Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) Phospho-RTK array results showing the inhibitory effects of siROR1 treatment on the phosphorylation state of multiple RTKs in NCI-H1975 cells (left panel). Averages of the mean pixel densities in two independent experiments are given for each of the representative RTKs (right panel). See for data in PC-9 cells. ( b ) The impairment of growth factor-induced phosphorylation in multiple RTKs in NCI-H1975 cells knocked down for ROR1. See for data in PC-9 cells. ( c ) Phospho-RTK array results showing the inhibitory effects of siCAV1 treatment on the phosphorylation state of multiple RTKs in NCI-H1975 cells (left panel), and averages of the mean pixel densities of the representative RTKs in two independent experiments (right panel). The siControl blot of a is re-displayed for ease of comparison. See for data in the PC-9 NSCLC cell line. Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques:

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) Decreased CAV1 but not of CAV2 expression with the use of three independent ROR1 siRNAs. See , which shows similar effects in other cell lines. ( b ) Reduced CAV1 expression by knocking down ROR1 but not EGFR, ERBB2 or MET in NCI-H1975 cells. Note that cavin-1 expression is not affected. ( c ) Rescue of CAV1 expression by treatment with a lysosome inhibitor (left panel) but not by treatment with a proteasome inhibitor (right panel) in NCI-H1975 cells knocked down for ROR1. ( d ) Schematic diagram of immunoelectron microscopy of SDS-treated freeze-fracture replica. ( e ) Decreased number of typical caveolae structures in the plasma membranes of the siROR1-treated NCI-H1975 cells were observed in the electron microscopic examination. More than 18 random plasma membrane areas of 17–50 μm 2 were examined for both samples. (average±s.e.m.; Student’s t -test; * P <0.001.) ( f ) Representative results of the freeze-fracture immunoelectron microscopy. CAV2 labels made clusters, but the labelled membrane was either flat or superficially depressed in the siROR1-treated NCI-H1975 cells. By contrast, the CAV2 labelling in the siControl-treated NCI-H1975 cells showed intact caveolae with indentations measuring 50–75 nm in diameter. Also refer to . Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Expressing, Immuno-Electron Microscopy

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) Sucrose density-gradient centrifugation confirmed the presence of ROR1 and cavin-1 in the DRM fractions containing CAV1 and CAV2 in the NCI-H1975 cells. ( b ) ROR1 and CAV1 colocalization shown by two-colour immunofluorescence staining in NCI-H1975 cells. Colocalization was quantified using ImageJ software. Also see . ( c ) The colocalization of ROR1 with CAV1 and cavin-1 shown by three-colour immunofluorescence staining using super-resolution structured illumination microscopy in NCI-H1975 cells. ( d ) Sucrose density-gradient centrifugation showing the loss of CAV1 as well as marked changes of cavin-1 subcellular distribution in NCI-H1975 cells knocked down for ROR1. Also see . ( e ) Two-colour immunofluorescence staining showing markedly impaired colocalization between cavin-1 and CAV2 induced by ROR1 knockdown in NCI-H1975 cells. Colocalization was quantified using ImageJ software. Also see . Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Gradient Centrifugation, Immunofluorescence, Staining, Software, Microscopy

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) Sustainment of CAV1 expression in the presence of siROR1 by introduction of both siRNA-resistant, wild-type (wt) and kinase-dead ROR1 in the NCI-H1975 cells. Also see . ( b ) Sucrose density-gradient assay showing that ROR1 kinase activity is dispensable for the retention of cavin-1 in DRM fractions. NCI-H1975 cells stably transfected with either wt or kinase-dead forms of siRNA-resistant ROR1 were subjected to siROR1 treatment in this analysis. Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Expressing, Activity Assay, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) IP–WB analysis using octylglucoside as a detergent showed association of ROR1 with cavin-1 and CAV1. See . Also see for pull-down assay showing their mutual associations. ( b ) Pull-down assay using purified proteins of ROR1, cavin-1, and CAV1 showing physical associations of ROR1 with cavin-1 and CAV1. ( c ) Identification of C-terminal two-thirds of the ROR1 kinase domain as the cavin-1 binding region by IP-WB analysis using various ROR1 deletion mutants. Also see . ( d ) Identification of the membrane association domain of cavin-1 as its ROR1-binding region by IP–WB analysis using cavin-1 deletion mutants. Also see . ( e ) Mapping of the CAV1 binding region to the C-terminal serine/threonine-rich domain by IP–WB analysis using various ROR1 deletion mutants. Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Pull Down Assay, Purification, Binding Assay

