Fig. 3: NUCKS1 transcriptionally upregulated ASNS expression.

A Heatmap showing the altered genes identified by RNA sequencing analysis in NUCKS1-depleting MNNG/HOS cells. B The differentially expressed mRNAs for NUCKS1 knockdown versus the control were shown (absolute log2-fold change >1 and q value < 0.05). The blue indicates the downregulated genes, and the pink indicates the upregulated genes. C The mRNA levels of ASNS are shown from RNA sequencing data. D, E The protein and mRNA levels of ASNS were detected by western blot (D) and qRT‒PCR (E) in osteosarcoma cells with or without NUCKS1 knockdown. F, G The protein and mRNA levels of ASNS were detected by western blot (F) and qRT‒PCR (G) in osteosarcoma cells with or without NUCKS1 overexpression. H Schematic illustration of pGL3-based reporter constructs used in luciferase assays to examine the transcriptional activity of ASNS named P1, P2, P3, and P4. I P1, P2, P3, and P4 together with the Renilla luciferase plasmid were transfected into MNNG/HOS cells with or without NUCKS1 overexpression. The Renilla luciferase construct was used to control for transfection efficiency, and dual luciferase activity was measured. J Schematic illustration of pGL3-based reporter constructs used in luciferase assays to examine the transcriptional activity of ASNS named P5 (Up). P5 together with the Renilla luciferase plasmid was transfected into MNNG/HOS cells with or without NUCKS1 overexpression. Dual luciferase activity was measured. K P5 together with the Renilla luciferase plasmid was transfected into MNNG/HOS with or without NUCKS1 knockdown, and the cells were collected. Dual luciferase activity was measured. L, M ChIP analysis showed the binding of NUCKS1 to the promoter of ASNS in MNNG/HOS cells with or without NUCKS1 knockdown. Isotype-matched IgG was used as a negative control. Data in E, G, I–M were analyzed by Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001.