Fig 1: Upregulation of Twist1 by KLF16 mediates the promoting effect of SF3B4 on ccRCC progression.A–C Caki-1 and 769-P cells were transfected with indicated constructs, and then western blot and RT-qPCR detected Twist1 expression. D The potential KLF16-binding motif on the proximal promoter of Twist1 gene. E ChIP-PCR was used to verify the binding site of KLF16 on the Twist1 promoter. F Dual-luciferase reporter gene examined the role of SF3B4 and KLF16 in regulating Twist1 promoter activity by co-transfection with indicated constructs. G, H Caki-1 cells were transfected with shTwist1 and oeKLF16, alone or together, and then transwell assay was used to examine cell migration and invasion. Scale bar = 50 µm. I The expression of E-cadherin and vimentin were detected by double immunofluorescence staining in cells transfected with indicated constructs. Scale bar = 20 µm. J, K Caki-1 cells were transfected with shSF3B4 and oeKLF16, alone or together, and then the expression of EMT-related genes was detected by western blot. L, M 769-P cells were transfected with oeSF3B4 and shTwist1, alone or together, and then the expression of EMT-related genes was detected by western blot. All data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. their corresponding controls.
Fig 2: SF3B4 upregulates KLF16 expression by promoting its mRNA export from the nucleus.A, B Western blot analysis examined KLF16 expression in MG132-treated cells after oeSF3B4 or shSF3B4 transfection. C The KLF16 mRNA was examined by RT-qPCR in actinomycin D-treated cells after transfection with the indicated constructs. D Caki-1 cells were transfected with oeSF3B4 or empty vector, and SF3B4 in the anti-SF3B4 immunoprecipitates was measured by Western blotting. E RNA binding protein immunoprecipitation assay (RIP) detected KLF16 mRNA enrichment by SF3B4 or RBM25 antibody. F Biotinylated RNA probes of KLF16 mRNA were used to pull down the RNA-protein complex, and the Western blot analysis detected SF3B4 enrichment. G Caki-1 cells were transfected with oeSF3B4 (oe) or pWPI (pW) and then RNA was isolated from the nucleus (Nuc) and cytoplasm (Cyt), mRNA of S14, KLF16, and NEAT RNA was detected by RT-qPCR. S14 acts as plasma-specific RNA and NEAT1 RNA as nucleus-specific RNA. H Caki-1 cells were treated as in G, and the expression of SF3B4 protein and KLF16 mRNA as well as their distribution in the nucleus and cytoplasm were detected by immunofluorescence combined with FISH. SF3B4 antibody (red) was used to detect SF3B4 protein, while the FITC-probe (green) was used to detect KLF16 mRNA. I Quantitative analysis of the KLF16 mRNA distribution in the nucleus and cytoplasm. Scale bar = 5 µm. All data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. their corresponding controls.
Fig 3: SF3B4-KLF16-Twist1 axis promotes the progression of ccRCC in vivo.A Caki-1 cells, engineered to stably knock down KLF16 or stably overexpress SF3B4, separately or together, were implanted into nude mice to establish xenograft tumors. The tumor sizes in each group were presented after 28 days. B Tumor volumes were measured by direct measurement with calipers. C Xenograft tumor wet weight was determined after the resection of tumors. D The expression of SF3B4, KLF16, Twist1, E-cadherin, and vimentin was examined in xenograft tumors by western blotting. E Quantitative analysis of B. F Double immunofluorescence staining of vimentin and E-cadherin in xenograft tumors. Scale bar = 20 µm. All data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. their corresponding controls.
Fig 4: KLF16 mediates SF3B4 upregulation of Twist1 expression.A Potential target genes of SF3B4 were analyzed from the data of gene transcriptome of SF3B4-depleted cells (Profile), genes expressed in association with SF3B4 in ccRCC tissues in TCGA data (Relation) and transcription factor (TF), and are shown by a Venn diagram. B RT-qPCR detected the Twist1 mRNA expression in Caki-1 cells transfected with the indicated siRNAs for candidate transcription factors. C, D Western blot examined the KLF16 expression in Caki-1 and 769-P cells transfected with the indicated constructs. E Cells were transfected as in C and then RT-qPCR detected the KLF16 mRNA expression. F, G Caki-1, and 769-P cells were transfected as indicated, and then RT-qPCR detected the expression of Twist1 mRNA. H, I Caki-1 cells were transfected with pWPI-KLF16 (oeKLF16) and shSF3B4, alone or together, and then the protein levels of KLF16, Twist1, and E-cadherin were examined by western blot. J, K 769-P cells were transfected with oeKLF16 and oeSF3B4, alone or together, and then KLF16, Twist1, and E-cadherin were examined by western blot. L KLF16 mRNA expression was detected by RT-qPCR in normal (N, n = 53) or ccRCC (T, n = 53) tissues. M KLF16 protein level was examined by western blot from three pair of randomly selected clinical ccRCC samples. N Quantitative analysis of M. O The expression level of KLF16 was downloaded and then analyzed from data of the TCGA database in normal (n = 72) and ccRCC (n = 533) tissues. P The correlation between SF3B4 and KLF16 mRNA level in ccRCC tissues was analyzed by Pearson correlation analysis of our clinical data (R = 0.3661, P = 0.0078). Q According to the TCGA database, the overall survival for ccRCC patients with low and high Twist1 levels were analyzed by using the Kaplan–Meier method. All data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. their corresponding controls.
Fig 5: Proposed model for SF3B4-KLF16-Twist1 regulation of pRCC progression.
Supplier Page from Abcam for Anti-KLF16 antibody