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 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

The Impact of Chain Length and Flexibility in the Interaction between Sulfated Alginates and HGF and FGF‑2

Alginate is a promising polysaccharide for use in biomaterials as it is biologically inert. One way to functionalize alginate is by chemical sulfation to emulate sulfated glycosaminoglycans, which interact with a variety of proteins critical for tissue development and homeostasis. In the present work we studied the impact of chain length and flexibility of sulfated alginates for interactions with FGF-2 and HGF. Both growth factors interact with defined sequences of heparan sulfate (HS) at the cell surface or in the extracellular matrix. Whereas FGF-2 interacts with a pentasaccharide sequence containing a critical 2-O-sulfated iduronic acid, HGF has been suggested to require a highly sulfated HS/heparin octasaccharide. Here, oligosaccharides of alternating mannuronic and guluronic acid (MG) were sulfated and assessed by their relative efficacy at releasing growth factor bound to the surface of myeloma cells. 8-mers of sulfated MG (SMG) alginate showed significant HGF release compared to shorter fragments, while the maximum efficacy was achieved at a chain length average of 14 monosaccharides. FGF-2 release required a higher concentration of the SMG fragments, and the 14-mer was less potent compared to an equally sulfated high-molecular weight SMG. Sulfated mannuronan (SM) was subjected to periodate oxidation to increase chain flexibility. To assess the change in flexibility, the persistence length was estimated by SEC-MALLS analysis and the Bohdanecky approach to the worm-like chain model. A high degree of oxidation of SM resulted in approximately twice as potent HGF release compared to the nonoxidized SM alginate. The release of FGF-2 also increased with the degree of oxidation, but to a lower degree compared to that of HGF. It was found that the SM alginates were more efficient at releasing FGF-2 than the SMG alginates, indicating a greater dependence on monosaccharide identity and charge orientation over chain flexibility and charge density

Heparin-Like Properties of Sulfated Alginates with Defined Sequences and Sulfation Degrees

Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-d-mannuronic acid (M) and α-l-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values

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

PDL1 Expression on Plasma and Dendritic Cells in Myeloma Bone Marrow Suggests Benefit of Targeted anti PD1-PDL1 Therapy

<div><p>In this study we set out to investigate whether anti PDL1 or PD–1 treatment targeting the immune system could be used against multiple myeloma. DCs are important in regulating T cell responses against tumors. We therefore determined PDL1 and PDL2 expression on DC populations in bone marrow of patients with plasma cell disorders using multicolour Flow Cytometry. We specifically looked at CD141⁺ and CD141^- myeloid and CD303⁺ plasmacytoid DC. The majority of plasma cells (PC) and DC subpopulations expressed PDL1, but the proportion of positive PDL1+ cells varied among patients. A correlation between the proportion of PDL1⁺ PC and CD141⁺ mDC was found, suggesting both cell types could down-regulate the anti-tumor T cell response.</p></div

Expression of PDL1 on PC and monocytes in myeloma bone marrow.

<p>(A) PDL1 on plasma cells: Bone marrow cells were stained with antibodies against CD45, CD138, CD38, CD19, and CD274 (PDL1). Gates were set on FSC and SSC and doublets and CD19+ cells were excluded. Gating strategy is shown in Fig A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139867#pone.0139867.s002" target="_blank">S2 File</a>. The distribution of % PDL1⁺ PC in the bone marrow of patients (n = 14) is shown. (B) Proportion of PDL1⁺ PC does not increase with tumor load. The % PDL1⁺ gated CD38⁺CD19^- PC versus % bone marrow plasma cells is plotted. Each dot represents one patient. P values were calculated from a Spearman’s test (n = 14). (C) PDL1 on monocytes and DCs: Bone marrow cells were stained with antibodies against lineage (CD3, CD19, CD56, CD138, CD15, CD34, and CD235a), CD45, HLADR, and CD11c. The gating strategy is shown in Supplementary S1B Fig. Gates were set on FSC and SSC, doublets excluded, and gates further set on lineage- CD45⁺cells. Figure shows distribution of % PDL1+ monocytes/DC in the bone marrow of patients (n = 14). (D) Correlation of % PDL1+ PC and monocytes/DC; % PDL1⁺CD11c⁺DR⁺ monocytes/DC versus % PDL1⁺CD38⁺CD19^- plasma cells is plotted. Each dot represents one patient. P value was calculated from a Spearman’s test.</p

DC subtypes express PDL1 in myeloma bone marrow.

<p>Bone marrow and blood were stained with antibodies against CD141, lineage (CD3, CD19, CD56, CD138, CD15, CD34, and CD235a), CD45, HLADR, CD303, CD1c, and CD11c. The gating strategy is shown in Fig D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139867#pone.0139867.s002" target="_blank">S2 File</a>). Three DC populations were analysed; CD141⁺ (CD141⁺DC) (panels A-C), CD141^- (CD141^-DC) (panels D-F), and CD303⁺DC (pDC) (panels G-I). PDL1 staining on one representative patient (panels A, D, G). Fluorescence minus one (FMO), (dotted line), was used as negative control and the percentage indicates PDL1⁺ cells of the gated DC population. Panels B, E, and H show percentage of PDL1⁺ cells within the (B) CD141⁺ DC, (E) CD141^- DC and (H) CD303⁺ pDC populations in the bone marrow (n = 19), blood (n = 8) from patients, or blood from age matched (median age 61) healthy controls (n = 9). (median age of patients 61). Statistical analysis was performed with Mann Whitney Test. Panels C, F, and I show concomitant expression levels on bone marrow DC subtypes and plasma cells in individual patients. Each dot represents one patient. P values were calculated from Spearman’s tests.</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