lamp1 flag x2 mrfp  (Addgene inc)

Journal: Cell

Article Title: Acid suspends the circadian clock in hypoxia through inhibition of mTOR

doi: 10.1016/j.cell.2018.05.009

Figure Lengend Snippet: A. U2OS cells immunostained for lysosomal protein LAMP2. Nuclei and cytoplasm outlined in lower panel. B. Live imaging of U2OS cells with lysosomes labeled with LysoTracker. Lower panel merged with phase-contrast image. C. U2OS cells immunostained for LAMP2 and mTOR after amino acid starvation for 115 min and rescue (+AA) or not (-AA) for 25 min. D./E. U2OS cells immunostained for LAMP2 after in media of pH 7.4 or 6.3 for 2.25 h (D) or pH 6.3 for 105 min followed by media pH 7.4 (rescue, left) or 6.3 (mock rescue, right two fields) for 25 min (E). F. Live imaging of U2OS cells expressing LAMP1-mRFP in pH 7.4 or 6.3 media for 4 h. Lower panels: merged mRFP and phase-contrast images. G. U2OS cells immunostained for LAMP2 and α-tubulin after in pH 7.4 or 6.3 media for 2.25 h. H. Model: the perinuclear aggregate of lysosomes disperses peripherally upon acidification. I. Mean intensity of DAPI (nuclear), a-tubulin, and LAMP2 staining as a function of distance from the nucleus center (i.e. radial coordinate) after 2.75 h in pH 7.4 or 6.3 media. (See Figure S6E.) n=15 cells each pH. Mean±SEM normalized to each channel’s maximum. T-test of pH (unpaired, 2-tailed, equal variance) unadjusted p<0.05 (*) at 0, 2, and 117 of 228 data points, respectively. J./K. U2OS cells immunostained for LAMP2 and mTOR after amino acid starvation for 130 min in pH 7.4 or 6.3 media and restimulation with amino acids for 10 min in the same pH (J) or after incubation in pH 6.3 media for 2 h with media change (same pH) 15 min prior to processing (K). L. Quantification of mTOR lysosomal enrichment in U2OS cells immunostained for LAMP2 and mTOR after amino acid starvation for 155 min and rescue (+AA) or not (-AA) for 8 min or in pH 7.4 or 6.3 media for 165 min. n =11 fields (≥86 cells) per condition. Mean±SEM superimposed with raw data. T-tests (unpaired, 2-tailed) ****p<0.0001, ns = p>0.05. RE of 3. M. Live imaging of U2OS cells treated with vehicle or 40 uM ciliobrevin D (CbD) for 10.5 h. Lysosomes and polymerized tubulin labeled with LysoTracker and TubulinTracker. N. Live imaging of U2OS cells expressing LAMP1-GFP (red pseudocolor) after treatment with vehicle or 60 uM CbD for 55 min (inset) and 9 h (different fields). Right: merged GFP and phase-contrast images. O. mTORC1 signaling in U2OS cells over 8 h of vehicle (veh.) or 40 uM CbD treatment. RE of 3. P. Luminescence of U2OS Arntl::dLUC cells synchronized and treated with 50 uM CbD or vehicle. Mean of 2 BR. RE of 2, 2–3 BR each. Representative fields of ≥3 BR for all microscopy. RE = representative experiment. BR = biological replicates. See also Figure S6.

Article Snippet: The following day, cells were transfected with 0.5–1.0 ug of LAMP1-FLAG(x2)-mRFP (gift from David Sabatini, Addgene plasmid #34611) or LAMP1-mGFP (gift from Esteban Dell’Angelica, Addgene plasmid #34831. mGFP is a non-dimerizing GFP variant that reduces aberrant aggregation of overexpressed protein).

Techniques: Imaging, Labeling, Expressing, Staining, Incubation, Microscopy

Journal: Cell

Article Title: Acid suspends the circadian clock in hypoxia through inhibition of mTOR

doi: 10.1016/j.cell.2018.05.009

Figure Lengend Snippet: 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.

Article Snippet: The following day, cells were transfected with 0.5–1.0 ug of LAMP1-FLAG(x2)-mRFP (gift from David Sabatini, Addgene plasmid #34611) or LAMP1-mGFP (gift from Esteban Dell’Angelica, Addgene plasmid #34831. mGFP is a non-dimerizing GFP variant that reduces aberrant aggregation of overexpressed protein).

Techniques: Western Blot, Infection, Produced, Inhibition, Activation Assay, CRISPR, Knock-Out, Expressing, Immunohistochemical staining, Staining