CCNE1 over-expression.

<p>RPMI8226 a low-CCNE1-expressing cell line was stably transfected with pRCCMV2hCyclin E1. (A) Stable clones have been selected by addition of G418, and cell extracts of selected cloned were subjected to immunoblotting, utilizing CCNE1 antibodies. CDK2 expression serves as an internal loading control. (B) Stable clones have been selected by addition of G418, and cell extracts of selected cloned were subjected to qRT-PCR. Presented is the relative amount of CCNE1 as compared to the levels of GAPDH<b>.</b> Data are represented as mean±standard deviation<b>.</b> (C) CCNE1-overexpressing stable cell lines as well as paternal cell line were incubated in the absence or presence of increasing concentrations of seliciclib for 3 days. Cell viability was determined by MTT assay. Data are represented as mean±standard deviation. Experiments were performed twice and one representative result is presented.</p

Survival of ToI-β pancreatic islet grafts in allogenic SJL- mice.

<p>500 ToI-β islets were transplanted under the kidney capsule of allogeneic SJL mice. Rejection/graft survival was determined by tail blood glucose measurements. Graft rejection was defined as consecutive measurements of glycemia >200 mg/dl. (<b>A</b>) Untreated control mice (diamonds, n = 10); mice that were exposed to Dox 24 hours after transplantation (triangles, n = 9); and mice that were exposed to Bortezomib 24 hours after transplantation (circles, n = 8). Statistical analysis was done by Kaplan-Meier estimation and comparison of survival curves by the MedCalc logrank test. p = 0.004 Dox <i>vs</i> control, p = 0.001 BZB <i>vs</i> control. (<b>B</b>) Blood glucose of ToI-β islet grafts in untreated SJL control mice (black diamonds); mice exposed to Dox (triangles) and mice treated with Bortezomib (BZB, squares). Blood glucose areas under the curve (AUC) were smaller in treated <i>vs</i> untreated recipient mice: Dox <i>vs</i>. Control <i>p</i> = 0.025, BZB <i>vs</i> Control <i>p</i> = 0.01.</p

Real-time PCR analysis of NF-κB target genes in islet grafts 24 hours after transplantation.

<p>CXCL-10/IP-10 (*p = 0.03 Dox <i>vs</i> control) (<b>A</b>), MCP-1 (<b>B</b>), cIAP2 (<b>C</b>)<b>,</b> iNOS (<b>D</b>), A20 (<b>E</b>) and XIAP (<b>F</b>). mRNA was extracted from ToI-β islet grafts retrieved from the kidney capsule 24 hours after syngeneic transplantation. Prior to transplantation, islet grafts were exposed to Dox in the culture media for 48 hours (D) or untreated controls (C). The right columns represent relative gene expression in isolated, untreated islets (ISLETS). Results are shown as fold induction normalized to HPRT values. Only retrieved grafts with less than 5% kidney contamination were included in the study, as assessed by expression of the kidney tissue-specific NKT (novel kidney transcript) gene. Results are the mean ± SEM of three to five independent experiments.</p

Seliciclib downregulates MCL1 expression in hMMCLs.

<p>(A) The indicated multiple myeloma cell lines in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing MCL1 antibodies. In all experiments CDK2 expression serves as an internal loading control. Experiments were performed at least 3 times and one representative result is presented. (B–C) MCL1 downregulation by seliciclib. Cells were extracted and subjected to immunoblotting. (B) Various hMMCLs were incubated in the presence of seliciclib 50 µM or DMSO for 6 hours and the level of MCL1 expression was analyzed. (C) RPMI8226, CAG and NCI H929 cells were incubated in the absence or presence of seliciclib 50 µM for the indicated time points, or in the presence of increasing concentrations of seliciclib for 8 hours. (D) Reduction in MCL1 phosphorylation by seliciclib. Cells were treated with 50 µM or DMSO for 8 hours and the levels of total and phosphorylated MCL1 were analyzed by immunoblotting using specific antibodies. β-actin was used to confirm equal protein loading. (E) CAG cells were incubated in the absence or presence of seliciclib or MG132 (10 µM) exclusively or combined. MCL1 level of expression was verified by immunoblotting. β-actin was used to confirm equal protein loading.</p

Heterogenous resistancy to seliciclib in hMMCLs.

