Fig 2: CS-E-elicited invasiveness is enhanced by the absence of DKK1. (A) Raw sensor grams. ROR1 was immobilized in a flow cell of a CM5 sensor chip. DKK1 alone (a), DKK1 premixed with CS-E at a 1:7 molar ratio (b), and DKK1 premixed with CS-A at a 1:7 molar ration (c) were used as analytes. (B) Response-unit quantification of binding. (C) DKK1 mRNA expression in MDA-MB-231 cells transfected with siDKK1 or control siRNA (siCont) measured using qPCR (n=4). Expression data were normalized to those of GAPDH. (D) Invasiveness of DKK1 knocked down MDA-MB-231 cells (siDKK1) or control cells (siCont) treated with or without CS-E (n>5). Data were analyzed using a Tukey–Kramer multiple comparison.

Fig 3: RA modulation of ROR1 regulates P13K/AKT/GSK3β. (A) Immunoblots of the ROR1 pathway under normal RA treatment over 96 h. (B) Densitometry of ratioed baseline-corrected proteins to their respective loading controls (ANOVA with Tukey's multiple comparison test to all conditions, n=3). **P<0.005, ****P<0.0001. (C) Luciferase assay of total β-catenin levels during differentiation over 96 h (ANOVA multiple comparisons to 0 h, n=3). *P<0.05, ***P<0.0005, ****P<0.0001. (D) Immunoblots of the ROR1 pathway under RA treatment and knockdown of ROR1 with shRNA over 96 h. (E) Densitometry of ratioed baseline-corrected proteins to their respective loading controls (ANOVA with Tukey's multiple comparison test to compare each condition with every other condition in the experiment, n=3). *P<0.05, ****P<0.0001. RA, retinoic acid; ROR1, receptor tyrosine kinase-like orphan receptor 1; NT, not treated.

Fig 4: ROR2 not only transduces non-canonical Wnt signaling and regulates HFSC migration, but also modulates canonical Wnt signaling via regulating GSK3β stability.a Ror2+/+ and Ror2−/− HFSCs were treated with Wnt5a or Wnt3a for 30 and 60 min before harvested for immunoblotting. Arrowhead denotes the phosphorylated form of Dvl2 protein. Values of fold change in the protein levels are shown under indicated bands. Data were normalized to β-Actin or α-Tubulin and calculated by comparing to Ror2+/+ HFSCs at 0’. Quantification of phosphorylated to total proteins in a time course manner is shown below. b Ror2+/+ and Ror2−/− HFSCs were treated with Wnt5a for 2 h, and GTP-bound Rac1 or Cdc42 were pulled down and identified using a small GTPase activation assay. Quantification of protein levels is shown. Data of total proteins were normalized to β-Actin. c ROR2 is required for the transcriptional activation of canonical Wnt signaling. HFSCs were starved overnight and then treated with Wnt3a for 6 h. Folds of Wnt3a induction were calculated by comparing Wnt3a-treated HFSCs to their vehicle-treated counterparts. Folds of reduction are shown above the bars. Data are reported as average ± s.d. d Inhibition of GSK3β activity restores the expression of canonical Wnt target genes in Ror2−/− HFSCs. Real-time PCR analyses of Ror2+/+ and Ror2−/− HFSCs treated with DMSO (vehicle) or CHIR-99021 (GSK3i) for 24 h. e Ror2−/− HFSCs are not responsive to Wnt5a-induced cell migration. Migration abilities of Ror2+/+ and Ror2−/− HFSCs were examined by transwell cell migration assay with Wnt5a or serum as a chemoattractant. f Real-time PCR analyses of Ror2+/+ and Ror2−/− HFSCs for Ror2, Ror1, HFSC stemness, and HF fate-related genes. Note that except for Nfatc1 which is related to quiescent state of HFSCs, Ror2−/− HFSCs expressed lower levels of HFSC stemness and HF fate-related genes. Data in d–f are reported as average ± s.d.; n = 3 independent experiments; *p < 0.05, **p < 0.005, ***p < 0.0005. All p values were calculated using unpaired two-sided t-test. All immunoblotting results are representative of at least two independent experiments. Source data are provided as a Source Data file.

Fig 5: RA promotes NB differentiation via ROR1. (A) Immunoblotting revealed the changes in ROR1 and synaptophysin levels under normal differentiation with RA. (B) Densitometry of immunoblots from A, baseline-corrected to GAPDH (ANOVA with Tukey's multiple comparison to all conditions, n=3). **P<0.005, ***P<.0005, ****P<0.0001. (C) Phase contrast images of SK-N-SH neuroblastoma cells undergoing RA or DMSO treatment over 96 h under shRNA test Lipofectamine transfection, knocking down ROR1 expression. Yellow arrows indicate marked neurite extensions that suggest development. (D) Visualization of the average neurite length over time following knockdown of ROR1 (n=3). (E) Immunoblots revealed the changes in ROR1 and synaptophysin levels after Lipofectamine transfection, knocking down ROR1 expression. (F) Densitometry of immunoblots from E, baseline-corrected to GAPDH (ANOVA with Tukey's multiple comparison test to compare each condition with every other condition in the experiment, n=3). ****P<0.0001. (G) Immunofluorescence revealed the effects of RA on synaptophysin (green) levels with or without shRNA knockdown of ROR1 (red). DAPI (blue) highlights the nucleus of the neurons. Yellow arrows indicate marked neurite extensions. RA, retinoic acid; NB, neuroblastoma; ROR1, receptor tyrosine kinase-like orphan receptor 1; NT, not treated.