Fig 1: Immunohistochemistry on tissue microarrays of different grades of astrocytomas (A) and MS/MS spectra of representative peptides (B) for six selected proteins - NUCKS1, SMARCA5, PARP1, PTBP1, HMGB2 and NFIB. IHC analysis of the proteins tested using commercially available tissue microarrays (US BioMax), confirmed overexpression of these proteins in multiple tumor specimens. The details of tissue microarrays used and IHC procedure are described in the Methods and the staining scoring details are shown in Supplementary Table S9. MS/MS spectra acquisition is described under Methods.
Fig 2: NFIB was highly expressed and was associated with poor prognosis in KIRC.(A) Venn plot diagram of differentially expressed genes in TCGA and GEO (GSE83999 and GSE53757) data sets. (B) Top10 mRNA expression profiles (FPKM) and identification of NFIB as differentially expressed between KIRC and normal tissues based on the TCGA data set. (C) Volcano plot of differentially expressed mRNAs between KIRC and normal tissues based on the TCGA data set. Up, upregulation; Down, downregulation. The levels of NFIB were significantly decreased in KIRC (LoMet-ccRCC, 786-0) compared with the human renal tubular epithelial cell line HKC-5 as indicated by (D) qRT-PCR and (E) western blot assay respectively. The high expression of NFIB in KIRC tissues compared with matched adjacent noncancerous tissues via (F) IHC (Scale bars = 100 µm) and (G) qRT-PCR. Kaplan–Meier curves of KIRC patients stratified based on the expression of NFIB in KIRC tissues (high or low) based on (H) TCGA data set, (I) the online data sites GEPIA (http://gepia.cancer-pku.cn). The data D were presented as means ± SD of three independent experiments. Values are significant at * P < 0.05, as demonstrated by paired Student’s t test.
Fig 3: PINK1 is the critical downstream of NFIB in KIRC.After co-transfection with NFIB sgRNA and PINK1 vector or NC vector respectively, the expression of PINK1 protein in LoMet-ccRCC and 786-0 cells were measured by (A) western blot assay. The proliferative capacity in 786-0 cells were analyzed by (B) MTT and (C–D) colony formation assay (1×). The migration ability in 786-0 cells were analyzed by (E–F) transwell assay, (G–H) invasion assay and (I–J) wound healing assay (200×), Scale bars = 100 µm. All data were presented as means ± SD of three independent experiments. Values are significant at *P < 0.05, as demonstrated by paired Student’s t test.
Fig 4: The high-expression of NFIB promotes the proliferation and migration of KIRC cells.(A) The promoting efficiency of NFIB vector was evaluated by RT-qPCR and western blotting in LoMet-ccRCC and 786-0 cells respectively. (B) LoMet-ccRCC and 786-0 cells were transfected with NFIB vector or negative control (NC) vector for 24 h, and the proliferative ability was assessed by MTT assay over a 5-day period. (C–D) Colony formation assay (1×) was performed in LoMet-ccRCC and 786-0 cells transfected with NFIB vector or NC vector for 15 days. Migration ability was assessed in LoMet-ccRCC and 786-0 cells transfected with NFIB vector or NC vector by (E–F) transwell assay, (G–H) invasion assay, and (I–J) wound healing assay (200×), Scale bars = 100 µm. All data were presented as means ± SD of three independent experiments. Values are significant at * P < 0.05, as demonstrated by paired Student’s t test.
Fig 5: PINK1 was the pivotal downstream of NFIB to promote the progress of KIRC.(A) Important KEGG pathways of differentially expressed genes in three datasets. (B) Potential binding sites of NFIB on the PINK1 gene promoter. After being transfected with NFIB vector or NC vector, respectively, the expression of PINK1 mRNA and protein in LoMet-ccRCC and 786-0 cells were analyzed by (C) qRT-PCR and (D) western blot assay respectively. After being transfected with NFIB siRNA or NC siRNA, respectively, the expression of PINK1 mRNA and protein in LoMet-ccRCC and 786-0 cells were analyzed by (E) qRT-PCR and (F) western blot assay respectively. (G) CHIP experiment was performed by using the NFIB and IgG antibodies to probe LoMet-ccRCC cells extracts, and the level of the co-precipitated RNAs were determined by using qRT-PCR. The data C, E and G were presented as means ± SD of three independent experiments. Values are significant at *P < 0.05, as demonstrated by paired Student’s t test.
Supplier Page from Abcam for Anti-NFIB / NF1B2 antibody [EPR14122]