B7651

Chemical induction of ER stress in zebrafish embryos was accomplished by using a final concentration of 0.5 mM DTT (NEB #B1034S; Invitrogen Y00147), 1 µM thapsigargin (Sigma-Aldrich #T9033), 1 µg/ml tunicamycin (Sigma-Aldrich #T7765), and 5 µM brefeldin A (Sigma-Aldrich #B7651) in 10 ml phenylthiourea (PTU) in a 16 mm×50 mm Petri dish. For heat stress, Petri dishes containing embryos in 5 ml PTU and sealed using parafilm were incubated in a water bath set to 39°C for 1 h.

BFA (Sigma-Aldrich B7651) was solubilized in DMSO to 50 mM stock concentration and added to liquid ½MS media for treatments. Beta-estradiol (Sigma-Aldrich E8875) was solubilized in 100% ethanol to 5 mg/mL stock concentration and added to ½MS media during preparation of solid media to a final concentration of 2.5 µg/mL. Induction of XVE»amiCHCa was performed approximately 48 hr before CLSM imaging by transferring 3-day-old seedlings to media supplemented with Beta-estradiol.

Leaves transiently expressing each construct 48 hr post-agroinfiltration were infiltrated with a dH ₂ O solution of Brefeldin A at a concentration of 50 µg / mL (B7651 SIGMA); from a DMSO stock solution; for 3 hr before observation. Mock conditions contain the same volume of DMSO alone. Leaves were then observed with a Zeiss LSM 880 confocal fluorescence microscope with an oil-immersion 63x lense using the appropriate excitation wavelengths for each fluorescent fusion proteins.

Smooth muscle cells were isolated from cystectomized human bladders as described [4 (link)],[24 (link)]. Cells in passages 2–6 were cultured in DMEM/Ham’s F12 supplemented with antibiotics (penicillin and streptomycin) and 10% fetal calf serum (FCS). The cells were treated for 24h with the following agents: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 0.16 μM 48599, Sigma Aldrich), forskolin (10 μM, F6886, Sigma Aldrich), brefeldin A (5 μg/ml, B7651 Sigma Aldrich), tunicamycin (5 μg/ml, T7765, Sigma Aldrich), Phorbol 12-myristate 13-acetate (PMA, 1μM, P8139, Sigma Aldrich) or fetal calf serum (FCS). Cells were harvested and RNA isolated as described above. MiR-132 and miR-212 inhibitors (AM10166, AM10340, Ambion, Thermo Scientific, Pittsburgh, PA, USA; 10 nM and 100 nM), mimics (Mission miRNA: Sigma-Aldrich, St. Louis, MO, USA; 10 and 100 nM) and negative control (HMC0002, Mission miRNA: Sigma-Aldrich, St. Louis, MO, USA; 10 and 100 nM) were transfected using Oligofectamine reagent (Life Technologies). Successful transfection of miR-132/212 mimics and inhibitors was confirmed using real-time quantitative PCR. Experiments with human cells are from 3–7 independent replicates using cells from both males and females.