JCS037051 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A-C) Characterization of STxB-incoming vesicles in EHD3 siRNA-treated cells. 20 minute uptake of 546-STxB (37°C) and fixation were followed by staining of the retromer subunit VPS35 (A), the early endosomal marker EEA1 (B), and Rab11 to visualize recycling endosomes (C). (D-E) Characterization of γ-adaptin-containing vesicles in EHD3-siRNA-treated HeLa cells. Fixed cells were stained with anti γ-adaptin and anti-EEA1 (D) or anti-VPS35 (E). (F-G) Characterization of CI-M6PR vesicles retained upon treatment with EHD3-siRNA. Fixed cells were stained with anti-CI-M6PR and anti-EEA1 (F), or anti-TGN46 (G). (H) The table summarizes colocalizations assessed. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Cytoskeleton and microtubule organizing center (MTOC) in cells treated with EHD3 or rabenosyn-5 (R5) siRNA. HeLa cells were treated for 48 hours to reduce EHD3 (C-D, I-H) or Rabenosyn-5 (E-F) and fixed. All cells were stained with giantin to depict the Golgi complex. Actin filaments were visualized with Rhodamine-Phalloidin (A-F). Anti-γ-tubulin antibodies were utilized to observe microtubules and the MTOC.

• Supplemental Figure S3 -

  Fig. S3. Dynamin II and PLA2 are not implicated in the vesiculation of Golgi caused by depletion of EHD3 or rabenosyn-5. (A) Endogenous dynamin II (Dyn II) and the cis-Golgi marker giantin (B) were co-stained with specific antibodies in fixed cells that were mock-treated (A-C and inset), treated with EHD3-siRNA (D-F and inset) or rabenosyn-5-siRNA (R5-siRNA) (G-I and inset). (J-M) Cells transfected with the dominant-negative mutant GFP-dynamin II (K44A) were also treated with EHD3-siRNA (L-M) or mock treated (J-K). The Golgi stacks were visualized with anti-GM130 and the nuclei were stained with DAPI. Mock-treated (N and O) and EHD3-siRNA-treated HeLa cells (P and Q) were incubated with 100 µM cytosolic PLA2 inhibitor MAFP, for 14 hours at 37°C (O and Q) or left without the drug (N and P). After fixation, the Golgi was visualized by immunostaining with anti-GM130, followed by Alexa Fluor 568 goat anti-mouse. Note that a more ‘compact’ Golgi is seen after MAFP treatment in the mock-treated cells (O). Scale bar: 10 µm