α glycyrrhetinic acid

The following reagents were used: NAC (Sigma-Aldrich, Cat# A7250), tunicamycin (Enzo Life Sciences, Cat# BML-CC104), glycine (VWR, Cat# 10119CU), tBHQ (Sigma-Aldrich, Cat# 19986), ML385 (Sigma-Aldrich, Cat# SML1833), Rapamycin (Sigma-Aldrich, Cat# R0395), Torin 2 (Selleckchem, Cat# S2817), serine (Sigma-Aldrich, Cat# S4500), asparagine (Sigma-Aldrich, Cat# A4159), VEGF-A 165 (R&D, Cat# 293-VE), Asparaginase (Sigma-Aldrich, Cat# A3809), l-Histidinol (Sigma-Aldrich, Cat# H6647), FFA (Sigma-Aldrich, Cat# F9905), α-glycyrrhetinic acid (Sigma-Aldrich, Cat# G10105), A438079 (Sigma-Aldrich, Cat# A9736), Oligomycin A (Sigma-Aldrich, Cat# 75351), CCCP (Sigma-Aldrich, Cat# C2759), antimycin A (Sigma-Aldrich, Cat# A8674), rotenone (Sigma-Aldrich, Cat# R8875), formate (Sigma-Aldrich, Cat# 06473), folate (Enzo Life Sciences, Cat# ALX-460-006). The following antibodies were used: pS6 (Cell Signaling, Cat# #2215, dilution 1:1000), S6 (Cell Signaling, Cat# 2317, dilution 1:1000), MTHFD2 (Proteintech, Cat# 12270-1-AP, WB dilution 1:1000, IF dilution 1:200), SHMT2 (Bethyl, Cat# A305-351A, dilution 1:2000), β-actin (Cell Signaling, Cat# 4970S, dilution 1:1000), Pecam-1 (BD Biosciences, Cat# 553370, dilution 1:200), FLAG (Sigma-Aldrich, Cat# F3165, dilution 1:1500). Primers for qRT-PCR are listed in Supplementary Table 9.

Primary cultures of adult female pituitary glands were prepared as described (Featherstone et al., 2011 (link)). Cells were resuspended in medium (DMEM + 4.5 g/l glucose, 10% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM ultraglutamine) and plated (1 × 10⁵/well) in white plastic 96-well plates pre-treated with poly-L-Lysine at. Cells were allowed to recover for 24 hr (37°C, 5% CO₂) after which the medium was replaced with serum-deprived medium (DMEM + 4.5 g/l glucose, 0.25% (w/v) BSA, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM ultraglutamine) supplemented with 1 mM luciferin (Biosynth, Switzerland) and cells were incubated for a further 24 hr (37°C, 5% CO₂). Cells were then treated with either: DMSO (control), 20 µM α-glycyrrhetinic acid (Sigma, UK), 50 µM Palmitoleic acid (Sigma, UK), or 5 µM forskolin (Sigma, UK). Cell responses were measured using the FLUOstar Omega CO₂- and temperature-controlled luminometer plate reader (BMG Labtech) with photon counts collected for 10s per well every 15 min for 24 hr. Three independent cultures were analysed with 3-4 replicates per treatment group.