Extended Data Fig. 3: Pharmacological inhibition of PERK deviates cellular metabolism in M2 macrophages.
From: PERK is a critical metabolic hub for immunosuppressive function in macrophages
a, Basal OCR of naïve (M0) and M1 (LPS + IFN-γ) BMDMs from PERK sufficient or deficient mice (n = 3; mean ± s.e.m). Data collected from three independent experiments. b, Basal ECAR of naïve (M0), M1 (LPS + IFN-γ), and M2 (IL-4) BMDMs from PERK wild-type or null animals (n = 3; mean ± s.e.m). Data are collected from three independent experiments. c,d,e Basal OCR (c), ECAR (d), or ATP production (e) from wild-type BMDMs treated with IL-4 in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Dashed line indicates wild-type M0. Data are collected from three independent experiments. f, Representative histogram (left) and quantitative analysis (right) of BODIPY FL C16 staining in BMDMs treated with IL-4 in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Data representative of three independent experiments. g, Representative histogram (left) and the frequency (right) of BODIPY (493/503) staining in BMDMs stimulated with IL-4 in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Data representative of three independent experiments. h, Representative TEM images of PERK wild-type and PERKcKO M2 (IL-4) macrophages. Red arrows indicate mitochondria. Data representative of three biological replicates. i, Representative histogram (left) and the frequency (right) of MitoTracker Green+ staining in BMDMs treated IL-4 (n = 3; mean ± s.e.m). Data representative of three independent experiments. j, Representative histogram (left) and the frequency (right) of MitoTracker Orange+ staining in BMDMs treated with IL-4 (n = 3; mean ± s.e.m). Data representative of three independent experiments. k, RNA-seq analysis of genes associated with mitochondrial calcium transport. l, Mitochondrial calcium flux (Rhod-2) from BMDMs treated with IL-4 in the presence or absence of GSK2656157 was determined and normalized by wild-type naïve M0 macrophages. Arrow, stimulation using 10 μM ionomycin (n = 6 for wild-type M0, M2; n = 4 for GSK-treated cells; mean ± s.e.m). Data are representative of three independent experiments. All data were analyzed using two-tailed unpaired Student’s t-test (a,b,c,d,e,f,g,i,j,l) or two-tailed paired Student’s t-test (l).