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) Indispensable involvement of the cavin-1-binding region of ROR1 in sustaining CAV1 expression shown in PC-9 cells reconstituted with ROR1 lacking the cavin-1-binding region. ( b ) Indispensable involvement of the CAV1 binding region of ROR1 for sustained CAV1 expression shown in PC-9 cells reconstituted with ROR1 lacking the CAV1-binding region. Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Binding Assay, Expressing

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ( a ) IGF-I-induced, IGF-IR-mediated bypass-signalling inhibition and the consequent gefitinib resistance by siROR1, sicavin-1 or siCAV1 treatment in the A431 vulval epidermoid carcinoma cell line. Also see . ( b ) HGF-induced, MET-mediated, bypass-signalling inhibition and consequent gefitinib resistance following knockdown of ROR1, cavin-1 or CAV1 in the PC-9 lung adenocarcinoma cell line. ( c ) Inhibition of HGF-induced bypass signalling and consequent CL-387, 785 resistance by knocking down of ROR1, cavin-1 or CAV1 in the NCI-H1975 lung adenocarcinoma cell line. All data are represented as the mean±s.e.m. ( n =3). * P <0.001 versus siControl, as determined by Student’s t -test. Uncropped images of blots are shown in .

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Inhibition

Journal: Nature Communications

Article Title: ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1

doi: 10.1038/ncomms10060

Figure Lengend Snippet: ROR1 facilitates the interaction of cavin-1 and CAV1 at the plasma membrane in a kinase activity-independent manner, which in turn sustains caveolae formation and prosurvival signalling towards AKT through multiple RTKs via its scaffold function for cavin-1 and CAV1 in human cancer cells. Our results also provide mechanistic insight into how ROR1 inhibition can overcome EGFR–TKI resistance caused by bypass signalling via diverse RTKs.

Article Snippet: The fixed cells were incubated with primary antibodies (anti-CAV1 (mouse), anti-ROR1 (goat) and anti-cavin-1 (rabbit) antibodies) diluted in PBS containing 1.0% BSA for 1 h, washed three times with PBS and then incubated with the appropriate secondary antibodies conjugated to the specified Alexa dyes (Alexa-488 (A11055), Alexa-568 (A11031), Alexa-350 (A11046)) for 1 h before mounting with Vectashield H-1,000 (Vector Laboratories, CA).

Techniques: Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: 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 .

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques: Expressing, Control, Negative Control, Western Blot, Phospho-proteomics, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: Frequency of ROR1 positive melanoma cells.

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques:

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: Frequency (%) of apoptotic/necrotic cells in Annexin-V + /PI + (A) and XTT cytotoxicity assay (B) induced by anti-ROR1 mAbs in the absence of complement or immune effector cells [anti-ROR1 mAb clones 1A8 (□), 1E9 ( ) and 5F1 ( )3H9 (▪)]. Dot plot diagrams of apoptosis induced by anti-ROR1 mAbs (clones 1A8 and 3H9) in melanoma cells and ROR1 negative cell line T47D (Annexin-V/PI) (C). Western blot for cleaved PARP, caspase 8, 9 and MCL-1 expression in apoptotic ESTDAB049 and ESTDAB112 cells induced by the anti-ROR1 mAb clone 5F1 (D). (−) cells treated with a non-relevant isotype control mAb (mouse IgG1 isotype). (+) cells treated with the anti-ROR1 mAb clone 5F1. (S) cells treated with staurosporine.

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques: Cytotoxicity Assay, Clone Assay, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: 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. * P = 0.01; ** 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.

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques: Comparison, Control

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: 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).

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques: Control

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: 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.

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: PLoS ONE

Article Title: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

doi: 10.1371/journal.pone.0061167

Figure Lengend Snippet: 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. 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.

Article Snippet: Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS.

