sgrna scaffold vector  (Addgene inc)

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: A The percent of SLCO4C1 expression in different types of liver cells from CTR and MASLD patients. B Western blot analysis of the relative levels of hepatic SLCO4C1 protein expression between control subjects ( n = 6) and MASLD patients ( n = 6). * p < 0.05 vs. the control subjects. C Multiplex IF analysis of SLCO4C1(Red), Na + /K + -ATPase (Green) expression in human livers. (Scale bars:50 μm). The relative levels of Slco4c1 mRNA transcripts ( D ) and protein expression ( E ) in individual liver tissues from the chow diet-fed control ( n = 6) and GAN diet-fed mice ( n = 8). The relative levels of Slco4c1 mRNA transcripts ( F ) and protein expression ( G ) in individual liver tissues from the chow diet-fed control ( n = 5) and MCD diet-fed mice ( n = 7). B, D-G: Each dot represents one liver sample; Plotted: mean ± SD; Statistics: unpaired two-tailed Student’s t test; 95% confidence interval. * p < 0.05, ** p < 0.01vs. the Chow diet-WT mice.

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Expressing, Western Blot, Control, Multiplex Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: A Slco4c1 -/- mice were generated using Cas9/gRNA gene-editing techniques. Slco4c1 -/- mice were characterized by mouse tail genotyping ( B ), Western blot ( C ) and IF analyses ( D ). E The strategies for the establishment of Slco4c1 -/- and WT MASLD mice by feeding with GAN diet. F The percent of body weight (BW) changes in the GAN diet-fed Slco4c1 -/- mice ( n = 8 per time point) and WT mice ( n = 6 per time point). G The liver weights (LW) and the ratio of liver weights to body weights (LW/BW) in the GAN diet-fed Slco4c1 -/- mice ( n = 8) and WT mice ( n = 6). H WAT and BAT weights in the GAN diet-fed Slco4c1 -/- mice( n = 8) and WT mice ( n = 6). (I) serum ALT and AST levels in the GAN diet-fed Slco4c1 -/- mice( n = 8) and WT mice ( n = 6). J The levels of liver TG, FFA, and TC in the GAN diet-fed Slco4c1 -/- mice ( n = 8) and WT mice ( n = 6). K H&E (Scale bars:100 μm), Sirius red (Scale bars:100 μm), and Oil red O staining (Scale bars:50 μm) of liver sections from the GAN diet-fed WT ( n = 6) and Slco4c1 -/- ( n = 8) mice. L Liver histologic assessments of steatosis, lobular inflammation, hepatocyte ballooning, fibrosis and NAS score from the GAN diet-fed WT ( n = 6) and Slco4c1 -/- ( n = 8) mice. B: n = 3, biologically independent experiments, the results are similar. C , D An experiment was conducted on mice identified in Figure2B, yielding results consistent with those in Figure2B. F Plotted: mean ± SEM; Statistics: Multiple unpaired t test; *p < 0.05, ** p < 0.01, *** p < 0.001vs. the GAN diet-fed WT mice. G – J , L Each dot represents one mouse sample; Plotted: mean ± SD; Statistics: unpaired two-tailed Student’s t test; 95% confidence interval. *P < 0.05, ** p < 0.01, *** p < 0.001vs. the GAN diet-fed WT mice.

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Generated, Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: A Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) of the lipidomic data from the livers of GAN diet-fed WT ( n = 6) and Slco4c1 -/- mice ( n = 6). B The lipid category ratios in mouse liver tissues. C The major components of liver lipid categories in the GAN diet-fed WT and Slco4c1 -/- mice. The relative levels of hepatic Fasn, Acc1, Scd1 mRNA transcripts ( D ) and protein expression ( E ) in the GAN diet-fed WT ( n = 6) and Slco4c1 -/- mice ( n = 8). F A diagram illustrates how SCLO4C1 inhibits lipid synthesis in the liver (Created with MedPeer (medpeer.cn). The relative levels of Fasn, Acc1, Scd1 mRNA transcripts ( G ) and protein expression ( H ) in primary mouse hepatocytes. The levels of TG ( I ) and TC ( J ) in primary mouse hepatocytes. K Oil red O staining in the BSA or PA-treated primary mouse hepatocytes from WT and Slco4c1 -/- mice. The relative levels of Fasn, Acc1, Scd1 mRNA transcripts ( L ) and protein expression ( M ) in different groups of HepG2 cells. The levels of TG ( N ) and TC ( O ) in different groups of HepG2 cells. P Oil red O staining of different groups of HepG2 cells. C The abscissa is the fold change after log2 conversion, and the ordinate is the lipid Sub Class. Each point in the figure represents a different lipid, the color of the point corresponds to different lipid Sub Class, and the size of the point is p after -log10 conversion. D , E Each dot represents one mouse sample; Plotted: mean ± SD; Statistics: unpaired two-tailed Student’s t test; 95% confidence interval. G n = 3 biologically independent experiments; Plotted: mean ± SD; Statistics: unpaired two-tailed Student’s t test; 95% confidence interval. H – P n = 3 biologically independent experiments; Plotted: mean ± SD; Statistics: one-way ANOVA test, Multiple comparisons, 95% confidence interval. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (PS Phosphatidylserine; PI Phosphatidylinositol; PG Phosphatidylglycerol; PE Phosphatidylethanolamine; PC Phosphatidylcholine; LPS Lyso-phosphatidylserine; LPG Lyso-phosphatidylglycerol; LPE Lyso-phosphatidylethanolamine; LPC Lyso-phosphatidylcholine; dMePE Dimethylphosphatidylethanolamine; SM Sphingomyelin; MGDG Monogalactosyldiacylglycerol; Cer Ceramides; DG Diglyceride).

