Fig. 3. The relationship between ER shape and PB biogenesis.

(A) Representative images of Edc3 IF studies performed in U-2 OS cells transiently transfected with mCh-Sec61β or Rtn4a-mCh. Edc3 IF gray-scale images were inverted to highlight Edc3 puncta (left), Edc3 IF in green merged with the nuclear stain Hoechst in blue (middle), and the middle panels merged with mCh-tagged ER markers in red (right). The dashed lines indicate the cellular boundary as estimated by the outer reaches of ER signal. (B) The mean quantified in U-2 OS cells exogenously expressing mCh-Sec61β or Rtn4a-mCh from three biological replicates. (C) Representative merged images of U-2 OS cells overexpressing either mCh-Sec61β or Rtn4a-mCh together with the PB marker GFP-Dcp2. Insets show movement of the two organelles through space and time over a 2-min time-lapse movie with frames captured every 5 s. PBs were binned as in Fig. 1C. The arrows show PBs that maintain contact with the ER for the entire movie. (D and E) Cells were imaged for each condition from three biological replicates and quantified for the mean number of PBs per cell (D) and the degree of association between the ER and PBs (E). (F) Immunoblot analyses of protein expression levels for Rtn4 and PB factors (Edc3, Dcp1b, Dcp2, and PatL1) in whole-cell lysates of wild-type and RTN4 KO U-2 OS cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (G) Representative images of IF studies performed in wild-type and RTN4 KO U-2 OS cells against Edc3 and calreticulin (Calr) to label PBs (green) and ER (red), respectively. Edc3 IF gray-scale images were inverted to highlight Edc3 puncta (left), Edc3 IF in green merged with the nuclear stain Hoechst in blue (middle), and the middle panels merged with calreticulin IF in red (right). (H) The mean numbers of PBs were quantified in wild-type and RTN4 KO U-2 OS cells from three biological replicates. (I) Representative images of Edc3 IF studies performed in U-2 OS cells transiently transfected with mCh-Sec61β or mCh-CLIMP63. Edc3 IF gray-scale images were inverted to highlight Edc3 puncta (left), Edc3 IF in green merged with the nuclear stain Hoechst in blue (middle), and the middle panels merged with mCh-tagged ER markers in red (right). (J) The mean numbers of endogenous PBs were quantified in U-2 OS cells exogenously expressing mCh-Sec61β or mCh-CLIMP63 from three biological replicates. In (B), (D), (H), and (J), statistical significance was determined by Student’s t test, and error bars indicate SEM; ****P < 0.0001. In (A), (C), (G), and (I), scale bars are 5 and 2 μm in full cell and inset images, respectively.