Journal: Oncology Reports

Article Title: Retinoic acid induces differentiation in neuroblastoma via ROR1 by modulating retinoic acid response elements

doi: 10.3892/or.2020.7681

Figure Lengend Snippet: RA promotes NB differentiation via ROR1. (A) Immunoblotting revealed the changes in ROR1 and synaptophysin levels under normal differentiation with RA. (B) Densitometry of immunoblots from A, baseline-corrected to GAPDH (ANOVA with Tukey's multiple comparison to all conditions, n=3). **P<0.005, ***P<.0005, ****P<0.0001. (C) Phase contrast images of SK-N-SH neuroblastoma cells undergoing RA or DMSO treatment over 96 h under shRNA test Lipofectamine transfection, knocking down ROR1 expression. Yellow arrows indicate marked neurite extensions that suggest development. (D) Visualization of the average neurite length over time following knockdown of ROR1 (n=3). (E) Immunoblots revealed the changes in ROR1 and synaptophysin levels after Lipofectamine transfection, knocking down ROR1 expression. (F) Densitometry of immunoblots from E, baseline-corrected to GAPDH (ANOVA with Tukey's multiple comparison test to compare each condition with every other condition in the experiment, n=3). ****P<0.0001. (G) Immunofluorescence revealed the effects of RA on synaptophysin (green) levels with or without shRNA knockdown of ROR1 (red). DAPI (blue) highlights the nucleus of the neurons. Yellow arrows indicate marked neurite extensions. RA, retinoic acid; NB, neuroblastoma; ROR1, receptor tyrosine kinase-like orphan receptor 1; NT, not treated.

Article Snippet: The primary antibodies used were as follows and diluted 1:1,000 unless specifically specified differently under manufacturer recommendations: ROR1 (D6T8C; product no. 16540; Cell Signaling Technology, Inc.), GAPDH (D16H11; product no. 5174; Cell Signaling Technology, Inc.), p-AKT-ser473 (product no. 9271; Cell Signaling Technology, Inc.), AKT (C67E7; product no. 4691; Cell Signaling Technology, Inc.), p-GSK3β-Ser9 (D17D2; product no. 8566; Cell Signaling Technology, Inc.), and GSK3β (D75D3; product no. 5676; Cell Signaling Technology, Inc.), β-catenin (cat. no. MA1-301; Invitrogen; Thermo Fisher Scientific, Inc.), p-β-catenin-Ser33/37 (product no. 2009; Cell Signaling Technology, Inc.), stabilized-β-catenin (product no. 9562; Cell Signaling Technology, Inc.), Wnt-5a (cat. no. NBP2-24752SS; Novus Biologicals, Ltd.), Synaptophysin (cat. no. 100298-T40; Sino Biological, Inc.), β-tubulin (product code ab21057; Abcam) and Lamin B1 (product code ab65986; Abcam).

Techniques: Western Blot, shRNA, Transfection, Expressing, Immunofluorescence

Journal: Oncology Reports

Article Title: Retinoic acid induces differentiation in neuroblastoma via ROR1 by modulating retinoic acid response elements

doi: 10.3892/or.2020.7681

Figure Lengend Snippet: Effects of RA on ROR1 and differentiation is RAR-dependent. (A) Light-microscopic images (×100) after various treatments modulating the degree of ROR1 in the cell at 0 and 96 h. Yellow arrows indicate marked neurite lengths and neurite clusters that suggest progression of development. (B) Visualization of average neurite length after ROR1-modulating treatments over 96 h (n=3). *P<0.05. (C) Immunoblots of synaptophysin and ROR1 after ROR1-modulating treatments. (D) Densitometric analysis of immunoblots in C assessing ROR1 and synaptophysin levels (ANOVA with Tukey's multiple comparison test to compare each condition with every other condition in the experiment, n=3). **P<0.005, ****P<0.0001. RA, retinoic acid; ROR1, receptor tyrosine kinase-like orphan receptor 1; RAR, RA receptor.

