The protein concentration of PI incubation medium influences the effect of PI on prelamin A processing in PBMC.

<p>PBMC were incubated for 24 hours with increasing concentrations of lopinavir (0, 2, 20, 40, 200 µM) diluted in plasma (total protein concentration: 60–80 g/L), in RPMI culture medium supplemented with 10% FBS and 2 mM L-glutamine (total protein concentration: 4 g/L), or in the RPMI culture medium supplemented with BSA (total protein concentration: 40 g/L). (<b>A</b>) Percentage of viable cells in plasma (white), culture medium (black), and BSA supplemented culture medium (gray) (error bars = SD, n ≥3, at least 200 cells counted in each experiment). **p<0.01. No viability changes were observed when PBMC were incubated in plasma containing lopinavir. A decrease in cell viability was apparent when PBMC were incubated in culture medium containing 20 µM or 40 µM PI. Only a slight decrease in viability was observed in cells incubated in BSA supplemented culture medium containing 200 µM PI. (<b>B</b>) Western blotting experiments. Both plasma and BSA supplemented culture medium containing 2 to 40 µM lopinavir had no effect on prelamin A maturation in PBMC. Prelamin A (anti-lamin A/C H110) was detected (*) in culture medium containing 20 µM and 40 µM lopinavir, and in BSA supplemented culture medium containing 200 µM lopinavir. Fibroblasts were cultured in the presence or absence of either 20 µM lopinavir (farnesylated prelamin A positive control) or 60 µM ZoPra (unfarnesylated prelamin A positive control) for 72 hours. GAPDH was used as total cellular protein loading control. (<b>A</b>) Western blot comparing the three lamin A/C antibodies used (N18, sc6215; H110, sc20681; Jol2, MAB3211). All antibodies recognized both lamin A and lamin C. Different amounts of farnesylated prelamin A were detected by N18 and H110, as shown by the ratio of prelamin A reported to the total prelamin A+lamin A signal. In the same conditions, Jol2 did not detect prelamin A. (<b>B</b>) Western blot comparing the two prelamin A antibodies tested (sc6214, ANT0045). The sc6214 antibody exhibited a higher affinity for both farnesylated and unfarnesylated prelamin A than the ANT0045 antibody. (<b>C</b>) Prelamin A, lamin A and lamin C protein domains and antibody epitopes. Lamin A/C N18 (sc6215, green); lamin A/C H110 (sc20681, blue); lamin A/C Jol2 (MAB3211, purple); prelamin A sc6214 (pink); prelamin A ANT0045 (orange).</p

Lack of prelamin A in PBMCs from compliant patients receiving the 2NRTI+1PI/r regimen.

<p>(<b>A)</b> Plasma concentrations of lopinavir, atazanavir and ritonavir from ANRS EP45 “Aging” patients reported at M0 (circles), M12 (square) and M24 (triangle). Colors indicate the durations between last drug uptake and venipuncture (Cmax, red; Cmin, green, Cintermediate, brown) and PI therapeutic concentration range (Cmax, red; Cmin, green). The majority of patients receiving lopinavir treatment were assayed at Cmin and remained within the therapeutic range. Samples from patients being treated with the atazanavir regimen were mainly assayed at mid-dose. (<b>B</b>) Representative western blots of PBMCs from patients receiving lopinavir or atazanavir treatment using three different lamin A/C-specific antibodies and one prelamin A-specific antibody. No prelamin A was detected in PBMCs from patients. Control PBMCs from healthy controls incubated with ZoPra were used as positive control cells. PI plasma concentrations in the patients shown: lopinavir 10.7 µM (M0), 12.3 µM (M12) and 9.3 µM (M24); atazanavir 1.5 µM (M0), 0.7 µM (M12) and 0 µM (M24).</p

Correlation between Lamin A expression in metastatic lung adenocarcinoma cells and number of metastatic sites and WHO Performance status.

<p>(A) The number of metastasis sites was inversely correlated with lamin A expression (p = 0.04; Pearson test). (B) The patients repartition was statistically different according the lamin A expression group (p = 0.037; Chi-square test). PS = Performance status. (C) The WHO Performance status was inversely correlated with lamin A expression (p = 0.01; Spearman test).</p

Expression pattern of lamins in lung adenocarcinoma cells from metastatic pleural effusions.

<p>(A) Representative results of western blot analysis of nuclear extracts from patients (Pt) 5, 9, 10, 11, and 16 and from total proteins extracts of control dermal fibroblasts using a mouse anti-lamin A/C antibody (Jol2). (B) Analyses of lamin A levels quantified after normalization with the total amount of lamins A and C showed two lamin A expression groups (p<0.001; Mann–Whitney test).</p

Immunofluorescence staining of lamins A and C in cells from metastatic pleural effusions.

