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. Author manuscript; available in PMC: 2009 Oct 1.

Published in final edited form as: Cancer Biol Ther. 2008 Oct 12;7(10):1648–1662. doi: 10.4161/cbt.7.10.6623

Panel a. HEPG2 cells were transfected to knock down expression of CD95 or FADD. Cells were treated 24h after transfection with vehicle (DMSO), sorafenib (3.0 μM), vorinostat (500 nM) or both sorafenib and vorinostat. Six hours after drug exposure, cells were isolated and subjected to SDS PAGE followed by immunoblotting to determine the expression of FADD, CD95, ATG5 and GAPDH, and in parallel the phosphorylation of PERK and eIF2α. Data are from a representative study (n = 3). Lower right: In parallel as a positive control, 6h after drug exposure, CD95 was immunoprecipitated and the formation of a DISC complex containing pro-caspase 8 demonstrated. Panel b. HEPG2 cells were plated on glass slides and transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. In parallel, cells were transfected with a construct to express LC3-GFP. Cells were treated 24h after transfection with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Twenty four hours after drug exposure, cells were visualized at 40X using an Axiovert 200 fluorescent microscope under fluorescent light on the Zeiss Axiovert 200 microscope using the FITC filter. The mean number of autophagic vesicles per cell from random fields of 40 cells were counted (± SEM, n = 3). Panel c. Left blotting section: HEPG2 cells were transfected to express dominant negative PERK or to knock down expression of FADD. Cells were treated 24h after transfection with vehicle (DMSO), sorafenib (3.0 μM), vorinostat (500 nM) or both sorafenib and vorinostat. Six hours after drug exposure, cells were isolated CD95 was immunoprecipitated and the formation of a DISC complex containing pro-caspase 8 demonstrated. The association of other proteins: BiP/Grp78 and ATG5 was determined. The cytosolic fraction was analyzed for GAPDH expression (n = 2). Right blotting section: HEPG2 cells were transfected with either MYC-tagged wild type PERK or MYC-tagged dominant negative PERK. Cells were treated 24h after transfection with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Six hours after drug exposure, cells were isolated, CD95 was immunoprecipitated and the association of PERK proteins with CD95 determined by immunoblotting the MYC tag using an anti-Myc 9E10 antibody (a representative study, n = 2 is shown). Panel d. HEPG2 cells were plated on glass slides and infected with a recombinant adenovirus to express c-FLIP-s. In parallel, cells were transfected with a construct to express LC3-GFP. Cells were treated 24h after transfection with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Twenty four hours after drug exposure, cells were visualized at 40X using an Axiovert 200 fluorescent microscope under fluorescent light on the Zeiss Axiovert 200 microscope using the FITC filter. The mean number of autophagic vesicles per cell from random fields of 40 cells were counted (± SEM, n = 2).

Panel a. HEPG2 cells were transfected to knock down expression of CD95 or FADD. Cells were treated 24h after transfection with vehicle (DMSO), sorafenib (3.0 μM), vorinostat (500 nM) or both sorafenib and vorinostat. Six hours after drug exposure, cells were isolated and subjected to SDS PAGE followed by immunoblotting to determine the expression of FADD, CD95, ATG5 and GAPDH, and in parallel the phosphorylation of PERK and eIF2α. Data are from a representative study (n = 3). Lower right: In parallel as a positive control, 6h after drug exposure, CD95 was immunoprecipitated and the formation of a DISC complex containing pro-caspase 8 demonstrated. Panel b. HEPG2 cells were plated on glass slides and transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. In parallel, cells were transfected with a construct to express LC3-GFP. Cells were treated 24h after transfection with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Twenty four hours after drug exposure, cells were visualized at 40X using an Axiovert 200 fluorescent microscope under fluorescent light on the Zeiss Axiovert 200 microscope using the FITC filter. The mean number of autophagic vesicles per cell from random fields of 40 cells were counted (± SEM, n = 3). Panel c. Left blotting section: HEPG2 cells were transfected to express dominant negative PERK or to knock down expression of FADD. Cells were treated 24h after transfection with vehicle (DMSO), sorafenib (3.0 μM), vorinostat (500 nM) or both sorafenib and vorinostat. Six hours after drug exposure, cells were isolated CD95 was immunoprecipitated and the formation of a DISC complex containing pro-caspase 8 demonstrated. The association of other proteins: BiP/Grp78 and ATG5 was determined. The cytosolic fraction was analyzed for GAPDH expression (n = 2). Right blotting section: HEPG2 cells were transfected with either MYC-tagged wild type PERK or MYC-tagged dominant negative PERK. Cells were treated 24h after transfection with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Six hours after drug exposure, cells were isolated, CD95 was immunoprecipitated and the association of PERK proteins with CD95 determined by immunoblotting the MYC tag using an anti-Myc 9E10 antibody (a representative study, n = 2 is shown). Panel d. HEPG2 cells were plated on glass slides and infected with a recombinant adenovirus to express c-FLIP-s. In parallel, cells were transfected with a construct to express LC3-GFP. Cells were treated 24h after transfection with vehicle (DMSO) or with sorafenib (3.0 μM) and vorinostat (500 nM). Twenty four hours after drug exposure, cells were visualized at 40X using an Axiovert 200 fluorescent microscope under fluorescent light on the Zeiss Axiovert 200 microscope using the FITC filter. The mean number of autophagic vesicles per cell from random fields of 40 cells were counted (± SEM, n = 2).