Article Snippet: The primary antibodies used were as follows and diluted 1:1,000 unless specifically specified differently under manufacturer recommendations: ROR1 (D6T8C; product no. 16540; Cell Signaling Technology, Inc.), GAPDH (D16H11; product no. 5174; Cell Signaling Technology, Inc.), p-AKT-ser473 (product no. 9271; Cell Signaling Technology, Inc.), AKT (C67E7; product no. 4691; Cell Signaling Technology, Inc.), p-GSK3β-Ser9 (D17D2; product no. 8566; Cell Signaling Technology, Inc.), and GSK3β (D75D3; product no. 5676; Cell Signaling Technology, Inc.), β-catenin (cat. no. MA1-301; Invitrogen; Thermo Fisher Scientific, Inc.), p-β-catenin-Ser33/37 (product no. 2009; Cell Signaling Technology, Inc.), stabilized-β-catenin (product no. 9562; Cell Signaling Technology, Inc.), Wnt-5a (cat. no. NBP2-24752SS; Novus Biologicals, Ltd.), Synaptophysin (cat. no. 100298-T40; Sino Biological, Inc.), β-tubulin (product code ab21057; Abcam) and Lamin B1 (product code ab65986; Abcam).

Techniques: Western Blot

Journal: Oncology Reports

Article Title: Retinoic acid induces differentiation in neuroblastoma via ROR1 by modulating retinoic acid response elements

doi: 10.3892/or.2020.7681

Figure Lengend Snippet: RA pathway model. The proposed proliferation and differentiation modulation pathway of RA. RA binds to RAR in the cytoplasm and migrates into the nucleus to act on RARE regions. Normal transcription and translation result in increased ROR1 and Wnt5a synthesis and the ROR1 signaling pathway cascades to synaptophysin as a node of differentiation as well as β-catenin-related growth and proliferation factors. The image was created using BioRender. RA, retinoic acid; ROR1, receptor tyrosine kinase-like orphan receptor 1; RAR, RA receptor; RARE, RA response element.

Article Snippet: The primary antibodies used were as follows and diluted 1:1,000 unless specifically specified differently under manufacturer recommendations: ROR1 (D6T8C; product no. 16540; Cell Signaling Technology, Inc.), GAPDH (D16H11; product no. 5174; Cell Signaling Technology, Inc.), p-AKT-ser473 (product no. 9271; Cell Signaling Technology, Inc.), AKT (C67E7; product no. 4691; Cell Signaling Technology, Inc.), p-GSK3β-Ser9 (D17D2; product no. 8566; Cell Signaling Technology, Inc.), and GSK3β (D75D3; product no. 5676; Cell Signaling Technology, Inc.), β-catenin (cat. no. MA1-301; Invitrogen; Thermo Fisher Scientific, Inc.), p-β-catenin-Ser33/37 (product no. 2009; Cell Signaling Technology, Inc.), stabilized-β-catenin (product no. 9562; Cell Signaling Technology, Inc.), Wnt-5a (cat. no. NBP2-24752SS; Novus Biologicals, Ltd.), Synaptophysin (cat. no. 100298-T40; Sino Biological, Inc.), β-tubulin (product code ab21057; Abcam) and Lamin B1 (product code ab65986; Abcam).

Techniques:

Journal: PLoS ONE

Article Title: The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

doi: 10.1371/journal.pone.0066290

Figure Lengend Snippet: GDNF increases mRNA expression of synaptophysin in rat enteric nerve cell cultures. Expression levels were measured after one week and are normalized to expression of the house-keeping gene HPRT. Data are shown as mean +/− SEM, n = 11–12 per experimental group, *p<0.05 vs. control.

Article Snippet: After fixation and permeabilization as described above, samples were incubated with a rabbit anti-synaptophysin antibody (1∶1000, Biozol, Echingen, Germany) and a mouse anti-PGP 9.5 antibody (1∶1000, Acris, Herford, Germany) for 1 h. After 30 min incubation with the secondary antibodies, goat anti-rabbit-AlexaFlour488 (1∶250, Invitrogen, Karlsruhe, Germany) and goat-anti-mouse-AlexaFluor546 (1∶250, Invitrogen, Karlsruhe, Germany), cells were counterstained with DAPI (Roche, Mannheim, Germany) to visualize cell nuclei.