<p>Cells from metastatic pleural effusions of lung adenocarcinoma from high (Patient (Pt) 51) and low (Pt 58) lamin A expression group were stained with lamin A and lamin C antibodies. DAPI: counterstained nuclei. White stars: dyskaryotic nuclei of cancer cells. Orange arrows: cancer cells with a high lamin A expression. White arrows: cancer cells with a low lamin A expression. Asterisk: normal leukocytes.</p

Lamin A and EMA expression in adenocarcinoma cells.

<p>Lamin A and EMA expression in adenocarcinoma cells.</p

Correlation between mitochondrial functional parameters and viral load in cells from ART naive patients.

<p>(<b>A</b>) ROS production by high-ROS lymphocytes (r = 0.355, p = 0.029, n = 38). (<b>B</b>) ΔΨm of high-ΔΨm lymphocytes (r = −0.417, p = 0.007, n = 40). (<b>C</b>) Percentage of low-ΔΨm lymphocytes (r = 0.457, p = 0.003, n = 40). (<b>D</b>) ROS production by high-ROS monocytes (r = 0.164, p = 0.288, n = 44) (<b>E</b>) ΔΨm of monocytes (r = −0.455, p = 0.003, n = 40).</p

Mitochondrial morphological parameters: differential effects of HIV-1 infection and ART on lymphocytes and monocytes.

<p>(<b>A, E</b>) Discriminant analysis using three mitochondrial morphological parameters. The five cohort populations are delineated by their 95% confidence circles around the means. The two perpendicular axes describe the combined variance of the parameters analysed (lymphocytes: 90.66%, monocytes: 98.73%). The contributions of each parameter to the variances on the X and Y axes are shown in the insets. Inset: The 3 mitochondrial parameters used were: Vv%: Volume density (lymphocytes: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041129#pone-0041129-g005" target="_blank">Figure 5B</a>; monocytes: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041129#pone-0041129-g005" target="_blank">Figure 5F</a>). Frag: Fragmentation (lymphocytes: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041129#pone-0041129-g005" target="_blank">Figure 5C</a>; monocytes: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041129#pone-0041129-g005" target="_blank">Figure 5G</a>). Bran: Branching (lymphocytes: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041129#pone-0041129-g005" target="_blank">Figure 5D</a>; monocytes: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041129#pone-0041129-g005" target="_blank">Figure 5H</a>). (<b>A</b>) Lymphocytes ART naive patients and control subjects are statistically different. Mitochondrial changes induced by HIV infection are partially reduced by ART. (<b>E</b>) Monocytes ART naive patients exhibit no variations compared to control subjects, while significant differences are observed for the three ART groups. (<b>B to D; F to H</b>) Box plots: *p<0,05, **p<0,01.</p

Pathological and clinical characteristics of patients, according lamin A expression.

<p>Pathological and clinical characteristics of patients, according lamin A expression.</p

Flow cytometric analysis of ROS production revealed two subpopulations of both CD14+ monocytes and lymphocytes.

<p>(<b>A to C</b>) Monocytes. (<b>A</b>) CD14+/SSC monocyte gating. (<b>B</b>) Representative zebra plot based on side scatter and ROS production showing low- and high-ROS producing cell subpopulations. (<b>C</b>) Histogram illustrating ROS production as a function of cell count (low-ROS production, gray; high-ROS production, red). ROS production levels (MFI ± SD) are indicated as percentages relative to CD14+ monocytes. The percentages of high-ROS monocytes were not statistically different between the groups: Control: 75.8±21.7%; ART naive: 70.7±19.4%; 2NRTI+1PI/r: 71.1±21.1%; 2NRTI+1NNRTI: 71.0±22.8%; 3NRTI: 66.2±23.7%. (<b>D to F</b>) Lymphocytes. (<b>D</b>) FSC/SSC lymphocyte gating. (<b>E</b>) Representative zebra plot based on side scatter and ROS production showing low- and high-ROS producing cell subpopulations. (<b>F</b>) Histogram illustrating ROS production as a function of cell count (low-ROS production, gray; high-ROS production, red). ROS production levels (MFI ± SD) are indicated as percentages relative to FSC/SSC-selected lymphocytes. The percentages of high-ROS lymphocytes were not statistically different between the groups: Control: 55.4±12.7%; ART naive: 48.0±14.1%; 2NRTI+1PI/r: 55.1±13.8%; 2NRTI+1NNRTI: 50.1±14.1%; 3NRTI: 47.6±15.0%. ROS production by the low- and high-ROS subpopulations was statistically different in both HIV and ART patients.</p