Journal: bioRxiv

Article Title: FoxO transcription factors couple the urea cycle and gluconeogenesis by controlling Ass1

doi: 10.64898/2025.12.22.695746

Figure Lengend Snippet: ( A ) Blood glucose levels in ICR mice with hepatic Ass1 knockdown (n = 4 per group). ( B - G ) Plasma levels of urea cycle related amino acids, urea, and ammonia (NH3) in ICR mice with hepatic Ass1 knockdown (n = 4 per group). ( H ) Immunoblot analysis of Ass1 protein using liver total lysate from mice in the fasted state (Samples were pooled from 3 mice). GAPDH was detected as an internal loading control. ( I - Q ) Relative gene expression in ICR mice with hepatic Ass1 knockdown (n = 4 per group). Ass1 knockdown was performed using adenovirus mediated RNAi (Ad- Ass1 i-1, Ad- Ass1 i-2). As the correction of the gene expression level for each sample Cyclophilin A was used. All groups of mice were sacrificed at the same period in the light cycle and blood and liver samples were collected in the fasted state following 24-h fasting. All values are presented as the mean with error bars representing the SEM. Datasets were assessed by Student’s t -test for unpaired samples. The differences were considered to be significant if P < 0.05 (* P < 0.05 and ** P <0.01).

Article Snippet: Mutated Ass1 - enhancer firefly luciferase reporter plasmids were generated using PrimeSTAR Mutagenesis Basal Kit (Cat# R046A, TaKaRa Bio, Tokyo, Japan).

Techniques: Knockdown, Clinical Proteomics, Western Blot, Control, Gene Expression, IF-P

Journal: bioRxiv

Article Title: FoxO transcription factors couple the urea cycle and gluconeogenesis by controlling Ass1

doi: 10.64898/2025.12.22.695746

Figure Lengend Snippet: ( A ) Top, the results of ChIP-seq with anti-FoxO1 antibody in livers of unfed mice obtained from ChIP-Atlas (ID: GSM3381273); bottom, simplified representation of the structure of the full fragment, fragment 1, fragment 2, fragment 3, and the subdivisions of fragment 1 i.e. small fragment 1A and small fragment 1B. ( B and C ) Enhancer analysis of FoxO1 and FoxO3a on Ass1 enhancer and its fragments. FoxO1 and FoxO3a expression plasmids were co-transfected with the indicated Ass1 enhancer firefly luciferase reporter plasmids in HepG2 cells. ( D and E ) Electrophoretic mobility shift assay (EMSA) using radiolabeled probe for ( D ) the two JASPAR predicted FoxO binding sites (Figure S5) in the smallest fragment 1A from the Ass1 enhancer and FoxO binding site in the promoter of Pck1 as a positive control; ( E ) the wild-type (WT) and mutant (Mut) predicted FoxO binding site1 incubated with GST and GST-FoxO recombinant proteins. BS, binding site; WT, wild-type; Mut, mutant. ( F ) Enhancer analysis of FoxO1 and FoxO3a on mutant Ass1 enhancer and its fragments. FoxO1 and FoxO3a expression plasmids were co-transfected with the indicated Ass1 enhancer firefly luciferase reporter plasmids in HepG2 cells. All values are presented as the mean with error bars representing the SEM. Datasets were assessed by Student’s t -test for unpaired samples. The differences were considered to be significant if P < 0.05 (* P < 0.05 and ** P <0.01).

Article Snippet: Mutated Ass1 - enhancer firefly luciferase reporter plasmids were generated using PrimeSTAR Mutagenesis Basal Kit (Cat# R046A, TaKaRa Bio, Tokyo, Japan).

Techniques: ChIP-sequencing, Expressing, Transfection, Luciferase, Electrophoretic Mobility Shift Assay, Binding Assay, Positive Control, Mutagenesis, Incubation, Recombinant, IF-P

Journal: bioRxiv

Article Title: FoxO transcription factors couple the urea cycle and gluconeogenesis by controlling Ass1

doi: 10.64898/2025.12.22.695746

Figure Lengend Snippet: ( A ) The structure of the Ad-Luc showing the identified FoxO binding site in the enhancer region of Ass1 linked to the Luc reporter using Ass1 native promoter. ( B - F ) In vivo Ad-Luc enhancer analyses using Ad-FoxOBS- Ass1 Promoter-Luc both wild-type and mutant. Representative images ( B ) and hepatic luciferase activities ( C - F ) of mice injected with Ad-FoxOBS- Ass1 Promoter-Luc both wild-type and mutant are shown (n = 5). ( D ) Enhancer activity in the liver for wild-type binding site after 24-h fasting is expressed relative to activity before fasting, to adjust for mouse-to-mouse differences in enhancer expression in Ad-treated mice. ( E ) Enhancer activity in the liver for mutant binding site after 24-h fasting is expressed relative to activity before fasting, to adjust for mouse-to-mouse differences in enhancer expression in Ad-treated mice. ( F ) Enhancer activity in the liver after 24-h fasting for wild-type binding site is expressed relative to activity of mutant binding site, to adjust for mouse-to-mouse differences in enhancer expression in Ad-treated mice. BS, binding site; WT, wild-type; Mut, mutant. Data were assessed using the paired two-tailed Student’s t -test. The differences were considered to be significant if P < 0.05 (** P < 0.01). Error bars mean SEM.

Article Snippet: Mutated Ass1 - enhancer firefly luciferase reporter plasmids were generated using PrimeSTAR Mutagenesis Basal Kit (Cat# R046A, TaKaRa Bio, Tokyo, Japan).

Techniques: Binding Assay, In Vivo, Mutagenesis, Luciferase, Injection, Activity Assay, Expressing, Two Tailed Test, IF-P

Journal: bioRxiv

Article Title: FoxO transcription factors couple the urea cycle and gluconeogenesis by controlling Ass1

doi: 10.64898/2025.12.22.695746

Figure Lengend Snippet: ( A - D ) Chromatin immunoprecipitation (ChIP) assay to determine the interaction of hepatic FoxO proteins with Ass1 DNA enhancer region using anti-FoxO1 antibody and normal mouse anti-IgG antibody as a control. ( E - H ) Chromatin immunoprecipitation (ChIP) assay to determine the interaction of hepatic FoxO proteins with Ass1 DNA enhancer region using anti-FoxO3a antibody and normal mouse anti-IgG antibody as a control. ( A , E ) FoxO proteins binding to Ass1 enhancer in liver detected by using primer set designed around the identified FoxO binding site in . ( B , F ) Pck1 promoter FoxO transcription factor binding site and ( C , G ) G6pc promoter FoxO transcription factor binding site used as a positive control. ( D , H ) Used as a negative control. ( I ) Immunoblot analysis of FoxO1 and FoxO3a proteins using liver nuclear extracts from mice in ad libitum-fed and fasted state (Samples were pooled from 3 mice). Lamin A/C was detected as an internal loading control for nuclear protein. All groups of mice were sacrificed in the light cycle and liver samples were collected in the fasted state following 24-h fasting or ad libitum-fed as indicated. Data were assessed using the unpaired two-tailed Student’s t -test. The differences were considered to be significant if P < 0.05 (** P < 0.01). Error bars mean SEM.

Article Snippet: Mutated Ass1 - enhancer firefly luciferase reporter plasmids were generated using PrimeSTAR Mutagenesis Basal Kit (Cat# R046A, TaKaRa Bio, Tokyo, Japan).

Techniques: Chromatin Immunoprecipitation, Control, Binding Assay, Positive Control, Negative Control, Western Blot, Two Tailed Test, IF-P