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: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Identification and characterisation of ROR1 as a LEC marker. (A) Schematic diagram showing the three-stage lens differentiation protocol, with modification to enable ROR1-based purification of LECs. (B,C) ROR1 + cells cultured at high cell densities showed uniform polygonal morphologies that formed tightly packed monolayers (B). When cultured at low cell densities or passaged in medium containing only FGF2 (C), ROR1 + cells became large and vacuolated (arrow) with stress fibres (arrowheads; cells shown 18 days after plating; n =3). Scale bars: 100 µm. (D-G) Flow cytometry data showing expression levels of: ROR1 prior to (D) and after (E) ROR1-based purification; CRYAB after ROR1-based purification (F); and average expression levels before and after purification (G). (H) Relative mRNA transcript expression levels for PAX6, CRYAB and the lens fibre-specific gene CRYBB3 after ROR1 + cell separation (* P <0.05). (I) Pearson correlation showing high similarity (>0.96) between RNA-seq libraries generated from two independent ROR1 + cell samples. (J) Principal component analysis shows the ROR1 + RNA-seq transcriptomes are most similar to primary human LECs (circled). (K) Representative data from the ROR1 + RNA-seq libraries shows key genes required by LECs are expressed ( CRYAA , CRYAB , PAX6 , PROX1 , SOX2 , MEIS1 , MAB21L1 , BMP7 ). In contrast, genes expressed by lens fibre cells ( CRYBA1 , CRYBA2 ) or various endodermal cells ( GDF3 , VWF ), mesodermal cells ( T , GSC ), non-lens ectodermal cells ( RPE65 , NEUROD1 ) and pluripotent cells ( NANOG , POU5F1 ) are not expressed. Data shown in B,C and D-H are representative of 50 and four (respectively) independent differentiation experiments using four different hPSC lines; data are mean±s.e.m. in G,H.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Marker, Modification, Purification, Cell Culture, Flow Cytometry, Expressing, RNA Sequencing, Generated

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Combinatorial growth factor screening identified media for ROR1 + cell expansion and differentiation. (A) Schematic diagram showing composition of the test media. FGF2 was included in the basal medium (TM32), with all combinations of the five other test pathways (eight growth factors) tested as shown [B, BMP4, BMP7; E, EGF, TGFα, H, HGF; I, insulin, IGF1; P, PDGF-AA; green square represents factor(s) present; red square represents factor(s) absent]. (B-I) Data from Hoechst-stained ROR1 + cells cultured in TM17 (B,E), TM30 (C,F) and TM32 (D,G) after seeding at low (B-D) and high (E-G) cell density, as well as average Hoechst-stained nuclei counts for all media (H,I). These data reveal that TM17 promoted expansion of ROR1 + cell cultures while maintaining expression of α- but not β-crystallins (see supplementary material Fig. S3 ). Scale bar: 20 µm. (J,K) Flow cytometry and light microscopy data show ROR1 + cells expanded, frozen, thawed and cultured for 6 days in TM17 retain high levels of CRYAB expression (J) with expected morphology (K) but without detectable expression of β-crystallins (see Table S1 and Fig. S4 ). Scale bar: 40 µm. (L,M) Light microscopy images show spontaneous production of lentoid-like structures after being expanded, frozen and thawed in TM17, then cultured in stage 2 lens differentiation medium. Cells in these cultures expressed α- and β-crystallins (see Table S2 and Fig. S3 ). Scale bars: 200 µm in L; 40 µm in M. The data shown in B-I are each representative of three independent differentiation experiments; data are mean±s.e.m. in H,I

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Staining, Cell Culture, Expressing, Flow Cytometry, Light Microscopy

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: ROR1 + cell aggregation leads to transparent and light-focusing micro-lenses. (A-Q) Light microscopy data from ROR1 + -cell aggregates and the maximal focal point below them. After 3 days of culture, the aggregates transmitted less light than the surrounding culture medium (A) and did not focus light (B). As culture progressed, the aggregates transmitted more light (C, day 7; E, day 14) and began focusing light (D, day 7; F, day 14). More-detailed characterisation of a single aggregate shows it had limited transparency (G) and focusing ability (I,K,M,O) on day 3 of culture, but by day 27 it transmitted the same amount of light as the surrounding culture medium (H) and had developed significant focusing ability (J,L,N,P). Quantification of the light transmittance and focusing ability confirms these findings (Q). Scale bar: 40 µm. The images are representative of five micro-lenses from two biological replicates; data in Q are mean±s.e.m.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Light Microscopy

