Figure 3
Figure 3. Apoptosis induced by xTRAIL1 in Xenopus A6 cells. (A) Real-time PCR analysis of xDR-M1 and xDR-M2 mRNAs in A6 cells. The cDNAs were amplified by PCR using gene-specific primer pairs (see Table 1). EF1α was used for normalization. The quantitative RT-PCR data represent the mean (n = 3) and SD. (B) A6 cells were treated with FLAG-xTRAIL1 (500 ng/mL), which was cross-linked to an anti-FLAG antibody (1 μg/mL). After 24 hours, the cells were observed by phase contrast microscopy (top panel), or fixed, stained with Hoechst 33258, and observed by fluorescence microscopy (bottom panel). Scale bars, 20 μm. (C) A6 cells were treated with FLAG-xTRAIL1 alone or cross-linked with anti-FLAG. After 24 hours, cell viability was quantified using the cell counting kit-8 (Dojindo). Cell viability is shown as the percentage of the value obtained using xTRAIL untreated cells. The data represent the mean and SD from3 separate experiments; *P < .01 and **P < .005 compared with no treatment with xTRAIL1; #P < .01 compared with ‘non–cross-linked.‘ (D) A6 cells mixed with a morpholino antisense oligonucleotide for xDR-M1 (xDR-M1 MO; see Supplementary Methods) were cultured for 24 hours and then treated with cross-linked xTRAIL1 (100 ng/mL). After 24 hours, cell viability was quantified. The data represent the mean and SD from3 separate experiments; P < .05 compared with control. (E) A6 cells were treated with cross-linked xTRAIL1 (100 ng/mL) in the presence or absence of each caspase inhibitor (20μM), as indicated. After 24 hours, cell viability was measured using the cell counting kit-8. Cell viability is shown as the percentage of the value obtained using xTRAIL1 untreated cells. The data represent the mean and SD from 3 separate experiments; P < .01 compared with control in ‘cross-linked FLAG-xTRAIL1.‘ (F) A6 cells were cotransfected with 0.5 μg of pFLAG-xCaspase-3(DN)-Myc and 0.5 μg of pEF1-Myc-His (vector), pEF1 xDR-M-LBR-Myc-His, pcDNA3 FLAG-xFADD-DN, or pEF1 CrmA (top panel), or were mixed with control MO or xDR-M1 MO (bottom panel). After 30 hours, the cells were treated with cross-linked FLAG-xTRAIL1 for 1 hour. The cell lysates were prepared and procaspase-3 and processed caspase-3 (processed) were detected by Western blotting with the anti-FLAG antibody.

Apoptosis induced by xTRAIL1 in Xenopus A6 cells. (A) Real-time PCR analysis of xDR-M1 and xDR-M2 mRNAs in A6 cells. The cDNAs were amplified by PCR using gene-specific primer pairs (see Table 1). EF1α was used for normalization. The quantitative RT-PCR data represent the mean (n = 3) and SD. (B) A6 cells were treated with FLAG-xTRAIL1 (500 ng/mL), which was cross-linked to an anti-FLAG antibody (1 μg/mL). After 24 hours, the cells were observed by phase contrast microscopy (top panel), or fixed, stained with Hoechst 33258, and observed by fluorescence microscopy (bottom panel). Scale bars, 20 μm. (C) A6 cells were treated with FLAG-xTRAIL1 alone or cross-linked with anti-FLAG. After 24 hours, cell viability was quantified using the cell counting kit-8 (Dojindo). Cell viability is shown as the percentage of the value obtained using xTRAIL untreated cells. The data represent the mean and SD from3 separate experiments; *P < .01 and **P < .005 compared with no treatment with xTRAIL1; #P < .01 compared with ‘non–cross-linked.‘ (D) A6 cells mixed with a morpholino antisense oligonucleotide for xDR-M1 (xDR-M1 MO; see Supplementary Methods) were cultured for 24 hours and then treated with cross-linked xTRAIL1 (100 ng/mL). After 24 hours, cell viability was quantified. The data represent the mean and SD from3 separate experiments; P < .05 compared with control. (E) A6 cells were treated with cross-linked xTRAIL1 (100 ng/mL) in the presence or absence of each caspase inhibitor (20μM), as indicated. After 24 hours, cell viability was measured using the cell counting kit-8. Cell viability is shown as the percentage of the value obtained using xTRAIL1 untreated cells. The data represent the mean and SD from 3 separate experiments; P < .01 compared with control in ‘cross-linked FLAG-xTRAIL1.‘ (F) A6 cells were cotransfected with 0.5 μg of pFLAG-xCaspase-3(DN)-Myc and 0.5 μg of pEF1-Myc-His (vector), pEF1 xDR-M-LBR-Myc-His, pcDNA3 FLAG-xFADD-DN, or pEF1 CrmA (top panel), or were mixed with control MO or xDR-M1 MO (bottom panel). After 30 hours, the cells were treated with cross-linked FLAG-xTRAIL1 for 1 hour. The cell lysates were prepared and procaspase-3 and processed caspase-3 (processed) were detected by Western blotting with the anti-FLAG antibody.

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