Journal: Cancer Science

Article Title: Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma

doi: 10.1111/cas.12858

Figure Lengend Snippet: Apoptosis signal‐regulating kinase 1 (ASK1)‐mediated signaling is involved, at least in part, in receptor tyrosine kinase‐like orphan receptor 1 (ROR1) siRNA‐induced growth inhibition. Effects of co‐treatment with siASK1 and siROR1 in (a) PC‐9 and (b) NCI‐H1975 cells. Colorimetric (top panel) and Western blot (bottom panel) analyses were carried out at 5 and 3 days, respectively, after co‐transfection. Data are shown as the mean ± SEM ( n = 3). * P < 0.05.

Article Snippet: Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Western Blot, Cotransfection

Journal: Cancer Science

Article Title: Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma

doi: 10.1111/cas.12858

Figure Lengend Snippet: Receptor tyrosine kinase‐like orphan receptor 1 (ROR1) suppresses reactive oxygen species‐induced apoptosis signal‐regulating kinase 1 (ASK1) and p38 phosphorylation. (a) Western blot (WB) analysis of ASK1 and p38 phosphorylation in MSTO‐211H cells stably transfected with an empty vector (VC #1 and VC #2), or an expression construct of wild‐type ROR1 (ROR1‐WT #1 and ROR1‐WT #2). (b) WB analysis of ASK1 and p38 phosphorylation in the presence or absence of H 2 O 2 in MSTO‐211H cells stably transfected with an empty or ROR1 expression vectors. (c) WB analysis of tert‐butyl hydroperoxide (TBHP)‐induced ASK1 and p38 phosphorylation in vector control or ROR1‐introduced MSTO‐211H cells. Arrowheads indicate non‐specific bands.

Article Snippet: Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Construct

Journal: Cancer Science

Article Title: Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma

doi: 10.1111/cas.12858

Figure Lengend Snippet: Receptor tyrosine kinase‐like orphan receptor 1 (ROR1) inhibits apoptosis signal‐regulating kinase 1 (ASK1) kinase activity. Results of in vitro ASK1 kinase assays with use of myc‐tagged ASK1 immunoprecipitated from ASK1‐transfected 293T cells, as well as recombinant GST‐ROR1 and maltose‐binding protein–MAPKK 6 (MBP‐MKK6) proteins purified from Escherichia coli .

Article Snippet: Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, In Vitro, Immunoprecipitation, Transfection, Recombinant, Binding Assay, Purification

Journal: Cancer Science

Article Title: Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma

doi: 10.1111/cas.12858

Figure Lengend Snippet: Receptor tyrosine kinase‐like orphan receptor 1 (ROR1) physically interacts with apoptosis signal‐regulating kinase 1 (ASK1). (a) Immunoprecipitation–Western blot (IP‐WB) analysis of interaction between ROR1 and ASK1 co‐introduced into 293T cells. (b) IP‐WB analysis of interaction of endogenous ASK1 with ROR1 introduced into MSTO‐211H cells. (c) IP‐WB analysis of interaction between endogenous ROR1 and ASK1 using lysates of PC‐9 and NCI‐H1975 cells. (d) IP‐WB analysis of interaction of endogenous ROR1 and ASK1 in presence or absence of H 2 O 2 in PC‐9 cells. VC, empty vector.

Article Snippet: Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: Cancer Science

Article Title: Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma

doi: 10.1111/cas.12858

Figure Lengend Snippet: Fine mapping of apoptosis signal‐regulating kinase 1 (ASK1)‐interacting domain of receptor tyrosine kinase‐like orphan receptor 1 (ROR1). (a) Schematic diagram of deletion mutants of ROR1. Immunoprecipitation (IP)–Western blot analysis of COS‐7 cells co‐introduced with ASK1 and various forms of ROR1. ΔP, mutant lacking a proline‐rich region; ΔS/T1 and ΔS/T2, mutants lacking one of the two serine/threonine‐rich regions; TK∆1, TK∆2, and TK∆3, mutants lacking one‐third of the kinase domain; WT, wild‐type ROR1. (b) IP–Western blot analysis for fine mapping of ASK1‐interacting domain of ROR1.

