SMAD-activation by recombinant GDF15 in myeloma cell lines.

<p>A. Phosphorylation of SMAD1/5 or SMAD2 was determined using immunoblotting in IH-1 cells treated with BMP-9 (0.5 ng/mL), activin A (25 ng/mL) or indicated concentrations of GDF15 (100–400 ng/mL) for 1 hour. B. INA-6 cells were treated with GDF15 (200 ng/mL) and the inhibitor SB431542 (0–2.5 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. C. INA-6 cells were transiently transfected with siRNAs targeting <i>ACVR1B/ALK4</i>, <i>ACVR1C/ALK7</i>, <i>TGFBR1/ALK5</i> and a non-targeting control siRNA. Two days after transfection the cells were treated with GDF15 (200 ng/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. D. Knock-down of receptors by siRNA in cells used in (C) as shown by QRT-PCR. Gene expression was calculated with the comparative ΔCt-method with <i>GAPDH</i> as housekeeping gene. The error bars indicate SEM of three independent experiments. Asterisks above bars indicate the degree of significance for downregulation of each gene compared to control (*, P≤0.05; **, P≤0.01; and ***, P≤0.001). E. INA-6 cells were treated with GDF15 (100 ng/mL) and a neutralizing TGFBR2 antibody (10–15 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. F. INA-6 cells were treated with GDF15 (100 ng/mL) and the indicated soluble receptors (5 μg/mL for all except endoglin, which was 1 μg/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. Antibody staining towards GAPDH was used as loading control for all Western blots. The experiments were performed 2–3 times each. GDF15 used in this figure was from R&D Systems, Lot# EHF1713081.</p

Activation of SMAD2 by recombinant GDF15 was caused by TGF-β.

<p><i>In vitro</i> differentiated macrophages (A) or THP-1 cells (B) were treated with increasing doses of recombinant GDF15 (R&D Systems, Lot# EHF0914051) or TGF-β for four hours. C. INA-6 cells were treated with increasing doses of TGF-β for 1 hour. D. INA-6 cells were treated for 1 hour with the indicated doses of TGF-β, GDF15 (Abcam) or GDF15 (Peprotech). E. INA-6 cells were treated for 1 hour with GDF15 (Peprotech) or TGF-β pre-treated with neutralizing antibodies targeting GDF15 or TGF-β. For C-E, the experiments were performed in RPMI with 0.1% bovine serum albumin (BSA). Phosphorylation of SMAD2 was determined using immunoblotting and GAPDH, ERK1/2 or SMAD2/3 antibodies were used as loading controls. All experiments were performed at least three times, except for D and E, which were performed twice.</p

SMAD-activation by recombinant GDF15 in THP-1-cells and <i>in vitro</i> differentiated macrophages.

<p>A. Monocytic THP-1 cells were treated with GDF15 (50, 100 or 200 ng/mL), BMP-9 (50 ng/mL) or activin A (100 ng/mL) for 4 hours. B. THP-1 cells were treated with GDF15 (100 ng/mL) for various time-points. C. <i>In vitro</i> differentiated macrophages were treated with indicated soluble receptors in the presence of TGF-β (1 ng/mL) or GDF15 (200 ng/mL) for four hours. Phosphorylation of SMAD2 was determined using immunoblotting and GAPDH was used as loading control for all Western blots. Each experiment was performed once. GDF15 used in this figure was from R&D Systems, Lot# EHF1713081.</p

APIM and PIP-box peptides have overlapping binding site on PCNA.

<p>(A) Protein sequence and structural model of PCNA (PDB entry 1vym) with M40 highlighted in red and the center loop (CL) in yellow (upper panel). Live cell (HeLa) confocal fluorescence images of CFP-PCNA wild type (WT) and CFP-PCNA M40 mutants. Bar, 5 µm (lower panel). (B) Normalized FRET (N_FRET) measurements between WT and mutated CFP-PCNA M40/APIM-YFP (light grey diamonds, PCNA WT−/PCNA M40A−/PCNA M40N−/PCNA M40R−/PCNA M40S- APIM) and WT and mutated CFP-PCNA M40/PIP-YFP (dark grey diamonds, PCNA WT−/PCNA M40A−/PCNA M40N−/PCNA M40R/PCNA M40S- PIP). CFP/YFP (vectors only) was used as background control (open diamonds). Data is from three independent experiments (mean ± SEM, n = 72–214). P-values were calculated by the unpaired Student’s t-test.</p

ATX-101, a cell-penetrating APIM-peptide, targets PCNA.

