Journal:

Article Title:

doi:

Figure Lengend Snippet: A . Pictorial representation of the Sphk1 promoter, in which multiple E-boxes reside, including positions -1800 and -300 base pairs from the transcription start site (TSS) (Adapted from Ross and Cowart, et al., 2013). B . Sphk1 mRNA expression in gWAT over time in 12hr-light:12hr-dark-entrained wildtype mice. C . Sphk1 mRNA expression in primary adipocytes with concomitant expression of Reverbα over 24 hours. D . Presence of BMAL1 and CLOCK at the promoter E-box loci in Sphk1 DNA. E . H3K27ac ChIP at the Sphk1 promoter E-boxes reveals fluctuations in the occupancy by transcriptionally active chromatin. F . Following synchronization, wildtype adipocytes 17 C-S1P production as measured by LC/MS/MS over a circadian time course when fed 5 µM 17 C-sphingosine for exactly 30 minutes prior to each harvest. CT, circadian time (CT0 is the time at which synchronization serum shock media is administered); ZT, Zeitgeber time

Article Snippet: WT primary matured, synchronized adipocytes were allowed to cycle for 24 hours, after which, cells were treated with a final concentration 5 µM 17 C-sphingosine (Avanti Polar Lipids, 860640) (dissolved in 100% ethanol) for exactly 30 minutes, collected immediately with addition of phosphatase inhibitors, for an exact volume of media in a 5-mL borosilicate glass tube and for a cell pellet in minimal PBS in a separate 5-mL borosilicate glass tube, capped, and stored at -80° C until analysis by tandem liquid chromatography and mass spectrometry lipidomics.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy

Journal:

Article Title:

doi:

Figure Lengend Snippet: A . mRNA rhythms of Bmal1 (positive core loop gene), Dbp (canonical clock-controlled gene (ccg)), Nr1d1 (also known as Rev-erbα; negative accessory loop gene), Per2 (negative core loop gene), Per3, and Rorγ (positive accessory loop gene) in synchronized primary matured WT and SPHK1 -/- adipocytes over a 48-hour time course. Refer to on page 13 for the molecular circadian clock. B . Western blot of BMAL1 protein expression in primary WT and SPHK1 -/- matured adipocytes whole cell lysates across the circadian time course. C . Expression of BMAL1 protein (by Western blot) in nuclear- and cytoplasmic-enriched fractions of synchronized primary WT and SPHK1 -/- matured adipocytes through a time course of 24 hours. D . Nuclei of primary WT and SPHK1 -/- matured adipocytes were isolated, and CLOCK protein expression over 24 hours was measured by Western blot. # denotes posttranslational modifications. Experiments were performed 3 times in 3 separate preparations of primary cells for culture, and representative blots are shown for the trends that were observed. Bmal1, brain and muscle ARNT-like protein 1; Dbp, D-site of albumin promoter binding protein; Nr1d1, nuclear receptor subfamily 1 group D member 1; Per2, 3, period 2, 3; Rorγ, retinoic acid receptor-related orphan receptor gamma; SK1 -/- , sphingosine kinase 1 knockout (for brevity in figures).

Article Snippet: WT primary matured, synchronized adipocytes were allowed to cycle for 24 hours, after which, cells were treated with a final concentration 5 µM 17 C-sphingosine (Avanti Polar Lipids, 860640) (dissolved in 100% ethanol) for exactly 30 minutes, collected immediately with addition of phosphatase inhibitors, for an exact volume of media in a 5-mL borosilicate glass tube and for a cell pellet in minimal PBS in a separate 5-mL borosilicate glass tube, capped, and stored at -80° C until analysis by tandem liquid chromatography and mass spectrometry lipidomics.

Techniques: Western Blot, Expressing, Isolation, Binding Assay, Knock-Out