Article Snippet: Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Mutagenesis

Journal: Cancer Science

Article Title: Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma

doi: 10.1111/cas.12858

Figure Lengend Snippet: Receptor tyrosine kinase‐like orphan receptor 1 (ROR1) kinase activity is required for inhibition of reactive oxygen species‐elicited phosphorylation of apoptosis signal‐regulating kinase 1 (ASK1) and p38. (a) Western blot analysis of ASK1 and p38 phosphorylation in the presence or absence of H 2 O 2 in MSTO‐211H cells stably expressing wild‐type (WT) or kinase‐dead (KD) ROR1. Arrowheads indicate non‐specific bands. (b) Colorimetric assay of cell proliferation in MSTO‐211H transfectants in the presence or absence of H 2 O 2. (c) Representative results of flow cytometric analysis of apoptosis induction in ROR1‐transfected MSTO‐211H cells treated with H 2 O 2 . (d) Flow cytometric analysis of sub‐G 1 cells. Representative data from five independent experiments are shown as the mean ± SEM. * P < 0.05, ** P < 0.01 vs empty vector (VC), as determined by Student's t ‐test. PI, propidium iodide.

Article Snippet: Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Inhibition, Western Blot, Stable Transfection, Expressing, Colorimetric Assay, Transfection, Plasmid Preparation

Journal: Clinical and Translational Medicine

Article Title: WNT5B drives osteosarcoma stemness, chemoresistance and metastasis

doi: 10.1002/ctm2.1670

Figure Lengend Snippet: ROR1 is a therapeutic target for osteosarcoma stem cells. (A) Expression array correlation data showing a significantly positive correlation between WNT5B and ROR1 in osteosarcoma samples. Data were analysed using the R2 genomics analysis and visualization platform, and data generated by Kuijjer et al. ¹⁸ n = 127, p < 3.5 × 10 −14 . (B) Representative images of immunohistochemistry on an osteosarcoma tissue microarray stained for ROR1 and WNT5B. Matched images from two samples, one low for both WNT5B and ROR1 and one high for both WNT5B and ROR1. n = 40 cores in duplicate, scale bar = 400 µm. (C) Correlation graph of osteosarcoma tissue microarray depicted in (B. Average immune reactive scoring of all WNT5B and ROR1 cores indicates a positive correlation between WNT5B and ROR1 in osteosarcoma patient samples. n = 40 cores, p = .0007. (D) Immunofluorescence of 143B adherent cells and spheres. 10× microscopy images, WNT5B = green, ROR1 = red, DAPI nuclear stain = blue. White arrows indicate areas of WNT5B/ROR1 colocalisation (yellow), scale bar = 400 µm. (E) 143B spheres treated with siRNA to ROR1 or scrambled siRNA for 72 h. Sphere size quantified using ImageJ. n = > 380 spheres per group, **** p < .0001. (F) 143B spheres treated with 100 µg/mL D10 and/or 50 ng/mL rWNT5B for 48 h. The sphere area was quantified using ImageJ. n ≥ 80 spheres per group, * p < .05. (G) 143B spheres treated with a low dose of (40 µg/mL) D10 and/or high dose (10 µg/mL) MTX for 48 h to assess reduction in chemoresistance. The sphere area was quantified using ImageJ. n ≥ 110 spheres per group, * p < .05, **** p < .0001. (H) PDX‐derived spheres treated with increasing doses (0, 10, 40, 100 µg/mL) of D10. Sphere area quantified using ImageJ. n ≥ 210 spheres per group, ** p < .01. (I) SOX2 western blot of 143B spheres control, spheres treated with 40 µg/mL D10 for 48 h, and 143B WNT5B KO spheres. (J) ImageJ quantification of the western blot in (I). * p < .05, ** p < .01, ns = not significant. Normalised to β‐Actin, n = 3 experimental replicates. PDX, Patient‐derived xenograft; MTX, methotrexate.

Article Snippet: Cells were treated with 100 nM of either a scrambled control or ROR1 (Santa Cruz: sc‐76424) siRNA combined with Lipofectamine Reagent for 15 min and then added to the cells for transfection.

Techniques: Expressing, Generated, Immunohistochemistry, Microarray, Staining, Immunofluorescence, Microscopy, Derivative Assay, Western Blot, Control