Cay10594

For Western blotting, anti-Flag (M2) (F3165-5MG, 1:5000 dilution) and anti-α-tubulin (T6199–200UL, 1:5000 dilution) monoclonal antibodies were obtained from Sigma-Aldrich. An anti-Myc (sc-40, 1:500 dilution) monoclonal antibody was purchased from Santa Cruz Biotechnology. An anti-hemagglutinin (HA) monoclonal antibody (MMS-101P, 1:3000 dilution) was obtained from BioLegend. Anti-PJA2 (#40180, 1:1000 dilution), anti-MOB1 (# 3863S, 1:2000 dilution), and anti-phospho-p70 S6 kinase (Thr389) (#9234S,1:1000 dilution) polyclonal antibodies were obtained from Cell Signaling Technology. An anti-S1P (Z-P300; 1:500 dilution) monoclonal antibody was obtained from Echelon Biosciences.
For immunostaining, anti-HA (3724S, 1:3000 dilution) and anti-LAMP1 (9091S, 1:400 dilution) polyclonal antibodies were obtained from Cell Signaling Technology. An anti-Flag (F7425-.2MG, 1:5000 dilution) polyclonal antibody was purchased from Sigma-Aldrich. An anti-TOM20 (612278, 1:200 dilution) monoclonal antibody was purchased from BD Biosciences.
For chemicals, FIPI hydrochloride hydrate (#F5807) was obtained from Sigma-Aldrich. CAY10594 (#13207) was obtained from Cayman Chemical.

The following antibodies were used: anti-MICAL-L1 (ab220648, Abcam, for immunoblots; H00085377, Abnova, for immunofluorescence), anti-Rab10 (ab237703, Abcam, for immunoblots and immunofluorescence), anti-EHBP1 (NBP1-93615, Novus, for immunoblots), anti-Syndapin2/Pacsin2 (SAB1300127, Sigma, for immunoblots), anti-EEA1 (NBP1-05962, Novus for immunoblots), anti-pPKCα (06-822, Millipore, for immunoblots), anti-GAPDH-horseradish peroxidase (HRP) (HRP-60004, Proteintech, for immunoblots), mouse anti-rabbit IgG light chain–HRP (211-032-171, Jackson, for immunoblots), Alexa Fluor 568–conjugated goat anti-rabbit (A11036, Molecular Probes, for immunofluorescences), Alexa Fluor 568–conjugated goat anti-mouse (A11031, Molecular Probes, for immunofluorescence), and Alexa Fluor 488–conjugated donkey anti-mouse (A21202, Molecular Probes, for immunofluorescence). The PLD inhibitors CAY 10593 (also known as VU0155069) and CAY 10594 were purchased from Cayman Chemical Co and were typically used at 50 μM for 30 min at 37 °C.

HeLa, HEK and U2OS cells were from American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 5 U ml⁻¹ penicillin and 50 μg ml⁻¹ streptomycin. The HEK 293A GFP-LC3 cell line53 (link) was a kind gift from S. Tooze, Cancer Research UK, London, UK. The HEK GFP-DFCP1 cell line6 (link) was a kind gift from N. Ktistakis, Babraham Institute, Cambridge, UK. The HEK GFP-p62 cell line was a kind gift from G. Bjørkøy, HiST, Trondheim, Norway. All cell lines have been tested negative for mycoplasma. Bafilomycin A1 (Enzo Lifesciences) was used at 100 nM. CI 976 (Tocris Bioscience), Cay10594 (Cayman Chemical) and R59022 (Tocris Bioscience) were used at 20 μM. Glass support was coated by 20 μg ml⁻¹ fibronectin (Sigma) before plating HEK cell lines to avoid the cells from detaching from the surface. For starvation in nutrient-deplete medium, the cells were incubated in Earls Balanced Salt Solution (EBSS; Invitrogen), with the exception of the HEK GFP-DFCP1 cells that were starved as described previously6 (link) in 140 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose and 20 mM Hepes, pH 7.4.