Techniques: Control, Negative Control

Journal: Theranostics

Article Title: WNT signaling inducing activity in ascites predicts poor outcome in ovarian cancer

doi: 10.7150/thno.37423

Figure Lengend Snippet: WNT/PCP components are differentially expressed during HGSC progression. A) WNT/PCP components are differentially expressed in HGSC. Summary of meta-analysis of datasets from Oncomine database, P values > 0.05 are in grey. Detailed information and whole analysis is provided in Supplementary B-C. B-C) Verification of the Oncomine transcriptomics results on primary patient samples on the protein level by WB. WNT/PCP components (FZD10, CELSR3, DVL3 and WNT11) are overexpressed in HGSC in comparison to healthy ovarian tissue ( B ). C) VANGL2 and WNT5A levels were decreased in metastatic tissue in comparison to primary site, while ROR1 and PRICKLE1 levels were upregulated. Quantification of WBs is available in Supplementary . D) Univariate and multivariate results of Cox proportional hazard models and Kaplan-Meier curves of overall survival in patients of TCGA Ovarian dataset for genes upregulated in HGSC. Detailed analysis is provided in Supplementary Table ST3.

Article Snippet: The antibodies used were rabbit anti β-ACTIN [cs-4970; Cell Signaling Technology (CST)], goat anti CELSR3 (sc-46849, SCBT), rabbit anti DVL2 (cs-3216; CST), mouse anti DVL3 (sc-8027; SCBT), rabbit anti FZD10 (ab71987, Abcam), rabbit anti LRP6 (cs-3395, CST), rabbit anti LRP6 phospho-S1490 (cs-2568, CST), rabbit anti PRICKLE1 (ab15577, Abcam), rabbit anti ROR1 (gift from Henry Ho ), rabbit anti ROR2 (gift from Henry Ho ), mouse anti ROR2 (sc-374174, SCBT), sheep anti VANGL2 (AF4815, RnD Systems), mouse anti α-TUBULIN (TG199, Sigma Aldrich), goat anti WNT5A (AF645, RnD Systems), rat anti WNT5A (MAB645, RnD Systems), rabbit anti WNT11 (LS-C185754, LifeSpan BioSciences).

Techniques: Comparison

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: Proteomics analysis of ROR1, ROR2, and PTK7 transfected BaF3 cells. a Principal component analysis (PCA) showing phenotypic similarity between BaF3-ROR1 and BaF3-ROR2 biological replicates, but not among the parental BaF3 and BaF3-PTK7 clones. b Heatmap of differentially expressed proteins (DEP) log 2 fold change with respect to the parental BaF3 samples. The DEPs shared among BaF3-ROR1, -ROR2, and -PTK7 are shown. c Volcano plots reporting the log 2 fold change of the DEPs between the parental BaF3 cells and BaF3 clones. Adjusted p value < 0.05 and log 2 FC >|0.5|. d Similarity matrix (shown as a heatmap) among GO biological processes related to DEPs of each BaF3 clone. The apoptotic- and metabolic-related processes are highlighted. The heatmaps in b and d are clustered using complete-linkage hierarchical clustering based on Euclidean distances

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Transfection, Clone Assay

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: Functional analysis of BaF3-ROR1, BaF3-ROR2, and BaF3-PTK7 clones. a Migration assay of BaF3 clones toward CXCL12 chemokine for 6 h. The graph shows the relative number of migrated cells normalized to BaF3 parental migrated cells. Data are represented as mean ± standard deviation (SD) and statistical comparison was performed with Student’s t test ( n = 3). b Relative survival of BaF3 cells cultured in starvation media (no serum and no IL-3) in the presence or absence of Wnt5a (100 ng/ml) for 24 h. The graph shows mean ± SD ( n = 3). In a and b, the statistical significance is indicated as * p < 0.05 and *** p < 0.001. c Immunoblotting analysis of the intracellular signaling in BaF3 parental cells and BaF3 clones expressing ROR1, ROR2, or PTK7. β-tubulin was used as a loading control. d Immunoblotting analysis of the cytoplasmic and nuclear lysates of BaF3-ROR1i clones that acquired IL-3 independence. Antibodies against the effectors of the pro-survival signaling were used to detect the protein levels and β-tubulin was used as a loading control for the cytoplasmic lysates, whereas PARP was used as a loading control for the nuclear lysates. e The relative proliferation of BaF3 parental and BaF3-ROR1i clone stimulated with 100 ng/ml Wnt5a or Wnt16b. Proliferation was measured at the indicated time points and the values were normalized to non-stimulated cells (set as value 1). The graph shows the mean ± SD ( n = 3). Statistical comparison between the non-stimulated and Wnt-stimulated BaF3-ROR1i cells was done using Student’s t test as * p < 0.05

