Changes in the number of EPO-R molecules per cell, <i>EPOR</i> gene expression and Sp1 expression in CD4⁺ lymphocytes before and after stimulation with anti-CD3 antibody.

<p>The figure shows results from 3 different individuals, MFI – mean fluorescence intensity, RFU – relative fluorescence units.</p

Comparison of GATA1, GATA3 and Sp1 expression determined by Western Blot in isolated CD4⁺ lymphocytes before and after stimulation with anti-CD3 antibody.

<p>Figure A shows representative results. Figure B presents mean expression of GATA1, GATA3 and Sp1 in arbitrary densitometric units. Figures C, D and E present expression of GATA1, GATA3 and Sp1, respectively. Midpoints of figures present medians, boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data, *p<0.05, Friedman ANOVA and Post Hoc test.</p

Fluorescence analysis of EPO-R and Sp1 expression measured by flow cytometry in CD4⁺ lymphocytes before and after stimulation with anti-CD3 antibody.

<p>CD4⁺ cells were selected on the basis of forward and side scatter characteristics of lymphocytes, <i>Gate 1</i> (A, E) and expression of CD4 antigen in gated lymphocytes, <i>Gate 2</i> (B, F). Figures C and D present the expression of EPO-R and Sp1 in CD4⁺ cells (cells from <i>Gate 1</i> and <i>Gate 2</i>) before stimulation, figures G and H present the expression of EPO-R and Sp1 in CD4⁺ cells stimulated with anti-CD3 antibody for 48 hours, respectively. For individual samples, expression of EPO-R and Sp1 was estimated as mean fluorescence shift (black line) toward isotype control (gray histogram): δMFI = MFI of the population of interest – MFI of the appropriate isotype control. Quantitative fluorescence analysis assessing the number of EPO-R molecules per cell based on δMFI was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060326#pone.0060326-Lisowska1" target="_blank">[3]</a>.</p

Similar proportions of m-calpain-positive cells and amounts of m-calpain in ALL blasts and control cells.

<p>A. Representative two-parameter plots (dot plots) resulting from simultaneous staining of BM samples (left panel–ALL, right panel–control) with anti-CD19 and anti-m-calpain antibodies. Actual corrected MFI values for calpain signal in CD19⁺ cells are shown. Details in Materials and Methods. B, C. No significant difference between the proportion of m-calpain-positive cells among ALL blasts and nonmalignant BM B cells (B) and between amount (MFI) (C). Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. N(ALL) = 6, N(control) = 6. For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

The amounts of calpastatin differ between CD19⁺ ALL blasts and non-malignant B cells.

<p>A. Representative two-parameter plots (dot plots) resulting from simultaneous staining of BM samples (left panel–ALL, right panel–control) with anti-CD19 and anti-calpastatin antibodies. Actual corrected MFI values for calpastatin signal in CD19⁺ cells are shown. B. No significant difference between the proportions of calpastatin-positive cells among ALL blasts and nonmalignant BM B cells. C. Significantly lower calpastatin amount (MFI) in the blasts (C). Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. N(ALL) = 30, N(control) = 17. For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Expression of μ-calpain but not other CCS genes is different in ALL blasts and nonmalignant BM B cells.

<p>A. Significantly higher expression of <i>CAPN1</i> (μ-calpain) gene in ALL blasts compared to control B cells. B,C. No differences between expression of <i>CAPN2</i> (m-calpain) and <i>CAST</i> (calpastatin) genes in ALL blasts vs non-malignant B cells. Please mark huge variability of expression of both the <i>CAPN1</i> and especially <i>CAST</i> genes. CCS gene expression is shown as proportion of the expression of <i>GAPDH</i> housekeeping gene considered 1. Box-and-whisker plots depict the means, SEM and SD respectively. P values (Kruskall-Wallis test) are given in the graphs; N(ALL) = 6, N(control) = 6. For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Endogenous calpain activity is present in ALL blasts.

<p><b>A</b>. Representative result of western blot determinantion of calpastatin and its immunoreactive fragments resulting from calpain activity in non-malignant BM CD19⁺ cells (lane 1), ALL blasts from a 12-year old patient (lane 2) and blasts from the same patient as in lane 2, but incubated in vitro for 24 hours with 4 μM calpain inhibitor IV (lane 3). Actin was used as a reference protein. For further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>. B. Endogenous calpain activity in ALL blasts measured by degree of calpastatin degradation (loss of the native form) is significantly higher in the children more than 10 years old. Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. Asterisk signifies p = 0.01; N (1–10 years old ALL patients) = 27, N(>10 years old ALL patients) = 10.</p

Proportions of μ-calpain-positive cells and relative amounts of μ-calpain are elevated among ALL blasts.

<p>A. Representative two-parameter plots (dot plots) resulting from simultaneous staining of BM samples (left panel–ALL, right panel–control) with anti-CD19, anti-CD34 and anti-μ-calpain antibodies. Actual corrected MFI values for calpain signal in CD19⁺ cells are shown. B. Significant difference between the proportion of μ-calpain-positive cells among ALL blasts and nonmalignant BM B cells. Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. Asterisk signifies p = 0.02; N(ALL) = 20, N(control) = 9. C. Amount of μ-calpain is significantly higher in ALL blasts than in nonmalignant B lymphocytes. Comparison of relative intensities (MFI) of μ-calpain–bound antibody in CD19⁺ ALL blasts (ALL) and non-malignant B cells (control). Asterisk denotes p = 0.03; N(ALL) = 16, N(control) = 9). For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Levels of spontaneous apoptosis of ALL blasts depend on patient age and μ–calpain amount.

<p>Apoptosis was determined as the proportion of AnnexinV+ blasts and plotted against the amount of μ–calpain in the blasts (A, N = 17, r = -0.54, p< 0.05) and against patients’ age (B, N = 39, r = -0.31, p< 0.05). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a> for details. When the patients were subdivided into below and above 10 years of age subgroups, the latter were characterized by significantly lower apoptosis (C, asterisk signifies p = 0.01) and significantly higher proportion of the μ–calpain positive blasts (D, asterisk signifies p = 0.04). N (1–10 years old ALL patients) = 29, N(>10 years old ALL patients) = 10.</p

Baltic cyanobacteria – a source of biologically active compounds

<div><p>Cyanobacteria are effective producers of bioactive metabolites, including both acute toxins and potential pharmaceuticals. In the current work, the biological activity of 27 strains of Baltic cyanobacteria representing different taxonomic groups and chemotypes were tested in a wide variety of assays. The cyanobacteria showed strain-specific differences in the induced effects. The extracts from <i>Nodularia spumigena</i> CCNP1401 were active in the highest number of tests, including protease and phosphatase inhibition assays. Four strains from Nostocales and four from Oscillatoriales increased proliferation of mitogen-stimulated human T cells. In antimicrobial assays, <i>Phormidium</i> sp. CCNP1317 (Oscillatoriales) strongly inhibited the growth of six fouling Gammaproteobacteria. The growth of monocotyl <i>Sorghum saccharatum</i> was inhibited by both toxin-producing and ‘non-toxic’ strains. The Baltic cyanobacteria were also found to be a rich source of commercially important enzymes. Among the 19 enzymatic activities tested, alkaline phosphatase, acid phosphatase, esterase (C4 and C8), and naphthol-AS-BI-phosphohydrolase were particularly common. In the cyanobacterial extracts, different peptides which may have been responsible for the observed effects were identified using LC-MS/MS. Their structures were classified to microcystins, nodularins, anabaenopeptins, cyanopeptolins, aeruginosins, spumigins and nostocyclopeptides.</p></div