Repeat UVA exposure of human skin fibroblasts induces both a transitionary & recovery methylation response - Supplementary material

Supplementary figures 1 and 2</p

Comparison of PBMC assay with the automated readout of the plaque reduction assay.

<p>Plaques, identified as GFP-expressing cells, were evaluated by use of an AxioVision Z1 Microscope with automated reading platform. The 96-well plates were screened through with illumination time of 200 ms throughout experiments. To reduce auto fluorescence, medium was removed and PBS was gently added pre-microscopy. Plaque quantity was measured with CellProfiler software ( <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036438#pone.0036438-Carpenter1" target="_blank">[32]</a> (<a href="http://www.cellprofiler.org" target="_blank">www.cellprofiler.org</a>), version r10997. Image analysis was performed using fifteen 5× mosaic images per well. Results presented are the means of 2–3 experiments. Black dots, IC50 obtained by individual laboratories in the PBMC assay; red squares, IC50 obtained in the plaque reduction assay.</p

Neutralization assays and their characteristics.

<p>Cell target: PBMC, peripheral blood mononuclear cells; the cell lines GHOST, U87 and HeLa are stably transfected with CD4 and CCR5 or CXCR4. MR, multiple round infection; SR, single round infection. The fusion assay is limited to cell surface-viral envelope interaction. Ab persistence: time of incubation of the inhibitory reagent with virus and cells before washout. Day: time of read-out, numbers indicate days; hr, hours. Env plasmid, Env expression plasmids obtained through NIBSC.</p

Mean inhibitory concentration (IC) 50 values for duplicate assays performed with TriMab and virus as indicated in the NeutNet Phase I (P1) and Phase II (P2) study.

<p>The cells are colour coded: green, poor or no neutralization, IC50>25 µg/ml; yellow, IC50 5–25 µg/ml; orange, IC50 1–5 µg/ml; red, IC50<1 µg/ml; white, no results available. Assays are grouped on the basis of several criteria: (1) the use of plasmids or culture supernatants as a source of HIV-1; (2) fusion based assays or infection based assays, either with pseudotyped virus or replication competent virus; and (3) the use of cell lines or PBMC. Laboratories performing the assays are numbered (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036438#pone-0036438-g001" target="_blank">Figure 1</a> for reference) and colour coded: blue, TZMbl assay or PSV/plasmid assays; green, PBMC assays using extracellular p24 as readout; pink, plaque reduction assay. In the listing of viruses, to the left, the cells of X4 viruses are labelled grey, the cells of R5 viruses are white.</p

Inter-Laboratory comparisons IC50 values.

<p>Values of the IC50s are expressed as reciprocal dilutions for plasma and as µg/ml for TriMab. N lab; Number of laboratories involved.</p

List of inhibitory reagents selected.

<p>List of inhibitory reagents selected.</p

Comparison of PSV and VI assays across viruses.

<p>(A) circular “radar” plots. Lines from the centre represent an axis for each virus. The geometric mean IC value for PSV (blue lines) and PBMC (red lines) against each virus is plotted, and the points joined. The scale is set such that the centre represents no neutralization and the concentric grid-lines are 2-fold dilution steps moving out to highest neutralization at the edge. (B) and (C) Ranking of viruses for relative sensitivity to neutralization was done by calculating geometric mean IC50s across laboratories (grouping PSV and PBMC separately). (B) Ranking by TriMab and (C) ranking by plasma (means over ARP515-522). The scale is set such that the most neutralization sensitive viruses are at the top.</p

Comparison of PSV and VI assays across plasma by circular “radar” plots.

<p>The scales were adjusted such that no neutralization (IC50<20) is at the centre, and the outer ring is strong neutralization (IC50>1280). The concentric grid-lines are 2-fold dilution steps. Lines from the centre represent an axis for each plasma. The geometric mean IC value for PSV (blue lines) and PBMC (red lines) against each plasma is plotted, and the points joined.</p