SIMS imaging of boron in B16 cells exposed <i>in vitro</i> to <i>cis</i>-ABCPC.

<p>The cells were treated with 30 ppm boron equivalent of <i>cis</i>-ABCPC for 2.5 hr (A-C) and for 1 hr in presence of 50 µM bromodeoxyuridine (BrdU) for the detection of DNA-synthesizing S-phase cells among the asynchronous population by imaging of ⁸¹Br⁻ signals with SIMS (D-F). The positive secondary ion SIMS images of ³⁹K, ⁴⁰Ca, and ¹¹B represent the subcellular distributions of potassium, calcium, and boron, respectively, in individual B16 cells (A-C). The ²³Na image also was recorded (not shown) and it revealed the K/Na ratio of ∼10∶1 in these cells. D-F represent SIMS analyses in <i>cis</i>-ABCPC and BrdU treatment. For the imaging of S-phase cells, the SIMS instrument was first operated in the negative secondary detection mode for imaging of ⁸¹Br⁻ signals and then the mode of detection was changed to the positive secondaries in the same field of analysis. The ³⁹K images were integrated on the CCD camera for 0.2 sec. each. The ⁴⁰Ca, ¹¹B, and ⁸¹Br⁻ images were integrated for 2 min. each.</p

SIMS imaging analysis of ³⁹K and ¹¹B distributions in B16 melanoma.

<p>The B16 melanoma bearing mouse was euthanized 1 hr after i.p. administration of <i>cis</i>-ABCPC. The photomicrograph is an H&E stained 4µ cryosection of the B16 melanoma. SIMS images were recorded from the adjacent 4µm thick cryosections. The SIMS ³⁹K⁺ image was integrated on the CCD camera for 0.2 sec and ¹¹B⁺ image for 2 min.</p

Quantitative SIMS imaging analysis of boron concentrations in tumor cells from B16 melanoma bearing mice.

a<p>Four mice in each treatment received 24 mg boron/kg b.w. <i>cis</i>-ABCPC <i>via</i> injection. After 1, 2.5, and 4 hr. post injection, the mice were euthanized and samples of tumor i.p. were frozen for SIMS studies. The boron concentrations from SIMS images, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075377#pone-0075377-g002" target="_blank">Fig. 2</a>, are expressed in µg/g wet wt. (mean ± SD).</p>b<p>In the 1 hr treatment group, observations represent 31 regions of interest (ROIs) in 5 SIMS imaging fields. A region of interest within a SIMS imaging field is defined as a clump of 10-15 individual cells taken together for quantification of SIMS images and, therefore, representing a large sampling of the imaging data. In 2.5 hr treatment, observations represent 47 ROIs in 7 SIMS imaging fields. In 4 hr treatment, observations represent 51 ROIs in 7 SIMS imaging fields. The superscript “c” denotes the significant difference (P<0.05) in boron concentrations from treatments designated with superscript “d”.</p

SIMS analyses of brain from F98 glioma bearing rats.

<p>Rats received <i>cis</i>-ABCPC by i.p. at a dose of 250 mg/kg b.w. and were euthanized 2.5 hr. later. The morphology of F98 glioma is shown in H & E stained cryosections (A-B). The main tumor mass (TM), normal brain tissue (BT), clusters of tumor satellite cells (TS) and individual infiltrating tumor cells (TC) are discernible in normal brain tissue (A). The optical image (B) shows a vessel (V) which is surrounded by TC and BT. SIMS analyses were made in adjacent cryosections. SIMS imaging analyses of boron from two different tissue regions are shown in C-D and E-F, respectively. In C-D, a subtle gradient of ³⁹K⁺ in the SIMS image identified the boundary of the TM from the BT which contains a TS and TC’s in panel C. The respective boron microdistribution from <i>cis</i>-ABCPC is shown in the ¹¹B⁺ boron SIMS image in panel D: the higher concentrations of boron in TM, TS, and TC’s are revealed in comparison to the BT. In a second example of the SIMS imaging of another tissue region (E-F), a gradient of ²⁴Mg⁺ is used for the identification of TS and infiltrating TC’s in the normal brain tissue which also contains a vessel (V) surrounded by TC’s (E). The respective ¹¹B⁺ SIMS image from this region, shown in F, revealed higher concentrations of boron in TS as well as the infiltrating TC’s in the normal brain tissue surrounding the vessel (V). The image integration times on the CCD camera for SIMS images of ³⁹K⁺ and ¹¹B⁺ in C and D were 0.2 sec and 2 min, respectively. The image integration times on the CCD camera for SIMS images of ²⁴Mg⁺ and ¹¹B⁺ in E and F were 1 min and 2 min, respectively.</p

IDB induces megakaryocytic differentiation of human CD34+ progenitors whereas PMA does not.

