Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Endogenous L1CAM expression in RB cell lines and RB tumor specimens. Depiction of endogenous L1CAM expression in human retina and RB cell lines as revealed by quantitative real‐time PCR (A) and western blot (L1–220) (B) analyses. The indicated intensity ratios relative to ß‐actin, used as a loading control in (B), were calculated using micro manager 1.4 software (University of California, San Francisco, CA, USA). (C) L1CAM expression levels in enucleated RB patient eyes after treatment with chemotherapeutics (treated) and without prior treatment (untreated) in comparison with a hRet pool. A total of 16 RB tumor specimens were analyzed, 13 untreated and 3 treated specimens. Values are means of at least three independent experiments ± SEM. ns P > 0.05; * P < 0.05; and *** P < 0.001 statistical differences compared to the control group calculated by Student's t ‐test or one‐way ANOVA with Newman–Keuls post‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Software, Comparison

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Effects of L1CAM knockdown on cell growth, apoptosis levels, and colony formation capacity of RB cells. (A) Verification of an efficient stable, lentiviral L1CAM knockdown (shL1) in RB355 and WERI‐Rb1 cells as revealed by western blot analysis. The indicated intensity ratios relative to ß‐actin, used as a loading control, were calculated using micro manager 1.4 software. Stable L1CAM knockdown significantly reduces cell growth of RB355 and WERI‐Rb1 cells reducing cell viability and proliferation levels compared to control cells (ctr) as revealed by growth curves (B, C), WST‐1 assays (D), and BrdU stains (E). L1CAM‐depleted RB355 RB cells show higher apoptosis levels as revealed by DAPI cell counts (F), and both RB cell lines show significantly reduced colony sizes as revealed by soft agarose assays (G, H). Values are means of three independent experiments ± SEM. ns P > 0.05; ** P < 0.01; and *** P < 0.001 statistical differences compared to the control group calculated by Student's t ‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Knockdown, Western Blot, Control, Software

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Effects of L1CAM overexpression on cell growth, apoptosis levels, and colony formation capacity of RB cell lines. (A) Transient L1CAM overexpression (L1OE) leads to a significant increase in L1CAM protein (L1–220) levels in Rbl30 and RB247 cells as revealed by western blot analysis. The indicated intensity ratios relative to ß‐actin, used as a loading control, were calculated using micro manager 1.4 software. (B, C) L1CAM overexpression results in significantly increased cell growth of Rbl30 and RB247 cells, accompanied by increased cell viability (D) and proliferation levels (E) compared to control cells (ctr). (F) L1CAM‐overexpressing Rbl30 and RB247 cells show significantly decreased apoptosis levels as revealed by DAPI cell counts. (G, H) As revealed by soft agarose assays, L1CAM overexpression results in increased colony sizes in both RB cell lines. Values are means of three independent experiments ± SEM. * P < 0.05; ** P < 0.01; and *** P < 0.001 statistical differences compared to the control group calculated by Student's t ‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Over Expression, Western Blot, Control, Software

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Effects of lentiviral L1CAM knockdown on viability of etoposide‐resistant RB cell lines. (A–C) WST‐1 assays showing that stable, lentiviral knockdown of L1CAM (shL1 + ) leads to significantly reduced cell viability of the etoposide‐resistant RB cell lines RB355‐Etop (A), WERI‐Rb1‐Etop (WERI‐Etop; B), and Y79‐Etop (C) simultaneously treated with indicated concentration of etoposide (Etop + ). Cell viability was normalized to the respective untreated control group (Etop − ). (D, E) Effective reduction of L1CAM expression levels upon lentiviral knockdown as revealed by quantitative real‐time PCR (D) and western blot analysis (E). The indicated intensity ratios relative to ß‐actin, used as a loading control, were calculated using micro manager 1.4 software. Values are means of three independent experiments ± SEM. ns P > 0.05; ** P < 0.01; and *** P < 0.001 statistical differences compared to the control group calculated by paired Student's t ‐test or one‐way ANOVA with Newman–Keuls post‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Knockdown, Concentration Assay, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Effect of lentiviral L1CAM knockdown on CAM tumor formation of etoposide‐resistant RB cell lines upon etoposide treatment in vivo . (A–C) Photographs of ruler measurements (in cm) of excised CAM tumors (left column) and CAM tumors in situ (right column) 7 days after grafting the etoposide‐resistant RB cell lines RB355‐Etop (A), WERI‐Rb1‐Etop (WERI‐Etop, B), and Y79‐Etop (C). Compared to etoposide‐treated control cells (ctr), lentiviral L1CAM knockdown (shL1) results in the development of significantly smaller CAM tumors from all three etoposide‐resistant RB cell lines upon single etoposide treatment.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Knockdown, In Vivo, In Situ, Control

