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Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

In Mol Cancer on 11 October 2024 by Ryu, K. J., Lee, K. W., et al.

Fig.4.E

Evasion of CMA-mediated Snail degradation induces EMT and increases metastatic abilities in luminal-type breast cancer cells. (A) Immunoblot analysis of NL-treated WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (upper). Relative Snail levels were quantified using ImageJ (lower). *, P < 0.05: **... more

Evasion of CMA-mediated Snail degradation induces EMT and increases metastatic abilities in luminal-type breast cancer cells. (A) Immunoblot analysis of NL-treated WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (upper). Relative Snail levels were quantified using ImageJ (lower). *, P < 0.05: **, P < 0.01: ***, P < 0.001 as determined by t-test. ns, not significant. (B) Immunoblot analysis in LAMP2A-depleted WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (left). Relative Snail levels were quantified using ImageJ (right). **, P < 0.01 as determined by t-test. ns, not significant. (C) WT-Snail or 58AAAA-Snail-expressing MCF-7 cells were treated with CHX (100 µg/ml) for the indicated times before harvest. Cell lysates were immunoblotted with the indicated antibodies (left), and data quantified using ImageJ (right). Normalized to α-tubulin. **, P < 0.01: ***, P < 0.001: ****, P < 0.0001 as determined by t-test. ns, not significant. (D) Morphological changes of WT-Snail or 58AAAA-Snail-expressing MCF-7 cells. WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were stained with TRITC-conjugated phalloidin and visualized by confocal microscopy. Scale bars, 40 μm–20 μm. (E) Expression levels of EMT marker proteins in WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were analyzed by immunoblotting. (F) WT-Snail, 58AAAA-Snail-expressing MCF-7 and control cells were analyzed in wound-healing assays by visualizing wound closure via phase-contrast microcopy (left). Wound areas were measured using WimScratch software (Wimasis). The data shown represent the percentage of the wound area and are expressed as the means ± SD of three individual experiments (right). **, P < 0.01: ****, P < 0.0001 as determined by t-test. (G) WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were seeded onto Matrigel matrix-coated top chambers, and the fold-changes of invading cells were measured after 30 h. The data shown are expressed as the means ± SD of three individual experiments, each performed in triplicate. **, P < 0.01: ****, P < 0.0001 as determined by t-test. (H) The indicated cells were seeded in a 6-well plate at a concentration of 1 × 105 cells per well. After incubation for 1 to 4 days, the viable cells were counted with a hemocytometer after trypan blue staining. (I) About 2 × 106 of the MCF-7 cells stably expressing WT-Snail, 58AAAA-Snail, or control cells were injected into the nude mice by tail-vein injection. Representative pictures of HE staining of lung sections (left). Scale bars, 200 μm. The number of metastatic lung nodules in individual mice was quantified at 6 weeks after tail-vein injection (right). The data are shown as the means ± SD of 7 mice/group. **, P < 0.01: ***, P < 0.001 as determined by t-test less

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