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

Journal: bioRxiv

Article Title: SUMOylation targets shugoshin to stabilize sister kinetochore biorientation

doi: 10.1101/840157

Figure Lengend Snippet: SUMO ligases promote Sgo1 inactivation to allow timely anaphase progression. (A) Overexpression of SIZ2 rescues the slow growth phenotype of SGO1 overexpression. A pGAL-SGO1 (AMy870) strain carrying empty vector (AMp67), or with yEP13-( SLP1 ) ISN1 SIZ2 ( PUP1 ) (AMp1435) was streaked onto medium containing galactose. (B and C) SIZ2 overexpression partially rescues the metaphase delay of SGO1- overexpressing cells. (B) Schematic of live cell imaging experiment. Cells carrying Spc42-tdTomato and Cdc14-GFP were synchronized in G1 in media containing 2% raffinose. 25 μM copper sulfate was added to induce pCUP1-SIZ2 expression. After releasing from G1, 0.2% galactose was added to induce pGAL-SGO1 expression. The duration of metaphase was estimated by the time taken between the separation of the spindle pole bodies (two Spc42-tdTomato foci) and the dispersal of Cdc14-GFP from the nucleolus. (C) Metaphase duration is shown for wild type (AMy24115), pGAL-SGO1 (AMy27596), pGAL-SGO1 pCUP1-SIZ2 (AMy27738) and pCUP1-SIZ2 (AMy27952) strains. (D and E) Siz1 and Siz2 are required for timely anaphase onset. Metaphase duration was determined as the time between formation of a short bipolar spindle (YFP-Tub1) and release of Cdc14-GFP from the nucleolus from live cell imaging. (D) Schematics and representative images are shown. (E) Metaphase duration is shown for wild type (AMy24174) and siz1 Δ siz2 Δ (AMy24313) strains. (F) and (G) The metaphase delay of siz1 Δ siz2 Δ cells is partially rescued by SGO1 deletion. Wild type (AMy1290), sgo1 Δ (AMy8466), siz1 Δ siz2 Δ (AMy8465) and siz1 Δ siz2 Δ sgo1 Δ (AMy12110) strains carrying PDS1-6HA were released from a G1 arrest. Spindle morphology was scored after anti-tubulin immunofluorescence and the percentages of short (metaphase) spindles are shown (top) and Pds1 levels were analysed by anti-HA Western blot (bottom). Pgk1 is shown as a loading control.

Article Snippet: Rat anti-α-tubulin (Abd Serotec) antibody was used at 1:50 and anti-Rat FITC (Jackson ImmunoResearch) was used at 1:16.7.

Techniques: Over Expression, Plasmid Preparation, Live Cell Imaging, Expressing, Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: SUMOylation targets shugoshin to stabilize sister kinetochore biorientation

doi: 10.1101/840157

Figure Lengend Snippet: Sgo1 is SUMOylated, depending on its association with pericentromeres. (A and B) Sgo1 is SUMOylated in a Siz1/Siz2-dependent manner. (A) Scheme describing purification of SUMOylated proteins. (B) Extracts from untagged (AMy1176), SGO1-6HA (AMy906) and siz1 Δ siz2 Δ SGO1-6HA (AMy7911) strains carrying empty vector (pRS426), or with 7 × HIS-SMT3 (AMp773) were purified over Ni-NTA resin and anti-HA immunoblot was performed on both input and elute. Arrows and asterisks indicate SUMO-Sgo1-6HA and unmodified Sgo1-6HA, which binds non-specifically to the resin, respectively. (C) Sgo1 is SUMOylated by Siz1 and Siz2 in vitro . Purified Sgo1 was incubated with 1 μM E1, E2, E3, SUMO and ATP or missing one component as indicated. Reaction was incubated at 30°C for 3 h. (D) Sgo1 SUMOylation occurs in metaphase. Cells carrying SGO1-6HA and 7xHIS-SMT3 (AMy7655) were released from G1, harvested at the indicated intervals, and SUMOylation was analysed as described in (A). Cell cycle stage was monitored by scoring spindle morphology after anti-tubulin immunofluorescence. (E) Chromatin association promotes Sgo1 SUMOylation. Sgo1 SUMOylation was determined in wild type (AMy7654), bub1 Δ (AMy10098), bub1-KD (catalytically inactive Bub1 kinase, AMy10102), sgo1-100 (AMy26334) and sgo1-700 (AMy26336) strains. (F) Sgo1 SUMOylation is lost upon the establishment of tension between sister kinetochores. Cells carrying pMET-CDC20 and either 7xHIS-SMT3 (AM9641) or empty vector (AMy26342) were arrested in metaphase by depletion of Cdc20 either in the presence of benomyl and nocodazole (no tension) or DMSO (tension).

Article Snippet: Rat anti-α-tubulin (Abd Serotec) antibody was used at 1:50 and anti-Rat FITC (Jackson ImmunoResearch) was used at 1:16.7.

Techniques: Purification, Plasmid Preparation, Western Blot, In Vitro, Incubation, Immunofluorescence