Fig 2: Western blotting for proteins related to canonical and non-canonical Wnt-signaling.ST2 cells with serial periods of MG treatment were subjected to Western blotting. Hela cell lysate was used as a control. While Wnt5a/b, Ror1, Ror2, whole- and phospho-SAPK/JNK, whole- and phospho-c-jun (factors related to non-canonical Wnt-signaling) were almost constant, the β-catenin protein level decreased and the phospho-β-catenin protein level increased reciprocally at 12 hr after treatment.

Fig 3: DOCK2 factors in Wnt5a-induced activation of ERK1/2, but not AKT.a Immunoblot analysis of lysates prepared from freshly-isolated primary CLL cells (representative of 3 patients) transfected 72 h previously with control siRNA or siRNA targeting DOCK2, then treated without (-) or with (+) Wnt5a; expression of DOCK2, total ERK1/2, AKT, and activated pERK1/2, pAKT was measured, as indicated on the left. The numbers between two lanes are ratios of band IOD of pERK1/2 or pAKT versus total ERK1/2 or AKT respectively, and normalized with respect to that of untreated cells. b Immunoblot analysis of lysates prepared from MEC1-ROR1 cells transfected 72 h previously with control siRNA or siRNA targeting DOCK2; expression of DOCK2, total ERK1/2, AKT, and activated pERK1/2, pAKT was measured, as indicated on the left. The numbers between two lanes are ratios of band IOD of pERK1/2 or pAKT versus total ERK1/2 or AKT respectively, and normalized to that of untreated cells.

Fig 4: Wnt5a induces ROR1-dependent phosphorylation of DOCK2 in CLL.a Tyrosine phosphorylation of DOCK2 was confirmed by immunoblot analysis of anti-DOCK2 immune precipitates (ip), using lysates of MEC1-Ctrl, MEC1-ΔPRD, MEC1-ROR1 (W/T) or MEC1 cells transfected with each of the various mutated forms of ROR1, as indicated on the top; the membranes were probed with anti-DOCK2 or anti-phospho tyrosine antibody (pDOCK2), as indicated on the left. b Immunoblot analysis of anti-DOCK2 ip, using lysates prepared from MEC1-ROR1 cells that had been treated with Wnt5a neutralizing antibody (2 µg/ml, R & D) for the times indicated at the top (in hours); membranes were probed with anti-phospho tyrosine (pDOCK2) or anti-DOCK2 antibody, as indicated on the left. c Immunoblot analysis of anti-DOCK2 ip, using lysates prepared from MEC1-ROR1 cells that had been treated with cirmtuzumab (20 µg/ml) for the times indicated at the top (in hours); membranes were probed with anti-phospho tyrosine (pDOCK2), anti-DOCK2 antibody, as indicated on the left. d Immunoblot analysis of anti-DOCK2 ip, using lysates prepared from overnight, serum-starved primary CLL cells (representative of 3 patients) that subsequently were treated for 5 min without (-) or with (+) Wnt5a (100 ng/ml), as indicated on the top; the membranes were probed with anti-DOCK2 or anti-phospho tyrosine antibody (pDOCK2), as indicated on the left. e Immunoblot analysis of anti-DOCK2 ip, using lysates prepared from overnight, serum-starved primary CLL cells (representative of 3 patients) that subsequently were treated with Ctrl-IgG or cirmtuzumab (20 μg/ml), without (-) or with (+) Wnt5a (100 ng/ml), as indicated on the top; the membranes were probed with anti-DOCK2 or anti-phospho tyrosine antibody (pDOCK2), as indicated on the left.

Fig 5: a Immunoblots showing the expression of ROR1 and ROR2 in MCF-7 and SK-BR-3 cells transfected with either an empty vector (ctl) or a ROR2 overexpression plasmid (pROR2). B In vitro microinvasion assays of ROR2-overexpressing MCF-7 and SK-BR-3 cells compared to wildtype cells (wt) or cells transfected with an empty vector (ctl) (mean ± SD, n = 3, *p < 0.001). C Immunoblot for c-Jun and HDAC of MCF-7 and SK-BR-3 empty vector (ctl) or ROR2-overexpressing cells for cytoplasmic (C) and nuclear (N) cell lysates. D Immunofluorescence staining of c-Jun and F-actin (phalloidin) in MCF-7 empty vector (ctl) and ROR2 cells as well as β-catenin and Dapi in MCF-7 wt and ROR2 cells. E Immunoblot of ROR1 in MDA-MB-231 cells transfected with nonsense control (ctl) or ROR1 (shROR1) shRNA. F In vitro microinvasion assay of MDA-MB-231 wildtype (wt), non-sense control (ctl) and ROR1 knockdown (shROR1) cells (mean ± SD, n = 3, *p < 0.001)