Fig 1: The DEF6 mRNA expression levels in ccRCC samples. (A) The mRNA expression levels of DEF6 in ccRCC tissues and adjacent normal renal tissues was compared. Nine databases were employed, namely, the GSE4282, GSE14994, GSE15641, GSE40435, GSE46699, GSE53757, GSE68417, GSE71963 and GSE76351 databases. The colored lines surrounding the data show the densities at the various expression levels in a violin chart. The mRNA expression levels of DEF6 in ccRCC tissues and adjacent normal renal tissues was compared in the (B) TCGA database and (C) 20 paired clinical samples. (D) The mRNA expression levels of DEF6 in various kidney cell lines was explored. (E) A forest plot of nine GEO databases was created. (F) The mRNA levels of 20 paired clinical samples are shown. *P<0.05, **P<0.01, ***P<0.001. #P<0.01, ##P<0.001 vs. HK-2 cells. ccRCC, clear cell RCC; CI, confidence interval; DEF6, DEF6 guanine nucleotide exchange factor; d.f., degrees of freedom; ns, not significant; RCC, renal cell carcinoma; SMD, standardized mean difference.
Fig 2: Spearman's correlation of expression of DEF6 with expression of 4 most significant lymphocytes and immunomodulators in the TISIDB database. (A) Correlations between DEF6 and the top 4 significant lymphocytes expression levels. (B) Correlations between DEF6 and the top 4 significant immunoinhibitors expression levels. (C) Correlations between DEF6 and the top 4 significant immunostimulators expression levels. (D) Correlations between DEF6 and the top 4 significant MHC molecules expression levels. Red and blue cells indicate positive and negative correlations, respectively. The color intensity is directly proportional to the strength of the correlations. DEF6, guanine nucleotide exchange factor; MHC, major histocompatibility complex. KIRC, kidney renal clear cell carcinoma.
Fig 3: Kaplan-Meier survival curves for the OS of patients related to DEF6 in the (A) TCGA database and (B) the present clinical database. Furthermore, the correlation between DEF6 expression levels and clinicopathological characteristics in patients with ccRCC was explored in the TCGA database, including (C) histologic grade, (D) pathologic T stage, (E) pathologic M stage and (F) pathologic stage. *P<0.05, ***P<0.001. DEF6, DEF6 guanine nucleotide exchange factor; OS, overall survival.
Fig 4: The DEF6 protein expression levels in ccRCC samples. (A) Representative positive DEF6 staining in adjacent normal renal tissue and ccRCC tissue. Scale bar, 200 µm. (B) Western blot analysis of DEF6 expression levels in ccRCC cell lines and normal cell lines. (C) Four representative images of the DEF6 expression profile as interpreted by IHC as negative, weak, moderate and strong. Scale bar, 200 µm. (D) Frequency distributions of DEF6 protein expression profiles across the cohort and the average scores from IHC. ***P<0.001. ccRCC, clear cell renal cell carcinoma; DEF6, DEF6 guanine nucleotide exchange factor; IHC, immunohistochemistry; IHC-P, immunohistochemistry-paraffin.
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