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. 2014 Mar;85(3):408–419. doi: 10.1124/mol.113.090043

Fig. 6.

Fig. 6.

Sildenafil and chemotherapy-induced lethality is mediated through RIP-1 and increased autophagy. (A) (Bottom) T24 cells were transfected with a plasmid to express LC3-GFP. Twenty-four hours after transfection cells were treated with vehicle, sildenafil (SIL, 2 μM), mitomycin C (MITO, 100 nM), DOX (200 nM), Gemzar (50 nM) as indicated. Cells were examined 6 and 24 hours after treatment using a fluorescent microscope, and the mean number of LC3-GFP+ vesicles was determined in >40 cells (n = 3, ± S.E.M.). (Top) T24 cells were treated with vehicle, SIL (2 μM), DOX (200 nM) as indicated. Cells were isolated 6 and 24 hours after treatment, and immunoblotting was performed to examine the phosphorylation/expression of the indicated proteins (n = 3). (B) T24 cells were treated with vehicle, SIL (2 μM), MITO (100 nM), DOX (200 nM) as indicated. In parallel, cells were treated with vehicle, necrostatin (1.0 μM) or 3-methyl adenine (3MA, 10 mM). Cells were isolated after 6 hours, and viability was determined by trypan blue exclusion (n = 3, ± S.E.M.). *P < 0.05 less than corresponding value in vehicle control. (C–E) Bladder cancer cells (HT-1376; J82; T24) were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of RIP-1 or Beclin1 (siRIP-1, siBeclin1). Thirty-six hours after transfection, cells were treated with vehicle, SIL (2 μM), MITO (100 nM), or DOX (200 nM) as indicated. Cells were isolated after 12 hours, and viability was determined by trypan blue exclusion (n = 3, ± S.E.M.). *P < 0.05 less than corresponding value in siSCR control. (F) T24 cells were transfected to express LC3-GFP and with siSCR or siRNA molecules to knock down expression of RIP-1 (siRIP-1). Thirty-six hours after transfection cells were treated with vehicle, SIL (2 μM), MITO (100 nM), or DOX (200 nM) as indicated. Cells were examined 24 hours after treatment using a fluorescent microscope, and the mean number of LC3-GFP+ vesicles was determined in >40 cells (n = 3, ± S.E.M.). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.