Fig 2: STAT3 mediates the ERp57 regulating ILF3. a SW839 cells were transfected shSTAT3 or A498 were cells transfected with STAT3 overexpression vector pWP-STAT3. ILF3 mRNA level was measured by qRT-PCR. *P < 0.05, **P < 0.01 vs. corresponding control. b SW839 cells were transfected shSTAT3 or shERp57 respectively or co-transfected them together. A498 cells were transfected with shSTAT3 or pWPI-ERp57 respectively or co-transfected them together. qRT-PCR detected ILF3 mRNA level. *P < 0.05 vs. corresponding control. Western blot performed to detect ERp57, STAT3, ILF3 and Cyclin E1 protein level. c SW839 cells were transfected shSTAT3 and shERp57 respectively or transfected them together. Cell proliferation was measured by BrdU stain. Scale bar = 20 μm. Right panel showed analysis for BrdU positive cell number. *P < 0.05 vs corresponding control. d A498 cells were transfected with pWPI-ERp57 or empty vector and then cell lysates were immunoprecipitated with antibody against ERp57. Western blot detected ERp57 and STAT3 protein level in lysates. e A498 were treated as (d), PLA analysis was used to test the interaction between ERp57 and STAT3. Analysis of number of PLA positive cells. *P < 0.05 vs. empty vector. f Potential binding site of STAT3 in ILF3 promotor. g ChIP-qPCR was used to test STAT3 and ERp57 binding to the ILF3 promoter region in 293A cells. *P < 0.05, **P < 0.01 vs. IgG. h ILF3 promoter-luciferase reporter were co-transfected with pWPI-ERp57 or co-transfected pWPI-ERp57 combination of shSTAT3 vector into 293A cells, and then luciferase reporter assays were performed. *P < 0.05 vs. corresponding control

Fig 3: Proposed model for ERp57/STAT3/ILF3 in ccRCC feedback loop in regulating ccRCC progression

Fig 4: ILF3 regulates ERp57 by promoting ERp57 mRNA stability. a SW839 cells transfected with shILF3 or A498 cells transfected with ILF3 overexpression vector pWP-ILF3. ERp57 mRNA level was measured by qRT-PCR. *P < 0.05 vs. corresponding control. b Cells were prepared as (a) and then exposed to Actinomycin D for 0, 2, 4, and 8 h. ERp57 mRNA level was detected using qRT-PCR. *P < 0.05, vs. corresponding control. c A498 cells were transfected with pWPI-ILF3 or empty vector. Western blot was used to measure the precipitation efficiency of ILF3 antibody. (d and e) RIP PCR and agarose gel electrophoresis was used to test the interaction between ILF3 protein and ERp57 mRNA. f A498 cells were transfected with ILF3-probe. Western blot analysis was used to test the ERp57, Cyclin D1, Cyclin E1 in pulldown precipitate. g SW839 cells were transfected with shERp57 or pWPI-ILF3 alone, or co-transfected with shERp57 and pWPI-ILF3. A498 cells were transfected with shILF3 or pWPI-ERp57 alone or co-transfected them together. Colony formation assay tested cell ability of proliferation. Right panel shows the analysis of colony formation number. *P < 0.05, ** P < 0.01 vs. corresponding control

Fig 5: ILF3 is positively regulated by ERp57. a SW839 cells transfected with shERp57 or A498 cells transfected with pWP-ERp57. Gene mRNA level was tested by qRT-PCR. *P < 0.05 vs. corresponding control. b Cells were prepared as A, IL3 protein level was measured by western blotting. c ILF3 mRNA level in ccRCC tissues (n = 35) and normal kidney tissues (n = 35) was detected by qRT-PCR. d ILF3 level from ccRCC tissues and normal kidney tissues was analyzed in TCGA database. e and f Western blot analysis and immunohistochemistry stain were performed to measure the ILF3 protein level in ccRCC tissues and normal kidney tissues. g Kaplan–Meier analysis was used to analyze the overall survival of ccRCC patients with low or high ILF3 level from TCGA database. h Pearson correlation analysis was used to analyze the relationship between ILF3 and ERp57 (R = 0.7351, P < 0.0001). i Immunofluorescence staining was used to locate and detect the expression of ERp57 and ILF3 in A498 cells. Scale bars = 100 μm