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. Author manuscript; available in PMC: 2014 Apr 25.
Published in final edited form as: Sci Transl Med. 2011 Nov 16;3(109):109ra117. doi: 10.1126/scitranslmed.3003182

Fig. 3.

Fig. 3

Blockade of CMA in human cancer cells induces changes in cellular metabolism that limit cell proliferation and induce cell death. (A) Concentrations of oxidized proteins in human lung cancer cell lines {control (ctr) or after LAMP-2A knockdown by RNAi [L2A(−)]} detected by the presence of carbonyl groups. (B) Pattern of polyubiquitinated proteins in the same cells. Stacking of the gel is shown to visualize possible protein aggregates that do not enter the gel. (C) Immunoblot for the indicated effectors and regulators of macroautophagy in the same cells. (D) Autophagic flux in the same cells was determined as the increase in LC3-II levels upon blockade of lysosomal hydrolysis with protease inhibitors (P.I.). Top: representative immunoblots. Increased levels of p53 in the knockdown cells are shown as a reference. Bottom: quantification of steady-state levels of LC3-II (left) and autophagic flux (right) defined as the ratio of LC3-II in cells treated with PI or untreated (n = 4). (E) Concentrations of intracellular ATP (left) and of lactate (right) released in the culture media of H460 human cancer cell lines, either control or cells in which LAMP-2A was knocked down by RNAi [L2A(−)] (n = 4; *P = 0.047 and 0.001, t test). (F) ECAR in A549 and H460 cells, control (ctr) or with LAMP-2A knocked down [L2A(−)]. Left: representative ECAR plots. Where indicated, rotenone was added to the culture media to block mitochondrial oxidative phosphorylation. Right: mean values of ECAR before rotenone addition (basal acidification, left) and average increase of ECAR values after rotenone addition (inducible acidification, right) (n = 6; *P = 0.0002, 0.0001, 0.00008, and 0.0001, t test). (G) OCR in the same cells. Left: representative OCR plots. Right: mean values of OCR before addition of rotenone, and percentage of OCR that is sensitive to rotenone (mitochondrial oxidative phosphorylation) (n = 6; *P = 0.0020 and 0.0066, t test). (H) Rates of β-oxidation of radiolabeled fatty acids in the same cells (n = 3; *P = 0.0001, t test).