Fig 1: CPEB4 inhibits RCC cell proliferation in vivo. (A) The efficiency of CPEB4 knockdown in ACHN cells. (B) Images of the tumors of CPEB4 knockdown groups and the control group. (C) Tumor weights were measured after the tumors were surgically dissected. (D) Time course of tumor growth in mice. CPEB4 knockdown and the control cells were injected into nude mice, and tumor volumes were measured every 3 days (E) The efficiency of CPEB4 overexpression in 786O cells. (F) Images of the tumors of CPEB4 overexpression groups and the control group. (G) Tumor weights were measured after the tumors were surgically dissected. (H) Time course of tumor growth in mice. CPEB4 overexpression and the control cells were injected into nude mice, and tumor volumes were measured every 3 days.
Fig 2: MiR-874-3p targets CPEB4 in EC cells. a Venn diagram suggested that 2 genes might be targets of miR-874-3p. b, c The relative RNA and protein levels of CRCP and CPEB4 were determined by qRT-PCR and Western blot analyses, respectively, after transfection with miR-874-3p mimic and inhibitor in Ishikawa cells and RL952 cells. d The relative RNA levels of CPEB4 in EC tissues and normal tissues were determined by qRT-PCR. e The luciferase reporter assay functionally validated the interaction between miR-874-3p and CPEB4 in Ishikawa cells and RL952 cells. f The CPEB4 expression in Ishikawa cells and RL952 cells after transfection of shRNA was determined by qRT-PCR analysis. g The impacts of CPEB4 knockdown on the migration and invasion of Ishikawa cells and RL952 cells were examined by using Transwell assays. h The expression of EMT-related proteins was detected by Western blot analysis. Scale bar: 100 µm, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 3: CircESRP1 enhances tumour growth and metastasis in vivo. a, e Images of subcutaneous injection of BALB/c nude mice. b, f Images of xenograft tumours of each group. c, g Tumour volume and weight measurement in BALB/c nude mice. d, h The relative protein levels of E-cadherin, Vimentin, and CPEB4 were determined in subcutaneous xenograft tumours by IHC. Scale bar, 100 µm. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 4: CPEB4 induces G1 cell cycle arrest. (A,B) Cell cycle distribution was measured by flow cytometry in ACHN cells with CPEB4 stable knockdown. (C,D) Cell cycle distribution was measured by flow cytometry in 786O cells with CPEB4 stable overexpression. Indicated cells were starved in medium containing no serum for 48 h, after which they were released into complete growth medium for 24 h. After release the cells were harvested and analyzed by flow cytometry. The dataset of (A, C) is representative example of triplicate experiments. Column graph of (B,D) was mean ± SD of three independent experiments.
Fig 5: CPEB4 upregulates the expression of p21 by increasing p21 mRNA stability. (A,B) Western blot of p21 expression in CPEB4 knockdown or overexpression 786O cells. (C) The localization of Flag-tagged CPEB4 assayed by indirect immunofluorescence in CPEB4 overexpression 786O cells. (D) The nuclei and cytoplasm of 786O cells were isolated, and CPEB4 levels were assayed by western blot. (E) RT-PCR of p21 mRNA expression in CPEB4 knockdown 786O cells. (F) The levels of p21 transcript was measured by RT-PCR in CPEB4 knockdown 786O cells treated with actinomycin D for various times. (G) RT-PCR of p21 mRNA expression in CPEB4 overexpression 786O cells. (H) The levels of p21 transcript was measured by RT-PCR in CPEB4 overexpression 786O cells treated with actinomycin D for various times. (I) de novo synthesis of the p21 protein was analyzed by CHX removal assay. CPEB4 stable knockdown and control cells were pretreated with 100 mM cycloheximide for 12 h. After washing out cycloheximide, cells were incubated for the indicated times. p21 synthesis levels were detected by Western blot and quantified using ImageJ. (J) CPEB4 interaction with p21 transcript in vivo. The lysates of CPEB4 overexpression 786O cells were immunoprecipitated with Flag antibodies or control IgG, and RT-PCR was used to measure the transcript levels of p21 and GAPDH precipitated by Flag or IgG immunocomplexes.
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