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Assessing methods to quantitatively validate TGF?-dependent autophagy.

ABSTRACT: Transforming growth factor beta (TGF?) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGF? may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGF?1 modulates autophagy related (ATG) gene expression and ATG protein levels. We also assessed microtubule-associated protein light chain 3 (LC3) lipidation, LC3 puncta formation and autophagosome-lysosome co-localization in non-small cell lung cancer (NSCLC) cell lines. These experimental approaches were validated using pharmacological autophagy inhibitors (chloroquine and spautin-1) and an autophagy activator (MG132). We found that TGF?1, chloroquine and MG132 had little effect on ATG protein levels but increased LC3 lipidation, LC3 puncta formation and autophagosome-lysosome co-localization. Since similar outcomes were observed using chloroquine and MG132, we concluded that several techniques employed to assess TGF?-dependent autophagy may not differentiate between the activation of autophagy versus lysosomal inhibition. Thus, NSCLC cell lines stably expressing a GFP-LC3-RFP-LC3?G autophagic flux probe were used to assess TGF?-mediated autophagy. Using this approach, we observed that TGF?, MG132 and serum starvation increased autophagic flux, whereas chloroquine and spautin-1 decreased autophagic flux. Finally, we demonstrated that ATG5 and ATG7 are critical for TGF?-dependent autophagy in NSCLC cells. The application of this model will fuel future experiments to characterize TGF?-dependent autophagy, which is necessary to understand the molecular processes that link, TGF?, autophagy and tumorigenesis.

SUBMITTER: Trelford CB

PROVIDER: S-EPMC7710024 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Assessing methods to quantitatively validate TGFβ-dependent autophagy.

Trelford Charles B CB Di Guglielmo Gianni M GM

Biology open 20201123 11

Transforming growth factor beta (TGFβ) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGFβ may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGFβ1 modulates autophagy related (<i>ATG</i>) gene expression and ATG protein levels. We also assessed microtubule-associated protein light chain 3 (LC3) lipidation, LC3 ...[more]

PMID: 33168592

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Assessing methods to quantitatively validate TGF?-dependent autophagy.

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Assessing methods to quantitatively validate TGFβ-dependent autophagy.

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