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Functional Assay, Clone Assay, Migration, Standard Deviation, Comparison, Cell Culture, Western Blot, Expressing, Control

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: Characterization of the C-terminal cytoplasmic region of ROR1 and ROR2. a Schematic representation of amino acid boundaries of ROR1 and ROR2 C-terminal deletions (Δ1–Δ4) and the full-length (FL) receptors stably transfected into BaF3 cells. b Immunoblot analysis of the HA-tagged ROR1 and ROR2 FL and Δ1–Δ4 deletions expressed in BaF3 cells cultured for 24 h in starvation media. β-tubulin was used as a loading control and the signal quantifications shows the normalized values using BaF3 (set as 1). c Immunoblotting analysis of downstream signaling of BaF3-ROR1 and BaF3-ROR2 (FL and Δ1–Δ4) clones. β-tubulin was used as a loading control and signal quantifications show the normalized values using BaF3 samples. d Left: schematic representation of the cytoplasmic ROR1 constructs used for recombinant protein production. Expression levels of the His-tagged constructs were verified by immunoblotting. Right: DSF analysis of recombinant ROR1 constructs. Melting temperatures (T m ) are also indicated, and the data represent the mean ± SD ( n = 3). JAK2 PK domain and MuSK kinase domain was used as a reference control. e Cellular thermal shift assay (CETSA) showing stabilization of ROR1 (FL and Δ1–Δ4) upon treatment with GZD824 (10 µM). Cell lysates were heated to the indicated temperatures and immunoblotted with anti-HA for ROR1 detection. β-tubulin was used as a loading control. The graph shows the signal quantification of 3 independent experiments (mean ± SD). Band intensities were normalized to the 42 °C untreated samples for each ROR1 construct (set as 1)

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Stable Transfection, Transfection, Western Blot, Cell Culture, Control, Clone Assay, Construct, Recombinant, Expressing, Thermal Shift Assay

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: ROR1 interactome detected by BioID and AP-MS. a Venn diagram showing the number of specific ROR1 interactors identified with BioID (red), AP-MS (pink), or both (white). The respective physical interactomes and enrichment p values were obtained with STRING (reference as reported in the methods chapter). b List of selected GO biological processes and molecular functions associated with the ROR1 interactors and identified with both BioID and AP-MS

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Protein-Protein interactions

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: GZD824 inhibits downstream signaling of ROR1. a Schematic representation of GZD824 binding to ROR1 PK domain, which induces inhibition of its downstream PI3K/AKT, STAT3, and NF-κB signaling. b Immunoblot analysis of the BaF3-ROR1 cells left untreated of pre-treated with Src inhibitor dasatinib or GZD824 (1 µM) for 2 h before the addition of Wnt5a (100 ng/ml) as indicated. Downstream ROR1 signaling levels or pERK/ERK, pAKT/AKT, and pSrc/Src are shown. β-tubulin was used as a loading control. c Venn diagram showing the number of shared and specific interactors of ROR1 before and after GZD284 treatment as identified by AP-MS, BioID, or both. For each set of interactors, the protein-coding genes involved in MAPK signaling (red), Rho GTPases signaling (yellow), or actin/cytoskeleton signaling (blue) are indicated. d , e Bar graphs of the top non-redundant enriched ontology clusters from multiple functional annotation databases in ROR1 interactomes identified with both AP-MS and BioID when before ( d ) and after ( e ) GZD824 treatment. The color scale represents statistical significance

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Binding Assay, Inhibition, Western Blot, Control, Protein-Protein interactions, Functional Assay

Journal: Molecular Oncology

Article Title: Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease

doi: 10.1016/j.molonc.2015.12.009

Figure Lengend Snippet: ROR1 expression in Ewing sarcoma (A) Upper panels: ROR1 gene expression. qPCR DDCt‐method relative to GAPDH and to 697 ALL positive control cells. Graphs and error bars show mean and standard deviation of at least three independent experiments. ...

Article Snippet: Flow cytometry Cells were stained with 0.4 μg of ROR1 polyclonal antibody (R&D Systems, Minneapolis, MN) or IgG isotype control (Jackson Immunoresearch, Baltimore, PA) for 30 min at 4 °C.

