Table 1.
The proteins upregulated in human renal cancer cell with induced DIO1 expression.
Table 2.
The proteins downregulated in human renal cancer cell with induced DIO1 expression.
Fig 1.
Functional annotation of proteins that were differently expressed in DIO1(+) cells when compared with DIO1(-) cells.
The pie charts show results of analysis performed using PANTHER (http://pantherdb.org). The largest categories of biological processes, molecular function, and cellular components related to the identified proteins are shown with arrows.
Fig 2.
Metabolic processes related to proteins affected by DIO1 expression in ccRCC cells.
Analysis was performed using PANTHER (www.pantherdb.org).
Fig 3.
Classification of proteins that were differently expressed in DIO1(+) cells when compared with DIO1(-) cells.
The graph shows results of analysis performed using PANTHER (http://pantherdb.org).
Table 3.
Enrichment analysis of proteins affected by DIO1 expression in human renal cancer cells.
Fig 4.
The network of proteins affected by DIO1 expression.
A. Network of expression correlations; the proteomic data of protein expression levels were analyzed using Metscape/Cytoscape application. Blue lines: negative correlations, red lines: positive correlations. Thickness of lines indicates strength of correlations. B. Protein interaction network generated with STRING v. 10.5 accessed on 2017.11.06. with default settings (minimum required interaction score: medium confidence 0.4). Three major clusters are labelled as I (cytoskeleton remodeling and intracellular trafficking), II (cellular adhesion), and III (metabolism).
Fig 5.
Validation of proteomic data using qPCR.
The plots show mean ± SEM results of three independent biological experiments performed on KIJ265T-DIO1(+) cells when compared with KIJ265-DIO1(-) cells. Statistical analysis was performed using t-test. *p<0.05, **p<0.01.
Fig 6.
The transcript expression of genes affected by DIO1 restoration is disturbed in renal cancer.
The plots show results of qPCR analysis performed in 30 matched pairs of tumor (TUMOR) and control (CONTROL) tissue samples. Statistical analysis was performed using Wilcoxon matched pairs signed test. * p<0.05; **p<0.01; **** p<0.0001.
Fig 7.
Matrix of correlations between the transcript expression of DIO1 and DIO1-affected genes in renal tumors.
The upper table shows Spearman's rank correlation coefficient values for gene expressions analyzed in 30 RCC tumors and 30 paired-matched controls. Dark red: rs≥0.85, red: 0.85>rs≥0.7, orange: 0.7>rs≥0.3, green rs<-0.3. The lower table shows p values (yellow: p<0.05).
Fig 8.
Altered transcript expression of DIO1-affected genes correlates with poor survival of renal cancer patients.
Kaplan-Meyer analysis for DIO1-affected genes identified in the study. The analysis was performed on independent cohort of 468 patients with ccRCC, basing on transcriptomic data published by The Cancer Genome Atlas Network Consortium. The red and green lines depict patients with high and low risk of death, respectively. The numbers of patients in each group are shown below graphs. Censored observations are shown with +. Log-rank p values, hazard ratio (HR) and confidence intervals (CI) are shown above each graph. Expression of genes in each risk group is given in S4 Fig.
Fig 9.
Increased T4 concentration in renal cancer cells with re-expressed DIO1.
Intracellular T4 concentration in renal cancer cells with (DIO1+) or without (DIO1-) ectopic DIO1 expression. The plots show mean ± SEM results of three independent biological experiments performed on KIJ265T-DIO1(+) cells and KIJ265-DIO1(-) cells. Statistical analysis was performed using t-test. T3 measurements were below the detection limit. *p<0.05.
Fig 10.
The model depicting the proteomic effects of DIO1 restoration in renal cancer.
Changes in protein levels are illustrated with green (decreased level) and red (increased level) colors. Induction of DIO1 expression in renal cancer cells results in robust downregulation of oncoproteins that are well known inhibitors of apoptosis and promoters of ccRCC proliferation, migration and invasion (left side of the drawing). Simultaneously, restoration of DIO1 in ccRCC cells leads to enhanced expression of proteins that contribute to metabolic reprogramming of renal tumors and affect PPP, TCA cycle, metabolism of amino acids and lipids. This may be associated with prominent induction of ROS that in turn trigger antioxidative response and results in enhanced levels of proteins of Nrf2 pathway (right side of the drawing). On the other hand, induced DIO1 expression can also possibly result in attenuation of ROS-scavenging system by decreasing GLUD1 and inducing FAH (middle part of the drawing). Altogether, this may possibly result in ROS levels that exceed the compensatory buffering systems of ccRCC cells and trigger mechanisms leading to apoptosis or autophagy.