L histidinol

L-Histidinol (HisOH), PP242, Integrated Stress Response Inhibitor (ISRIB), SP600125, Cycloheximide (CHX) were from Sigma; Salubrinal was from Tocris Bioscience; U0126 was from Promega. Drugs were used at the following final concentrations: HisOH at 4–16 mM; PP242 at 1–3 μM; ISRIB at 100 nM; SP600125 at 20 μM in HepG2 cells and 50 μM in HeLa cells; Cycloheximide (CHX) at 50 ug/ml in HepG2 cells and 100 ug/ml in HeLa cells; Salubrinal at 75 μM; U0126 at 50 μM. Vehicle was used as mock control. Treatments with drugs to be tested for their ability to inhibit transgene reactivation (ISRIB, SP600125 and U0126) were initiated 1h before the subsequent addition of L-Histidinol (ISRIB) or the subsequent depletion of Met/Cys (SP600125 and U0126).

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.