Techniques: Expressing, Gene Expression, Positive Control, Standard Deviation

Journal: Molecular Oncology

Article Title: Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease

doi: 10.1016/j.molonc.2015.12.009

Figure Lengend Snippet: Functional effects of ROR1 silencing A673 and 5838 cells were transfected with siRNA targeting ROR1 (siROR1) or non‐silencing control (siCTRL). (A) Left panel: Viable cells were counted by trypan blue exclusion 96 h after transfection. ...

Article Snippet: Flow cytometry Cells were stained with 0.4 μg of ROR1 polyclonal antibody (R&D Systems, Minneapolis, MN) or IgG isotype control (Jackson Immunoresearch, Baltimore, PA) for 30 min at 4 °C.

Techniques: Functional Assay, Transfection, Control

Journal: Molecular Oncology

Article Title: Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease

doi: 10.1016/j.molonc.2015.12.009

Figure Lengend Snippet: Wnt5a is a potential ROR1 ligand (A) Correlation plot of ROR1 and Wnt5a gene expressions. Wnt5a gene expression was analyzed in cell lines and 12 Ewing sarcoma specimens by qPCR and ΔΔCt‐method compared to GAPDH and MSC cultures ...

Article Snippet: Flow cytometry Cells were stained with 0.4 μg of ROR1 polyclonal antibody (R&D Systems, Minneapolis, MN) or IgG isotype control (Jackson Immunoresearch, Baltimore, PA) for 30 min at 4 °C.

Techniques: Gene Expression

Journal: iScience

Article Title: EMT alterations in the solute carrier landscape uncover SLC22A10/A15 imposed vulnerabilities in pancreatic cancer

doi: 10.1016/j.isci.2022.104193

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ROR1 , Cell Signaling Technology , Cat# 4102; RRID: AB_2180136.

Techniques: Virus, Plasmid Preparation, shRNA, Control, Microarray, Recombinant, Saline, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Glutathione Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Isolation, Biomarker Discovery, Software, Membrane, Sterility, Transfection

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

doi: 10.1161/JAHA.120.019904

Figure Lengend Snippet: Cells expressing Ror2 in cardiac tissue were visualized in Ror2‐LacZ mice 28 days after ( A ) sham surgery or ( B ) TAC surgery. C , Protein localization was visualized in cardiac tissue 7 days after TAC by immunohistochemistry for Ror2. D , Ror1 and Ror2 protein expression was measured by western blot at various days after TAC surgery (β‐actin as a loading control). Images are representative of >3 replicates per experiment. E , Endothelial cells, leukocytes, and fibroblasts were isolated 7 days after sham or TAC surgery, and RNA expression of relevant genes was quantified (bars represent mean±SD, sample size=4 per group, Kruskal‐Wallis P values: Fsp1=0.0013, Acta2=0.0004, Postn=0.0014, Ror1=0.0016, Ror2=0.0002). A.U., arbitrary units; ICH, immunohistochemistry; LacZ, β‐galactosidase; Ror, receptor tyrosine kinase–like orphan receptor; and TAC, transverse aortic constriction. *p<0.05.

Article Snippet: Protein lysates from TAC heart samples were isolated from cardiac tissue by 1% Triton‐X supplemented with protease inhibitors and probed for rabbit antimouse polyclonal Ror1 (provided by Dr Michael Greenberg, ) rabbit antimouse monoclonal Ror2 (Cell Signaling Technology Catalog #88639), or β‐actin (Cell Signaling Technology Catalog #4970) by western blot.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control, Isolation, RNA Expression

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

doi: 10.1161/JAHA.120.019904

Figure Lengend Snippet: Murine cardiac fibroblasts were isolated from healthy cardiac tissue of C57Bl6 mice. A , RNA expression of genes associated with fibroblast identity, fibroblast activation, Ror1/2 receptors, and planar cell polarity was quantified over time (bars represent mean±SD, sample size=3 per group, Kruskal‐Wallis P values: Fsp1=0.0003, Slug=0.0014, Snail=0.0003, Ror1=0.0014, Ror2=0.0014, Ptk7=<0.0001, Prickle=0.0020, Vangl2=0.0137). Friedman test P values for overall effect of time: Fsp1=0.002, Slug=0.017, Snail=0.002, Ror1=0.017, Ror2=0.148, Ptk7=0.002, Prickle=0.017, Vangl2=0.054. Cardiac myofibroblasts at passage 2 were stained for ⍺‐smooth muscle actin fibers after ( B ) control and ( C ) TGF‐β1 treatment. D , RNA expression of genes associated with myofibroblast differentiation, inflammation, and Ror1/2 receptors was quantified in control and TGF‐β1 treated fibroblasts (bars represent mean±SD, sample size=3 per group, Student t test P values: Acta2=0.0078, Postn=0.0379, IL‐6=0.0487, Tnfa=0.0939, Ror1=0.9921, Ror2=0.2313). A.U., arbitrary units; DAPI, 4′,6‐diamidino‐2‐phenylindole; Ror indicates receptor tyrosine kinase–like orphan receptor; and TGF‐β1, transforming growth factor beta. *p<0.05.