<p>(A) The indicated hMMCLs were incubated in the absence or presence of increasing concentrations of seliciclib for 3 days. Cell viability was determined by MTT assay. Data are represented as mean±standard deviation. Experiments were performed at least 3 times and one representative result is presented. Seliciclib resulted in a decrease in cell viability with an IC50 ranging from 25 to 90 µM. (B) Cell cycle analysis by PI staining was performed on hMMCLs incubated in the absence or presence of 50 µM seliciclib for 12 hours. Cells were collected, fixed, stained with propidium iodide (PI) and analyzed by flow cytometry. DNA distribution in the cells is presented. (C) Quantification of cell cycle stage analysis of control and seliciclib-treated (50 µM, 12 hours culture) hMMCLs. Analysis of representative lines of highly sensitive (NCI H929), a moderately-sensitive (ARP1) and a resistant (ARH77) lines are displayed. The subdyploid DNA peak (subG1) represents apoptotic cell fraction. Data are represented as mean±standard deviation of 3 different experiments. Probability values of t-test are presented **p<0.01.</p

CCNE1 expression profile in hMMCLS.

<p>(A) Various multiple myeloma cell lines (NCI H929, RPMI8226, CAG, ARP1 and U266) and plasma cell leukemia cell line ARH77 in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing CCNE1 antibodies. CDK2 expression served as an internal loading control. (B) Various multiple myeloma cell lines (NCI H929, RPMI8226, CAG, ARP1 and U266) and plasma cell leukemia cell line ARH77 in logarithmic growth phase were extracted and subjected to qRT-PCR in quadruplicates. Data are represented as mean±standard deviation of the ratio of CCNE1 and GAPDH between all hMMCLs and ARP1. (A–B) Experiments were performed at least 3 times and one representative result is presented.</p

Inhibition of NF-κB activation is associated with an increased endocrine/total graft area ratio in the islet graft.

<p><b>A.</b> The recipient graft-bearing kidneys from normoglycemic untreated mice, and Dox- or Bortezomib (BZB)-treated animals were removed 7 days after allogeneic transplantation, fixed in formaldehyde, thin-sliced and stained with hematoxylin/eosin solution. The border between the kidney and the graft is marked (Upper panel). Paraffin sections were stained for insulin (Lower panel). <b>B.</b> Using a fixed grid, the percentage of endocrine area was calculated from the total graft area. Results are the average of at least five non-consecutive sections incorporating the whole graft area. *p = 0.033 Dox <i>vs</i> control; **p = 0.0001 BZB <i>vs</i> control n = 3.</p

Seliciclib induces apoptosis in sensitive hMMCLs.

<p>hMMCLS were incubated in the absence or presence of 50 µM seliciclib. Following 5 hours or 16 hours of culture cells were collected, fixed, stained using annexin V/PI and analyzed by flow cytometry. The extent of apoptosis is expressed as a percentage of cells positively stained using annexin V. (A) Seliciclib-sensitive NCI H929 and seliciclib-resistant ARH77 were incubated in the presence or absence of seliciclib for the indicated time points. Experiments were performed at least 3 times and one representative result is presented. (B) Representative analysis of seliciclib-induced apoptosis following a 16 hour incubation time in control, DMSO and seliciclib treated cells. The percentage of live (annexin V/PI-negative) and apoptotic (annexin V-positive) cells is given. Data represents the mean and standard deviation of 3 different experiments. Probability values of t-test are presented *p<0.05 **p<0.01.</p

Medium nitrite levels secreted from islets exposed in vitro to IL-1β (50 units/ml) and IFN-γ (1,000 units/ml) for 48 h in the presence or absence of Bortezomib (BZB- 100

<p> <b>nM).</b> Nitrite data are pooled from two separate experiments incorporating 5–6 repeats of each treatment, presented as the mean ± SEM. *p value = 0.023 for BZB+Cytokines <i>vs</i> Cytokines.</p

Decreased IP-10 in doxycycline- or Bortezomib-treated islet grafts.

<p>Representative anti-CXCL10/IP-10 stained graft-bearing kidney sections from normoglycemic untreated mice (control), Dox- or Bortezomib (BZB)-treated animals, 7 days after allogeneic transplantation (Upper panel- Graft). As noted in control the cytoplasmic IP-10 staining is more intense in control islets than in Dox- or Bortezomib-treated mice. The lower panel represents IP-10 staining of kidney sections of the corresponding graft adjacent areas.</p