Journal: Cells

Article Title: N-SREBP2 Provides a Mechanism for Dynamic Control of Cellular Cholesterol Homeostasis

doi: 10.3390/cells13151255

Figure Lengend Snippet: Sterol depletion protects nuclear N-SREBP2 from degradation and sequestration into PML nuclear bodies. ( A ) Western Blot analysis of N-SREBP2 in total cell lysates prepared from McArdle-RH7777 cells incubated in the basal medium containing 10 μM cycloheximide (CHX) for specified durations. Data (mean ± SEM) for this pilot analysis are from two independent experiments, each containing three technical replicates. ( B ) Western Blot analysis of N-SREBP2 in nuclear extracts prepared from McArdle-RH7777 cells incubated in a 10 μM CHX-containing medium for specified durations. At the 4 h time point, one set of cells was switched to the lipoprotein-depleted medium (LPDS) for 1 h (5 h CHX + 1 h LPDS): CHX was present throughout the 5 h experiment. Data (mean ± SEM) expressed relative to no CHX are from three independent experiments, each with three technical replicates. Significance between 5 h CHX + 1 h LPDS versus 5 h CHX was determined by an unpaired one-tailed Student’s t -test. ***, p < 0.001; ns: no significant difference. ( C , D ) The co-occurrence of N-SREBP2 with PML nuclear bodies. HepG2 cells cultured under basal conditions were fixed and subject to indirect immunofluorescence using PML and N-SREBP2 antibodies. Cells were counterstained with DAPI. An expanded view of the region marked by a white square ( D ) shows the separate channels (panels 1, 2) and orthogonal views (XZ axis, right-hand side; YZ, top) of merged images through the centre of the nucleus. ( E ) Proximity ligation assay images of DAPI-stained nuclei overlaid with the PLA dots (arrowheads) generated by the same antibody pairings used in ( C , D ). ( F ) Fewer PML nuclear bodies in HepG2 cells switched to the lipoprotein-depleted medium (LPDS) for 1 h. ( G ) The co-occurrence of N-SREBP2 labelling with PML nuclear body staining in the same nuclei analysed in F, irrespective of PML numbers. For ( F , G ), the data (mean ± SEM) are from 250 cells per independent experiment (n = 6) for each experimental condition. Significance was determined by a one-tailed paired Student’s t -test: * and **, p < 0.05 and 0.01, for a difference from the basal condition.

Article Snippet: McArdle-RH7777 (CRL 1601) and HepG2 cells (HB-8065™) were obtained from ATCC.

Techniques: Western Blot, Incubation, One-tailed Test, Cell Culture, Immunofluorescence, Proximity Ligation Assay, Staining, Generated

Journal: Toxicology

Article Title: Hazard and Risk Characterization and Grouping of 56 Structurally Diverse PFAS Using a Targeted Battery of Broad Coverage Assays in Six Human Cell Types

doi: 10.1016/j.tox.2024.153763

Figure Lengend Snippet: Cell types and cell type-specific phenotypes evaluated in this study. See Supplemental Text for experimental details and culture conditions.

Article Snippet: Selected cell types included: (i) iCell hepatocytes 2.0 (iCell Heps, Cat# C1023, FujiFilm Cellular Dynamics, Madison, WI), (ii) iCell GABAneurons (iCell Neurons, Cat# C1012, FujiFilm Cellular Dynamics), (iii) iCell cardiomyocytes (iCell Cardios, Cat# C1006, FujiFilm Cellular Dynamics), (iv) human umbilical vein endothelial cells (HUVECs, Cat# C2517A, Lonza, Walkersville, MD), (v) primary human hepatocytes (PHHs, Cat# HMCPIS; Gibco, Waltham, MA), and (vi) HepG2 cells (HepG2s, Cat# HB-8065; ATCC, Manassas, VA).

Techniques: Functional Assay