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> However none of the previous studies used Puma knock-out or knock-down systems to prove the involvement of Puma in virus-induced apoptosis
post Aug 22, 2017, 08:00 AM
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Additionally, really lately, Puma protein amounts were demonstrated to be elevated right after an infection with influenza A virus [forty five]. Nonetheless none of the previous scientific studies utilized Puma knock-out or knock-down systems to confirm the involvement of Puma in virus-induced apoptosis. Listed here we show that a few diverse cell lines, mouse embryo fibroblasts, issue-dependent monocytes as properly as human colon carcinoma cells all demand Puma to succumb to HSV-1- and SFV-induced mobile loss of life. This is not only shown with recognized Puma-/- cells, which might have obtained mutations in other genes major to apoptosis resistance, but also in freshly well prepared cells in which Puma was downregulated by shRNA. Moreover, the simple fact that Puma-/- cells are as resistant to virus-induced mobile demise as Fig 9. SFV-induced caspase-3 activation/processing and apoptosis need Puma and to a lesser extent Bmf. (A) Annexin-V/PI FACS and (B/C) anticaspase-three (pro-caspase-3 and cleaved caspase-3) western blot analyses of the different SV40 TAg-reworked or 3T9-immortalized WT and knock-out MEF mobile traces/extracts infected with ten moi of SFV for , fourteen, 24, 36 or forty eight h (hpi). Anti-actin as loading and anti-SFV-C as infection controls in (cool.gif. The anti-cleaved caspase-three bands in (cool.gif are quantified by densitometric scanning, and the knowledge are depicted in ©. Information in (A) and © are the implies of at minimum 3 unbiased experiments using two clones of WT and each knock-out cell line SEM. The p values are the following: (A) SV40 TAg Bid-/- vs . SV40 TAg WT: not important SV40 TAg Bmf-/- vs . SV40 TAg WT: p = .05 for fourteen and 24 h, not important for 36 h 3T9 Puma-/- vs . 3T9 WT and 3T9 Bax/Bak-/versus 3T9 WT: p < 0.001 for 14, 24 and 36 h, n = 4. © 3T9 Puma-/- and 3T9 Bax/Bak-/- versus 3T9 WT: p < 0.001 for 14 and 24 h SV40 TAg Bmf-/- versus WT: p = 0.01 for 24 h, not significant for 48 h, n = 6.Bax/Bak-/- cells indicates that Puma is the major BH3-only protein mediating Bax/Bak activation in response to HSV-1 and SFV infections. The only other BH3-only protein, which seems to partially contribute to this process is Bmf since Bmf-/- cells displayed a slight protection to apoptosis by both viruses during early phases of infection (24 h). Munger and Roizman previously suggested the BH3-only protein Bad as a mediator of HSV-1-induced apoptosis due to the fact that the US3 protein kinase of HSV-1 could phosphorylate and inactivate Bad and protect cells from Bad-induced cell death [15]. However, Bad-/- cells were not studied in their report and as shown here Bad-/- and WT MEFs died in a similar way after HSV-1 infection. Another controversial issue has been to what extent death receptor signalling contributes to HSV-1- and SFV-induced apoptosis. While in one case inhibition of FasL by soluble Fas did not prevent apoptosis caused by HSV [46], another report found that HSV-induced apoptosis Fig 10. Puma knock-down in MEFs and its knock-out in FDMs also markedly diminish SFV-induced caspase-3 activation and apoptosis. (A) Caspase-3/-7 (DEVDase) activity assay of total extracts of puromycin selected, mixed populations of SV40 TAg-transformed and 3T9-immortalized MEFs infected with lentiviruses carrying either a scrambled shRNA (sh-Ctrl) or a shRNA of mouse Puma (sh-Puma), infected with SFV for 0, 6, 14 or 24 h (hpi). As a control the data of 3T9 Puma-/- MEFs are shown.

In addition, the caspase activities are compared to those from cells taken care of with ten ng/ml FasL for fourteen h. Information are the means of at least 3 unbiased experiments SEM. The p values are the following: 3T9 sh-Puma compared to 3T9 sh-Ctrl: p = .008 for six h, p < 0.001 for 14 and 24 h SV40 TAg sh-Puma versus SV40 TAg sh-Ctrl and 3T9 Puma-/- versus 3T9 WT: p < 0.001 for 6, 14 and 24 h, n = 4. (cool.gif Annexin-V/ PI FACS analysis of WT, Puma-/- and Bax/Bax-/- FDM cells infected with 10 moi of SFV for 0, 14, 24 or 36 h (hpi). Data are the means of at least three independent experiments using three different clones of WT, Puma-/- and Bax/Bak-/- cells SEM. The p values are the following: Puma-/- versus WT: p = 0.03 for 14 h, p < 0.001 for 24 and 36 h. Bax/Bak-/- versus WT: p = 0.01 for 14 h, p < 0.001 for 24 and 36 h, n = 4. was suppressed by antibodies directed towards Fas or FasL [47,48]. Moreover, gD and gJ of HSV-1 were shown to protect against FasL-induced apoptosis [17,18] and dendritic cells seem to die after HSV-1 infection due to the downregulation of the caspase-8 inhibitor c-FLIP [28]. However, all these findings provided only indirect evidence for or against a role of FasL/Fas signalling in HSV-1-induced apoptosis and the other death receptor signalling systems have not been studied. Here we used neutralizing antibodies or recombinant Fc proteins to clearly show that neither FasL, TNF, TRAIL nor their receptors were required for the death of HSV-1-infected cells. We previously published that this was also not the case for SFV [32]. Finally, we used the RIP1 inhibitor necrostatin-1 and shRNA-mediated downregulation of RIP3 to exclude Fig 11. SFV enhances Puma mRNA and protein levels in MEFs and FDM cells, but mRNA increase is late and Bax/Bak-dependent. (A) Quantitative/ real time reverse transcriptase PCR (qRT-PCR) of Puma mRNA isolated from SV40 TAg WT and Bax/Bak-/- MEFs infected with 10 moi of SFV for 0, 1, 2, 4, 6, 8, 10, 14, 18 or 24 h (hpi). The mRNA values were normalized to the ribosomal housekeeping 18S gene and depicted as 2-Ct relative to mock cells (see Materials and Methods for details). Data are the means of at least three independent experiments SEM. The p values are the following: SFV-treated WT versus untreated: p = 0.005 for 6 h, p = 0.01 for 8 h, p = 0.008 for 10 h. SFV-treated Bax/Bak-/- versus untreated: not significant, n = 5. (cool.gif Anti-Puma, anticaspase-3 (pro-caspase-3 and cleaved caspase-3) and anti-PARP western blot analysis of total cell extracts of SV40 TAg WT MEFs infected with SFV for 0, 2, 6, 8, 10 or 18 h. Anti-actin as loading control the participation of necroptosis in HSV-1-induced cell death. We can therefore not confirm a recent report by Wang et al. [49] that HSV-1 triggers necrosis/necroptosis via RIP3, at least not in our cellular systems. Our study therefore shows that Puma is the major sentinel/sensor of incoming viruses to convey an apoptotic signal to MOMP. But how does Puma sense viral infection and/or what is the viral component, if any, which engages Puma One possibility is via the transcriptional regulation of Puma. We previously reported that the gD envelope protein of HSV-1 induces the transcription factor NFB [18,19,23].
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