Fig. 3.

Enrichment in peri-LD cages is an evolutionarily conserved property of seipin. a The SEIP-1::Venus protein was stably expressed in COS7 cells. The cells were incubated with oleic acid, fixed and immunostained for endogenous PDI (the ER) and TOM20 (mitochondria). Alexa Fluor (AF)-405 and 594 conjugated secondary antibodies were used for visualizing PDI and TOM20, respectively. AF594 signals were pseudocolored yellow. FAS was used to stain LDs and pseudocolored magenta. A projection of 4.5 μm z stack is shown. The boxed area was magnified 3× and shown in the inset. Scale bar = 10 μm. b Time-lapse monitoring of peri-LD cages maturation. The SEIP-1::Venus protein and a nascent LD marker, mRuby::LiveDrop were stably co-expressed in COS7 cells. mRuby signals were pseudocolored magenta. The cells were incubated with oleic acid for 1.5 h before live imaging for another 1 h at 30-s intervals. Arrows with the same color mark the same LiveDrop (+) LDs over time. Scale bar = 5 μm. c Quantification of LD size in COS7 cells stably expressing SEIP-1::Venus. A scatter plot of data from 48 LDs of each category, with median and interquartile range is shown. d Transmission electron micrograph of a COS7 cell stably expressing SEIP-1::Venus protein. The cell was incubated with oleic acid prior to fixation, and stained with diaminobenzidine (DAB), uranyl acetate, and osmium tetroxide prior to electron microscopy. No dark deposits were detected in this negative control. Scale bar = 1 μm. e As in (d), but with SEIP-1::V5-APEX2 fusion protein stably expressed. f The boxed area in (e) was magnified 5×. Dark deposits indicate the localization of SEIP-1::V5-APEX2 at ER tubules. The LD is indicated by an asterisk. Scale bar = 0.2 μm. g As in (e), but with five consecutive sections (150-nm thick) shown. Scale bar = 0.5 μm