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Expressing, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: A Luminescence Diagram of the “Persistently Open” Fluorescent Probe Jat-cAMP: The fluorophore was chemically conjugated to cAMP to form the Jat-cAMP probe, which was used for visualizing the transport of cAMP by SLCO4C1 protein. B Chemical synthesis of the Jat-cAMP probe. C The 2D highlighted the key interaction sites. D The 3D depiction illustrated the spatial binding conformation. E The strategies for determining the importance of SLCO4C1 Gln463 in transporting cAMP. F The CCK-8 analysis of the impact of Jat-cAMP on the viability of HepG2 cells. ( n = 6 Biological replicates; Plotted: mean ± SD; Statistics: one-way ANOVA test, Multiple comparisons, 95% confidence interval.) G , H Live cell imaging of Jat-cAMP treated HepG2 cells. I ELISA analyses of cAMP concentrations in different groups of primary mouse hepatocytes. J ELISA analyses of cAMP concentrations in liver tissues of the GAN diet-fed WT( n = 6) and Slco4c1 -/- mice( n = 8). K ELISA analyses of cAMP concentrations in serum samples of the GAN diet-fed WT( n = 6) and Slco4c1 -/- mice( n = 8). L Correlation between hepatic Slco4c1 mRNA transcripts and cAMP levels in the GAN-fed WT mice. (Person r, two-tailed, 95% confidence interval) ( M ) Relative levels of PKAc and p-Creb in different groups of primary mouse hepatocytes. N Relative Srebp1 mRNA transcripts in different groups of primary mouse hepatocytes. ( n = 6 Biological replicates; Plotted: mean ± SD; Statistics: unpaired two-tailed Student’s t test; 95% confidence interval.) O Relative Srebp1c protein levels in different groups of primary mouse hepatocytes. P Relative Epac1 protein levels in the different groups of primary mouse hepatocytes. H , I n = 5 Biological replicates; Plotted: mean ± SD; Statistics: one-way ANOVA test, Multiple comparisons, 95% confidence interval. J-K: Each dot represents one mouse sample; Plotted: mean ± SD; Statistics: unpaired two-tailed Student’s t test; 95% confidence interval. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. M , O , P n = 3 per group, Biological replicates; Plotted: mean ± SD; Statistics: one-way ANOVA test, Multiple comparisons, 95% confidence interval. * p < 0.05 vs. the BSA-treated cells from WT mice; # p < 0.05 vs. the PA-treated cells from WT mice. $ p < 0.05 vs. the BSA-treated cells from Slco4c1 -/- mice.

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Binding Assay, CCK-8 Assay, Live Cell Imaging, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: A Cytokines stimulated Slco4c1 mRNA transcription in primary mouse hepatocytes ( B ) FGF21 expression levels in different hepatic cell types. C Hepatic Fgf21 mRNA levels in GAN diet fed mice. D Correlation analysis between hepatic Fgf21 and Slco4c1 mRNA levels ( n = 6). E , F Treatment with FGF21 stimulated Slco4c1 mRNA and protein expression in primary mouse hepatocytes ( G ) Schematic illustration of the predicted transcription factors. H The predicted transcription factor mRNA transcripts in the FGF21-treated primary mouse hepatocytes. I , J Hepatic Egr1/2/3 mRNA transcripts and protein expression in the indicated groups of mice. (* p < 0.05, vs. Chow diet-WT) ( K ) Correlation between hepatic Slco4c1 and Egrs mRNA levels in the GAN diet-fed WT mice ( n = 6). L A diagram illustrating the potential EGRs binding sites in human SLCO4C1 proximal promoter and the reporter constructs. M Luciferase activities in 293 T cells co-transfected with truncated SLCO4C1 promoters with, or without, plasmids for EGRs overexpression. N A schematic diagram displayed the EGR1-binding site in the SLCO4C1 promoter and its mutant. O Luciferase activity in 293 T cells co-transfected with the mutant Slco4c1 promoter and the plasmid for EGR1 overexpression. Relative levels of Egr1 mRNA ( P ) and protein ( Q ) expression in primary mouse hepatocytes treated with, or without, an ERK/MAPK inhibitor, and after stimulated by FGF21 were examined. R ChIP assay revealed that FGF21 increased Egr1 binding to the Slco4c1 -59 promoters in primary mouse hepatocytes. Relative levels Slco4c1 mRNA (S) and protein (T) expression in primary mouse hepatocytes treated with, or without, an ERK/MAPK inhibitor, and after stimulated by FGF21 were examined. A n = 5 and M , O n = 3, Biological replicates; C , I , J Each dot represents one mouse sample; Statistics: unpaired two-tailed Student’s t test. R: n = 3 and E , F , H , P , Q , S , T n = 5, Biological replicates; Statistics: one-way ANOVA test, Multiple comparisons. *p < 0.05, * *p < 0.01, *** p < 0.001, **** p < 0.0001. F: * p < 0.05 vs. FGF21 0 μg/ml; # p < 0.05 vs. FGF21 0.1 μg/ml. Q, T: * p < 0.05 vs. FGF21 0 μg/ml; # p < 0.05 vs. FGF21 1 μg/ml. All data statistics are presented as mean ± SD and 95% confidence interval.

