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: Gastroenterology Report

Article Title: TRUB1 is a novel biomarker for promoting malignancy in colorectal cancer via NFκB signaling

doi: 10.1093/gastro/goaf027

Figure Lengend Snippet: Potential downstream targets of TRUB1 in CRC. (A) Scatter plot showing genes with significant changes in RNA-seq after TRUB1 knock-down; (B) GSEA analysis showing enrichment of TNFα signaling via NFκB in cells with high TRUB1 expression; (C) Western blot results for TRUB1, P65, and pP65 in control and TRUB1-knock-down HCT116 cells; (D) intersection analysis of gene sets between HCT116-sh-TRUB1-1 and HCT116-sh-TRUB1-2 (FC ≥ 1.5, FDR ≤ 0.05); (E) intersection analysis of TNFα signaling via NFκB gene sets between HCT116-sh-TRUB1-1 and HCT116-sh-TRUB1-2 ( P ≤ 0.05); (F) intersection analysis of gene sets between GSEA_NFκB signaling (61 genes), RNA-seq (261 genes), and apoptosis via NFκB (33 genes); (G) expression of TRUB1 and BIRC3 mRNA in control and TRUB1-knock-down HCT116 cells. GSEA = Gene Set Enrichment Analysis, pP65 = phosphorylated P65, BIRC3 = Baculoviral IAP repeat-containing protein 3.

Article Snippet: The antibodies that were used in the Western blot were TRUB1 (1:2,000; #PA536003; Thermo, Waltham, MA, USA); TRUB2 (1:2,000; #K110029P; Solarbio, Beijing, China); p65 (1:2,000; #a22331; ABclonal, Woburn, MA, USA); phosphorylated-P65 (pP65; 1:2,000; #af2006; Affinity, Nottingham, UK); GAPDH (1:1,000; #BM3876; BOSTER, Pleasanton, CA, USA); Cytochrome C (1:2,000; #A13430; ABclonal); Bcl 2 (1:2,000; #A0208; ABclonal); Caspase-8 (1:2,000; #A0215; ABclonal); Caspase-3 (1:2,000; #A02156; ABclonal); Caspase-9 (1:2,000; #A11910; ABclonal); cleaved Caspase-3 (1:2,000; #9661 T; CST, Danvers, MA, USA); and β-Actin (1:2,000; #AC038; ABclonal).

Techniques: RNA Sequencing, Knockdown, Expressing, Western Blot, Control

Journal: Frontiers in Nutrition

Article Title: Curcumin attenuates liver injury by modulating the AGE–RAGE axis and metabolic homeostasis in high-fat diet/streptozotocin-induced type 2 diabetic mice

doi: 10.3389/fnut.2025.1710380

Figure Lengend Snippet: CUR modulates AGE/RAGE signaling and its downstream targets PI3K/Akt and NFκB pathways. (A,B) The levels of plasma (A) and hepatic AGEs (B) were measured by ELISA ( n = 5). (C) The mRNA expression levels of RAGE in the liver were analysed ( n = 3). (D–F) Representative bands and quantitative expression levels of p-PI3K, PI3K, p-Akt, Akt, p-NFκB p65, and NFκB p65 proteins in the liver from Western blot were presented. β-actin was served as an internal reference ( n = 3). For quantitative analysis (D–F) , the band intensity of each target protein was normalized to its corresponding internal control. The normalized values were presented relative to the mean of the NC group, which was set to 1. * p < 0.05, ** p < 0.01 and *** p < 0.001; ns, not significant difference.

Article Snippet: Antibodies against PI3K (CAT#AF6241), phospho-PI3K (Tyr 607 ) (CAT#AF3241), Akt (CAT#AF6261), phospho-Akt (Ser 473 ) (CAT#AF0016), phospho-NFκB p65 (Ser 536 ) (CAT#AF2006), TNF- α (CAT#AF7014), and IL-1 β (CAT#AF5103) were purchased from Affinity Biosciences (OH, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control