Frequency of ROR1 positive melanoma cells.

<p>Frequency (%) of ROR1 positive cell lines and Geometric Mean Fluorescence Intensity, stained by 4 anti-ROR1 mAbs in flow cytometry.</p

Transfection of melanoma cells (n = 6) using ROR1 suppressing siRNA.

<p>Downregulation of ROR1 mRNA (RT-PCR) (A). Downregulation of the ROR1 protein (130 kDa) expression (B). (−) untreated cells, (C) control siRNA treated cells, (+) ROR1 siRNA treated cells.</p

Protein expression of the receptor tyrosine kinase ROR1 in melanoma cell lines.

<p>Representative experiment (IF) showing the expression of ROR1 on the ESTDAB112 cell line using the anti-ROR1 (clone 3H9) mAb (40×). Nuclei were counterstained with DAPI (blue). A non-relevant isotype control mAb (mouse IgG1 isotype) was used as a negative control (A). Western blot analysis of ROR1 protein expression and phosphorylation in melanoma cells detected by a goat anti-ROR1 antibody, anti-p-tyrosine (PY99) and anti-p-serine (clone 4A4) mAbs (B). ROR1 protein was shown to be phosphorylated in all cell lines using immunoprecipitation of ROR1. A 130 kDa band corresponding to the fully glycosylated/phosphorylated ROR1 was observed. The T47D cell line was used as a ROR1 negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061167#pone.0061167-Gentile1" target="_blank">[16]</a>.</p

Apoptosis of melanoma cells treated with siROR1.

<p>Dot plot (frequency) of apoptotic/necrotic melanoma cells (Annexin-V⁺/PI⁺) treated with siRNA, control siRNA and untreated. Within each quadrant the frequency of apoptotic cells is shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061167#s3" target="_blank">Results</a> are presented for the ESTDAB049, ESTDAB075, A375, ESTDAB112 (sensitive to apoptosis by anti-ROR1 mAbs) and ESTDAB081 (resistant to apoptosis by anti-ROR1 mAbs), The cell lines T74D cell line was used as a ROR1 negative control.</p

Cytotoxic effects of anti-ROR1 mAbs in the presence of NK cells (ADCC).

<p>Frequency (%) (mean+SEM) of apoptotic/necrotic cells (Annexin-V⁺/PI⁺) induced by 4 anti-ROR1 mAbs and a non-relevant isotype control mAb (mouse IgG1 isotype) in the presence of NK cells at different target: effector ratios. Target cells: ESTDAB049 (□), 075 (), DFW (▪), A375 () (A) and ESTDAB081 (□), 094 (), 112 (▪) melanoma cells and T47D () as a ROR1 negative cell line (B). ADCC of the melanoma cells induced by the anti-ROR1 mAbs compared to the non-relevant isotype control mAb (mouse IgG1 isotype) as wells as to the T47D cell line was statistically significant (P = 0.05-0.0001).</p

Anti-ROR1 mAbs in complement dependent cytotoxicity (CDC).

<p>Frequency (%) (mean+SEM) of apoptotic/necrotic cells (Annexin-V⁺/PI⁺) induced by 4 anti-ROR1 mAbs with (▪) or without (□) human complement using various ESTDAB (A, B), DFW and A375 melanoma cell lines (C). The T47D cell line did not express ROR1. <b>^*</b>P = 0.01; <b>^**</b>P = 0.001. P-values refer to comparison with and without complement for the respective mAbs. NR mAb: non-relevant isotype control mAb (mouse IgG1 isotype), C: Complement.</p

Characteristics of CLL and multiple myeloma patients.

<p>Characteristics of CLL and multiple myeloma patients.</p

wtKLF6 mRNA expression in purified CD4+ and CD8+ T cells of CLL patients (n = 29), B cells of CLL patients (n = 6), CD4+ and CD8+ T cells of myeloma patients (n = 6) and healthy donors (n = 10) as well as in the 293 T cell line.

<p>wtKLF6 mRNA expression in purified CD4+ and CD8+ T cells of CLL patients (n = 29), B cells of CLL patients (n = 6), CD4+ and CD8+ T cells of myeloma patients (n = 6) and healthy donors (n = 10) as well as in the 293 T cell line.</p

ROR1 isoforms and phosphorylation in CLL patients.

<p>Representative experiments using non-immunoprecipitated CLL cell lysates from 5 CLL patients showing phosphorylated dimerized ROR1 (260 kDa), fully glycosylated (130 kDa) and non-glycosylated (105 kDa) ROR1 molecules (A). NP indicates non-progressive disease and P progressive disease. As controls, CLL cell lysates immunoprecipitated with a non-relevant mAb (mouse IgG1 isotype) were used. No bands could be seen. Furthermore, in PBMC of healthy donors, no bands were detected (data not shown). Representative experiments of PBMC using surface staining for ROR1 (left panel) and intracytoplasmic staining for pROR1 (right panel) in three CLL patients (B). Expression of ROR1 isoforms in the cytoplasm (C), nucleus (N) and total cell lysate (TCL) of leukemic CLL cells (C). Confirmation of protein localization was done using antibodies against α/β –tubulin, histone H3 and nucleolin before analysing the expression pattern of ROR1. </p

KLF6-SV1 protein expression (Western blot) in purified CLL T (CD3⁺) cells of CLL patients (n = 6) as well as PBMC (n = 4) and purified T cells (n = 2) of healthy donors.

<p>Relative intensity was measured in relation to β-actin.</p