Sirna targeting

The CMECs at 60% confluence in a 24-well plate were transfected with siRNA targeting HSP72 (Santa-Cruz Biotechnology, Santa-Cruz, CA, USA) or siRNA-scramble (siRNA-control) with Lipofectamine LTX Reagent (Invitrogen) and Opti-MEM TM (Invitrogen). To determine the knockdown efficiency, real-time PCR and immunoblotting analyses were performed to confirm the HSP72 downregulation at 3 days after transfection (Li et al., 2018c (link)).

CBD was bought from Biomol GmbH (Hamburg, Germany). JWH-133 and THC were bought from Bio-Techne GmbH (Wiesbaden, Germany) and Lipomed GmbH (Herne, Germany), respectively. R(+)-methanandamide, capsazepine and NAC were obtained from Sigma-Aldrich (Taufkirchen, Germany). AM251 and AM630 were obtained from Biozol (Eching, Germany). O-1602 and O-1918 were purchased from Tocris Bioscience (Bristol, UK) and Cayman Chemicals (Ann Arbor, Michigan, USA), respectively. SnPPIX was obtained from Enzo Life Sciences (Lörrach, Germany). The transfection reagent Lipofectamine™ RNAiMAX, OptiMEM and hPDGF-BB were purchased from Thermo Fisher Scientific Inc. (Schwerte, Germany). siRNA targeting HO-1 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany; sc-35554). Negative control siRNA was from Qiagen (Hilden, Germany; cat. no. 1022076). Accutase cell detachment solution was obtained from Merck Chemicals GmbH (Darmstadt, Germany).

The plasmids used for ectopic expression of TET1 and HIF1α were established by inserting the full-length cDNA into pRetroX-tight and pCDNA3.1 vectors, respectively. HRE luciferase reporter plasmid was established based on pGL3-promotor vector (Promega) as described previously [60 (link)]. CK2B luciferase reporter plasmid was established by inserting its sequence from +14 to −1525 into pGL3-basic (Promega). pCI-K48-Ub-6×His plasmid was kindly provided by Prof. Wuhan Xiao (Chinese Academy of Sciences, Hubei, China), and used for in vitro protein ubiquitination assay. siRNA targeting HIF1α and control siRNA were purchased from Santa Cruz. Cells were transfected with the above plasmids and siRNAs following a standard protocol with Lipofectamine 3000 (Thermo Fisher Scientific, USA).