A. mCherry-SEpHluorin-derived intracellular pH
(pHi) of U2OS cells treated for 24 h with the indicated pH media
or with 500 uM DMOG in low buffer media. Extracellular pH (pHe) at 33
h. Mean pHi ± standard deviation (SD) based on ≥3
10× fields per condition (see Figures S4A-C). T-tests (unpaired,
2-tailed, unequal variances) of pHi ****p≤0.0001,
***p≤0.001, **p≤0.01. RE of 2–3 per condition.
B. Timecourse immunoblots of core clock proteins in U2OS cells
in normoxic high buffer or hypoxic (1% O2) low or high buffer
conditions. C/D. Timecourse immunoblots for HIFIα and clock
proteins in U2OS cells in normoxic high buffer or hypoxic (1% O2)
low buffer conditions (C) or in pH 7.4 or 6.5 media (D). E/F.
Immunoblots of lysate collected in C (E) and D (F) for phosphorylated sites
(Ser2448 (mTOR), Thr389 (S6K), Ser235/236(S6), Thr37/46(4EBP1)) or total levels
of mTORC1 substrates and downstream signaling components. Tubulin shared by E,
C. G. Media pH over the 48 h in C-F. H. Immunoblots
for core clock proteins and mTORC1 signaling in U2OS cells in normoxia or
hypoxia (1% O2) or treated with vehicle or 300 uM DMOG in low or high
buffer conditions or in media of pH 7.4 or 6.3 for 8 h. I. Luminescence of U2OS
Arntl::dLUC cells treated with vehicle or 100 nM Torin1.
Mean±SEM of 3 BR. RE of 2. J. Normalized ratio of the
intensities of p4EBP1 to total 4EBP1 at 27 h (quantified from K) and the
mean±SEM Arntl::dLUC amplitude over 4 days (see Methods)
as functions of Torin1 dose. Y-axis scaled log([Torin]+1). K.
Immunoblot for mTORC1 signaling in U2OS cells after 1 and 27 h of treatment with
vehicle or 1–1000 nM Torin1. Unrelated intervening lanes cropped.
L. Luminescence of U2OS Arntl::dLUC cells
treated with 10 nM control (siCtl) siRNA or siRNA against
EIF4EBP1 or EIF4EBP2 prior to 300 uM DMOG
in low buffer media. Mean of 2 BR. RE of 2. M/N. Luminescence of
U2OS Arntl::dLUC EIF4EBP1 CRISPR knockout
(4EBP1 −/−) and editing control clonal lines treated with vehicle
or 500 uM DMOG in low buffer conditions (M) or pH 7.4 or 6.3 media (N). Mean of
2–3 BR. RE of ≥3. Black rectangles enclose immunoblots from same
gel. Yellow lines for readability only. All cells synchronized except A, K.
Hypoxic medias pre-deoxygenated. RE = representative experiment. BR = biological
replicates. See also Figure
S4.