Fig. 4

Effects of laylin-KD on apoptosis-related proteins and DNA fragmentation. A172 cells were transfected with control siRNA (siC) and 2 kinds of layilin siRNA (siL1 and siL2). After 24 h, the culture medium was replaced with RPMI containing 50 µM STS. (A) Three hours later, cells were subjected to Annexin V and PI assay. The intensity of cell membrane-bound Annexin V and DNA-bound PI were expressed in RLU (relative luminescence units) and RFU (relative fluorescence units), respectively. (B) Four hours later, the protein samples extracted from the whole cells were subjected to western blotting. (C and D) Intensity of detected bands was measured by densitometry. The measured intensity of the layilin, cleaved CASP-3, cleaved CASP-6, cleaved CASP-7, and cleaved PARP1 bands was normalized using that of β-actin bands. The average of the normalized intensity of the layilin, cleaved CASP-3, cleaved CASP-6, cleaved CASP-7, and cleaved PARP1 bands in the ‘siC, STS (-)’ samples was defined as 1.0. Mean values with SD are presented. * p < 0.05, ** p < 0.01. (E) A172 cells, transfected with siL-1, siL-2 or siC (1.0 × 10⁶ cells) in RPMI containing 10% FBS, were plated on Φ100 mm dishes. The cells were cultured with RPMI containing 400 µM STS for 4 h. Then, fragmented DNA isolated from the cells was subjected to agarose gel electrophoresis (left). DNA in the gels was visualized under ultraviolet light after staining with ethidium bromide and photographed. The intensity of the detected DNA bands was measured by densitometry. The average of the DNA fragmentation levels in the ‘siC, STS (-)’ samples was defined as 1.0 (n = 3 in each condition). Mean values with SD are shown. * p < 0.05, ** p < 0.01 (siC vs. siL1, closed), † p < 0.05, †† p < 0.01 (siC vs. siL2, closed)