Fig. 2

Spy1 negatively regulates the transcriptional activity of CLIP3 by CDK2/NRF1 signaling. A mRNA and protein levels of Spy1 and CLIP3 were analyzed by real-time qRT-PCR and Western blot, respectively, upon Spy1 siRNA treatment in U87MG and T98G cells. B A possible NRF1 binding site on the CLIP3 promoter was predicted using GeneCards database (https://www.genecards.org/). NRF1 was expected to bind − 24 to − 14 (GCGCATGCGCA) from the transcription start site (TSS) of CLIP3. C mRNA and protein levels of NRF1 and CLIP3 were analyzed by real-time qRT-PCR and Western blot, respectively, upon NRF1 siRNA treatment in U87MG and T98G cells. D Luciferase activity was measured upon transfection of pGL3-NFAT-luc plasmids, including wild-type (CLIP3-Luc) or mutant CLIP3 (CLIP3 mut-Luc) promoter linked to luciferase gene, in the absence or presence of transfection of NRF1 gene in U87MG and T98G cells. E Western blot with CDK2 or HA antibodies after immunoprecipitation of Spy1 from U87MG and T98G whole cell lysates at 12 and 24 h after IR (3 or 6 Gy) using a HA antibody. Protein levels of CDK2, Spy1, and β-actin in the whole cell lysates were analyzed at 12 and 24 h after IR (3 or 6 Gy) by western blot. F A schematic diagram illustrates that Spy1 negatively regulates CLIP3 through CDK2/NRF1 signaling. G Luciferase activity was measured upon transfection of the pGL3-NFAT-luc plasmids with Spy1 gene, CDK2 siRNA, or NRF1 siRNA treatment in U87MG and T98G cells. Statistical analysis was performed with Student’s t-test for (A) and (C), and one-way ANOVA plus a Tukey’s multiple comparisons test for (D) and (G). *p < 0.05, **p < 0.01, ****p < 0.0001