Fig 1: Analysis of ER scramblases and lumenal lipid droplets.A) Confidence score for CLCC1, TMEM41B, and VMP1 from two genome wide CRISPR screens, the neutral lipid (i.e., BODIPY 493/503) screen in the current manuscript and a previous PLIN2-GFP screen9. The sign, positive or negative, indicates the effect of gene depletion on neutral lipids or PLIN2-GFP levels.B) Immunoblot of the indicated proteins in control and CLCC1KO Huh7 cells.C) Immunoblot of TMEM41B in control and TMEM41BKO Huh7 cells.D) Immunoblot of VMP1 in control and VMP1KO Huh7 cells.E) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control and TMEM41BKO and VMP1KO cells. Scale bar represents 10 μm.F) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control and TMEM41BKO cells treated with MTP inhibitor (MTPi) for 72 h. Zoom images of the boxed regions are shown on the right. Scale bar represents 10 μm.G) Quantification of LDs and LD PLIN2 staining of control and TMEM41BKO cells incubated in the presence and absence of MTP inhibitor (MTPi) as in panel F.
Fig 2: Analysis of ER scramblases in CLCC1KO cells.A) Immunoblot of VMP1 overexpression in control and CLCC1KO cells.B) Immunoblot of TMEM41B overexpression in control and CLCC1KO cells.C) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control and CLCC1KO cells overexpression TMEM41B and VMP1, as indicated. Scale bar represents 20 μm.
Fig 3: Analysis of ER scramblases and lumenal lipid droplets.A) Confidence score for CLCC1, TMEM41B, and VMP1 from two genome wide CRISPR screens, the neutral lipid (i.e., BODIPY 493/503) screen in the current manuscript and a previous PLIN2-GFP screen8. The sign, positive or negative, indicates the effect of gene depletion on neutral lipids or PLIN2-GFP levels. B) Immunoblot of the indicated proteins in control and CLCC1KO Huh7 cells. C) Immunoblot of TMEM41B in control and TMEM41BKO Huh7 cells. D) Immunoblot of VMP1 in control and VMP1KO Huh7 cells. E) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control, TMEM41BKO and VMP1KO Huh7 cells. Scale bar represents 10 µm. F) Representative image of PLIN2 (green) and apoB (purple) crescents on an LD in TMEM41BKO Huh7 cells. Scale bar represents 20 µm. G) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control and TMEM41BKO Huh7 cells treated with 50 nM CP-346086 (MTPi) for 72 h. Zoom images of the boxed regions are shown on the right. Scale bar represents 10 µm. H) Quantification of LDs and LD PLIN2 staining of control and TMEM41BKO Huh7 cells incubated in with DMSO or 50 nM CP-346086 (MTPi) as in panel F. Data represent mean ± SD across three biological replicates. Source data
Supplier Page from DNASU for VMP1 (Homo sapiens) in pDONR201 (Gateway donor/master vector)