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. 2019 Jun 4;11(6):1269. doi: 10.3390/nu11061269

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Effect of dihydrocapsaicin on amino acid signaling. (A) TSC2^−/−, p53^−/− MEFs were treated as indicated for 30 min and lysed for immunoblot analysis. Media was changed to a serum-free media when dihydrocapsiain was added. (B) TSC2^+/+, p53^−/− MEFs were incubated with amino acid-deprived and dialyzed-FBS containing media for 5 h to provide amino acid starvation. Dihydrocapsaicin was pre-treated for 1 h and cells were subsequently re-stimulated with amino acid, using media containing amino acid and regular FBS for 1 h. TSC2^−/−, p53^−/− MEFs were serum starved overnight and then incubated with amino acid-deprived and serum-free media for 5 h. Dihydrocapsaicin was pre-treated for 1 h and cells were subsequently re-stimulated with amino acid, using serum-free media containing amino acid for 1 h. (C) Immunofluorescence was performed to examine the translocation of mTOR in TSC2^−/−, p53^−/− MEFs. Mechanistic targets of rapamycin (mTOR) is shown in green and the lysosomal marker, lysosomal-associated membrane protein 1 (LAMP1) is shown in red. DAPI (4′,6-diamidino-2-phenylindole) is shown in blue. (D) 24 h after seeding, media was changed to regular growth media, or media with reduced amino acid content as indicated. Cell death was measured 48 h after changing the media.

Effect of dihydrocapsaicin on amino acid signaling. (A) TSC2^−/−, p53^−/− MEFs were treated as indicated for 30 min and lysed for immunoblot analysis. Media was changed to a serum-free media when dihydrocapsiain was added. (B) TSC2^+/+, p53^−/− MEFs were incubated with amino acid-deprived and dialyzed-FBS containing media for 5 h to provide amino acid starvation. Dihydrocapsaicin was pre-treated for 1 h and cells were subsequently re-stimulated with amino acid, using media containing amino acid and regular FBS for 1 h. TSC2^−/−, p53^−/− MEFs were serum starved overnight and then incubated with amino acid-deprived and serum-free media for 5 h. Dihydrocapsaicin was pre-treated for 1 h and cells were subsequently re-stimulated with amino acid, using serum-free media containing amino acid for 1 h. (C) Immunofluorescence was performed to examine the translocation of mTOR in TSC2^−/−, p53^−/− MEFs. Mechanistic targets of rapamycin (mTOR) is shown in green and the lysosomal marker, lysosomal-associated membrane protein 1 (LAMP1) is shown in red. DAPI (4′,6-diamidino-2-phenylindole) is shown in blue. (D) 24 h after seeding, media was changed to regular growth media, or media with reduced amino acid content as indicated. Cell death was measured 48 h after changing the media.