On targetplus non targeting pool d 001810 10 20

To examine the effects of ER stress on TGF-β1 expression, granulosa-lutein cells were pre-incubated with the ER stress inhibitor TUDCA at 1 mg/mL for 24 h, followed by incubation with an ER stress inducer, 2.5 μg/mL tunicamycin (WAKO, Osaka, Japan) or 0.5 μM thapsigargin (Sigma-Aldrich), for 24 h. The optimal concentrations for the ER stress inhibitor and the ER stress inducers were determined according to our previous studies^{28 (link), 29 (link)}. To knock down XBP1(S), siRNA was obtained as the ON-TARGET plus SMART pool human XBP-1 siRNA (L-009552-00-020) from Dharmacon (GE Healthcare, Buckinghamshire, United Kingdom). The non-targeting siRNA control, ON-TARGET plus non-targeting pool (D-001810-10-20), was also obtained from Dharmacon. Granulosa-lutein cells were transfected with 50 nM siRNA for 24 h in Opti-MEM (Invitrogen, Carlsbad, CA, USA) using Lipofectamine RNAiMAX (Invitrogen). After transfection, the medium was removed, and the granulosa-lutein cells were treated with tunicamycin (2.5 μg/mL) for 24 h.

cDNAs encoding NTSR2 and TrkB in pCMV6-XL4 expression vectors were purchased from OriGene Technologies (Herford, Germany). For transient overexpression, cells were transfected using Amaxa Nucleofector 2b (Lonza, Levallois-Perret, France) and the Amaxa Nucleofector Kit-V (Lonza) according to the manufacturer’s instructions. For interference assays, B-CLL cells were transfected using INTERFERin (Polyplus transfection, Illkirch, France). In each transfection, 84 ng siRNA against NTSR2 (ON-TARGETplus Human NTSR2 [23620] siRNA – SMARTpool), or siRNA control (ON-TARGETplus Non-targeting pool D-001810-10-20) (Dharmacon, CO, USA), were used.