Journal: Cell death & disease

Article Title: Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist.

doi: 10.1038/s41419-021-03862-x

Figure Lengend Snippet: Fig. 1 Discovery of potent HNF4α agonists. A Structures of tested compounds. B Assay for HNF4a activity. Compounds from A were assayed as previously described10. Briefly, T6PNE cells treated with tamoxifen (0.5 μM) to induce the activity of the E47MER transgene10, were treated with the indicated compounds at 3 concentrations (5 μM, black, 10 μM, red, 20 μM, green) for 3 days, followed by fixation, staining with DAPI for nuclear visualization, and imaging for quantification of the percentage of cells expressing the insulin promoter-GFP transgene in the T6PNE cells (N =6) using a Celigo imaging cytometer. C Representative images of wells containing the DMSO negative control, alverine positive control, and the positive compounds NCT and NFT (green is from insulin promotor-GFP expression and blue is DAPI nuclear staining, scale bar = 500 μm). D Quantification of GFP-positive cells, reflecting activity of the human insulin promoter-GFP transgene in T6PNE cells, was done with multiple doses of NCT and NFT to demonstrate dose-responsiveness using a Celigo imaging cytometer (N = 14). E, F Insulin and HNF4α mRNA levels on compounds from A were measured by qPCR as an additional measure of HNF4α activity (compound concentration was 20 μM, N= 4). G, H Insulin and HNF4α mRNA levels were measured by qPCR with multiple doses of NCT and NFT (N = 4). Values represent the mean ± SE. *p < 0.05, **p < 0.01 (vs DMSO).

Article Snippet: Subsequently, sections were incubated with blocking buffer containing 5% normal donkey serum (Jackson Immuno Research) followed by incubation overnight at 4 °C with mouse monoclonal primary antibody against HNF4 (1:800, Cat# PP-H1415-00, R&D Systems).

Techniques: Activity Assay, Staining, Imaging, Expressing, Cytometry, Negative Control, Positive Control, Concentration Assay

Journal: Cell death & disease

Article Title: Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist.

doi: 10.1038/s41419-021-03862-x

Figure Lengend Snippet: Fig. 2 NCT and NFT are HNF4α agonists. A HNF4α siRNA blocked the effect of HNF4α agonists. Scrambled or HNF4α siRNAs were transfected into T6PNE cells 2 days before compound administration. Compounds at the indicated concentrations were treated for an additional 2 days. The cells were analyzed for the percentage of cells expressing the insulin promoter-GFP transgene (N = 6). B DARTS assay to detect effect of compounds on HNF4α protease sensitivity. For the DARTS assay, HepG2 cells were treated with DMSO (lane 1), BI6015 (lane 2), NCT (lane 3), or NFT (lane 4) at a concentration of 40 or 80 μM for 16 h. Total cell protein was extracted and each sample was split into two aliquots for proteolysis without (−) or with (+) subtilisin and analyzed by Western blotting for HNF4α as done previously7. After detection of HNF4α, the membrane was stained with Ponceau S (magenta color) as a control to ensure that the compounds did not induce nonspecific proteolysis (Lane M has MW markers). All compounds were run on the same gel. C The HNF4α level was quantified by ImageJ using the Western blots from panel B. Values represent the mean ± SE of 3 biological replicates, *p < 0.05, **p < 0.01 (vs scrambled siRNA or DMSO).

Article Snippet: Subsequently, sections were incubated with blocking buffer containing 5% normal donkey serum (Jackson Immuno Research) followed by incubation overnight at 4 °C with mouse monoclonal primary antibody against HNF4 (1:800, Cat# PP-H1415-00, R&D Systems).

Techniques: Transfection, Expressing, Concentration Assay, Western Blot, Membrane, Staining, Control

Journal: Cell death & disease

Article Title: Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist.

doi: 10.1038/s41419-021-03862-x

Figure Lengend Snippet: Fig. 6 NCT reverses hepatic steatosis in vivo. DIO mice (C57BL/6 J) were injected intraperitoneally with NCT (200 mg/kg bid) for two weeks, followed by harvesting of organs. A Red box indicates the area of the liver, demonstrating a marked difference in color. B Dissected liver from representative mice indicating difference in color and weight (quantified in C, N = 12 for each group). D Epididymal fat pads from representative mice showing increased weight with NCT (quantified in E, N = 12 for each group). F Hepatic triglyceride (TG) content normalized to hepatic protein (Normal chow control, N = 3, DMSO and NCT, N = 12). G Serum free fatty acid (FFA) level (Normal chow control, N = 5 and DMSO and NCT, N = 12). H Blood alkaline phosphatase (ALP) level (Normal chow control, N = 3 and DMSO and NCT, N = 12). I Representative photomicrograph of hepatic Oil Red O staining (scale bar = 200 μm). J Oil Red O quantification. The percent of the liver section positive for Oil Red O was measured using Image J with a consistent threshold setting and normalized to liver sections from mice fed normal chow. K Representative liver sections stained for Bodipy (green), HNF4α (red), DAPI (blue) and merged images in mice fed normal chow or HFD plus DMSO or NCT. L Quantification of HNF4α nuclear staining. HNF4α nuclear staining intensity with non-specific cytoplasmic staining from same cell subtracted. Normalized to livers from mice fed normal chow. M Quantification of hepatic HNF4α mRNA level. Dots indicate individual mice. Values represent the mean ± SE, Normal chow control, N = 3–5; DMSO and NCT, N = 12. *p < 0.05, **p < 0.01. Scale bar = 100 μm.

Article Snippet: Subsequently, sections were incubated with blocking buffer containing 5% normal donkey serum (Jackson Immuno Research) followed by incubation overnight at 4 °C with mouse monoclonal primary antibody against HNF4 (1:800, Cat# PP-H1415-00, R&D Systems).

Techniques: In Vivo, Injection, Control, Staining