Block solution

Cells (25,000 per coverslip) were seeded onto coverslips coated with Attachment Factor and incubated at 37°C and 5% CO₂ in EGM-2 until ∼80% confluence was achieved. The cells were then incubated for 4 hours with the indicated concentrations of compound 2. Staurosporine (SP; 1 µM) was used as a positive control. After the incubation, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature followed by three quick washes in Tris buffered saline pH 7.4 (TBS). The cells were permeabilized by incubating with 0.5% Triton X-100 for 10 minutes and then blocked in 10% block solution (DAKO, Glostrup, Denmark) in TBS plus 1% bovine serum albumin (BSA) for 1 hour. The cells were then incubated with cleaved caspase-3 (D175) antibody (1∶200 dilution) overnight at 4°C. Dylight 555 conjugated goat anti-rabbit secondary antibody (1∶400) was used to probe the cleaved caspase-3 antibody. The coverslips were mounted using Vectashield mounting medium containing DAPI (Vector Labs, Burlingame, CA, USA) for nuclear staining. The cells were imaged using an LSM 700 confocal microscope (Zeiss, Thornwood, NY, USA).

Cells (25,000 per coverslip) were seeded onto coverslips coated with Attachment Factor and incubated at 37°C and 5% CO₂ for 24 hours in EGM-2. The cells were starved in 0.1% serum-EBM-2 for 8 hours followed by 0.1% serum-EBM-2 medium for one hour in the presence of different concentrations of compound 2. The cells were induced with 10 ng/ml TNF-α for 20 minutes and fixed with 4% paraformaldehyde solution for 20 min at room temperature. Cells were quickly washed three times in TBS and permeabilized by incubating with 0.5% Triton X-100 for 10 minutes. The cells were blocked in 10% block solution (DAKO) in TBS plus 1% BSA followed by incubation with an antibody against NF-κB p65 (1∶50 dilution). Dylight 488-conjugated goat anti-mouse secondary antibody (1∶200 dilution) was used to probe the NF-κB p65 antibody. The coverslips were mounted using Vectashield mounting medium containing DAPI (Vector Labs) for nuclear staining. The cells were imaged using an LSM 700 confocal microscope (Zeiss).

Cells (25,000 per coverslip) were seeded onto coverslips coated with Attachment Factor and incubated at 37°C and 5% CO₂ for 24 hours in EGM-2. The cells were starved in 0.1% serum-EBM-2 for 8 hours followed by incubation in 0.1% serum-EBM-2 medium for an hour in the presence of different concentrations of compound 2. The cells were challenged with 10 ng/ml of TNF-α for 24 hours and fixed with 4% paraformaldehyde solution for 20 minutes at room temperature. The coverslips were quickly washed three times in TBS and blocked using 10% block solution (DAKO) prepared in 1× TBS-1% BSA buffer. The coverslips were incubated with the antibody against VCAM-1 (1∶100 dilution) for 16 hours at 4°C followed by three washes in TBS- 0.1% BSA buffer. Dylight 555-conjugated secondary antibody (1∶200) was used to probe for the VCAM-1 antibody. After three washes in TBS-0.1% BSA, the coverslips were mounted using Vectashield mounting medium containing DAPI nuclear stain. The cells were imaged using an LSM 700 confocal microscope. The image was analyzed for fluorescence signal intensity using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).