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

doi: 10.1371/journal.pone.0066290

Figure Lengend Snippet: Rat enteric nerve cells were cultured for 3 week without (A-C) or with 50 ng/ml GDNF (D-F). Dual label immunocytochemistry for synaptophysin (green, A, D) and the pan-neuronal marker PGP 9.5 (B, E) was performed. In the merged pictures (C, F) cellular nuclei are stained with DAPI (blue). Cell cultures treated with GDNF display punctuate and granular synaptophysin immunoreactivity along ramifying nerve fibers most likely resembling accumulated synaptic vesicles, whereas in untreated cell cultures immunoreactive signals were confined to neuronal somata. Magnification: 40×.

Article Snippet: After fixation and permeabilization as described above, samples were incubated with a rabbit anti-synaptophysin antibody (1∶1000, Biozol, Echingen, Germany) and a mouse anti-PGP 9.5 antibody (1∶1000, Acris, Herford, Germany) for 1 h. After 30 min incubation with the secondary antibodies, goat anti-rabbit-AlexaFlour488 (1∶250, Invitrogen, Karlsruhe, Germany) and goat-anti-mouse-AlexaFluor546 (1∶250, Invitrogen, Karlsruhe, Germany), cells were counterstained with DAPI (Roche, Mannheim, Germany) to visualize cell nuclei.

Techniques: Cell Culture, Immunocytochemistry, Marker, Staining

Journal: Chinese Journal of Lung Cancer

Article Title: 神经内分泌分化不是非小细胞肺癌高恶性度的指标

doi: 10.3779/j.issn.1009-3419.2011.08.03

Figure Lengend Snippet: 主要试剂、来源及工作浓度 Main reagents, sources and working concentrations

Article Snippet: Rabbit anti-human synaptophysin (Syn) monoclonal antibody , ZSGB-BIO , SP11 , 1:100.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neuropathic Pain Memory Is Maintained by Rac1-Regulated Dendritic Spine Remodeling after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.3142-08.2008

Figure Lengend Snippet: Western blot analysis of synapse-associated proteins in lumbar spinal cord tissue from intact, SCI and control, vehicle- or NSC23766-treated animals. Quantification was performed on three blots for each protein. To normalize for variation between blots, β-actin control protein level was set to 100% in each lane and proteins of interest were compared across treatment groups. A, Phosphorylated Rac1 protein was probed in the three treatment groups with a visible band at ∼21 kDa. B, Significantly increased levels of phosphorylated Rac1 were found after SCI plus veh treatment compared with uninjured controls. Post-SCI levels were reduced with intrathecal NSC23766. C, D, No significant difference in the levels of total Rac (∼21 kDa) was observed between the treatment groups. E, F, PSD-95 band is visible at ∼95 kDa (E), and significantly higher levels were observed in SCI plus veh compared with intact levels (F). NSC23766 treatment also significantly reduced PSD-95 levels below that of SCI plus veh and intact animals. G, A cortactin band was observed at ∼80 kDa along with breakdown products. H, No differences were observed for levels of cortactin between groups. I, J, We also observed a trend, which did not reach statistical significance, toward an increase in the levels of synaptophysin after SCI. Graphs are mean ± SEM. *p < 0.05; **p < 0.01.

Article Snippet: The following primary antibodies were used: rabbit anti-phospho Rac1 plus Cdc42 (Abcam; 1:250), mouse anti-Rac1 (Millipore), rabbit anti-cortactin (Santa Cruz Biotechnologies; 1:2000), mouse anti-PSD-95 (Abcam; 1:2000), rabbit anti-synaptophysin (GeneTex), and rabbit anti-β-actin (Abcam; 1:5000).

Techniques: Western Blot