PMC:5118436 / 26644-29688 JSONTXT

{"project":"TEST0","denotations":[{"id":"27920775-123-129-3789049","span":{"begin":123,"end":125},"obj":"[\"25952668\"]"},{"id":"27920775-113-119-3789050","span":{"begin":241,"end":243},"obj":"[\"21865393\"]"},{"id":"27920775-137-143-3789051","span":{"begin":383,"end":385},"obj":"[\"26876615\"]"},{"id":"27920775-13-19-3789052","span":{"begin":707,"end":709},"obj":"[\"26876615\"]"},{"id":"27920775-194-200-3789053","span":{"begin":1383,"end":1385},"obj":"[\"17437993\"]"},{"id":"27920775-103-109-3789054","span":{"begin":2067,"end":2069},"obj":"[\"19509260\"]"}],"text":"In many cancer cell lines, the expression of RIPK3 is lost due to genomic methylation near its transcriptional start site (42). Recently, it was reported that the methyltransferase EZH2 was overexpressed in HPV16 E6- and E7-transformed KCs (43). Since EZH2 is a core component of polycomb-repressive complex 2 (PRC2) and plays a role in promoter-targeted transcriptional repression (44), we hypothesized that EZH2 may also be involved in repressing the expression of RIPK3. Western blot analysis and RT-qPCR of primary KCs and hrHPV-infected KCs revealed that both gene and protein expression of EZH2 was higher at the basal level and after IFNγ/TNFα stimulation in hrHPV + KCs (Figures 4A,B). As expected (44), hrHPV-infected KCs display a higher methylation of H3K27 at the basal level (Figure S5A in Supplementary Material). Knockdown of the polycistronic mRNA of HPV16 in hrHPV16-positive KCs resulted in lower EZH2 protein levels indicating that EZH2 overexpression was induced by hrHPV in KCs (Figure 4C). This effect was even more pronounced after IFNγ/TNFα stimulation fitting with the observation that also the expression of the viral genes was further downregulated (Figure 2E). The use of 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase, is known to effectively deplete cellular levels of the PRC2 components, including EZH2 (45). Indeed, treatment of the hrHPV + KCs with DZNep resulted in a dose-dependent decrease in EZH2 protein levels (Figure 4D), decreased of H3K27 methylation (Figure S5B in Supplementary Material), and the concomitant increase in RIPK3 at the protein and gene expression level (Figures 4D,E). Treatment of hrHPV + KCs with the catalytic EZH2 inhibitor GSK503 did not have a clear effect on the expression of RIPK3 (Figure S5C in Supplementary Material), suggesting that the hrHPV-induced overexpression of EZH2 is indirectly responsible for suppressing RIPK3-mediated necroptosis. It should be noted that DZNep has been reported to function as a global histone methylation inhibitor (46). We, therefore, analyzed the expression of other methyltransferases and found that, in addition to EZH2, eight other methyltransferases were expressed at a significantly higher level in hrHPV + KCs (Figures S5D,E in Supplementary Material). Potentially, these methyltransferase may also play a role in downregulating the expression of RIPK3. To test if methyltransferases where involved in suppressing RIPK3-mediated necroptosis, KCs and hrHPV + KCs again were stimulated with IFNγ/TNFα, BV6, and zVAD-fmk, but, now, either in the absence or presence of DZNep. Cell death was determined both by flow cytometry in order to analyze larger numbers of cells (Figure 4F) and by immunofluorescence in cell cultures (Figure S6 in Supplementary Material). Clearly, the presence of DZNep increased the BV6/zVAD-fmk/IFNγ/TNFα-induced percentage of dead cells. Cells died by necroptosis, since cell death could be blocked by Nec-1s (Figures 4F,G; Figure S6 in Supplementary Material)."}

