ly3039478  (MedChemExpress)

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Drug-resistant cells were generated by treating the MB157 cells for 25 weeks with 1 µM γ-secretase inhibitor (GSI, Spirochem, LY3039478), replenishing the inhibitor every 3–4 days.

Techniques: Recombinant, Plasmid Preparation, Binding Assay, Polymer, SYBR Green Assay, Transfection, Protease Inhibitor, Reporter Assay, Gel Purification, Purification, Ligation, Software

Journal: Cell Death & Disease

Article Title: ALYREF promotes the metastasis of nasopharyngeal carcinoma by increasing the stability of NOTCH1 mRNA

doi: 10.1038/s41419-024-06959-1

Figure Lengend Snippet: A GSEA results of SUNE1 and SUNE2 cells with ALYREF knockdown (siALYREF) or siRNA control (siNC). B The relative Flag-RIP enrichment ratio of NOTCH1 mRNA in HONE1 cells with ALYREF overexpression or vector control. ACTB was used as the negative control. Fold enrichment was determined by calculating the 2 -ΔCt of each RIP sample relative to the corresponding input sample. The mRNA and protein levels of NOTCH1 in NPC cells with ALYREF overexpression ( C ) and ALYREF knockdown ( D ) were determined using qRT-PCR and western blotting, respectively. E The mRNA and protein levels of downstream targets (HES1, HEY1) of the Notch signaling pathway in SUNE1 cells with ALYREF knockdown or the siNC control were determined by qRT-PCR and western blotting, respectively. F Representative immunohistochemical images of lung tissues isolated from mice injected with vector control HONE1 cells (left) or ALYREF-overexpressing cells (right) via the tail vein, confirming the high expression of NOTCH1 in the ALYREF overexpression group. G The protein levels of NOTCH1 in lung metastatic nodules with ALYREF overexpression or vector control in nude mice ( n = 5 per group) were determined by western blot analysis. H Inhibition of the NOTCH1 pathway abrogated the ALYREF-mediated upregulation of NPC cell invasion and metastasis. HONE1-overexpressing and corresponding vector control cells were treated with 50 mg/L LY3039478 for 24 h. Scale bar: 100 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: To determine the effect of NOTCH1 on the role of ALYREF in NPC metastasis, 50 mg/L of the NOTCH1 inhibitor LY3039478 (Selleckchem Chemicals, USA) was used to treat the HONE1 cells with ALYREF overexpression or vector control cells for 24 h.

Techniques: Knockdown, Control, Over Expression, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Isolation, Injection, Expressing, Inhibition

Journal: Cell Death & Disease

Article Title: ALYREF promotes the metastasis of nasopharyngeal carcinoma by increasing the stability of NOTCH1 mRNA

doi: 10.1038/s41419-024-06959-1

Figure Lengend Snippet: A The m5C sites within NOTCH1 (at position chr9:13939412 and 13939452) were identified via RNA BS-Sanger sequencing. The cDNA was amplified by PCR using conventional primers for untreated mRNA and specific primers for bisulfite-treated mRNA. Knockdown efficiency of NSUN2 and NSUN6 in HONE1 cells was determined by qRT-PCR ( B ) and western blot analysis ( C ). D The relative Flag-RIP enrichment ratio of the NOTCH1 mRNA in ALYREF-overexpressing and vector control HONE1 cells transfected with siNSUN2, siNSUN6, or siNC, respectively. The RIP assays were performed using an anti-Flag antibody after 48 h of siRNA transfection. Fold enrichment was determined by calculating the 2 −ΔCt of the RIP sample relative to the corresponding input sample. E The relative IgG or anti-m5C antibody enrichment of the NOTCH1 mRNA in HONE1 cells transfected with siNSUN2 or siNC siRNA. The RIP assays were performed using an antibody specific for m5C or IgG control. ACTB was used as the negative control. Fold enrichment was determined by calculating the 2 −ΔCt of the RIP sample relative to the corresponding input sample. F The relative Flag-RIP enrichment of the NOTCH1 mRNA was measured in 293 T cells co-transfected with plasmids encoding wild-type or mutant NOTCH1 combined with the plasmid encoding ALYREF or vector control for 24 h. The fold Flag-RIP enrichment was determined by calculating the 2 −ΔCt of the RIP sample relative to the corresponding input sample. G Residual mRNA levels of NOTCH1 after termination of transcription via actinomycin D treatment in ALYREF overexpressed or vector control HONE1 cells. H 293 T cells were co-transfected the ALYREF plasmid and the wild-type or mutant NOTCH1 plasmids for 24 h. The relative mRNA levels were normalized to the housekeeping gene GAPDH. Experiments were independently repeated three times, and the results are presented as the means ± SD of n = 3 biological replicates. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: To determine the effect of NOTCH1 on the role of ALYREF in NPC metastasis, 50 mg/L of the NOTCH1 inhibitor LY3039478 (Selleckchem Chemicals, USA) was used to treat the HONE1 cells with ALYREF overexpression or vector control cells for 24 h.

Techniques: Sequencing, Amplification, Knockdown, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Control, Transfection, Negative Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: ALYREF promotes the metastasis of nasopharyngeal carcinoma by increasing the stability of NOTCH1 mRNA

doi: 10.1038/s41419-024-06959-1

Figure Lengend Snippet: A The correlation between NOTCH1 and NSUN2, as well as ( B ) NSUN2 and ALYREF protein levels was assessed using 92 paraffin-embedded human NPC tissues. C The correlation between NOTCH1 and ALYREF expression at the mRNA level in NPC tissues was assessed using a publicly available RNA profile data (HRA000035). D Working model of the proposed mechanism based on the results of this study. In NPC cells, NSUN2 mediates the m5C modification of NOTCH1 mRNA, after which ALYREF cats as an m5C reader protein that increases its stability. Finally, the increased NOTCH1 protein levels promote NPC cell metastasis through the activation of the Notch signaling pathway, leading to NPC progression. Notably, the oncogenic function of ALYREF could be abrogated using the NOTCH1 inhibitor LY3039478. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To determine the effect of NOTCH1 on the role of ALYREF in NPC metastasis, 50 mg/L of the NOTCH1 inhibitor LY3039478 (Selleckchem Chemicals, USA) was used to treat the HONE1 cells with ALYREF overexpression or vector control cells for 24 h.

Techniques: Expressing, Modification, Activation Assay