*2.3. Cells*

Human primary MDM were generated and purified as previously described [53–56]. MDM were derived from PBMCs of healthy donors. Briefly, PBMCs were separated by Ficoll-Hypaque gradient centrifugation and seeded in T25 flasks at a number of 50.10<sup>6</sup> cells in 7 mL Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 20% heat inactivated, mycoplasma- and endotoxin-free fetal bovine serum (FBS), L-glutamine (1 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL), without exogenous cytokines or growth factors, at 37 ◦C in a humidified atmosphere enriched with 5% CO2. After five days of culture, non-adherent cells were eliminated with caution by consecutive gentle washings with warmed RPMI 1640, leaving a monolayer of adherent cells which were finally incubated in complete medium [57–59].

The MDM obtained showed a purity exceeding 98% as tested by cytofluorimetric analysis. Expression of CXCR4 and CCR5 in all our MDM cultures was assayed by flow cytometric analysis (FCM) (FACScanTM, Becton Dickinson System, San José, CA) by means of CD184 (CXCR4/fusin) R-phycoerythrin (R-PE)-conjugated mouse anti-human monoclonal antibody and CD195 (CCR5) R-phycoerythrin (R-PE)-conjugated mouse anti-human monoclonal antibody both purchased from BD Pharmingen (Becton Dickinson biosciences, USA). Measurements were performed in at least 3 independent experiments. In each experiment, MDM derived from a single healthy donor.

#### *2.4. Drug Treatment, Infection and Virus Detection*

Six days after Ficoll-Hypaque, non-adherent cells were removed, and monocytes were further allowed to di fferentiate in MDM for four days. Their purity exceeded 98%. For exposure to inhibitor AMD3100 and TAK779, at least twenty-four hours before CXCR4 and CCR5 strain infection, MDM culture medium was removed and replaced with fresh media 20% serum. 45–60 min before infection; where needed, the drug was added to the cell supernatants at appropriate concentrations (0.4–2 μg/mL for TAK779, and 5 μM for AMD3100) and then MDM were reincubated at 37 ◦C in a humidified atmosphere enriched with 5% CO2.

Virus challenge was performed for at least 4 h to almost a week by exposing MDM to 3000 up to 7500 pg/mL of p24 (corresponding to, respectively, 400 and 1000 tissue cultures infectious doses 50% per ml (TCID50/mL) of the Laboratory-adapted strain HIV-1 Ba-L) of the all strains described above, followed by extensive washing to remove excess virus.

Virus production was assessed by the HIV-1 p24 gag antigen concentration in culture supernatants using a p24 gag antigen detection kit according to the instructions of the manufacturer (Abbott labs, Pomezia, Italy).

#### *2.5. Western Blotting of Cell Cultures*

Cells were challenged with IIIB, NL4-3, Ba-L and 81A strains of HIV (whole visions) in warmed media 20% serum at indicated times at 37 ◦C in a humidified atmosphere enriched with 5% CO2, incubated for the times indicated and then lysed and subjected to immunoblot analysis. Lysis was performed in ice-cold bu ffer Radio-Immunoprecipitation Assay (RIPA) (50 mM tris hydroxymethyl aminomethane ((Tris)-HCl), pH 7.4; 250 mM NaCl; 50 mM NaF, EDTA 5 mM; 0,15% Triton X-100) containing a protease and phosphatase inhibitor cocktails (1 mM phenylmethylsulfonyl fluoride; 10 μg/mL pepstatin; 10 μg/mL leupeptin and 1 mm sodium vanadate) and incubated for di fferent times at 4 ◦C. Cell lysates were then clarified by centrifugation at 13,000 rpm for 10 min at 4 ◦C.

Protein concentrations were determined by a spectrophotometric assay (Pierce). Immunoblot analysis was performed on cell lysates containing 30 μg protein mixed with Laemmli bu ffer and boiled for 5 min. Samples were subjected to 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked overnight with 5% Bovine serum Albumine (BSA) in TBS-tween.

A 1:1000 dilution of the lysates was used for the detection of activated MAPK/p38 a polyclonal antibody specific for Phospho-p38 (Thr202/Tyr204), and for total p38 (Cell Signaling Technology, Beverly, MA). (Jackson ImmunoResearch Laboratories). Then, membranes were treated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) (Jackson ImmunoResearch Laboratories). The immunoreactive bands were visualized using enhanced chemiluminescence Western blotting system (Immun-Star HRP Chemiluminescent Kit, Hercules, Ca, USA) according to the manufacturer's instructions (Biorad, Hercules, CA, USA).

