Fig 1: CRNDE was involved in epigenetic repression mediated by EZH2, SUZ12, and SUV39H1 on multiple tumor suppressor genes.qPCR was performed to measure CRNDE levels in immunoprecipitates in Bel-100 (a) and Huh-7 cells (b). CRNDE expression levels were displayed as fold changes relative to IgG immunoprecipitate. c RNA pull-down assay was applied to capture the proteins that bound CRNDE in Bel-100, and protein expression levels of HuR, EZH2, SUZ12, and SUV39H1 were determined by WB. HuR and AR were used as positive control. d, e qPCR analysis was performed to measure the relative expression levels of multiple tumor suppressor genes in Bel-100 (d) and Huh-7 cells (e). f, g HCC cells were transfected with lentiviruses encoding siRNAs against EZH2 (f), SUZ12 (g), SUV39H1 (h), or negative control and the protein expression levels of the silenced proteins together with BIK, LATS2, p27KIP1, and CELF2 were determined by WB. i–l HCC cells were transfected with shCRNDE or shNC, and ChIP-qPCR was used to identify the enrichment of SUZ12, SUV39H1, EZH2, H3K27me3, and H3K9me3 at promoter region of BIK (i), LATS2 (j), p27KIP1 (k), CELF2 (l), and GAPDH (m), respectively. The data were shown as Mean ± SD based on at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 2: CELF2 inhibited HCC cell proliferation, migration, and chemoresistance and was involved in CRNDE-mediated oncogenic effect.a Relative expression of CELF2 in HCC tumor tissues and corresponding adjacent tissues was determined by qPCR (n = 47). b GEPIA was accessed and CELF2 expression in healthy human individual (n = 160) and in HCC patients (n = 369) was compared. c Expression of CELF2 was detected in HCCC-9810, Bel-100, Bel-7402, Bel-7405, HUH-7, SMMC-7721, HepG2, WRL68, and THLE3. d CELF2 was cloned into pcDNA3.1(+) and overexpressed in Bel-100 and Huh-7 cells. CELF2 expression was determined by WB and empty vectors were used as a negative control. e, f Cell vitality was evaluated by MTT assay in Bel-100 (e) and Huh-7 cells (f). g, h Cell migration and invasive abilities were determined by transwell assays in in Bel-100 (g) and Huh-7 cells (h). i Huh-7 and Huh-7/ADR cells were transfected with pcDNA3.1(+)-CELF2 or empty vectors, and relative cell viability was detected by MTT assay combined with ADR. j ADR IC50 was determined in both Huh-7 and Huh-7/ADR cells. k Bel-100 cell were transfected with shCRNDE alone or co-transfected with shCRNDE and siCELF2, cell viability was measured by MTT assay. l Transwell assay was used to determine the cell migration and invasive abilities in Bel-100 transfected with shCRNDE and/or siCELF2. The data were shown as mean ± SD based on at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
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