alphascreen assays  (OriGene)

Journal: Advanced Science

Article Title: The Super Enhancer‐Driven Long Noncoding RNA PRKCQ‐AS1 Promotes Neuroblastoma Tumorigenesis by Interacting With MSI2 Protein and Is Targetable by Small Molecule Compounds

doi: 10.1002/advs.202412520

Figure Lengend Snippet: The small molecule compound NSC617570 suppresses the interaction between PRKCQ‐AS1 RNA and MSI2 protein and shows anticancer effects in vitro and in vivo. A) PRKCQ‐AS1 RNA fragment 1 (65‐306 bp at the 5′‐ end) was in vitro transcribed, biotin‐labelled, mixed with Flag‐tagged MSI2 protein, and subjected to AlphaScreen of 2932 small molecule compounds at 10 µM for inhibitors of the interaction between PRKCQ‐AS1 RNA and MSI2 protein. Eight‐five compounds were found to reduce the interaction between PRKCQ‐AS1 RNA and MSI2 protein by > 70% (above the red line). B) Structure of the “hit” compound NSC617570. C) SK‐N‐AS cells were treated with vehicle control or 10 µM NSC617570 for 6 h, followed by RNA immunoprecipitation assays with control IgG or MSI2 antibody and RT‐PCR with primers targeting PRKCQ‐AS1 or BMX. Data were shown as the mean ± standard deviation of three independent experiments and evaluated by Student's t ‐test. * indicated p < 0.05. D‐E) SK‐N‐AS and SK‐N‐SH cells were treated with vehicle control, 5 µM or 10 µM NSC617570 for 48 h, followed by RT‐PCR analysis of BMX mRNA (D) and immunoblot analysis of BMX protein, total ERK protein (ERK) and phosphorylated ERK protein (phos‐ERK) (E). F) SK‐N‐AS and SK‐N‐SH cells were treated with vehicle control or a range of doses of NSC617570 for 72 h, followed by Alamar blue assays. Data were shown as the mean ± standard deviation of three independent experiments and evaluated by ANOVA. *, ** and *** indicated p < 0.05, 0.01 and 0.001 respectively. G) SK‐N‐AS and SK‐N‐SH cells were treated with vehicle control, 5 µM or 10 µM NSC617570 for 14 (SK‐N‐AS) or 21 (SK‐N‐SH) days, followed by clonogenic assays. H‐I) SK‐N‐AS cells were xenografted into nude mice. When tumors reached 50 mm 3 , the mice were treated with NSC617570 at 8.75 mg/kg or vehicle control via i.p. injection, 5 times per week. Tumor growth was monitored (H) and mouse overall survival was analyzed (I). For survival analysis, P value was obtained from two‐sided log‐rank test.

Article Snippet: AlphaScreen assays were performed using purified human recombinant MSI2 protein (TP301003, Origene Technologies, Rockville, MD) and PRKCQ‐AS1 RNA fragment 1 (306 base pairs).

Techniques: In Vitro, In Vivo, Amplified Luminescent Proximity Homogenous Assay, Control, RNA Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Western Blot, Injection