Journal: Frontiers in Cell and Developmental Biology

Article Title: Pleiotropic effects of Syntaxin16 identified by gene editing in cultured adipocytes

doi: 10.3389/fcell.2022.1033501

Figure Lengend Snippet: Sx16 knockdown does not affect 3T3-L1 adipogenesis. (A) Lysates prepared from Sx16 KO or WT cells were immunoblotted for fatty acid synthase (FAS) and stained for total protein with Revert™ total protein stain. (B) Quantification of FAS levels was determined by comparison with total protein using densitometric analysis. Data shown represent the mean ± SD of three independent experiments. Data is presented as % relative to Sx16 wild type cell lysate, (C) The indicated populations of adipocytes were stained with Oil Red O and Mayers Hematoxylin. Representative images are shown (D) Oil Red O stain was eluted from stained cells and absorbance at 590 nm measured. Data shown represent the mean ± SD of three independent experiments (E) Sx16-KO or WT cells were incubated with or without insulin as indicated and lysates prepared as described. Shown are representative immunoblots of phospho-S471 AKT and total AKT. (F) Quantification of AKT phosphorylation was determined by comparison with total AKT using densitometric analysis. Data is presented as % relative to insulin stimulated Sx16 wild type from three experiments of this type, each point on the graph is a single biological replicate. **** p < 0.0001 as assessed by two-way ANOVA.

Article Snippet: Total protein was stained with Revert™ Total Protein Stain (Fisher Scientific, Loughborough, United Kingdom).

Techniques: Staining, Incubation, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Pleiotropic effects of Syntaxin16 identified by gene editing in cultured adipocytes

doi: 10.3389/fcell.2022.1033501

Figure Lengend Snippet: Sx16 knockout cells have altered lactate metabolism. Hyperinsulinemia was achieved by incubating cells with 300 nM insulin for 18 h in serum free media. Lactate concentration was determined from cell homogenates (A) or conditioned media (B) . (C) Cell lysates were immunoblotted for MCT1 and compared with total protein stain (D) Quantification of MCT1 was determined by comparison to the total protein stain using densitometric analysis from three biological replicates of each condition. Data shown represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA or Students t -test; n.s. = not significant.

Article Snippet: Total protein was stained with Revert™ Total Protein Stain (Fisher Scientific, Loughborough, United Kingdom).

Techniques: Knock-Out, Concentration Assay, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Pleiotropic effects of Syntaxin16 identified by gene editing in cultured adipocytes

doi: 10.3389/fcell.2022.1033501

Figure Lengend Snippet: Adipokine secretion in impaired in Sx16 knockout adipocytes. (A) Adipocytes were cultured in serum free DMEM for 18 h prior to media collection and preparation of cell lysates from Sx16-KO or WT cells as described. Shown is a representative immunoblot of adiponectin and total protein stain (B) Quantification of intracellular adiponectin relative to total protein stain by densitometric analysis (C) Quantification of secreted adiponectin by densitometric analysis (D) Adipocytes were stimulated with 500 ng lipopolysaccharide (LPS) for 18 h in serum free media, conditioned media was collected and MCP-1 content assessed by Quantikine ® ELISA. Data shown represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001; n.s. = non significant.

Article Snippet: Total protein was stained with Revert™ Total Protein Stain (Fisher Scientific, Loughborough, United Kingdom).

Techniques: Knock-Out, Cell Culture, Western Blot, Staining, Enzyme-linked Immunosorbent Assay