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Kinetics of FLIP Down-Regulation in Ren and NCI-H28 Cells Following SAHA Treatment

This study explores the kinetics of FLIP (FLIPL and FLIPS) down-regulation in Ren and NCI-H28 cells treated with 5 μM SAHA over various time points (3, 6, 12, and 24 hours). A combination of qPCR and Western blot analyses was employed to measure changes in FLIP mRNA levels and protein expression, showing significant down-regulation correlating with SAHA treatment. Additionally, pre-treatment with caspase and proteasome inhibitors revealed that proteasome inhibition reduces FLIP down-regulation, while caspase inhibition does not affect the SAHA-induced response.

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Kinetics of FLIP Down-Regulation in Ren and NCI-H28 Cells Following SAHA Treatment

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  1. Fig. S2 A NCI-H28 Ren ONE58 B Ren ONE58 NCI-H28 Time (hrs) 3 6 12 24 SAHA - + - + - + - + 3 6 12 24 - + - + - + - + 3 6 12 24 - + -+ - + - + PARP FLIPL FLIPS p18-caspase 8 β-actin C 10mM Z-VAD 1mM MG132 - + - + - + 5mM SAHA FLIPL FLIPS β-actin

  2. Fig. S2 - The kinetics of FLIP down-regulation by SAHA. Ren, ONE58 and NCI-H28 cells were treated with 5μM SAHA for 3, 6, 12 and 24 h. (A) qPCR analysis of total FLIP (FLIPL and FLIPS) mRNA levels following SAHA treatment, normalised to GAPDH mRNA expression. (B) Western blot analysis showing FLIPL and FLIPS down-regulation, caspase 8 processing and PARP cleavage in each cell line. (C) Ren cells were pre-treated with 10 µM z-VAD-fmk (pan-caspase inhibitor) or 1 µM MG132 (proteasome inhibitor) for 1 h, followed by treatment with 5 μM SAHA for 12 h. Immunoblot analysis shows that proteasome inhibition attenuates SAHA-induced FLIP down-regulation, however caspase inhibition fails to prevent SAHA-induced FLIP down-regulation.

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