Fig 1: Epithelial–mesenchymal transition inducer Snail augments fatty acid oxidation.(A) Clonogenic capacity under glucose-deprived condition followed by refreshment of normal culture medium. Immunoblot analysis (left) and clonogenic capacity (middle and right) were measured. (B) The cancer cells expressing control-shRNA or independent set of Snail-shRNA-2 were incubated in the presence of glucose (Glc+) or absence (Glc−) for 2 h, and the relative ATP levels were measured (n = 3, means ± SD) (left). Immunoblot analysis shows endogenous Snail protein abundance (right). (C) The cancer cells expressing Tet-inducible Snail (Dox+) were incubated in the presence of glucose (Glc+) or absence (Glc−) for 2 h, and the relative ATP levels were measured (n = 3, means ± SD). (D) The enzymatic activities of ACADVL, ACADM, and HADHA in breast cancer cells expressing Tet-inducible Snail (Dox+) were measured under glucose-starved condition (0.5 mM glucose, n = 3, means ± SD). (E) The CPT1 activities of breast cancer cells expressing Tet-inducible Snail (Dox+) under glucose starved condition were measured (0.5 mM glucose, n = 3, means ± SD). (F) Relative oxygen consumption rate (OCR) normalized to protein abundance over time in control (Dox−, n = 5) and inducible Snail (Dox+, n = 5) in MCF-7 (left) and MDA-MB-231 cells (right). (G) Malonyl-CoA abundances of breast cancer cells expressing Tet-inducible Snail for control (Dox−) or Snail overexpression (Dox+) were measured (n = 3, means ± SD). (H) Lipid (left) and free fatty acid synthesis (right) after inducible Snail for control (Dox−) or Snail overexpression (Dox+). Upper panels represent lipid droplets under Oil Red O staining. Scale bar, 100 μm.
Fig 2: Snail augments ATP levels via fatty acid metabolism promoting cancer cell survival under glucose starvation.(A) Clonogenic survival assay of cancer cells following glucose starvation as described in the Materials and Methods section (left). Colonies of more than 50 cells were counted after crystal violet staining (right). Data are expressed as means and SD. The double asterisks denote P < 0.01, one asterisk denoting P < 0.05 (n = 5, means ± SD, t tests). (B) The cancer cells expressing control-shRNA or Snail-shRNA were incubated in the presence (Glc+) or absence (Glc−) of glucose for 4 h, and the relative ATP levels were measured (n = 3, means ± SD, t tests). (C) The breast cancer cells in the absence of glucose (Glc−) were treated with BSA-Palmitate (100 μM) in combination with DMSO control or ETX (100 μM) for 4 h, and the relative ATP levels were measured (n = 3, means ± SD, t tests). (D) The MDA-MB-231 cells expressing control-shRNA or Snail-shRNA were treated with 13C-palmitate (100 μM) in the absence of glucose for 4 h. Mass isotopomer distribution of [U-13C]-palmitate–derived carbon into some TCA metabolites was determined by LC–MS. Filled blue circles represent 13C atoms derived from [U-13C]-palmitate (*P < 0.05, **P < 0.01, ***P < 0.001, t tests). (E) The enzymatic activities of ACADVL, ACADM, and HADHA in breast cancer cells expressing shRNA for control (shControl) or Snail (shSnail) were measured under glucose-starved condition (0.5 mM glucose, n = 3, means ± SD, t tests). (F) The CPT1 activities of breast cancer cells expressing shRNA for control (shControl) or Snail (shSnail) under glucose-starved condition were measured (0.5 mM glucose, n = 3, means ± SD, t tests). (G) Snail was induced by treatment of doxycycline (Dox) for 48 h and ATP (left), NADPH (middle), and cell death (right) in starved condition were measured in combination with CPT1 inhibitor etomoxir (ETX, 100 μM, n = 3, means ± SD, t tests). (H) Mitochondrial oxygen consumption rate (OCR) in Tet-inducible Snail in combination with ETX (n = 3, means ± SD, t tests). (I) Malonyl-CoA abundances in breast cancer cells expressing shRNA for control (shControl) or Snail (shSnail) were measured (n = 3, means ± SD, t tests). (J) A schematic diagram depicting a potential mechanism by which the Snail regulates fatty acid oxidation (FAO).
Fig 3: Co-culture of HCC cells with macrophage assays to verify the effect of FAO on phenotypic alteration in macrophages and the effect of FAO on macrophage alteration through the PPAR-α/EHHADH signaling pathway. (a) Representative BODIPY staining for co-cultured macrophages for 24 h and 72 h and associated statistical plots (n = 3). (b) The ACADVL, ACADM, and HADHA expression are shown in co-cultured macrophages after 24 h, respectively (n = 6). (c) The ACADVL, ACADM, and HADHA expression are shown in co-cultured macrophages after 72 h, respectively (n = 7). (d) The PPAR-α mRNA expression levels in macrophages co-cultured with MICA+ Huh-7 cells for 24 h and 72 h (n = 10). (e) The EHHADH mRNA expression levels in macrophages co-cultured with MICA+ Huh-7 cells for 24 h and 72 h (n = 8). (f) IF staining confirm increased EHHADH expression in macrophages co-cultured with MICA+ Huh-7 cells compared with NC cells for 72 h (n = 5). (g) Associated statistical plots confirm increased EHHADH expression in macrophages co-cultured with MICA+ Huh-7 cells compared with NC cells for 72 h (n = 5). (h) Western blotting confirm decreased EHHADH expression in macrophages treated with the PPAR-α inhibitor GW647 compared with DMSO. The statistically quantificational results are shown on the right (n = 3). The uncropped blots are shown in Supplementary File S1. (i) The CD206 mRNA expression levels in macrophages co-cultured with MICA+ Huh-7 cells for 24 h and 72 h after treated with GW6471 or DMSO are shown by qPCR (n = 8). (j) The IL-10 mRNA expression levels in macrophages co-cultured with MICA+ Huh-7 cells for 24 h and 72 h after treated with GW6471 or DMSO are shown using qPCR (n = 10). (k) The CD86 mRNA expression levels in macrophages co-cultured with MICA+ Huh-7 cells for 24 h and 72 h after treated with GW6471 or DMSO are shown using qPCR (n = 7). (l) The TNF-α mRNA expression levels in macrophages co-cultured with MICA+ Huh-7 cells for 24 h and 72 h after treated with GW6471 or DMSO are shown using qPCR (n = 9). (m,n) The TNF-α (n = 5) and IL-10 (n = 4) levels in supernatants from cultured macrophages were measured by ELISA. All images were taken at 400× magnification. p-value significance codes: * p < 0.05, ** p < 0.01, and *** p < 0.001.
Supplier Page from Abcam for Human Fatty Acid Oxidation In-Cell ELISA Kit