A. Immunoblots for mTORC1 signaling, HIFIα, and the
human cytomegalovirus (HCMV) proteins IE72 and IE86 in HCMV-infected or
uninfected (mock) U2OS cells treated with vehicle or 500 uM DMOG in low buffer
media each for 8 and 12 h prior to harvest at 27 and 59 hours post infection
(hpi), respectively, or in media pH 7.4 and 6.3 for 1 h prior to harvest.
B. mTORC1 signaling and kinesin-1 heavy chain (HC) in U2OS
cells at time points post delivery of 10 nM control (Ctl) siRNA or three
different siRNA against KIF5B (kinesin-1 HC) and in pH 7.4 and
6.5 media 1 h prior to harvest. RE of 2. C. Model. Acid produced
during hypoxic metabolic rewiring suppresses the circadian clock through
inhibition of mTORCI-governed translation as a consequence of centrifugal
redistribution of lysosome-bound mTORCI limiting mTOR activation by RHEB.
D. mTORCI signaling over 32 h in TSC2 CRISPR
knockout (−/−) or parental U2OS cells (+/+) treated with vehicle
or 50 uM ciliobrevin D. E/F. Parental and
RHEBN153T-expressing U2OS cells
immunostained for LAMP1, mTOR, RHEB and nuclei (DAPI) after 160 min in pH 7.4 or
6.3 media. White boxes in F enlarged in E. RE of 3. G. Mean
intensity of DAPI, mTOR, RHEB, and LAMP1 as a function of distance from the
nucleus in F. n=10–13 cells each pH per cell line. Mean±SEM
normalized to each channel’s parental pH 7.4 maximum. H.
Arntl::dLUC luminescence in TSC2 CRISPR
knockout (−/−), RHEBN153T-expressing,
and respective control U2OS cells synchronized and in pH 7.4 or 6.5 media. Mean
of 3 BR. RE of 3–4, 1–3 BR each. I. mTORC1 signaling
in parallel to H or treated with vehicle or 500 uM DMOG in low buffer conditions
for 20 h (TSC2) or 16 h (RHEBN163T). RE of 2. J. Model
of trans-endomembrane contact between lysosome-localized mTORC1 and
non-lysosomal RHEB disrupted upon acid-driven peripheral redistribution of
lysosome-bound mTOR. MTOC = microtubule organizing center K.
Immunohistochemical pS6 staining of HCT116 xenograft tumors hosted by mice
drinking tap water or 200 mM sodium bicarbonate ad libitum
throughout tumor hosting (up to 3 weeks). Representative high-power fields and
inset low-power images of entire tumor cross section. Positivity mask in lower
panels. Percent pS6 positive pixels quantified over entire viable area of tumor
cross section. Mean±SD n=4 mice each arm. 2-tailed Student’s
t-test. RE= representative experiment. Biological replicates = BR. See also
Figure S7.