TP53INP1 Antibody - #DF8731

Fig. 4. FOXO1 inhibits the G1- to S-phase progression in hypoxic GCs by activating transcription of TP53INP1. (A) A 2072 bp fragment of the TP53INP1 promoter was amplified by PCR using pig genomic DNA containing three FRE (TGTTTC) motifs and cloned into the pGL3-Basic plasmid as pGL3-TP53INP1(WT). pGL3-TP53INP1 (M1), pGL3-TP53INP1 (M2) and pGL3-TP53INP1 (M3) were individually constructed by introducing mutations (italics) into each of the FRE sites. (B) TP53INP1 reporter activities in GCs co-transfected with FOXO1 expression vectors and TP53INP1 promoter constructs for 36 h. The reporter activities were normalized to those of pRL-TK. RLA, relative luciferase activity. (C-E) Binding of FOXO1 to the TP53INP1 promoter in GCs was detected with ChIP assays following normoxia (20% O2) or hypoxia (1% O2) treatment for 12 h. DNA was isolated from the precipitated complexes as a template for qRT-PCR (C). The qRT-PCR products were then analysed on a 2% agarose gel (D) and quantified with densitometry using ImageJ 1.42q software (E). The amount of immunoprecipitated DNA for each ChIP reaction was presented as % input. The GAPDH gene promoter immunoprecipitated with RNA polymerase II antibody served as a positive ChIP control. IgG was used as the negative ChIP control. (F,G) GCs transfected with FOXO1 siRNA and/or TP53INP1 siRNAs (siRNA3, siRNA4) for 12 h were exposed to 1% O2 for 24 h, and then collected for cell cycle analysis using flow cytometry. The cell cycle distribution of GCs was quantified using the ModFit LT 3.2 software. (H) GCs transfected with FOXO1 siRNA and/or TP53INP1 siRNAs for 12 h were grown under hypoxia (1% O2) for 12 h, and then collected for western blot detection of TP53INP1, CDKN1A and FOXO1 levels in GCs. Data are represented as mean±s.e.m.; n=3. *P