Fig 1: DAPI staining and Annexin-V/PI assay were used to detect apoptosis. (A) Untransfected, Barkor-siRNA- and control-siRNA-transfected Saos-2 cells were treated with cisplatin (2 µM) for 48 h and then fixed and stained with DAPI. Morphological changes were visualized by laser confocal fluorescence microscopy. Nuclear fragmentation and apoptotic bodies were clearly apparent in Barkor-siRNA-transfected cells, while no nuclear fragmentation or apoptotic bodies were evident in the control-siRNA-transfected or untransfected Saos-2 cells treated with cisplatin (2 µM). (B) Annexin-V/PI apoptosis assay of control, Barkor-siRNA- and control-siRNA-transfected cells treated with 0 and 2 µM of cisplatin for 24 and 48 h, respectively. The relative percentage of live (lower-left quadrant), early apoptotic (lower-right quadrant), late apoptotic (upper-right quadrant) and necrotic (upper-left quadrant) cells is shown. DAPI, 4',6-diamidino-2-phenylindole; Barkor/ATG14, Beclin1-associated autophagy-related key regulator.
Fig 2: Knockdown of Barkor sensitizes Saos-2 cells to cisplatin. (A) Cytotoxicity of cisplatin in Saos-2 cells, Saos-2 Barkor-silenced cells and control-siRNA-transfected Saos-2 cells. The cells were treated with cisplatin at the indicated concentrations for 48 h. Percent survival was determined using the CCK-8 assay. (B) IC50 values for cisplatin in the different cells. The IC50 values were defined as the concentration causing 50% growth inhibition in treated cells, compared to that in control cells and were determined after 48 h of exposure to cisplatin. Bars show the average intensity of each band ± standard deviation (n=6) (*P<0.05). Barkor/ATG14, Beclin1-associated autophagy-related key regulator.
Fig 3: The effect of Barkor siRNA transfection on Saos-2 cell growth. Saos-2 cells were transfected with Barkor- or control-siRNA for 48 h, after which the Barkor expression level was analyzed by (A) western blotting and (B) qRT-PCR. Bars show the average intensity (relative quantity) of each band ± standard deviation (n=6) (*P<0.05). (C) The CCK-8 assay was used to measure the growth rates in Barkor-siRNA- and control-siRNA-transfected Saos-2 cells after transfection for 48 h. The proliferation rates were similar in the Barkor-siRNA- and control-siRNA-transfected and control cells. Bars show the average intensity of each band ± standard deviation (n=6) (P>0.05 vs. control). Barkor/ATG14, Beclin1-associated autophagy-related key regulator; qRT-PCR, quantitative real-time polymerase chain reaction.
Fig 4: Cisplatin enhanced the expression of Barkor mRNA and protein levels in Saos-2 cells. Cells were treated with cisplatin (10 µM) for 12, 24 and 48 h and prepared for (A) western blotting and (B) qRT-PCR. Quantification of band intensity in (B) normalized to that of mRNA (relative quantity) is shown (n=6). Bars show the average intensity (relative quantitiy) of each band ± standard deviation (*P<0.05). Barkor/ATG14, Beclin1-associated autophagy-related key regulator; qRT-PCR, quantitative real-time polymerase chain reaction.
Fig 5: Determination of caspase-12 by western blotting following treatment of Saos-2 cells with cisplatin. Saos-2 cells were treated with cisplatin (10 µM) and the caspase-12 levels were examined over a period of 12–48 h. Cisplatin treatment induced a significant increase in caspase-12 level in Barkor-siRNA-transfected Saos-2 cells. Barkor/ATG14, Beclin1-associated autophagy-related key regulator.
Supplier Page from Cell Signaling Technology for SignalSilence ® Atg14 siRNA I