<p><b>(A–C, upper panels)</b> Morphology of adult human CD34+ hematopoietic progenitors cultured for 7 days in medium containing TPO and SCF alone or with 10 nM IDB or 10 nM PMA on Wright-stained preparations. Note that relative to control cells, cells treated with IDB were larger and had polyploid nuclei. <b>(A–C, lower panels)</b> Adult human CD34+ progenitors were treated with TPO and SCF with or without 10 nM IDB or 10 nM PMA. After 7 days of culture, the cells were stained with a fluorescent antibody directed against CD41a (open profiles) or with an isotype matched control antibody (black profiles). All flow cytograms in this and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051059#pone-0051059-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051059#pone-0051059-g003" target="_blank">3</a> are representative of at least 3 independent runs. (D) Adult human CD34+ progenitors were treated with TPO and SCF with 25 nM IDB, 10 nM PMA, 100 nM PMA, or 1 µM Mezerein. After 7 days of culture, the cells were stained with a fluorescent antibody directed against CD41a (open profiles) or with an isotype matched control antibody (black profiles).</p

IDB induces early expression of promegakaryopoietic transcription factors.

<p>(<b>A</b>) CD34+ cells cultured in TPO/SCF or EPO/SCF for 12 days and subjected to Western blot analysis. (<b>B</b>)TPO/SCF +/− 100 nM IDB for 72 hrs and subjected to western blot analysis. Blots were then stripped and reprobed for each of the indicated proteins.</p

Inhibitor studies support that CD9 induction by IDB is mediated by PKCε.

<p>(<b>A</b>) CD34+ cells were cultured in TPO/SCF for 3 days with or without 100 nM IDB +/− 5 µM of GF109203X or 1 µM Go6976 and analyzed for CD9 (open panel) or isotype control (shaded panel) by flow cytometry. (<b>B</b>) CD34+ cells were cultured in TPO/SCF for 2 hours with or without 100 nM IDB +/− GF109203X or Go6976 and subjected to western blot analysis for the indicated antigens. (<b>C</b>) CD34+ cells were cultured for 48 hours in media containing TPO/SCF. Cells were then nucleofected as per the manufacturer's protocol for CD34+ cells with either scrambled or PKCε siRNA. After a 2 hour recovery, the cells were treated with either vehicle or 25 nM IDB for an additional 24 hours and mRNA was extracted and analyzed by PCR for CD9, PKCε, and GAPDH expression.</p

IDB induction of ERK activation, egr-1, and CD9 in CD34+ cells.

<p>(<b>A&B</b>) CD34+ cells were thawed and recovered overnight in serum-free medium supplemented with SCF (25 ng/ml). Cells were stimulated with TPO (40 ng/ml) with 25 nM IDB or vehicle. mRNA for CD9 and egr-1 were quantitated using SYBR green and real time PCR. The values shown in A and B represent the mean of 2–3 replicate determinations. (<b>C</b>) CD34+ cells were recovered overnight as described above. Cells were stimulated with TPO with or without IDB as indicated. Immunoblot analysis of phospho-ERK and total ERK then were performed on whole cell lysates. (<b>D</b>) CD34+ cells were cultured in serum-free media and TPO/SCF with or without 100 nM IDB+/− 10 µM of the MEK inhibitor U0126 for 3 days and then analyzed for CD9 expression by flow cytometry.</p

Boron concentration in wet weight in nuclei and cytoplasm of B16 melanoma cells <i>in vitro</i> determined by SIMS imaging analysis.

a<p>In each treatment, more than 30 cells were analyzed in 5–7 SIMS imaging fields for quantitative analysis.</p

Single i.p. injection of IDB increases platelet counts and mitigates thrombocytpoenia induced by irradiation.

<p>(<b>A</b>) A single i.p. injection of IDB increased platelet counts at day 7 compared with vehicle injected control animals (N = 5 per group; *p<0.01). No differences were observed in hemoglobin and white blood counts (p = NS). (<b>B</b>) Groups of BALB/c mice (N = 10/group/time point) were injected with a single i.p. dose of IDB (720 or 1080 µg/kg body weight) 3 hours before irradiation with 6 or 8 Gy. At two weeks the mice treated with 6 Gy were euthanized and complete blood counts were performed. At three weeks the mice treated with 8 Gy were euthanized and complete blood counts were taken (* showed statistical significance of at least p<0.01 or greater).</p