Figure S1.
Quantification of RE content. (A) CHO-k1 cell lines expressing GFP or LRAT-GFP were incubated in the presence or absence of 20 µM ROH. RE species were analyzed by LC-MS/MS. Amounts are expressed as pmol RE per μg protein (mean ± SD of a representative experiment). (B) Chromatogram showing in vitro LRAT activity of CHO-k1 homogenates with (red line) and without (gray line) expression of LRAT-GFP. After incubation with PC (7:0/7:0) and 10 µM ROH, ROH (peak a) and RE (7:0; peak b) were measured by LC-MS/MS. Multiple reaction monitoring transition 269/93 (retinoid backbones) is shown. (C) Relative contribution of RE species that were synthesized under the conditions described in A. (D) CHO-k1 cell lines with or without expression of LRAT-GFP were incubated overnight with 20 µM ROH in the presence or absence of 10 µM DGAT1 inhibitor T863. Total RE (m/z 269) and free cholesterol (m/z 369) were analyzed by LC-MS. Amounts are expressed as pmol RE per nmol cholesterol (mean ± SD of a representative experiment). (E) Confocal microscopy of transiently transfected LX-2 cells expressing LRAT-GFP (green) costained with DAPI (blue) and LipidTOX Red (red). Cells were incubated without (left) or with (right) 20 µM ROH and 200 µM OA for 4 h. OLE, oleic acid. (F) Confocal microscopy of transiently transfected LX-2 cells expressing LRAT-GFP stained with DAPI (blue) and LD540 (green). Cells were incubated without (left) or with 200 µM OA (middle panel) or 20 µM ROH (right panel) overnight. Scale bars indicate 10 µm.

Quantification of RE content. (A) CHO-k1 cell lines expressing GFP or LRAT-GFP were incubated in the presence or absence of 20 µM ROH. RE species were analyzed by LC-MS/MS. Amounts are expressed as pmol RE per μg protein (mean ± SD of a representative experiment). (B) Chromatogram showing in vitro LRAT activity of CHO-k1 homogenates with (red line) and without (gray line) expression of LRAT-GFP. After incubation with PC (7:0/7:0) and 10 µM ROH, ROH (peak a) and RE (7:0; peak b) were measured by LC-MS/MS. Multiple reaction monitoring transition 269/93 (retinoid backbones) is shown. (C) Relative contribution of RE species that were synthesized under the conditions described in A. (D) CHO-k1 cell lines with or without expression of LRAT-GFP were incubated overnight with 20 µM ROH in the presence or absence of 10 µM DGAT1 inhibitor T863. Total RE (m/z 269) and free cholesterol (m/z 369) were analyzed by LC-MS. Amounts are expressed as pmol RE per nmol cholesterol (mean ± SD of a representative experiment). (E) Confocal microscopy of transiently transfected LX-2 cells expressing LRAT-GFP (green) costained with DAPI (blue) and LipidTOX Red (red). Cells were incubated without (left) or with (right) 20 µM ROH and 200 µM OA for 4 h. OLE, oleic acid. (F) Confocal microscopy of transiently transfected LX-2 cells expressing LRAT-GFP stained with DAPI (blue) and LD540 (green). Cells were incubated without (left) or with 200 µM OA (middle panel) or 20 µM ROH (right panel) overnight. Scale bars indicate 10 µm.

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