Figure 1. RTN3 over-expression induces ER tubules fragmentation during starvation.
(A, B) Schematic representation of the protein structure and topology of FAM134B (A) and RTN3 long and short isoforms (B). (C) Immunofluorescence of HA and LC3B in U2OS TRex stable cell lines expressing FLAG-HA-RTN3L after 24 hr treatment with 1 µg/ml of doxycycline. Cells were kept in standard growing condition (DMEM with 10% FBS) or starved with EBSS for 6 hr. RTN3L was monitored using anti HA antibody, while autophagy induction was visualized using anti-LC3B antibody. Bafilomycin A1 was added at a final concentration of 200 ng/ml. Scale bars: 10 µm. (D) Quantification of U2OS TRex FLAG-HA-RTN1-4L cells with ER tubule fragment after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Number of cells >100 for each condition. Data are representative of three independent biological replicates. Error bars indicate s.d. (E) Super-resolution fluorescence microscopy (dSTORM) of ER fragments in U2OS TRex FLAG-HA-RTN3L cells stained with anti HA and anti LC3B antibodies after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Scale bar: 0.5 µm. (F) Schematic representation of ER tubules fragmentation and LC3 labeling in U2OS TRex FLAG-HA-RTN3L cells. The red square indicates the level of high resolution represented in panel E.
Figure 1—figure supplement 1. Reticulon family members.
Figure 1—figure supplement 2. ER tubules morphology in U2OS TRex cells after over-expression of full length RTN proteins.
Figure 1—figure supplement 3. ER tubules morphology in U2OS TRex cell after over-expression of short RTN variants.
Figure 1—figure supplement 4. ER tubules morphology in U2OS TRex RTNs cell lines after autophagy induction via EBSS starvation.
Figure 1—figure supplement 5. RTN3L over-expression promotes ER tubules fragmentation during autophagy induction.