<p>(A) Confocal fluorescence image of live HeLa cells 2 minutes after addition of fluorescently tagged ATX-101. Bar, 5 µm. (B) Cell growth measured by MTT assay of HeLa cells stably expressing YFP and APIM-(hABH2 _1–7 F4W)-YFP unexposed (♦ and×, respectively) and after continuous exposure to 0.5 µM cisplatin (▴ and •, respectively) (left panel) and parental HeLa cells unexposed (♦) and after continuous exposure to 8 µM ATX-101 (×), 0.5 µM cisplatin (▴), and combination of ATX-101 and cisplatin (•) (right panel). Data is from one representative experiment out of at least three. (C) Normalized FRET (N_FRET) measurements in HeLa cells between CFP-PCNA and APIM-YFP without and in the presence of ATX-101. The cells were treated with 8 µM ATX-101 8 h after transient transfection and incubated for 16 h before the N_FRET measurements. CFP/YFP (vectors only) was used as background control. Data is from three independent experiments (mean ± SEM, n = 36–40). P-value was calculated by the unpaired Student’s t-test. (D) Cell growth measured by MTT assay of HeLa cells unexposed (♦) and after continuous exposure to 8 µM ATX-A (—), 8 µM ATX-101 (×), 0.5 µM cisplatin (▴), and combination of ATX-A or ATX-101 and cisplatin (▪ and •, respectively). The confocal image shows fluorescently tagged ATX-A in HeLa cells as in (A). Bar, 5 µm. Data is from one representative experiment out of three.</p

ATX-101 induces apoptosis in the MM cell line JJN-3.

<p>(A–C) Flow cytometric measurement of the apoptotic cell population by annexin V-Pacific Blue labeling. (A) JJN-3 cells treated with 6 µM ATX-101 and 0.5 µM melphalan alone or combined were incubated for 1, 2, and 3 days. Control cells were left unexposed. (B and C) JJN-3 cells treated with 6 and 10 µM ATX-101 were incubated for 1, 2, and 4 h. In addition to annexin V labeling, cells were stained with DRAQ5 for DNA profile. (C) The histograms show the cell cycle distribution of live (blue) and apoptotic (pink) cells after 1 h of ATX-101 treatments. (A–C) show data from representative experiments out of three. (D) Flow cytometric measurement of caspase 8, 9, and 3/7 activity by Fluorescent Labeled Inhibitor of Caspases (FLICA) assay. JJN-3 cells were left unexposed and exposed to 8 µM ATX-101 for 2 and 4 h before the FLICA probe was added for staining. The FLICA probe binds irreversible only to the activated caspase and labels apoptotic cells. Data is from four independent experiments for caspase 8 activity and three independent experiments for caspase 9 and 3/7 activity (mean ± SD, ** P < 0.01, Student’s t-test).</p

ATX-101 inhibits cell growth of cancer cell lines.

<p>(A) Cell growth after ATX-101 addition in different cell lines measured by MTT assay. K562 (chronic myelogenous leukemia), CCRF-CEM (T-lymphoblast, acute lymphocytic leukemia), RPMI-8226 and JJN-3 (MM), HeLa (cervical cancer), PC3 and DU145 (prostate cancer), H460 (non-small cell lung carcinoma), HCT116 (colorectal carcinoma), A549 (non-small cell lung carcinoma), U2OS (osteosarcoma) and HaCaT (spontaneously immortalized keratinocyte) cells were left unexposed (♦) and exposed to 4, 6, 8, 10, and/or 12 µM of ATX-101 (▪, ▴, ×, —, and •, respectively). (B and C) Cell growth measured by MTT assay of the MM cell lines RPMI-8226 and JJN-3, respectively, unexposed (♦) and after continuous exposure to 6 or 4 µM of ATX-101 (×), 2 or 0.5 µM melphalan (▴), and combination of ATX-101 and melphalan (•). (A–C) Data is normalized to cell growth from untreated cells on day 1 and from one representative experiment out of at least three.</p

ATX-101 induces cancer cell specific apoptosis.

<p>Flow cytometric measurement of the apoptotic cell population by annexin V-Pacific Blue labeling. JJN-3 cells were treated with 4 and 8 µM ATX-101 for 2 h (left panel), and U937 cells were treated for 24 h (right panel). Lymphocytes freshly isolated from buffy coats (from blood donors) treated in parallel with JJN-3 and U937 are included as controls. Data is from representative experiments out of two.</p

DataSheet_1_Mutational analysis and protein profiling predict drug sensitivity in multiple myeloma cell lines.zip

IntroductionMultiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease.MethodsTo identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins.ResultsWe found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors.DiscussionTaken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.</p

Additional file 4: of Artesunate shows potent anti-tumor activity in B-cell lymphoma

Figure S3. The tumor load is evenly distributed among the treatment and control groups. (A) Tumor growth was monitored with IVIS luminescence measurements (physical units of surface radiance, photons/s/cm2/sr). Image was taken at day 0 when treatment was started (day 4 after injection of BL-41-luc). (B) Graphical distribution of luminescence measurements at treatment start. (C) Artesunate treatment does not affect the weight of the mice. An increased weight was observed for both groups during the treatment. Shown is weight (g) after treatment start (n = 10 for each group). Weight at start was 16.0-22.8 g and the mice were 6-10 weeks old. (EPS 3428 kb