Fig 1.
Expression of FTO gene in different tissues of mouse.
(A) FTO expression in various mouse tissues measured by Real-time PCR. The data were calculated as relative fold changes between each sample and the one with the lowest expression level of FTO. GAPDH gene is used as internal reference gene for the normalization. Data were presented as mean ± SD. (B) Validation of FTO expression in mouse tissues by Western blot. β-Actin was served as an internal control. (C) Semi-quantitative PCR analysis of FTO expression in endocrine pancreas and pancreas tissue. GAPDH is used as an internal control.
Fig 2.
The effect of FTO expression on insulin secretion.
(A) FTO overexpression in MIN6 cells via lentivirus delivery using Western blot. β-actin was loaded as an internal control. (B) Detection of FTO in MIN6 cells transfected with FTO shRNA and control shRNA. β-actin was loaded as an internal control. (C) Insulin secretion of MIN6 cells with FTO overexpression at 60 min after 2 mM, 20 mM glucose or 50 mM KCl stimulation. MIN6 cells were stably transfected with pLVX-IRES-ZsGreen (CON) or pLVX-IRES-ZsGreen-FTO (pLVX-FTO). (D) Effect of FTO silence on the insulin secretion in MIN6 cells. MIN6 cells transfected with FTO shRNA 2 or shRNA 3 were analyzed with insulin secretion at 60 min after the glucose or KCl stimulation. Data were presented as mean ± SD. The symbol ** denotes significant statistical difference (p < 0.01).
Fig 3.
FTO doesn’t affect the transcription of insulin 1/2.
The expression level of insulin 1 (A) and insulin 2 (B) in MIN6 cells delivered with FTO expression lentivirus or control vector using Real-time PCR.
Fig 4.
FTO promotes the ROS production and then induces NF-κB activation in MIN6 cells.
(A) Detection of ROS production in FTO-overexpressed cells or control cells with or without NAC treatment. (B) Total and phosphorylated IκBα immunoblotting in whole cell lysates. β-actin was used as an internal control. (C) Detection of p65 in nuclear fraction using Western blot. Lamin B was used as an internal control. Data were presented as mean ± SD. The symbol ** denotes significantly statistical difference (p < 0.01).
Fig 5.
NAC treatment partially rescues the effect of FTO overexpression on the inhibiton of insulin secretion.
Detection of ROS production in MIN6 with overexpressed-FTO or empty vector with NAC treatment or not in the presence of 2 mM glucose (A) or 20 mM glucose (B). Detection of insulin secretion with NAC treatment or not at 10 min (C) or 60 min (D) after the stimulation of 2 mM or 20 mM glucose. Data were presented as mean ± SD. The symbol* and ** denotes statistical difference p < 0.05 and p < 0.01, respectively.
Fig 6.
GO annotation of differential genes modulated by FTO overexpression in MIN6 cells.
GO categories (p<1.0×10–5) were ranked according to their p values and shown in the left. The data in the horizontal axis mean the minus logarithm based on 10 and they represent the significant degrees of annotated functions of differential genes.
Fig 7.
Pathway analysis of differential genes modulated by FTO overexpression in MIN6 cells.
(A) Pathway analysis of upregulated genes modulated by FTO overexpression. (B) Pathway analysis of downregulated genes modulated by FTO overexpression. The data in the horizontal axis mean the minus logarithm based on 10 and they represent the significant degrees of annotated pathways of differential genes.
Fig 8.
Gene expression analysis by Real-time PCR.
The expression of genes modulated by FTO overexpression was verified using qPCR. (A) Expression analysis of the upregulated genes in the microarray. (B) Expression analysis of the downregulated genes in the microarray. These genes are related to NF-κB signaling, metabolism and insulin secretion regulation. Data indicated mean ± SD. The symbol ** denotes statistical difference (p < 0.01).