The apoptosis rate rapidly increased as the CoCl2 concentrations and incubation Carfilzomib mechanism time increased, although the increase did not occur Inhibitors,Modulators,Libraries in a linear fashion. Clearly, 100 M CoCl2 had a minimal effect, as evidenced by persistently low apoptosis rates over time, while 600 M and 900 M CoCl2 induced high rates of cellular apoptosis earlier. When incubated with 300 M CoCl2, HUVECs showed a large transition in apoptosis rates, which increased from 14% to 55% between 24 and 36 hrs. We therefore considered the first 24 hrs of HUVECs with 300 M CoCl2 treatment as the early stage of apoptosis. Differentially expressed genes and functional analysis A comparison of the mimicked hypoxic and normoxic groups identified 1583 differentially expressed genes, consisting of 300 upregulated and 1283 downregulated genes.
The number of downregulated genes was 4. 28 times higher than the number of upregulated genes in response to stress. A different strategy of functional analy sis was performed on DEGs. First, function enrichments of DEGs were detected based on their protein annotations from the UniProt data base. Interestingly, 24% Inhibitors,Modulators,Libraries of the DEGs were catego rized as genes undergoing or regulating alternative splicing. It is equally notable that the other functional categories available for short term cellular response to hypoxia include nuclear protein, phosphor ylation, metal binding, and DNA binding, which are also prominent in enrichment. These categories demonstrate extensive responses of gene regulation to hypoxia. GO analysis was then carried out on the up and downregulated genes respectively.
Importantly, we found that programmed cell death is only significantly observed in the upregulated genes, which indicates that apoptosis is initiated in response to mimicked hypoxia in HUVECs. Alternative splicing events and protein domain Inhibitors,Modulators,Libraries analysis Using the Splicing Index algorithm described in the methods, 342 probe select regions labeled as core were identified as alternatively spliced exons, and these belonged to 293 alternatively spliced transcripts. Compared with normal HUVECs, 250 PSRs with higher expression were classified as general exon inclusion events, while the other 92 PSRs with lower expression were considered as general exon skip ping events. Thirty five percent of the alterna tively spliced transcripts are supported by experimental evidence based on the NCBI RefSeq database records.
Since it is possible for multiple alternative splicing events to occur in the same transcript, 13% of the tran scripts were found to contain 25% of the alter natively spliced exons. Therefore, there was an average of 2. 3 alternatively spliced exons per transcript under mimicked hypoxia conditions in our data. A typical example Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries is ubiquitin associated protein 2, for which five alternatively spliced exons were detected, indi cating a complicated Palbociclib CDK pattern of splicing regulation of UBAP2 in HUVECs under hypoxic stress.