HEK 293T cells were obtained from ATCC. the imply infection measured for a given computer virus (n = 3 determinations), expressed relative to contamination in the KD 5170 absence of chemokine (100%). One (C) and two (D) out of three impartial experiments are shown. Error bars symbolize the SD to the means. ns, not significant; **P .01 in the Mann-Whitney test. E Coreceptor usage of recombinant computer virus populations pseudotyped with the Envs depicted in Fig 1C, isolated at the time of diagnosis, at the chronic or late stage of contamination. Results (means SEM of triplicate determinations) represent infectivity of viruses measured 48 h post-inoculation of R5X4JT cells (with 20 ng of p24), in the KD 5170 presence or absence (black bars, 100%) of 10 M maraviroc (MVC, light green bars), 10 M AMD3100 (AMD, orange bars) or a mixture of both antagonists at 10 M each (pink bars). A representative experiment out of three is usually shown. Group-1 and group-2 refer to late Envs that are not, or are, significantly Rabbit polyclonal to APLP2 more resistant to inhibition by CXCL12, as compared to chronic Envs and NL4-3. F Correlation analysis (Spearman two-tailed test) between the IC50s of CXCL12 for inhibiting the plasma-derived Envs shown in Fig 1C and the CD4TL count in the blood of patients. Error bars are means SEM of 2 to 3 3 impartial determinations. ns, not significant. G Coreceptor usage of recombinant viruses pseudotyped with the Envs depicted in Fig 1D, isolated at the stage of PHI. Results (means SEM of triplicate determinations) represent infectivity of viruses measured 48 h post-inoculation of activated CD4TL (with 20 ng of p24), in the presence or absence (black bars, 100%) of 10 M maraviroc (MVC, light green bars), 10 M AMD3100 (AMD, orange bars) or a mixture of both antagonists at 10 M each (pink bars). Luciferase activity in the lysates of infected cells, expressed as relative light models (RLU) was used to quantify computer virus infectivity. One representative experiment out of three is usually shown. H Inhibition by CXCL12 of recombinant viruses pseudotyped with the indicated Envs isolated at the time of PHI. Data points (means SEM of triplicate determinations) are expressed as percent contamination of activated CD4TL relative to control infection measured in the absence of CXCL12 (100%) and were fitted to a sigmoidal dose-response model with a variable slope. IC50s were calculated using Prism 6. A representative experiment out of three impartial experiments carried out on CD4TL from unique healthy donors is usually shown. I and J Inhibition by CXCL12 of contamination of CD4TL with viruses pseudotyped with chimeric Envs made up of gp120 from the early (#1 and #16) or late (#6 and #28) viruses of P#39 (A) and P#208 (B), combined with NL4-3 gp41. Equivalent amounts of viruses were used (50 ng of Gag p24). Inhibition curves were fitted according to a sigmoidal dose-response model with a variable slope. Data points (means SEM of triplicate determinations) are expressed as percent contamination of CD4TL relative to control infection measured in the absence of CXCL12 (100%). Representative experiments out of three impartial experiments carried out on CD4TL from unique donors are shown. K Fusion of chimeric viruses containing late gp120s (reddish bars) with CD4TL is more resistant to CXCL12 inhibition, compared with viruses with early gp120s (green bars). Lactamase (BLaM)-Vpr-containing viruses (500 ng of Gag p24) were incubated with CD4TL loaded with KD 5170 the fluorescent BLaM substrate CCF2. Fusion was quantified by circulation cytometry by counting the number of cells with cleaved CCF2. Results are expressed as.