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Expressing, Binding Assay, Construct, Luciferase, Transfection, Over Expression, Mutagenesis, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: A Schematic illustrating of the experiment design of the GAN diet-induced MASL and MASH mouse models. B Bioluminescence images displayed the AAV8-mediated Slco4c1 expression in mice 12 weeks after inoculation. C The dynamic changes in body weights in the different groups of mice. (Plotted: mean ± SEM; Statistics: two-way ANOVA test, Multiple comparisons, 95% confidence interval.) D Liver weights and liver weight/body weight ratios (LW/BW) of MASL and MASH mice. E White and brown adipose tissue weights of MASL and MASH mice. H&E, Sirius red and Oil red O staining of liver tissue sections of MASL F and MASH ( G ) mice. Quantitative liver histologic assessment of the severity of MASL ( H ) and MASH ( I ) mice, including steatosis, lobular inflammation, hepatocyte ballooning, fibrosis and NAS score. The levels of serum ALT, AST ( J ) and TG, TC ( K ) in MASL and MASH mice. L Mice were injected with control virions or AAV8-TBG-Luciferase- Slco4c1 and their serum and hepatic cAMP levels were quantified using ELISA. M , N Western blot revealed that induction of Slco4c1 overexpression by injection with AAV8-TBG-Luciferase- Slco4c1 increased the relative levels of PKAc and p-Creb(S133) proteins ( M ), but decreased the relative levels of Srebp1 , Fasn , Acc1 , and Scd1 mRNA transcripts in the liver of mice, compared with that in the control mice ( N ). C – E , H – K n = 4 per group in MASL model. In MASH model, Chow diet group and GAN diet-AAV8-TBG-Luciferase-CTR group n = 5, AAV8-TBG-Luciferase- Slco4c1 group n = 6; D , E , H – K Each dot represents one mouse sample, Plotted: mean ± SD; Statistics: one-way ANOVA test, Multiple comparisons, 95% confidence interval. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. L , N Chow diet group and GAN diet-AAV8-TBG-Luciferase-CTR group n = 9, AAV8-TBG-Luciferase- Slco4c1 group n = 10, each dot represents one mouse sample, Plotted: mean ± SD; Statistics: one-way ANOVA test, Multiple comparisons, 95% confidence interval. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. M: * P < 0.05 vs. the Chow diet group, # P < 0.05 vs. the GAN diet-AAV8-TBG-Luciferase-CTR group.

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Expressing, Staining, Injection, Control, Luciferase, Enzyme-linked Immunosorbent Assay, Western Blot, Over Expression

Journal: Nature Communications

Article Title: Hepatocyte SLCO4C1 is a cAMP uptake transporter for inhibiting lipogenesis and a therapeutic target for MASLD

doi: 10.1038/s41467-026-70729-0

Figure Lengend Snippet: During the pathogenic process, metabolic disorder stimulates FGF21 expression, which through its receptors, activates the ERK/MAPK signaling to induce EGR1 expression. The transcription factor of EGR1 binds to the SLCO4C1 promoter and up-regulates its expression in hepatocytes. The SLCO4C1 functions as a cAMP uptake transporter through the interaction of its Gln463 with cAMP, increasing intracellular cAMP levels that activate the PKA-CREB signaling to feedback down-regulate SREBP1 and downstream ACC1, FASN and SCD1 expression, inhibiting fatty acid synthesis. Accordingly, induction of SLCO4C1 overexpression by AAV8-mediated hepatic SLCO4C1 expression and/or increasing intracellular cAMP levels by the Forskolin treatment effectively prevent and mitigate the progression of MASLD. Therefore, SLCO4C1 is a therapeutic target for MASLD. Conceivably, our findings may aid in the design of therapeutic strategies for treating MASLD.

Article Snippet: In addition, pGL3- SLCO4C1 and pCDNA3.1- SLCO4C1 ( SLCO4C1 o/e) were constructed by Genechem (Shanghai, China), and pcDNA3.1- EGR 1 ( EGR1 o/e) was constructed by Fenghui (Hunan, China).

Techniques: Expressing, Over Expression