Article Snippet: Protein lysates from TAC heart samples were isolated from cardiac tissue by 1% Triton‐X supplemented with protease inhibitors and probed for rabbit antimouse polyclonal Ror1 (provided by Dr Michael Greenberg, ) rabbit antimouse monoclonal Ror2 (Cell Signaling Technology Catalog #88639), or β‐actin (Cell Signaling Technology Catalog #4970) by western blot.

Techniques: Isolation, RNA Expression, Activation Assay, Staining, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

doi: 10.1161/JAHA.120.019904

Figure Lengend Snippet: Primary cardiac fibroblasts were isolated from healthy control and transgenic Ror1/2 double‐knockout mice transcriptional phenotype was assessed by bulk RNA sequencing. A , RNA transcript reads of specific genes related to planar cell polarity, inflammatory cytokines, proliferation and cell division, extracellular signal‐related kinase 1/2 signaling, matrix remodeling, natriuretic signaling, and myofibroblast differentiation were quantified as normalized Z score (4 samples for each genotype). B , Gene ontology analysis of differential gene expression between control and Ror1/2 knockout cardiac fibroblasts showed the top 10 upregulated and downregulated terms by q value, with terms grouped by color: cell activation in orange, inflammation in red, proliferation in green, and microtubule regulation in black (bars represent mean±SEM, q values obtained through Generally Applicable Gene‐set Enrichment for Pathway Analysis, or GAGE, statistical algorithms). C , Significance vs fold change of each gene between control and Ror1/2 knockout samples was visualized by volcano plot, with genes in each gene ontology term highlighted in corresponding colors (all other genes in gray), and specific genes related to planar cell polarity, inflammation, and proliferation were highlighted and labeled. D , 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation assay after 2 hours of EdU incubation determined rate of proliferation in control and Ror1/2 knockout cardiac fibroblasts (bars represent mean±SD, sample size=3 per group, Student t test P =0.0054). ERK, iextracellular signal‐regulated kinase; GO, gene ontology; KO, knock out; pos reg, positive regulation; and Ror indicates receptor tyrosine kinase–like orphan receptor. *p<0.05.

Article Snippet: Protein lysates from TAC heart samples were isolated from cardiac tissue by 1% Triton‐X supplemented with protease inhibitors and probed for rabbit antimouse polyclonal Ror1 (provided by Dr Michael Greenberg, ) rabbit antimouse monoclonal Ror2 (Cell Signaling Technology Catalog #88639), or β‐actin (Cell Signaling Technology Catalog #4970) by western blot.

Techniques: Isolation, Control, Transgenic Assay, Double Knockout, RNA Sequencing, Gene Expression, Knock-Out, Activation Assay, Labeling, Incubation

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

doi: 10.1161/JAHA.120.019904

Figure Lengend Snippet: Myofibroblast differentiation was induced by treatment with 10 ng/mL TGF‐β1 for 4 days. A , RNA expression of myofibroblast‐related (Acta2 and Postn) and inflammation‐related (interleukin‐6) genes was quantified in control and Ror1/2 knockout fibroblasts (bars represent mean±SD, sample size=3 per group, Kruskal‐Wallis P values: Acta2=0.0006, Postn<0.0001, Il6=0.0006). B , Protein levels of interleukin‐6 were visualized by Western blot in control and Ror1/2 knockout fibroblasts after TGF‐β1 induction (representative blot shown), quantification of 3 replicates in ( C ) (bars represent mean±SD, sample size=3 per group, Kruskal‐Wallis P =0.0006). D , ⍺‐Smooth muscle actin filaments were visualized by immunofluorescent staining of subconfluent cells in control and Ror1/2 knockout fibroblasts, and ( E ) alignment of ⍺‐smooth muscle actin filaments was quantified and normalized per cell (461 filament angles in control, 717 filament angles in Ror1/2 knockout, lines on graph represent contingency table of 10‐degree bins for 19 total bins, chi‐square P value for testing numbers in bins <0.0001). A.U., arbitrary units; KO, knock out; ns, not significant; Ror, receptor tyrosine kinase–like orphan receptor; and TGF‐β1, transforming growth factor beta. *p<0.05, ****p<0.0001.

