HCBP6 upregulates human SREBP1c expression by binding to C/EBPβ-binding site in the SREBP1c promoter

Figures

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  Fig. 1. HCBP6 upregulates SREBP1c expression and promotes TG synthesis in HepG2 cells. HepG2 cells were transfected with pcDNA-HCBP6. (A) HCBP6 mRNA expression was analyzed by performing qRT-PCR, which showed successful HCBP6 overexpression. (B) Next, qRT-PCR was performed to determine the expression of all the key genes involved in TG synthesis, including SREBP1c, ACC1, and FASN at 24 h after transfection. Results of qRT-PCR showed that HCBP6 overexpression increased the expression of the genes associated with TG synthesis. (C) Western blotting was performed to determine the protein expression of HCBP6, SREBP1c, ACC1, p-ACC1, and FASN at 48 h after transfection, with GAPDH as a loading control. Results of western blotting showed that HCBP6 overexpression upregulated the protein expression of SREBP1c, p-ACC1, and FASN. (D) Intracellular TG content was determined using the Adipogenesis Assay Kit at 48 h after transfection according to the manufacturer’s instructions, and was normalized to the total protein content. HCBP6 overexpression increased intracellular TG content. (E) After 24 h of pcDNA-HCBP6 transfection, HepG2 cells were treated with oleic acid (0.25 mmol/L) for 24 h and were…

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  Fig. 2. HCBP6 silencing decreases SREBP1c expression and suppresses TG synthesis in HepG2 cells. HepG2 cells were transfected with si-HCBP6 or si-NC. (A) HCBP6 mRNA expression was analyzed by performing qRT-PCR, which showed successful HCBP6 silencing. (B) qRT-PCR was performed to analyze the expression of all the key genes involved in TG synthesis, including SREBP1c, ACC1, and FASN at 24 h after transfection. Results of qRT-PCR showed that HCBP6 silencing decreased the expression of all the genes involved in TG synthesis. (C) Western blotting was performed to determine the protein expression of HCBP6, SREBP1c, ACC1, p-ACC1, and FASN at 48 h after transfection, with GAPDH as the loading control. Results of western blotting analysis showed that HCBP6 silencing down-regulated the protein expression of SREBP1c, p-ACC1, and FASN. (D) Intracellular TG content was determined using the Adipogenesis Assay Kit at 48 h after transfection according to the manufacturer’s instructions, and was normalized to the total protein content. HCBP6 silencing decreased the intracellular TG content. (E) After 24 h of si-HCBP6 transfection, HepG2 cells were treated with oleic acid (0.25 mmol/L) for 24 h and were…

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  Fig. 3. HCBP6 upregulates transcriptional activity of the SREBP1c promoter in HepG2 cells. (A) Deletion fragments of the SREBP1c promoter were cloned into luciferase reporter vector pGL4.10-Basic based on the findings of Promoter 2.0 and Promoter Scan prediction (left). HepG2 cells were cotransfected with containing the different deletion fragments of the SREBP1c promoter or the pGL4.10-Basic vector and Renilla luciferase vector (pRL-TK), which served as an internal control. Luciferase activity was measured after 24 h of transfection. Results of the luciferase reporter assay showed that the P, P2, and P3 promoter fragments were active, with the P2 fragment showing the highest transcriptional activity (right). (B) HepG2 cells were cotransfected with 60 ng vectors containing the different SREBP1c promoter fragments or the pGL4.10-Basic vector with or without 180 ng pcDNA-HCBP6 plasmid and pRL-TK. Luciferase activity was measured after 24 h of transfection. HCBP6 overexpression enhanced the activity of the P, P2 and P3 promoter fragments. (C) Site-directed mutagenesis of NF-κB-, C/EBPβ-, E-box-, Sp1-, and GATA-binding sites in the P2 promoter fragment. The basal activity of the mutation P2…

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  Fig. 4. HCBP6 directly binds to the C/EBPβ-binding site in the SREBP1c promoter. (A) Activities of the wild-type P2 promoter (P2) and P2 promoter with a mutated C/EBPβ-binding site (mut-C/EBPβ) were measured for 24 h in the absence or presence of pcDNA-HCBP6. Luciferase activity was measured after 24 h of transfection. C/EBPβ enhanced the activity of the P2 and mut-C/EBPβ promoter fragments; however, the activity of the mut-C/EBPβ promoter fragment was significantly lower than that of the wild-type P2 promoter fragment in presence of pcDNA-C/EBPβ. (B) C/EBPβ and HCBP6 is evolutionarily conserved in different mammals. (C) The presence of supershifted bands when an anti-His or anti-C/EBPβ antibody was added confirmed the specificity of interaction. (D) EMSA was performed using the LightShift chemiluminescence EMSA kit. Next, 6 μg nuclear protein extracts showing HCBP6 or C/EBPβ overexpression in the absence or presence of 100-fold excess of the unlabeled probe or mutant biotin-labeled probe (left and right, respectively) were incubated with biotin-labeled probes. Results indicate that HCBP6 and C/EBPβ interacted with the C/EBPβ-binding site in the SREBP1c promoter in vitro. (E) ChIP assay…