Figure 1.
Proteomic workflow and validation studies of the SILAC analysis of Jurkat-T cells expressing HIV-1 Tat.
A. Organellar proteomic workflow for the analysis of quantitative changes in Jurkat NTAP-Tat versus the Jurkat NTAP cells. B. Designed cassettes for NTAP and NTAP-Tat. GFP is cloned downstream the NTAP-Tat and NTAP genes and its translation is independently controlled by IRES. FACS analysis of the Jurkat NTAP-Tat and Jurkat NTAP showing a highly enriched population of polyclonal transduced cells. C. We transfected the Jurkat NTAP-Tat and Jurkat NTAP using 2 and 5 µg of pGL3-LTR plasmid. HIV-1 LTR luciferase reporter gene assay confirmed that the NTAP-Tat is functionally active. D. Validation of the subcellular fractionation from mixed Jurkat NTAP-Tat (R0K0) and Jurkat NTAP (R6K6) (1∶1). Each fraction (10 µg of protein per lane) was checked by Western-Blot using anti-nucleolin (C23), anti-fibrillarin, anti-α-tubulin and anti-PARP antibodies. The molecular weight (kDa) of each protein is indicated on the left. (Fractions: WC: whole cells, C: Cytoplasmic, N: Nuclear, Np: Nucleoplasmic and No: Nucleolar). E. Expression and subcellular distribution of NTAP-Tat in Jurkat T-cells using Western-Blot analysis. NTAP-Tat (36 kDa) was detected using anti-HIV-1 Tat antibody (ab43014, Abcam). For immunofluorescence analysis, Jurkat NTAP-Tat cells were stained for fibrillarin (green), HIV-1 Tat (red), and DAPI (Blue or in grey contrast on the top left panel). NTAP-Tat overlaps with fibrillarin in the nucleolar compartment. (Bar: 2 µm). F. GO biological processes and cellular components distribution of the nucleolar proteome of mixed cells. G. Distribution of the changes in protein abundance in the nucleolus of Jurkat T-cells upon HIV-1 Tat expression. Relative abundance is plotted as Log2 (SILAC ratios). Green denotes depletion, while red denotes enrichment.
Table 1.
List of proteins showing a significant change in abundance in the nucleoli of Jurkat T-cells following expression of HIV-1 Tat.
Figure 2.
Western-Blot validation of the SILAC quantitative analysis.
Western-Blot results comparing expression levels of selected 15 proteins to their SILAC ratios (right panel). 10 µg total protein of each subcellular fraction (WC: whole cells, C: Cytoplasmic, N: Nuclear, Np: Nucleoplasmic and No: Nucleolar) from Jurkat NTAP (NTAP) and Jurkat NTAP-Tat (TAT) were resolved by SDS-PAGE, blotted and probed with indicated antibodies. For HSP90B, as the nucleolar detection level was low, we increased separately the amount of total protein loaded to 20 µg.
Figure 3.
Network analysis of the Tat interactome in nucleoli of Jurkat T-cells. A.
The main network contains 416 nodes (proteins) connected with 5060 edges gathered from Protein-Protein interaction databases as described in the text. The central position HIV-1 Tat is highlighted in yellow. B. Subnetwork of HIV-1 Tat cellular partners. HIV-1 Tat interacts with 146 proteins out of the 416 proteins identified in the mixed nucleolar fraction. This subnetwork contains 651 edges. Red and green denotes proteins with increased or decreased nucleolar abundance, respectively.
Figure 4.
Overall profiles of changes in proteins abundance in the nucleolus of T-cells upon HIV-1 Tat expression.
Proteins were organized according GO biological processes and KEGG pathways identified by the ToppGene Suite tools (http://toppgene.cchmc.org/). Red and green denotes proteins with increased or decreased nucleolar abundance, respectively.