI-MS/MS. (g) MCF7 cells transfected with pEGFP-LC3 were treated with 10 μM S1P or DHS1P for 24 h and autophagy quantified by confocal microscopy. (h) Equal amounts of lysates from MCF7 cells treated without or with 10 μM S1P were immunoblotted with anti-LC3 or anti-p-eIF2α antibodies. Blots were re-probed for tubulin to insure equal loading and transfer. (i) MCF7 cells transfected with siControl or siSPP1 were transfected with vector or dnPERK. (j) MCF7 cells transfected with siControl or siSPP1 were treated without or with LY294004 (5 μM). Equal amounts of lysates were immunoblotted with anti-phospho-Akt (Ser473) and re-probed with anti-Akt antibody. (k) Wild-type (PERK+/+) and PERK null (PERK−/−) MEFs were transfected with siControl or siSPP1. Equal amounts of lysates were immunoblotted with the indicated antibodies. *P<0.01 compared with vehicle