8 br camp

Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Chicago (protocol no. ACUP 71811, approved on 27 February 2019). Studies were performed on age- and gender-matched (both males and females) adult (3 to 5 months old) WT, Olfr78-null (from J. Pluznick, the Johns Hopkins University) (40 (link)), HO-2 (Hmox-2)–null (from S. H. Snyder, the Johns Hopkins University; initially generated by S. Tonegawa) (41 (link)), TH-Cre/Adcy3 ^f/f (Adcy3 ^f/f from X. Chen, the University of New Hampshire and D. R. Storm, the University of Washington) (42 (link)), Cth-null (from R. Wang, York University Toronto, Ontario, Canada) (4 (link)), and Cnga2^+/− (Jackson Laboratory, stock no. 002905) mice. Olfr78-null mice were initially generated by Bozza et al. (40 (link)) and subsequently backcrossed with C57BL/6 mice by J. Pluznick. Hmox-2/Olfr78 double-null mice were generated by crossing Hmox-2– and Olfr78-null mice, as well as TH-Cre/Adcy3 ^f/f mice by crossing TH-Cre (Jackson Laboratory, stock no. 008601) and Adcy3 ^f/f mice at the University of Chicago. All mice were on C57BL/6 background. Experiments were performed by individuals blinded to the genotype. The following chemicals were used: NaHS (Sigma-Aldrich, MO, USA), 8-Br-cAMP and 8-Br-cGMP (Cayman Chemical), and forskolin (Cayman Chemical).

A schematic diagram of the culture procedure and protocol for decidualization of HESCs is depicted in Figure 1A. Isolated primary HESCs (between passages two and five) were cultured in control or decidualization media until confluent. The control medium consisted of basal DMEM/F12 supplemented with 2% charcoal-stripped FBS and 1% pen/strep. The decidualization medium included control medium plus 1 µM MPA (Pfizer Canada, Kirkland, QC, Canada) and 0.5 mM 8-br-cAMP (a stable, cell permeable analog of cAMP; Cayman Chemicals, Ann Arbor, MI, USA). Conditioned media (CM) was replenished every 48 h. Cells were allowed to grow until the end of experimental treatment. CM from decidualized and un-decidualized HESCs were collected and, using Western blot, IGFBP-1 levels were detected using rabbit polyclonal IGFBP-1 antibody (a kind gift from Dr. Robert Baxter, Sydney, Australia). Phospho IGFBP-1 was assessed using custom made IGFBP-1 phospho site-specific (Ser101, 119 and 169) antibodies, validated as described previously [22 (link),31 (link)].