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Figure 6 | Retrovirology

Figure 6

From: WFDC1 expression identifies memory CD4 T- lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions

Figure 6

siRNA-mediated knockdown of ps20 inhibits conjugate and multiple conjugate formation more effectively than siRNA-mediated knockdown of ICAM-1. (A) ps20 and ICAM-1 mRNA levels were measured in a selection of ps20high and ps20inter/low cells at 4 different time points. Data show a two-tailed non-parametric Spearman's r correlation of all data points. CD4 T-cell Jwt ps20inter were treated with either; a non-silencing (NS), ICAM-1 or WFDC1/ps20-silencing siRNA pool for 6 days. (B) After siRNA treatment the expression of ICAM-1, ps20 and GAPDH mRNA was analyzed by qRT-PCR and relative expression to β-actin was measured. Normalized relative expression was calculated in reference to siNS control. Data represent the mean of three replicate assays. (C) Surface expression of ICAM-1 in siRNA treated cells is shown as assessed by standard immunofluorescence. Normalized MFI was calculated in reference to siNS control. Data represent the mean of three replicate assays (D) 8 × 104 NS, ICAM-1, or ps20 siRNA-treated WT Jurkat cells were dye-labelled and co-cultured with donor 40% 2044-infected donor cells at a T:D ratio of 1:0.2. Mean percentage of Gag+ target cells after 4-hour co-culture is shown. Normalized % of Gag+ target cells was calculated in reference to siNS control. Data represent the mean of three replicate assays. (E) 5 × 105 siRNA treated cells were dye-labelled and co-cultured with 5 × 105 60% 2044-infected donor cells. Co-cultures were incubated for 1 hour on Poly-L-lysine coated glass cover slips, then fixed and stained with a FITC anti-Gag (Green) Ab. Conjugates and MCs were assessed as before in at least 500 random target cells per population across triplicate experiments. (F) The panel depicts the mean conjugate interface diameter (μM) between siRNA treated Jurkat cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across triplicate experiments. Asterisk denotes statistically significant data as calculated using a paired t-test (*P ≤0.05) in relation to NS siRNA control.

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