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Effect of L1CAM knockdown on size and weight of tumors forming from etoposide‐resistant, drug‐treated RB cell lines in vivo . (A–F) Quantification of size and weight of CAM tumors developing from etoposide‐resistant, L1CAM ‐depleted (shL1 + ) RB355‐Etop (A, B), WERI‐Rb1‐Etop (WERI‐Etop, C, D), and Y79‐Etop (E, F) cell lines treated once with etoposide (Etop + ) or left untreated (Etop − ). Values are means of four independent experiments ± SEM. ns P > 0.05; * P < 0.05; ** P < 0.01; and *** P < 0.001 statistical differences compared to the control group calculated by one‐way ANOVA with Newman–Keuls post‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Knockdown, In Vivo, Control

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Endogenous L1CAM, ADAM10, and ADAM17 expression in RB cell lines as well as ADAM10 and ADAM17 expression in RB tumor specimens. (A) Western blot analysis of protein expression levels of L1CAM (L1–220), ADAM10, and ADAM17 in different RB cell lines and detection of the soluble 200 kDa L1 ectodomain (L1–200) as well as the C‐terminal fragment (L1–32) in cell culture supernatants and cell pellets. ß‐actin was used as a loading control. An endogenous cleavage of L1CAM and the presence of the soluble L1–200 ectodomain is clearly detectable in WERI‐Rb1, Y79, and RB355 cell culture supernatant. The RB cell lines analyzed show differential expression patterns for the precursor and active form of ADAM10 and ADAM17. (B) ADAM10 and 17 expression levels in enucleated RB patient eyes in comparison with a hRet pool. Values are means of 16 independent RB tumor specimens ± SEM. ** P < 0.01 and *** P < 0.001 statistical differences compared to the control group calculated by Student's t ‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Expressing, Western Blot, Cell Culture, Control, Quantitative Proteomics, Comparison

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Analysis of ADAM activation by PMA and specific inhibition of ADAM10 by GI254023X and ADAM17 by TAPI‐1 in the RB cell lines RB355 (A, C, E) and Y79 (B, D, F). (A, B) Western blot analysis of ADAM‐mediated L1CAM ectodomain (L1–200) shedding in cell culture supernatant of RB355 (A) and Y79 (B) cells 48h after treatment with different concentrations of PMA. HeLA cell lysate was used as an antibody positive control (pos ctr). DMSO‐treated cells served as a vehicle control and DMEM as a negative control. (C, D) Representative western blots showing activation of L1 shedding upon PMA treatment (50 n m in C; 500 n m in D) and subsequent inhibition of L1 shedding by specific ADAM10 (2 µm GI254023X) and ADAM17 (5 µ m TAPI‐1) inhibitors. (E, F) Quantification of L1 ectodomain expression reveals a significant activation of L1 shedding by PMA treatment (50 n m in e; 500 n m in f) and its significant inhibition by administration of ADAM10 (2 µ m GI254023X) and ADAM17 (5 µ m TAPI‐1) inhibitors in the RB cell lines RB355 and Y79. Values are means of three independent experiments ± SEM. ns P > 0.05; * P < 0.05; ** P < 0.01; and *** P < 0.001 statistical differences compared to the control group calculated by one‐way ANOVA with Newman–Keuls post‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Activation Assay, Inhibition, Western Blot, Cell Culture, Positive Control, Control, Negative Control, Expressing

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: MiRNA‐146a‐5p and L1CAM expression levels in parental, chemosensitive, and etoposide‐resistant WERI‐Rb1, Y79, and RB355 RB cell lines as revealed by real‐time PCR (A) and western blot analysis (B). The indicated intensity ratios relative to ß‐actin, used as a loading control, were calculated using micro manager 1.4 software. Values are means of five independent experiments ± SEM. ns P > 0.05 and * P < 0.05 statistical differences compared to the control group calculated by paired Student's t ‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Software

Journal: Molecular Oncology

Article Title: Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

doi: 10.1002/1878-0261.13054

Figure Lengend Snippet: Ezrin, galectin‐3 (Gal‐3), and FGFb expression levels after shRNA‐mediated L1CAM knockdown (shL1) in the RB cell line RB355 (A, B) and stable L1CAM overexpression (L1OE) in RB247 cells (C, D) as revealed by real‐time PCR (A, C) and western blot analysis (B, D). The indicated intensity ratios relative to ß‐actin, used as a loading control, were calculated using micro manager 1.4 software. Values are means of three independent experiments ± SEM. ns P > 0.05; * P < 0.05; and *** P < 0.001 statistical differences compared to the control group calculated by paired Student's t ‐test.

Article Snippet: The RB cell lines Y79 [ ] and WERI‐Rb1 [ ], originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr. H. Stephan.

Techniques: Expressing, shRNA, Knockdown, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Control, Software

Journal: OncoTargets and therapy

Article Title: Long non-coding RNA homeobox A11 antisense RNA (HOXA11-AS) promotes retinoblastoma progression via sponging miR-506-3p

doi: 10.2147/OTT.S195404

Figure Lengend Snippet: HOXA11-AS was up-regulated in RB tissues and cell lines. (A) The relative expression levels of HOXA11-AS was examined in 30 RB tissues and 10 normal retina tissue samples by using qRT-PCR analysis. ( B ) The relative expression levels of HOXA11-AS was examined in four human RB cell lines (Y79, Weri-Rb1, SO-Rb50 and HXO-RB44) and human retinal epithelial cells ARPE-19 by using qRT-PCR analysis. ** P< 0.01. Abbreviation: RB, retinoblastoma.

