Utilizing BMP-2 muteins for treatment of multiple myeloma - Fig 5

<p>(A) A collection of different human multiple myeloma cell lines are Activin A-resistant. Ten different cell lines were incubated with or without 125 nM Activin A. After 72 h, cell proliferation was assessed by WST-1 measurements. (B) Expression profile of Activin A receptors in MM cell lines. RNA was isolated from the indicated MM cell lines, and the expression levels of the indicated receptors were determined by qRT-PCR. Expression levels were normalized to the housekeeping gene HPRT (= 100%). (C) Activin A sensitivity of primary CD138+ cells isolated from different MM patients. CD138+ cells were isolated from 8 different donors and stimulated with 125 nM Activin A for 72 h. The control cells were untreated. Cell numbers were assessed by WST-1 assay and related to the untreated cells (control). Assays were performed in duplicate (except for the values marked with a #). The figures show the mean values and standard deviation of duplicates.</p

BMP2 counteracts Activin A-induced inhibition of cell proliferation in INA6 cells.

<p>INA6 cells were incubated with Activin A alone (10 nM) or co-stimulated by the addition of Activin A and increasing concentrations of either wildtype BMP2 or BMP2-PKD. Cell numbers were assessed by WST-1 measurements. The black line represents the value obtained from the untreated cells (control).The dashed line indicates the IC50 (half maximal biological inhibitor activity. In the experiment the IC50 was determined as 250 nM for BMP2 and as 62.5 nM for BMP2-PKD. The figures show the mean values of triplicates with standard deviation. The measurements were performed as three independent experiments.</p

Activin A inhibits BMP2-mediated cellular responses.

<p>(A) KMS12-BM cells were stimulated with either 125 nM Activin A or 4 nM BMP2 alone or co-stimulated with both growth factors for 72 h. Cell growth was assessed by WST-1 measurements. (B) C2C12 cells were incubated without BMP2, 10 nM BMP2 and 10 nM BMP2 plus increasing concentrations of Activin A. After 72 h, ALP activity was measured by determining p-nitrophenylphosphate conversion using an ELISA reader. In the absence of BMP2 cells showed no ALP activity and the background signal was set as 0%. ALP activity derived from stimulation with 10 nM BMP2 was defined as 100% (dashed line). Addition of Activin A without BMP2 yielded no ALP activity (data not shown). The figures show the mean values and the standard deviation of triplicates. The assays were performed as three independent experiments.</p

Schematic representation of the BMP2 variants in terms of their receptor binding characteristics.

<p>The arrows demonstrate the relative binding affinities of the individual BMP2 variants to the indicated receptors. High-affinity binding is indicated by thick black arrows, while low-affinity binding is indicated by thin grey arrows. For both “P” variants, there was no binding (indicated by the grey cross) to the type I receptors BRIA and BRIB that could be detected by surface plasmon resonance (SPR) analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174884#pone.0174884.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174884#pone.0174884.ref028" target="_blank">28</a>]. Bold receptor names indicate binding preferences among the two receptor types.</p

BMP2, but not Activin A, downregulates relative cell number in the L363 and KMS12-BM myeloma cell lines.

<p><b>(A)</b> Cells were grown in the presence or absence of 125 nM BMP2 or Activin A for 72 h. Relative cell numbers were assessed by WST-1 measurements. (B) For a detailed analysis the dose response curve of BMP2 was determined for L363 (black circle line) and KMS12-BM (black circle) after incubation with BMP2 for 72h via WST1 assay. To allow comparison of the IC50 values, the dose-response curves were normalized with the value measured in the absence of BMP2 set to zero. The figures show the mean values and standard deviation of triplicates. The assays were carried out as three independent experiments.</p

Biological activities of the different BMP2 variants.

<p>(A) ATDC5 cells were stimulated with the indicated concentrations of either wildtype BMP2, BMP2-KD, BMP2-PKD or BMP2-P. ALP activity was determined after 72 h. (B) KMS12-BM and (C) L363 cells were stimulated with the indicated concentrations of the four BMP2 variants. Inhibition of proliferation was assessed after 96 h by WST-1 measurements. The figures show mean values and standard deviation of triplicates. The measurements were performed in three independent experiments.</p