PMC:4504148 / 31942-35671 JSONTXT

{"project":"2_test","denotations":[{"id":"26236191-21522185-32463237","span":{"begin":363,"end":367},"obj":"21522185"}],"text":"Association of DFNA5 with the MAPK-related mechanisms in HEK293T cells\nTo further elucidate the DFNA5-related pathways, a microarray experiment was performed in human HEK293T cells. As described previously, mutDFNA5 induced a growth defect in transfected HEK293T cells compared to wtDFNA5 and control (cells transfected with an empty vector) (Op de Beeck et al., 2011). These cell death events were evident from 9 h post-transfection and peaked at 12 h (data not shown). Therefore RNA samples of HEK293T cells were collected 12 h post-transfection. A transcriptomic analysis was performed on HEK293T cells transfected with either wtDFNA5 or mutDFNA5. Six biological replicates of every RNA sample were collected although one wtDFNA5-transfected sample did not survive quality control. Subsequent analyses, using wtDFNA5 as a reference, were therefore performed on five wtDFNA5- vs. six mutDFNA5-transfected samples. Analysis using “Beadarray” and “LIMMA” packages available in R identified 228 significantly up- and 222 significantly down-regulated genes after correction for multiple hypothesis testing (p \u003c 0.05). In addition to individual gene expression, GO analysis was performed to determine the biologically, cellularly, and molecularly enriched GO annotations linked to the differentially expressed genes.\nTable 3 shows the top 34 of the significantly up-regulated genes. It contains several genes related to the MAPK pathway such as EGR1/2, FOSB, andJUNB (indicated in bold). Interestingly, this list also contained the PMAIP1 gene. PMAIP1 encodes a BH3-only protein belonging to the BCL2 protein family, a family of important regulators of apoptotic cell death related to the mitochondria. The top 34 highest down-regulated genes are shown in Table 4 and contains several genes related to protein folding such as HSPA6, ATF3, and CTH (indicated in bold, Table 4).\nTable 3 Top 34 of the significantly up-regulated genes in mutDFNA5 transfected HEK293T cells. LogFC, logarithm of fold change, down-regulation in mutDFNA5 yeast samples using wtDFNA5 as a reference; adj.p.value, p-value adjusted for multiple hypothesis testing. The array address of the specific splice variant on the microarray is provided between parentheses. Genes involved in processes related to either the MAPK pathway, cAMP response or the mitochondria are indicated in bold.\nTable 4 Top 34 of the highest significantly down-regulated genes in mutDFNA5 transfected HEK293T cells. LogFC, logarithm of fold change, down-regulation in mutDFNA5 yeast samples using wtDFNA5 as a reference; adj.p.value, p-value adjusted for multiple hypothesis testing. The array address of the specific splice variant on the microarray is provided between parentheses. Genes involved in processes related to protein folding are indicated in bold. Subsequent GO analysis of the biological annotations revealed, in addition to the more general development processes, the up-regulation of the MAPK pathway (GO:0043407) and the cAMP response (GO:0051591) (Supplemental Data Table 6, indicated in bold). The response to protein folding (GO:0006986) and to topologically incorrect protein (GO:0035966) were the only two significantly down-regulated processes and both were related to protein folding (Supplemental Data Table 7). The most important genes that are involved in these processes were HSPA6, a heat shock protein and several chaperones proteins, such as DNAJB1 and DNAJB2.\nThese results demonstrate the association of mutDFNA5-induced cell death with the MAPK pathways. The identification of processes related to protein folding supports the results in yeast in which GO terms related to protein folding and the ER were significantly associated with mutDFNA5."}

{"project":"MyTest","denotations":[{"id":"26236191-21522185-32463237","span":{"begin":363,"end":367},"obj":"21522185"}],"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":"Association of DFNA5 with the MAPK-related mechanisms in HEK293T cells\nTo further elucidate the DFNA5-related pathways, a microarray experiment was performed in human HEK293T cells. As described previously, mutDFNA5 induced a growth defect in transfected HEK293T cells compared to wtDFNA5 and control (cells transfected with an empty vector) (Op de Beeck et al., 2011). These cell death events were evident from 9 h post-transfection and peaked at 12 h (data not shown). Therefore RNA samples of HEK293T cells were collected 12 h post-transfection. A transcriptomic analysis was performed on HEK293T cells transfected with either wtDFNA5 or mutDFNA5. Six biological replicates of every RNA sample were collected although one wtDFNA5-transfected sample did not survive quality control. Subsequent analyses, using wtDFNA5 as a reference, were therefore performed on five wtDFNA5- vs. six mutDFNA5-transfected samples. Analysis using “Beadarray” and “LIMMA” packages available in R identified 228 significantly up- and 222 significantly down-regulated genes after correction for multiple hypothesis testing (p \u003c 0.05). In addition to individual gene expression, GO analysis was performed to determine the biologically, cellularly, and molecularly enriched GO annotations linked to the differentially expressed genes.\nTable 3 shows the top 34 of the significantly up-regulated genes. It contains several genes related to the MAPK pathway such as EGR1/2, FOSB, andJUNB (indicated in bold). Interestingly, this list also contained the PMAIP1 gene. PMAIP1 encodes a BH3-only protein belonging to the BCL2 protein family, a family of important regulators of apoptotic cell death related to the mitochondria. The top 34 highest down-regulated genes are shown in Table 4 and contains several genes related to protein folding such as HSPA6, ATF3, and CTH (indicated in bold, Table 4).\nTable 3 Top 34 of the significantly up-regulated genes in mutDFNA5 transfected HEK293T cells. LogFC, logarithm of fold change, down-regulation in mutDFNA5 yeast samples using wtDFNA5 as a reference; adj.p.value, p-value adjusted for multiple hypothesis testing. The array address of the specific splice variant on the microarray is provided between parentheses. Genes involved in processes related to either the MAPK pathway, cAMP response or the mitochondria are indicated in bold.\nTable 4 Top 34 of the highest significantly down-regulated genes in mutDFNA5 transfected HEK293T cells. LogFC, logarithm of fold change, down-regulation in mutDFNA5 yeast samples using wtDFNA5 as a reference; adj.p.value, p-value adjusted for multiple hypothesis testing. The array address of the specific splice variant on the microarray is provided between parentheses. Genes involved in processes related to protein folding are indicated in bold. Subsequent GO analysis of the biological annotations revealed, in addition to the more general development processes, the up-regulation of the MAPK pathway (GO:0043407) and the cAMP response (GO:0051591) (Supplemental Data Table 6, indicated in bold). The response to protein folding (GO:0006986) and to topologically incorrect protein (GO:0035966) were the only two significantly down-regulated processes and both were related to protein folding (Supplemental Data Table 7). The most important genes that are involved in these processes were HSPA6, a heat shock protein and several chaperones proteins, such as DNAJB1 and DNAJB2.\nThese results demonstrate the association of mutDFNA5-induced cell death with the MAPK pathways. The identification of processes related to protein folding supports the results in yeast in which GO terms related to protein folding and the ER were significantly associated with mutDFNA5."}