{"project":"2_test","denotations":[{"id":"27920775-25952668-35314165","span":{"begin":123,"end":125},"obj":"25952668"},{"id":"27920775-21865393-35314166","span":{"begin":241,"end":243},"obj":"21865393"},{"id":"27920775-26876615-35314167","span":{"begin":383,"end":385},"obj":"26876615"},{"id":"27920775-26876615-35314168","span":{"begin":707,"end":709},"obj":"26876615"},{"id":"27920775-17437993-35314169","span":{"begin":1383,"end":1385},"obj":"17437993"},{"id":"27920775-19509260-35314170","span":{"begin":2067,"end":2069},"obj":"19509260"}],"text":"In many cancer cell lines, the expression of RIPK3 is lost due to genomic methylation near its transcriptional start site (42). Recently, it was reported that the methyltransferase EZH2 was overexpressed in HPV16 E6- and E7-transformed KCs (43). Since EZH2 is a core component of polycomb-repressive complex 2 (PRC2) and plays a role in promoter-targeted transcriptional repression (44), we hypothesized that EZH2 may also be involved in repressing the expression of RIPK3. Western blot analysis and RT-qPCR of primary KCs and hrHPV-infected KCs revealed that both gene and protein expression of EZH2 was higher at the basal level and after IFNγ/TNFα stimulation in hrHPV + KCs (Figures 4A,B). As expected (44), hrHPV-infected KCs display a higher methylation of H3K27 at the basal level (Figure S5A in Supplementary Material). Knockdown of the polycistronic mRNA of HPV16 in hrHPV16-positive KCs resulted in lower EZH2 protein levels indicating that EZH2 overexpression was induced by hrHPV in KCs (Figure 4C). This effect was even more pronounced after IFNγ/TNFα stimulation fitting with the observation that also the expression of the viral genes was further downregulated (Figure 2E). The use of 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase, is known to effectively deplete cellular levels of the PRC2 components, including EZH2 (45). Indeed, treatment of the hrHPV + KCs with DZNep resulted in a dose-dependent decrease in EZH2 protein levels (Figure 4D), decreased of H3K27 methylation (Figure S5B in Supplementary Material), and the concomitant increase in RIPK3 at the protein and gene expression level (Figures 4D,E). Treatment of hrHPV + KCs with the catalytic EZH2 inhibitor GSK503 did not have a clear effect on the expression of RIPK3 (Figure S5C in Supplementary Material), suggesting that the hrHPV-induced overexpression of EZH2 is indirectly responsible for suppressing RIPK3-mediated necroptosis. It should be noted that DZNep has been reported to function as a global histone methylation inhibitor (46). We, therefore, analyzed the expression of other methyltransferases and found that, in addition to EZH2, eight other methyltransferases were expressed at a significantly higher level in hrHPV + KCs (Figures S5D,E in Supplementary Material). Potentially, these methyltransferase may also play a role in downregulating the expression of RIPK3. To test if methyltransferases where involved in suppressing RIPK3-mediated necroptosis, KCs and hrHPV + KCs again were stimulated with IFNγ/TNFα, BV6, and zVAD-fmk, but, now, either in the absence or presence of DZNep. Cell death was determined both by flow cytometry in order to analyze larger numbers of cells (Figure 4F) and by immunofluorescence in cell cultures (Figure S6 in Supplementary Material). Clearly, the presence of DZNep increased the BV6/zVAD-fmk/IFNγ/TNFα-induced percentage of dead cells. Cells died by necroptosis, since cell death could be blocked by Nec-1s (Figures 4F,G; Figure S6 in Supplementary Material)."}

{"project":"MyTest","denotations":[{"id":"27920775-25952668-35314165","span":{"begin":123,"end":125},"obj":"25952668"},{"id":"27920775-21865393-35314166","span":{"begin":241,"end":243},"obj":"21865393"},{"id":"27920775-26876615-35314167","span":{"begin":383,"end":385},"obj":"26876615"},{"id":"27920775-26876615-35314168","span":{"begin":707,"end":709},"obj":"26876615"},{"id":"27920775-17437993-35314169","span":{"begin":1383,"end":1385},"obj":"17437993"},{"id":"27920775-19509260-35314170","span":{"begin":2067,"end":2069},"obj":"19509260"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"In many cancer cell lines, the expression of RIPK3 is lost due to genomic methylation near its transcriptional start site (42). Recently, it was reported that the methyltransferase EZH2 was overexpressed in HPV16 E6- and E7-transformed KCs (43). Since EZH2 is a core component of polycomb-repressive complex 2 (PRC2) and plays a role in promoter-targeted transcriptional repression (44), we hypothesized that EZH2 may also be involved in repressing the expression of RIPK3. Western blot analysis and RT-qPCR of primary KCs and hrHPV-infected KCs revealed that both gene and protein expression of EZH2 was higher at the basal level and after IFNγ/TNFα stimulation in hrHPV + KCs (Figures 4A,B). As expected (44), hrHPV-infected KCs display a higher methylation of H3K27 at the basal level (Figure S5A in Supplementary Material). Knockdown of the polycistronic mRNA of HPV16 in hrHPV16-positive KCs resulted in lower EZH2 protein levels indicating that EZH2 overexpression was induced by hrHPV in KCs (Figure 4C). This effect was even more pronounced after IFNγ/TNFα stimulation fitting with the observation that also the expression of the viral genes was further downregulated (Figure 2E). The use of 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase, is known to effectively deplete cellular levels of the PRC2 components, including EZH2 (45). Indeed, treatment of the hrHPV + KCs with DZNep resulted in a dose-dependent decrease in EZH2 protein levels (Figure 4D), decreased of H3K27 methylation (Figure S5B in Supplementary Material), and the concomitant increase in RIPK3 at the protein and gene expression level (Figures 4D,E). Treatment of hrHPV + KCs with the catalytic EZH2 inhibitor GSK503 did not have a clear effect on the expression of RIPK3 (Figure S5C in Supplementary Material), suggesting that the hrHPV-induced overexpression of EZH2 is indirectly responsible for suppressing RIPK3-mediated necroptosis. It should be noted that DZNep has been reported to function as a global histone methylation inhibitor (46). We, therefore, analyzed the expression of other methyltransferases and found that, in addition to EZH2, eight other methyltransferases were expressed at a significantly higher level in hrHPV + KCs (Figures S5D,E in Supplementary Material). Potentially, these methyltransferase may also play a role in downregulating the expression of RIPK3. To test if methyltransferases where involved in suppressing RIPK3-mediated necroptosis, KCs and hrHPV + KCs again were stimulated with IFNγ/TNFα, BV6, and zVAD-fmk, but, now, either in the absence or presence of DZNep. Cell death was determined both by flow cytometry in order to analyze larger numbers of cells (Figure 4F) and by immunofluorescence in cell cultures (Figure S6 in Supplementary Material). Clearly, the presence of DZNep increased the BV6/zVAD-fmk/IFNγ/TNFα-induced percentage of dead cells. Cells died by necroptosis, since cell death could be blocked by Nec-1s (Figures 4F,G; Figure S6 in Supplementary Material)."}