Article Snippet: Protein lysates from TAC heart samples were isolated from cardiac tissue by 1% Triton‐X supplemented with protease inhibitors and probed for rabbit antimouse polyclonal Ror1 (provided by Dr Michael Greenberg, ) rabbit antimouse monoclonal Ror2 (Cell Signaling Technology Catalog #88639), or β‐actin (Cell Signaling Technology Catalog #4970) by western blot.

Techniques: RNA Expression, Control, Knock-Out, Western Blot, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

doi: 10.1161/JAHA.120.019904

Figure Lengend Snippet: A , Transgenic Ror1/2 double‐knockout and control mice were subjected to TAC surgery, and cardiac output was imaged by echocardiography (n=9 mice were used per group in each experimental condition). B , Cardiac output factors were quantified: fractional shortening (FS), left ventricular posterior wall thickness at end diastole (LVPWTd), left ventricular diameter at end systole (LVDs), and left ventricular diameter at end diastole (LVDd) (graphs represent mean±SEM, sample size=5, Mann‐Whitney P values at day 3: FS, %=0.0079, LVPWTd, mm=0.8413, LVDs, mm=0.0079, LVDd, mm=0.0079). C , Survival of Ror1/2 double‐knockout mice and control mice after TAC surgery was recorded each day (sample size=10 per group, log‐rank [Mantel‐Cox] test P <0.0001). Ror indicates receptor tyrosine kinase–like orphan receptor; TAC, transverse aortic constriction; and Ubc‐Cre ERT2 , ubiquitin C‐driven Cre recombinase with tamoxifen‐inducible mutant human estrogen receptor. *p<0.05.

Article Snippet: Protein lysates from TAC heart samples were isolated from cardiac tissue by 1% Triton‐X supplemented with protease inhibitors and probed for rabbit antimouse polyclonal Ror1 (provided by Dr Michael Greenberg, ) rabbit antimouse monoclonal Ror2 (Cell Signaling Technology Catalog #88639), or β‐actin (Cell Signaling Technology Catalog #4970) by western blot.

Techniques: Transgenic Assay, Double Knockout, Control, MANN-WHITNEY, Ubiquitin Proteomics, Mutagenesis

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

doi: 10.1161/JAHA.120.019904

Figure Lengend Snippet: Inflammatory profile of control and transgenic Ror1/2 double‐knockout mice was assessed after TAC or sham surgery (n=5 mice were used per group in each experimental condition). A , Hematoxylin and eosin staining of cardiac tissues 3 days after TAC were imaged. B , Cells were isolated from cardiac tissue and relative quantity of leukocyte populations were determined by flow cytometry, ( C ) quantified by absolute number (bars represent mean±SEM, sample size=5 per group, Mann‐Whitney P values: neutrophil=0.0079, monocyte=0.0079, macrophage=0.0079). D , Gene expression of proinflammatory cytokines in cardiac lysate was measured (bars represent mean±SEM, sample size=5 per group, Kruskal‐Wallis P values: Il1b=0.0004, Il6=0.0030, Ccl2=0.0011). E , Vascular permeability at 1 day after TAC was assessed by Evans Blue dye injection, with vascular leakage visualized by blue dye in the cardiac tissue. Ror indicates receptor tyrosine kinase–like orphan receptor; TAC, transverse aortic constriction; and Ubc‐CreERT2, ubiquitous cre recombinase estrogen receptor (tamoxifen‐inducible). *p<0.05.

Article Snippet: Protein lysates from TAC heart samples were isolated from cardiac tissue by 1% Triton‐X supplemented with protease inhibitors and probed for rabbit antimouse polyclonal Ror1 (provided by Dr Michael Greenberg, ) rabbit antimouse monoclonal Ror2 (Cell Signaling Technology Catalog #88639), or β‐actin (Cell Signaling Technology Catalog #4970) by western blot.

Techniques: Control, Transgenic Assay, Double Knockout, Staining, Isolation, Flow Cytometry, MANN-WHITNEY, Gene Expression, Permeability, Injection