Article Snippet: The human retinal epithelial cells ARPE-19 and four human RB cell lines Y79, Weri-Rb1, SO-Rb50, and HXO-RB44 were bought from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin (Invitrogen; Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR

Journal: OncoTargets and therapy

Article Title: Long non-coding RNA homeobox A11 antisense RNA (HOXA11-AS) promotes retinoblastoma progression via sponging miR-506-3p

doi: 10.2147/OTT.S195404

Figure Lengend Snippet: Knockdown of HOXA11-AS suppressed proliferation, and induced apoptosis in RB cells. ( A ) The relative expression levels of HOXA11-AS was examined in Y79 cells transfected with si-HOXA11-AS or si-NC. Subsequently, ( B ) cell proliferation, ( C ) cell cycle distribution and ( D ) cell apoptosis were determined in Y79 cells transfected with si-HOXA11-AS or si-NC. * P <0.05, ** P< 0.01. Abbreviation: RB, retinoblastoma.

Article Snippet: The human retinal epithelial cells ARPE-19 and four human RB cell lines Y79, Weri-Rb1, SO-Rb50, and HXO-RB44 were bought from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin (Invitrogen; Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Knockdown, Expressing, Transfection

Journal: OncoTargets and therapy

Article Title: Long non-coding RNA homeobox A11 antisense RNA (HOXA11-AS) promotes retinoblastoma progression via sponging miR-506-3p

doi: 10.2147/OTT.S195404

Figure Lengend Snippet: HOXA11-AS acted as a molecular sponge for miR-506-3p in RB cells. ( A ) Schematic representation of the predicted binding sites between miR-506-3p and HOXA11-AS, and the mutagenesis design for the reporter assays. ( B ) Luciferase activity in Y79 cells co-transfected with miR-506-3p mimics or miR-NC, and luciferase reporters containing HOXA11-AS-WT or HOXA11-AS-MT. ( C ) The relative expression of miR-506-3p was determined in Y79 cells transfected with si-HOXA11-AS or si-NC. ( D ) The relative expression levels of HOXA11-AS was determined in Y79 cells transfected with miR-506-3p mimics or miR-NC. ( E ) The relative expression levels of miR-506-3p were examined in 30 RB tissues and 10 normal retina tissue samples using qRT-PCR analysis. ( F ) An inverse association between HOXA11-AS and miR-506-3p expression in RB tissues was identified by Pearson’s correlation analysis. ** P< 0.01. Abbreviation: RB, retinoblastoma.

Article Snippet: The human retinal epithelial cells ARPE-19 and four human RB cell lines Y79, Weri-Rb1, SO-Rb50, and HXO-RB44 were bought from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin (Invitrogen; Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: OncoTargets and therapy

Article Title: Long non-coding RNA homeobox A11 antisense RNA (HOXA11-AS) promotes retinoblastoma progression via sponging miR-506-3p

doi: 10.2147/OTT.S195404

Figure Lengend Snippet: miR-506-3p inhibition restored the si-HOXA11-AS-induced effects on cell proliferation, cycle arrest and apoptosis in RB cells. ( A ) The relative expression of miR-506-3p expression was determined in Y79 cells transfected with si-NC or si-HOXA11-AS, with or without the miR-506-3p inhibitor. ( B–D ) Cell proliferation, cycle arrest and apoptosis were determined in Y79 cells transfected with si-NC or si-HOXA11-AS, with or without the miR-506-3p inhibitor. * P <0.05, ** P< 0.01. Abbreviation: RB, retinoblastoma.

Article Snippet: The human retinal epithelial cells ARPE-19 and four human RB cell lines Y79, Weri-Rb1, SO-Rb50, and HXO-RB44 were bought from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin (Invitrogen; Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Inhibition, Expressing, Transfection

Journal: OncoTargets and therapy

Article Title: Long non-coding RNA homeobox A11 antisense RNA (HOXA11-AS) promotes retinoblastoma progression via sponging miR-506-3p

doi: 10.2147/OTT.S195404

Figure Lengend Snippet: HOXA11-AS regulated the expression of NEK6 through miR-506-3p in RB cells. ( A ) The relative expression of NEK6 was measured in Y79 cells transfected with si-NC or si-HOXA11-AS, with or without the miR-506-3p inhibitor. ( B ) The relative expression levels of NEK6 mRNA were examined in 30 RB tissues and 10 normal retina tissue samples by using qRT-PCR analysis. ( C ) An inverse association between NEK6 mRNA and miR-506-3p expression in RB tissues was identified by Pearson’s correlation analysis. ( D ) A positive association between NEK6 mRNA and HOXA11-AS expression in RB tissues was identified by Pearson’s correlation analysis. * P <0.05, ** P< 0.01. Abbreviation: RB, retinoblastoma.

Article Snippet: The human retinal epithelial cells ARPE-19 and four human RB cell lines Y79, Weri-Rb1, SO-Rb50, and HXO-RB44 were bought from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin (Invitrogen; Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Transfection, Quantitative RT-PCR