Fig 2: p17 drives the formation of a multiprotein complex containing CXCR-2, syn-2, RACK-1 and JAK-1 on LX2 cells.LX2 cells were serum starved and stimulated for 5, 15, 30 and 60 minutes with 2 µg/ml of p17. (A) Co-immuneprecipitation of CXCR2 with syndecan-2 and RACK-1. (B) Co-immuneprecipitation of RACK1 with syndecan-2, CXCR2 and JAK1. (C) Confocal immunofluorescence of LX2 cells left untreated (a) or exposed to p17 for 5 minutes and stained with anti-CXCR2 antibody. (D) Immunofluorescence analysis of syndecan-2 and CXCR2 localization on LX2 cells left untreated (NT) or stimulated 5 minutes with p17. Cells were stained with the indicated antibodies. Red: syn-2. Green: CXCR2. The red and green signals were electronically merged for co-localization analysis.

Fig 3: Progression of liver fibrosis is associated with a down-regulation of CXCR2 receptor.Liver biopsies from HIV/HCV coinfected and HCV monoinfected patients were serially sectioned and stained with the following antibodies: aSMA, syndecan-2 and CXCR2. (A) Patient n. 3 HCV positive. Diffusely and intensely sinusoidal walls staining with aSMA (Magnification 200x). Patient n.6 HIV/HCV positive. Focally and weakly sinusoidal walls staining with aSMA (Magnification 200x). (B) Patient n. 3 HCV positive and patient n. 6 HIV/HCV positive. In both cases strongly cytoplasmatic staining of stellate cells with syndecan-2 (Magnification 200x) (C) Patient n. 3 HCV positive. Focally and weakly sinusoidal walls staining with CXCR2 (Magnification 200x; inset 400x). Patient n.5 HIV/HCV positive. Absence of staining with CXCR2 (Magnification 200x)

Fig 4: HIV p17 regulates NRs and transcriptional coregulators in HepG2 cells.(A) Immunoblot of syndecan-2 (Syn-2), CXCR2, and Tubulin. (B) qRT-PCR of syn-2 and CXCR2. Data represent the mean of 4 experiments. Values are normalized relative to GAPDH mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P,0.05 versus not treated cells. (C,D) Microarray analysis of nuclear receptors and transcriptional coregulators in HepG2 cells stimulated with p17 (10 µg/ml). Out of 84 genes examined, nine genes were upregulated 1.5-fold or greater (LXRa, MED1, DDX5, TR2, TR3, RARa, ESR1, NCOA1, NRIP1) while six genes were down-regulated 1.5 -fold or greater (PPAR?, KAT5, BRD8, PSMC5, HDAC7, ARNT). (C) Clustergram and (D) Scatter plot. (E) Validation of microarray results by RT-PCR showing the relative mRNA expression of LXRa, MED1, ESR1, DDX5, PPAR?, BRD8 and KAT5. Values are normalized relative to GAPDH mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P,0.05 versus not treated cells.

Fig 5: p17 signals through RACK-1/Jak-1/STAT-1 pathway in THP-1 cells.(A) Schematic diagram showing putative interaction of p17 with syndecan-2 and the activation of RACK-1/Jak-1/STAT-1 signal transduction pathway. (B) Co-immuneprecipitation of RACK-1 with syndecan-2 and Jak-1 following stimulation of THP-1 cells with p17 recombinant protein (1 µg/ml) for 5, 15 and 30 minutes. (C) Analysis of Jak-1 protein (total and phosphorylated fraction) by immune blot in THP-1 cells stimulated with p17 (1 µg/ml) for 15, 30 and 60 minutes. (D) Analysis of STAT-1 protein (total and phosphorylated fraction) by immune blot in THP-1 cells stimulated with p17 (1 µg/ml) for 15, 30 and 60 minutes. (E) Chromatin Immunoprecipitation assay carried out in THP-1 cells left untreated or primed with 1 µg/ml p17 for 1, 6 and 18 hours. Real-Time PCR was performed on MCP-1 promoter. (F) Chromatin Immunoprecipitation assay carried out in THP-1 cells left untreated or primed with 1 µg/ml p17 for 18 hours. Real-Time PCR was performed on both MCP-1 promoter and intron-II of FXR gene. Values are normalized relative to input DNA concentration and are expressed relative to those of not treated cells immunoprecipitated with an anti IgG antibody, condition set as 1. Analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells. **P<0.05 versus p17 stimulated cells.