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Aggregation of ROR1 + cells induces lens fibre cell crystallin expression. (A) Real-time PCR analysis of aggregated ROR1 + cells results in decreased relative expression of PAX6 and CRYAB, and increased expression of CRYBB3 (* P <0.01; data obtained from four biological replicates and presented as mean±s.e.m.). (B-M) Immunofluorescence analysis shows that after 14 days of culture, αA-crystallin (C) was expressed uniformly throughout the bulk of the micro-lenses, whereas β-crystallin (E) and γ-crystallin (G) were not. After 24 days of culture, αA-crystallin (I), β-crystallin (K) and γ-crystallin (M) were all expressed relatively uniformly throughout the bulk of the micro-lens. The location of DAPI-stained nuclei within the day 14 (B,D,F) and day 24 (H,J,L) aggregates are shown. Scale bar: 40 µm. Each image is representative of five micro-lenses from two biological replicates.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Evidence of progressive lens fibre cell differentiation in ROR1 + cell aggregates. Electron microscopy data from cultured aggregates. (A,B) A micro-lens cultured for 14 days shows a monolayer of LEC-like cells at the periphery of the tissue (A), and cells with small, rod-shaped nuclei (asterisk) and numerous organelles within the bulk of the tissue (B). (C) LEC-like cell with numerous organelles present at the periphery of a micro-lens after 24 days of culture. (D-G) Ultrastructural indicators of lens fibre cell differentiation within a micro-lens cultured for 42 days. (D) Ball-and-socket type membrane interdigitations (arrows) between adjacent lens fibre-like cells (inset shows a higher magnification of the region indicated with an arrow and asterisk). (E) A swollen mitochondria (arrow). (F) An enlarged nuclei with spoke-like nucleolus (inset). (G) A degraded nuclei with nuclear membrane visible as a series of vesicles (arrowheads). Scale bars: 5 µm in A-C; 2 µm in D,F,G; 0.5 µm in E. Images are representative of seven micro-lenses obtained from two biological replicates.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Cell Differentiation, Electron Microscopy, Cell Culture, Membrane

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: The CFTR potentiator Vx-770 inhibits light focusing in ROR1 micro-lenses. (A-D) Light microscopy data showing ROR1 + micro-lenses treated with DMSO-only (A) or 200 ng/ml Vx-770 (B) transmitted light at similar levels to the culture medium after 24 days of culture, whereas a micro-lens treated with 2000 ng/ml Vx-770 transmitted less light (C). Scale bar: 40 µm. Quantitative data are shown in D. (E-H) Light microscopy data showing ROR1 micro-lenses treated with DMSO-only (E) or 200 ng/ml Vx-770 (F) had developed focusing ability after 24 days in culture, whereas micro-lenses treated with 2000 ng/ml Vx-770 had not (G). Scale bar: 40 µm. Quantitative data are shown in H. (I-O) Light microscopy data showing micro-lenses treated after they had developed focusing ability. Micro-lenses treated with DMSO-only (I,L), 200 ng/ml Vx-770 (J,M) or 2000 ng/ml Vx-770 (K,N) all transmitted similar levels of light after 7 days of treatment (L-N) compared with before treatment (I-K). Scale bar: 40 µm. Quantitative data are shown in O. (P-V) Light microscopy data showing micro-lenses treated after having developed focusing ability. Micro-lenses treated with DMSO-only (S) and 200 ng/ml Vx-770 (T) retained focusing ability after 7 days of treatment compared with before treatment (P,Q, respectively). A micro-lens treated with 2000 ng/ml Vx-770 focused light prior to treatment (R) but did not after 7 days of treatment (U). Scale bar: 40 µm. Quantitative data are shown in V. In D,O,H,V, * P <1×10 −4 . The data shown (mean±s.e.m.) were each obtained from measurements of 15 micro-lenses from three biological replicates.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Light Microscopy