Fig 2: Effect of USF1 on SLC25A13 gene expression. (A) ChIP-qPCR assay shows the amplification bands of PCR using the specific primers suitable to amplify the -498/-343 bp region of the SLC25A13 gene promoter. M: Marker, lane I: Input, lane USF1: amplification PCR band after ChIP with specific antibody for human USF1, lane NoAb: without the anti-USF1 antibody addition. (B) Luciferase activity was measured in HepG2 cells co-transfected with C5 construct (-442/-19 of the SLC25A13 gene promoter) and siRNA targeting human USF1 (+, black bar) or control siRNA (-, white bar). (C) Total RNA extracted from HepG2 cells treated without (white bar) or with (black bar) siRNA against human USF1 was used to quantify SLC25A13 mRNA by real-time PCR. Immunodecoration was performed with specific antibodies for AGC2 and ß-actin proteins in HepG2 cells treated under the conditions described. (D) Luciferase activity of HepG2cells co-transfected with pGL3 basic-LUC vector containing the -442/-19 SLC25A13 promoter region wild-type (W) or mutated (M) in USF1 site and pcDNA3-USF1 (+: black bar) or empty pcDNA3 (-: white bar). (E) Total RNA extracted from HepG2 cells transfected with pcDNA3-USF1 (+) or empty pcDNA3 (-) was used to quantify SLC25A13 mRNA by real-time PCR. AGC2 and ß-actin of HepG2 cells transfected with pc-DNA3-USF1 (+) or empty pcDNA3 (-) were immunodecorated with anti-AGC2 and anti-ß-actin antibodies. (F) Total RNA from liver foetal cells (white bar) and adult (black bar) was used to quantify USF1 and SLC25A13 mRNAs by real-time PCR. For panels (B–F), means ± SD of three duplicate independent experiments are shown; all differences between samples and relative controls were significant (*p < 0.05 or **p < 0.01 one-way ANOVA followed by Student’s t-test).

Fig 3: (A) Synergy between FOXA2 and USF1. HepG2 cells co-transfected with pGL3 basic-LUC vector containing C2 construct (-968/-19 of the SLC25A13 gene promoter region) wild-type (wt) or mutated (mutFOXA, mut USF and mut FOXA/USF) in combination with pcDNA3-FOXA2 and pcDNA3-USF1 (+: black bars) or empty pcDNA3 (-: white bars) were assayed for LUC activity. (B) FOXA2 and USF1 effect on AGC2 activity. Quantification of the NAD+/NADH ratio in HepG2 cells transfected with siRNA against FOXA1 and USF1 alone or in combination. siRNA against SLC25A13 was used as control. Where indicated, differences between samples and relative controls were significant (*p < 0.05 or **p < 0.01 one-way ANOVA followed by Student’s t-test).

Fig 4: (A) Deletion analysis of the 5'-flanking region of the human SLC25A13 gene. The deletion mutants named C1-C6 were cloned into the pGL3 basic-LUC vector and tested for LUC activity in transfected HepG2, HEK293 and SK-N-SH cells. pGL3 indicates the pGL3 basic-LUC vector alone. Numbering indicates the extent of fragments, while bars indicate LUC activity. The values of construct C1 transfected in HepG2 cells were set to 100%. Means ± SD of three duplicate independent experiments are shown. (B) Total RNAs extracted from HepG2, HEK293 and SK-N-SH cells were used to quantify SLC25A13 mRNA by real-time polymerase chain reaction (PCR). Means ± S.D. of four replicate independent real-time PCR experiments are shown. Where indicated, differences between samples and control were significant (**p < 0.01). AGC2 and ß-actin proteins of HepG2, HEK293 and SK-N-SH cells were immunodecorated with specific antibodies. The Western blot analysis is representative of three independent experiments with similar results.

Fig 5: Activation of SLC25A13 gene by FOXA2. (A) FOXA1, FOXA2 and FOXA3 mRNA levels in HepG2 and SK-N-SH cells. (B) HepG2 and SK-N-SH cells co-transfected with pGL3 basic-LUC vector containing C2 construct (968/-19 of the SLC25A13 gene promoter region) wild-type (W) or mutated (M) in FOXA2 binding site and with pcDNA3-FOXA2 (+: black bars) or empty pcDNA3 (-: white bar) were assayed for LUC activity. Where indicated, differences between samples and relative controls were significant (*p < 0.05 or **p < 0.01 one-way ANOVA followed by Student’s t-test).