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. 2020 Jul 8;11(7):515. doi: 10.1038/s41419-020-2713-8

Fig. 6. SNHG12 bound to and stabilised SP1, which activated CDCA3 transcription.

Fig. 6

a qRT-PCR for mRNA levels of SP1 and CDCA3 in transfected ACHN cells. b western blot assays for protein levels of SP1 and CDCA3 in transfected ACHN and 786-O cells. c The predicted positions of putative SP1 binding motif in −2000-bp human CDCA3 promoter. d ChIP-PCR assays were performed to show direct binding of SP1 to CDCA3 promoter regions in ACHN cells. e Luciferase reporter assays were performed by co-transfecting the wild type CDCA3 promoter or fragment E2-mutant CDCA3 promoter with SP1 overexpression vector or blank vector in 293T cells. f Anti-SP1 RIP-PCR assays were performed in ACHN and 786-O cells to show SP1 directly bound to SNHG12. g qRT-PCR and western blot for mRNA and protein levels of SP1 in transfected RCC cells. h, i SP1 protein levels were measured by western blot in RCC cells after transfected sh SNHG12 or SNHG12 overexpression vector and treated with cycloheximide (CHX) for a certain period of time. j Cells with SNHG12 knockdown were treated with vehicle (DMSO), MG132 (20 nM) or chloroquine (50 nM) for 24 h. Western blot assays were applied to show SP1 protein levels. k Immunoprecipitation with an anti-SP1 antibody were performed in SNHG12 knockdown or overexpression RCC cells, and analysed by western blotting with an anti-ubiquitin antibody. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate mean ± SD.