Fig 1: CASC15 depletion decreased the proliferation and promoted the apoptosis of ESCC cells by upregulating SIM2. (A) SIM2 expression was measured via reverse transcription-quantitative PCR in tumor and adjacent normal tissues isolated from patients with ESCC (n=45). (B) Linear correlation of CASC15 and SIM2 expression in ESCC tissues. (C) SIM2 protein expression was detected in HET-1A, Eca109 and KYSE450 cells via western blotting. (D) SIM2 protein expression was measured by western blotting at 48 h post transfection in Eca109 and KYSE450 cells transfected with si-NC or si-SIM2. Eca109 and KYSE450 cells were transfected with si-NC, si-CASC15#1+si-NC or si-CASC15#1+si-SIM2. (E) SIM2 protein abundance was examined via western blotting. (F) Cell proliferation was determined by performing an MTT assay in Eca109 and KYSE450 cells. Data analyzed via two-way ANOVA followed by Bonferroni's post-hoc test. (G and H) Apoptotic rate was detected via flow cytometry. (I) Relative caspase-3 activity was detected using the caspase-3 assay kit in Eca109 and KYSE450 cells. *P<0.05, **P<0.01 and ***P<0.001. CASC15, cancer susceptibility candidate 15; ESCC, esophageal squamous cell carcinoma; SIM2, single-minded 2; si, small interfering RNA; NC, negative control; OD, optical density.
Fig 2: CASC15 inhibited SIM2 expression and decreased SIM2 mRNA stability via FTO in esophageal squamous cell carcinoma cells. (A) RNA pull-down and western blotting assays were performed to determine the interaction of CASC15 and FTO protein. (B) Venn diagram of the possible mRNAs that interact with the FTO protein, as determined using the StarBase database and previous RNA-seq data. (C) SIM2 expression was assessed in ESCC tumor and adjacent normal tissues using the Gene Expression Profiling Interactive Analysis database. (D) CASC15 and SIM2 mRNA expression enriched by IgG or FTO antibodies were detected via RT-qPCR after a RIP assay in Eca109 cells. (E) Eca109 cells were transfected with pcDNA or the FTO overexpression vector. At 48 h post transfection, FTO protein expression was measured via western blotting, and m6A-enriched SIM2 and CASC15 mRNA expression was examined through RIP and RT-qPCR assays. Transfection efficiency of the (F) CASC15 overexpression vector and (G) si-FTO was measured in Eca109 and KYSE450 cell lines. (H) Eca109 cells were transfected with pcDNA, CASC15 overexpression vector, CASC15 overexpression vector + si-NC or CASC15 overexpression vector + si-FTO. At 48 h post transfection, RIP and RT-qPCR assays were conducted to measure SIM2 mRNA expression following m6A antibody enrichment. SIM2 mRNA stability was analyzed by performing an ActD assay in (I) Eca109 and (J) KYSE450 cells transfected with pcDNA, CASC15 overexpression vector, CASC15 overexpression vector + si-NC or CASC15 overexpression vector + si-FTO. Data analyzed via two-way ANOVA followed by Bonferroni's post-hoc test. SIM2 mRNA and protein expression was respectively examined via RT-qPCR and western blotting in (K) Eca109 and (L) KYSE450 cells transfected with pcDNA, CASC15 overexpression vector, CASC15 overexpression vector + si-NC or si-FTO. *P<0.05, **P<0.01 and ***P<0.001. CASC15, cancer susceptibility candidate 15; SIM2, single-minded 2; FTO, fat mass and obesity-associated; RIP, RNA immunoprecipitation; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; NC, negative control; m6A, n6-methyladenosine; ActD, Actinomycin D.
Supplier Page from Abcam for Anti-SIM2 antibody [EPR7878]