Extended Data Fig. 6: Related to Fig. 6. Evaluation of integrated stress responses in Tollip−/− mice and macrophages.
a–c) B6 and Tollip−/− peritoneal extract macrophages (PEM) were incubated with media or mycolic acid (MA; 10 µg/ml) for 72 hours, then infected with Mtb (MOI 1) overnight. mRNA transcripts of key regulatory genes of the cellular stress response were measured before and after Mtb infection. a) Ern1 (IRE1a; p = 0.001 for media, p = 0.049 for MA), b) Eif2ak3 (PERK; p = 0.002 for media, p = 0.007 for MA), and c) Atf6 (ATF6; p = 0.02 for media) were measured and displayed as their fold change from baseline after Mtb infection. FC = (Normalized mRNA expression after Mtb infection) / (Normalized mRNA expression after media control stimulation). N = 2/group and are representative of at least two independent experiments. d) Western blot of PEM incubated as above measuring pEIF2 and tubulin expression. e) Optical density at 600 nm (OD600) of Mtb H37Rv in 7H9 broth culture in the presence of raphin-1 (10 µM), ISRIB (250 nM), or vehicle control over time. N = 2 over two independent experiments; error bars – SEM. f–h) TNF concentrations in cellular supernatants from TOLLIP-deficient f) bone marrow-derived macrophages (BMDM), g) alveolar macrophages (AM; p = 0.04 for Mtb+MA, p = 0.003 for Mtb+ISRIB) and h) THP-1 cells (p = 0.002 for Mtb+MA, p = 0.005 for Mtb+ISRIB, p = 0.004 for Mtb+MA + ISRIB), after 24 hours of Mtb infection (MOI 5), mycolic acid (MA, 10 µg/ml), and ISRIB (250 nM), measured by ELISA. This experiment was performed twice, each with three technical replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 two-sided t-test.
