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. Author manuscript; available in PMC: 2020 Jun 8.
Published in final edited form as: Biochim Biophys Acta Mol Cell Biol Lipids. 2020 May 1;1865(8):158730. doi: 10.1016/j.bbalip.2020.158730

Fig. 7. LAL-deficient murine HSCs lose RE stores under serum-starvation.

Fig. 7

Primary HSCs were isolated from WT and LAL-ko mice by collagenase digestion and cultivation for 14 days in DMEM media containing 10% FCS and ROH (5 μM). (A–F) HSCs were plated in 6-well plates and cultivated in DMEM containing 10% FCS (= basal). The next day, cells were incubated with DMEM, containing 10% FCS, ROH (20 μM), and oleic acid (100 μM) for 24 h (loading period). Media were changed to DMEM low glucose (1 g/l) supplemented with 2% FA-free BSA for 8 h (serum-starvation period). (A) Lipids were extracted with n-hexane:2-propanol (3:2, v/v) and RP content was determined by HPLC-FD. (C) Lipid extracts were separated by thin-layer chromatography. Band intensities corresponding to TG were quantitated by Bio-Rad Image Lab Software and normalized to mg cell protein. (B, D) Amount of degraded RP and TG were calculated by subtracting the amounts after “serum-starvation” from that of “loaded” cells. (E, F) Protein expressions of the lipid droplet marker ADRP (E) and autophagy markers ATG7, p62, and LC-3 I and II (F) were analyzed by Western blot analyses. (H) Schematic presentation of pulse-chase experiment. Data are mean + S.D. and representative for three independent experiments (n = 3). Statistically significant differences were determined by Student's unpaired t-test (two-tailed; **, p < 0.01; ***, p < 0.001). n.d. = not detectable.