Fv1000 d ix81 confocal laser scanning microscope

After 24 h of transfection with DsRed-monomer-DGKη1 and/or EGFP-ASK3, COS-7 cells were incubated in DMEM with or without 500 mM sorbitol for 30 min. The cells were fixed in 3.7% paraformaldehyde. The coverslips were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescence imaging was performed using an Olympus FV1000-D (IX81) confocal laser scanning microscope (Olympus, Tokyo, Japan). Images were acquired using FV-10 ASW software (Olympus).
To observe stress granule markers, Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and T-cell intracellular antigen 1 related protein (TIAR), cells were fixed and then permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 and 1% bovine serum albumin. Coverslips were incubated with an anti-G3BP1 (Cat. #: 611126, BD Biosciences, Franklin Lakes, NJ, USA) or anti-TIAR (Cat. #: 610352, BD Biosciences) mouse monoclonal antibody for 1 h and then incubated with Alexa 594-conjugated anti-mouse IgG (Molecular Probe) for 1 h.

COS-7 cells seeded on coverslips were transiently transfected with plasmids using PolyFect reagent (Qiagen) as described by the manufacturer. After 20 h of transfection, the cells were incubated with the DGKα selective inhibitor CU-3 (29 (link)) (or DMSO alone as a control) in DMEM (final concentration: 10 μM) for 30 min to inhibit PA generation by DGKα-CA. The cells were then fixed in 4% paraformaldehyde. The coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were obtained with an Olympus FV1000-D (IX81) confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a UPLSAPO 60 × 1.35 NA oil at room temperature. EGFP fluorescence was excited at 488 nm, and mCherry fluorescence was excited at 543 nm. Images were obtained using FV-10 ASW software (Olympus).