Und to be 3-fold higher in 0 cells compared to the parental
Und to be 3-fold higher in 0 cells compared to the parental line. HIF-1 levels increased in the parental A549 line following treatment with cobalt acetate or incubation under hypoxic (1.5 O2) conditions. In A549 0 cells, HIF-1 levels were increased modestly following cobalt treatment but did not appear to be changed by hypoxic treatment. However, the level of HIF-1 protein does not solely determine its ability to induce gene expression, as post-transcriptional modifications are also known to modulate its activity [49].A549 ADaysFigure 2 Growth rates of A549 and A549 0 xenografts Growth rates of A549 and A549 0 xenografts. Median tumor volume over time in nude mice bearing tumors derived from A549 or A549 0 cells. Tumor volume measurements commenced at an initial average tumor volume of 155 mm3 and 208 mm3, respectively. Experiments were conducted using six or seven mice per cohort with error bars representing ?1 standard error of the mean (SEM).Expression profiles of HIF-1 responsive transcripts in A549 0 cells Next, we focused on identifying key transcription factors that could account for a significant number of overexpressed transcripts in A549 0cells. RG1662MedChemExpress RG1662 mtDNA-deficient cells have proven useful for dissecting the role that mitochondria play in HIF-mediated responses to oxygen levels (reviewed by [43]). In fact, increased baseline levels of HIF-1 activity in cultured 0 cell lines have been noted by others [44,45]. In our A549 0 cells, HIF-1 appeared to be an excellent candidate given the over-expression of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29072704 two well-established downstream genes (VEGFA and BNIP3). We began our analyses by focusing on a group of 95 probe sets representing 63 unique HIF-1 responsive genes highlighted in a recent comprehensive review [46] (Additional File 6). While no other HIF-1 responsive genes reached our statistical criteria for over-expression, four other wellestablished HIF-1 regulated transcripts (IGFBP1, IGFBP3, TF, and PTGS2) were less abundant in the 0 cells relative to their parental cells. This could reflect the influence of other transcription factors or accessory proteins that regulate HIF-1 activity.To further explore the possible functional consequences of HIF-1 over-expression, we measured the levels of several HIF-regulated gene products by Western blot (Fig. 3B). The MT-CO2 product was included in this analysis to demonstrate the absence of this mtDNA-encoded protein in A549 0 cells. In accordance with the expression data, we found that PGK1 and DDIT4 protein levels were increased in A549 0 cells (Fig. 3B). However, GLUT1 (aka SLC2A1) protein levels were essentially unchanged in A549 and A549 0 cells despite the fact that its transcript was 2.0-fold more abundant (corrected P = 0.017) in A549 0 cells. As could be expected, the incubation of either parental or 0 cells in the presence of cobalt or under hypoxic conditions led to increased levels of these HIF-regulated proteins. The effect of cobalt was not as significant as that of hypoxia under these conditions. These data demonstrate that although baseline HIF-1 activity is higher in 0 than parental cells, HIF-regulated activity can be induced further in both cases.Decreased icosanoid metabolism and cytoskeleton gene expression in cultured A549 0 cells In parallel, we conducted separate GO analyses on transcripts that were less abundant in A549 0 cells (P < 0.001 and at least four probes sets) (Additional File 5B). The blood pressure regulation (FGB, PTGS2, FGG, and FGA) and icosanoid met.