Plentilox3.7 vector

Lentivirus-mediated shRNA was generated by cloning oligonucleotides encoding shRNA into pLentiLox3.7 vector (Addgene#11795).
For virus production, vectors containing targeting sequences were transfected in HEK293T cells together with packaging vectors pMD2.G (Addgene #12259) and psPAX2 (Addgene#12260) using polyethylenimine (PEI) (23966-1, Polysciences). Viruses were harvested at 48 hours post-transfection and filtered through a 0.45 μm filter.
For infection, 250 μl lentivirus was added to the cells with 2 μg polybrene (sc-134220, Santa) per well. Media were changed 6 hours after infection.

Stable GR knockdown in HEK293 cells was established, as described previously [11 (link)]. shRNA sequences are provided in Table 1. The pLentiLox3.7 vector (Addgene plasmid #11795; http://n2t.net/addgene:11795, accessed on 13 November 2020; RRID:Addgene_11795) was used to generate shRNA constructs. AllStars Negative Control siRNA (Qiagen, Hilden, Germany) served as a negative control. Co-transfection of HEK293 cells with the lentiviral packaging plasmids pVSVG and pPAX2 by means of calcium phosphate precipitation yielded viral particles. Supernatants were collected after 48-72 h and supplemented with 8 µg/mL polybrene. HEK293 cells were infected by centrifugation for 90 min at 700× g and 37 °C. Successfully transduced cells were identified by expression of green fluorescent protein.