Caspase 6

Paraffin embedded tissue sections were used for staining. For deparaffinization, slides were heated for 45 min at 65 °C prior to rehydration step. In the rehydration step, slides were treated three times with 100% xylene and three times with 100% ethanol. For antigen retrieval, rehydrated slides were incubated with citrate buffer (pH 6.0) in a pressure cooker for 1 h. After cooling down to 27 °C, tissues were permeabilized in 0.5% Triton X-100 for 10 min. This step was followed by a blocking step, where slides were incubated in BSA for 45 min, then washed in 0.5% BSA diluted in PBS (PBB), three times. Then, slides were incubated overnight at 4 °C with primary antibodies for PCNA, E-cadherin, N-cadherin, connexin 37, connexin 43, cytochrome c, caspase 6 (Santa Cruz Biotechnology, Dallas, TX) or ceramide (Enzo Life Sciences, Farmingdale, NY) diluted 1:50. The following day, slides were washed three times with PBB and incubated for 60 min at room temperature with Alexa fluor 594-conjugated or Alexa fluor 488-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA) diluted 1:200. Slides were then washed three times in PBS, incubated with 4′, 6-diamidino-2-phenylindole (DAPI; 1:1000) (Life Technologies, Carlsbad, CA), and washed in PBS. Slides were stored at 4 °C until imaging.

Polyclonal antibodies specific to extracellular signal regulated kinase (ERK), phospho-ERK, p38MAPK, phospho-p38MAPK, c-Jun N-terminal kinase (JNK), phospho-JNK, AKT, and phospho-AKT were obtained from Cell Signaling (Danvers, MA, USA). U0126, SP600125, SB203580, and LY294002 were obtained from Calbiochem (San Diego, CA, USA). Polyclonal antibodies against cyclin D1, cyclin E, CDK2, CDK4, p53, p21WAF1, p27KIP1, and GAPDH were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Polyclonal antibodies specific to FAS, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), X-linked inhibitor of apoptosis protein (XIAP), poly(ADP-ribose) polymerase-1 (PARP-1), caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling (Danvers, MA, USA). Polyclonal antibodies to phospho-endothelial nitric oxide synthase (eNOS) (S1177) and eNOS were obtained from Cell Signaling (Danvers, MA, USA). Human recombinant VEGF was purchased from Research and Diagnostic Systems, Inc. (Minneapolis, MN, USA).