Figure 6.
Figure 6. IL-1α–induced atypical apoptosis with caspase-3 activation in fetal liver MKs. (A and B) Liver cells were obtained from fetal WT mice on embryonic day 13, cultured with TPO or IL-1α, and analyzed on day 7. (A) RT-PCR analysis of gene expression in the harvested cells. The values were normalized to a vehicle-treated control. n = 5 experiments. (B) Flow cytometric analysis of caspase-3 activation in Lin−CD41+CD42b+ MKs. (C) Fetal liver cells were cultured with TPO. siRNA-mediated knockdown was performed on day 4. On day 7, cells were washed, incubated with TPO, IL-1α, or IL-1β for an additional 1 d, and pAKT–pERK signaling in Lin−CD41+CD42b+ MKs was analyzed on day 8. The data shown are from a single representative experiment from among three repeats (B and C). (D) Western blotting of p53 and phospho-p53 in fetal liver cells cultured with TPO and IL-1α from day 0 to 7. MKs were enriched with discontinuous albumin density gradient centrifugation. (E) Fetal liver cells were differentiated using TPO for 7 d, and then stimulated with vehicle, TPO, IL-1α, or IL-1β for an additional 1 d, after which gene expression was analyzed using RT-PCR. (F) Flow cytometric analysis of T. orangehigh and T. orangelow platelets isolated from WT mice treated with TPO or IL-1α. NC denotes a negative control. The data shown are from a single representative experiment from among five repeats. (G–J) Immunofluorescence analysis of fetal liver MKs, which were cultured and differentiated from day 0 to 7 with TPO or IL-1α. Some cells were treated with Fas-ligand from day 6 to 7 after differentiation with TPO (FasL). Note that IL-1α–treated MKs were caspase-3–positive with release of von Willebrand factor–positive granules, but TUNEL staining was negative, which was different than typical FasL-induced apoptosis with blebbing. (K) Blood cell counts in WT mice treated with TPO, IL-1α, and/or Z-VAD (OMe)-FMK. (L) MK dynamics and platelet counts in WT, Casp3−/−, and Thpo−/− mice treated with vehicle or IL-1α. n = 3–8 mice. Bars, (red) 20 µm. *, P < 0.05 versus CTRL group.

IL-1α–induced atypical apoptosis with caspase-3 activation in fetal liver MKs. (A and B) Liver cells were obtained from fetal WT mice on embryonic day 13, cultured with TPO or IL-1α, and analyzed on day 7. (A) RT-PCR analysis of gene expression in the harvested cells. The values were normalized to a vehicle-treated control. n = 5 experiments. (B) Flow cytometric analysis of caspase-3 activation in Lin−CD41+CD42b+ MKs. (C) Fetal liver cells were cultured with TPO. siRNA-mediated knockdown was performed on day 4. On day 7, cells were washed, incubated with TPO, IL-1α, or IL-1β for an additional 1 d, and pAKT–pERK signaling in Lin−CD41+CD42b+ MKs was analyzed on day 8. The data shown are from a single representative experiment from among three repeats (B and C). (D) Western blotting of p53 and phospho-p53 in fetal liver cells cultured with TPO and IL-1α from day 0 to 7. MKs were enriched with discontinuous albumin density gradient centrifugation. (E) Fetal liver cells were differentiated using TPO for 7 d, and then stimulated with vehicle, TPO, IL-1α, or IL-1β for an additional 1 d, after which gene expression was analyzed using RT-PCR. (F) Flow cytometric analysis of T. orangehigh and T. orangelow platelets isolated from WT mice treated with TPO or IL-1α. NC denotes a negative control. The data shown are from a single representative experiment from among five repeats. (G–J) Immunofluorescence analysis of fetal liver MKs, which were cultured and differentiated from day 0 to 7 with TPO or IL-1α. Some cells were treated with Fas-ligand from day 6 to 7 after differentiation with TPO (FasL). Note that IL-1α–treated MKs were caspase-3–positive with release of von Willebrand factor–positive granules, but TUNEL staining was negative, which was different than typical FasL-induced apoptosis with blebbing. (K) Blood cell counts in WT mice treated with TPO, IL-1α, and/or Z-VAD (OMe)-FMK. (L) MK dynamics and platelet counts in WT, Casp3−/−, and Thpo−/− mice treated with vehicle or IL-1α. n = 3–8 mice. Bars, (red) 20 µm. *, P < 0.05 versus CTRL group.

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