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

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

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

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

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

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