Blots were stripped (2% SDS, 62.5 mM Tris, 100 mM mercaptoethanol) for 30 min at 56 ◦C and washed in PBS containing 0.05% Tween 20, before blocking and reprobing with primary antibody. For the quantification of the phosphorylated and total proteins, the bands on the films were first scanned by the Epson software program and then the images were processed through the Scion Image analysis program (Houston, TX, USA) for the IBM PC based on the popular NIH Image on the Macintosh platform.

#### *2.6. RNA Isolation and Microarray Analysis*

In di fferent experiments, 81A and NL4-3 infected MDM were incubated in parallel at a dose of 2000 pg/mL of viral p24 from 6 to 24 h of infection, then exposed to 4M guanidinium and total RNA was isolated by the guanidinium-phenol procedure. The isolation of polyA mRNA from each total RNA preparation was obtained by OLIGOTEX mRNA Purification System (Qiagen, Hilden, Germany). cDNA probes for microarray experiments were prepared from 0.5 μg of cellular mRNA by CyScribe post-Labelling Kit (Amersham Biosciences). The CyDye labelled cDNA was purificated by QIAquik PCR purification Kit (Qiagen). For dual colour hybridization we combined Cy3 and Cy5 labelled cDNAs in one tube. The solution protected from light was dried by using a rotary evaporator and adding tRNA (40 γ), Calf Thymus DNA (40 γ) and Cot-1 DNA (1 γ). The dry solution was dissolved in nuclease free water, denatured at 95 ◦C for 5 min and cooled in ice. Hybridization bu ffer 4X (supplied

in the CyScribe Post-labelling Kit) was added with 12 volume of 100% formammide. Hybridization was performed in an humid hybridization chamber (5X SSC) at 42 ◦C for 14–18 h, following washing with saline-sodium citrate (SSC) and SDS (*w*/*v*) pre-warmed to 37 ◦C. Fluorescent-array images were collected for both Cy3 and Cy5 by using a ScanArray Express, Microarray Analysis System Version 2.0 (Perkin-Elmer) and image intensity data were extracted and analysed by using QuantArray Pachard Biochips Software. In particular, QuantArray Software provides automated analysis of color microarray images (automatic scanning and quantitation to measure fluorescence signal at each spot on the array) before exporting data to bioinformatics software packages. Triplicate array positions are used for each gene to avoid signal noise. The human Cancer Chip version 4.0 (Takara) slides were used for microarray analysis and all spots were known. In order to evaluate the inhibition or enhancement of genes expression in terms of mRNA production, a comparison of Cy3 and Cy5 signals intensity was applied.

#### *2.7. Flow Cytometry Measurement of Apoptotic Cells*

At established time points of infection (see results), MDM were washed and detached from the 25 flasks with gentle scraping as previously described [60]. MDM were precipitated by centrifugation at 1500 rpm for 5 min at 4 ◦C. All MDM in the culture, both adherent and non-adherent, were considered in the final count for apoptosis analysis by Flow Cytometry measurement (FCM). Supernatants were removed, then aliquoted, and stored at −80 ◦C for p24 titration. After washing, MDM were kept in 3 mL of cold PBS, 0.02% EDTA in 10 min at 4 ◦C, then gently scraped and transferred to the respective tubes and precipitated by centrifugation at 1500 rpm for 5 min at 4 ◦C. Supernatants were removed and the pellet resuspended in 0.5 mL of Trypan-blue solution for cell count and viability check. Cells were washed with 2 mL PBS cold and centrifuged as described above.

The supernatants were completely removed and 2 mL of 70% ice ethanol was added to permeabilize for 40 min at 4 ◦C. After washing, cells were centrifuged (1600 rpm/5 min/4 ◦C), again washed and centrifuged as described above and gently resuspended in 1.0 mL hysotonic Propidium Iodide (PI) solution (Sigma) 50 μg/mL, in PBS and RNase 50 μg/mL) in polipropylen tubes. After rotating for 15 min at room temperature, the tubes were placed at 4 ◦C for 2 h in the dark. Cells were washed with 2 mL PBS cold, centrifuged at 1500 rpm for 5 min at 4 ◦C, resuspended in 0.4 mL cold PBS and kept in the dark at 4 ◦C for not more than 20 min before PI fluorescent measurement. The DNA specific fluorocrome Propidium Iodide (PI) recognized apoptotic cells as a distinct hyploploid cell population with a reduced staining below the G0/G1 population of normal diploid cells as results of cell shrinkage, nuclear condensation, internucleosomal DNA fragmentation [61]. The PI fluorescence was measured by Flow Cytometry in FL2-H (FACScanTM, Becton Dickinson System, San Josè, CA, USA) and registered on a logarithmic scale. All the tests were performed in duplicate.
