Tunicamycin

4T1 shSCR and shPHGDH#2 cells were treated for 72 h with 0.05 ug/ml tunicamycin (Merk Life Science, T7765). tunicamycin was washed out and the cells still attached to the flask were harvested and seeded – at 1×10⁵, 8×10⁴, 4×10⁴ per well for the 3 time points – on 10 mm round glass coverslip previously coated with fibronectin bovine plasma (1:1 in PBS) (Merk Life Science, F1141) and placed in 12-wells plates. At each time point cells adhering to coverslip were incubated for 10 min at 37°C with wheat germ agglutinin (WGA) Alexa Fluor 555 (Life Technologies, W32464) at final concentration of 5 μg/ml in Hank’s balanced salt solution (HBSS) without phenol red. Samples were then fixed in paraformaldehyde 4% in PBS for 20 min at RT and permeabilized in Triton X-100 0,2% in PBS for 15 min at RT. At last samples were incubated with 1 μg/ml of DAPI (Life Technologies, D1306) for 5 min at RT. Imaging was performed on a Leica TCS SP8 X confocal microscope equipped with a White Light Laser and UV lamp and a HCX PL APO CS 63x/1.40 OIL objective. Images were acquired and processed with LAS X software (Leica).

X.laevis oocytes were prepared and maintained in OR3 medium as described previously (8 (link)). Capped cRNAs were transcribed in vitro from linearized cDNAs with T7 polymerase using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion); cRNAs (2 ng) of UT-A3 alone or with ST6GalI in total volume of 23 nl water were injected into each oocyte. Three days later, healthy oocytes were selected for functional study (8 (link)). Urea flux data are expressed as means ± SDs. Oocyte biotinylation (~15 cells/group) was performed as described (13 (link)). For tunicamycin treatment, at 2 h prior to cRNA injection, oocytes were pre-injected with 10 ng tunicamycin (Sigma).

Mice were injected intraperitoneally with 1mg/kg tunicamycin (catalog #T7765; Sigma, St. Louis, Missouri) dissolved in DMSO (vehicle control). tunicamycin is known to cross blood brain barrier [31 (link)] and previous studies have shown that tunicamycin administration (1 mg/kg; IP) induces increases in ER stress markers in mouse brain [32 (link), 33 (link)]. The behavioral effects of ERB-041 are less well characterized than those of other ER subtype agonists, such as propyl pyrazole triol (PPT) and diarylpropionitrile (DPN). The few behavioral studies used 0.9 mg/kg of DPN and PPT; and showed that both drugs enhanced novel object recognition [34 (link)] and object placement [35 (link)]. Given the high affinity and selectivity of ERB-041 for ERβ [36 (link)], we chose 1 mg/kg to use in our study. It is known that ERB-041 reaches maximum brain levels 30 minutes after injection [36 (link)]. Therefore, ERB-041 (catalog #PZ0183; Sigma, St. Louis, Missouri) was administered 30 minutes before tunicamycin injection.

HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM low glucose, 1g/L glucose) (Wisent, Toronto, ON, Canada) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Antibiotic/Antimicotic (Wisent). HepG2 cells were seeded in wells 24-48hrs prior to treatments, and treated when cells reached 70% confluence. Medium was removed and replaced with treatments consisting of thapsigargin (25nM, 50nM, 100nM, 200nM) or tunicamycin (2.5ug/ml, 5ug/ml, 10ug/ml, 20ug/ml) (Sigma Aldrich, St. Louis, MO, USA) diluted in medium. 3T3L1 and C3H/10T1/2 adipocytes were maintained in DMEM (4.5g/L glucose) supplemented with fetal bovine serum and differentiated for 10 days following incubation with a differentiation protocol previously described (11 (link)). Differentiation was initiated two days post-confluency in DMEM containing 10% Fetal Bovine Serum in presence of a differentiation cocktail (insulin, Dexamethasone, IBMX) for two days. In the two following days, dexamethasone and IBMX were removed. In the following days, the cells were maintained in DMEM, 10% FBS until full differentiation was achieved (day 8) (11 (link)). Following differentiation, medium was removed and replaced with treatments consisting of thapsigargin (25nM, 50nM, 100nM) or tunicamycin (2.5ug/ml, 5ug/ml, 10ug/ml) (Sigma Aldrich, St. Louis, MO, USA) diluted in medium.