Figure 4
p65 protein accumulates in Snail-induced EMT process via impaired CMA activity. (a) MCF-10A cells were stably infected with lenti-NC and Snail-6SA, respectively, phase-contrast morphology observation (left) and expression of EMT marker proteins as indicated (right) were analyzed. Scale bar represents 20 μm. (b) Immunofluorescence staining of proteins as indicated together with re-staining of DAPI in MCF-10A-NC and MCF-10A-6SA cells. Scale bar represents 20 μm. (c) Transwell migration assay of MCF-10A-NC and MCF-10A-6SA cells. Left, representative images of migrated cells. Right, quantitative analysis of the migrated cell numbers in five different fields. Scale bar represents 20 μm. ***P<0.001 as compared with the migrated cell number of MCF-10A-NC cells. (d) Western blots of indicated proteins in MCF-10A-NC and MCF-10A-6SA cells. β-Actin served as internal control. LRRK2, IκBα, PKM2 and GAPDH were all reported CMA substrates, whereas wild-type p53 expressed in MCF-10A cells was utilized as a negative control. (e) Western blots of LAMP2A and HSC70 in MCF-10A-NC and MCF-10A-6SA cells. (f) Left: immunofluorescence staining of LAMP2 together with re-staining of DAPI in MCF-10A-NC and MCF-10A-6SA cells. Scale bar represents 20 μm. Right: the distance of LAMP2 from the nucleus in MCF-10A-NC and MCF-10A-6SA cells was measured using ImageJ. Twenty cells in each group were analyzed. Bar graphs represent the mean±s.d. The P-value was calculated using Student’s t-test. ***P<0.001 as compared with MCF-10A-NC group. (g) MCF-10A-NC and MCF-10A-6SA cells were stably infected with a KFERQ-PA-mCherry1 photoactivatable reporter, and after photoactivation by 405-nm light CMA was highly activated by serum starvation for 20 h. Left: representative images of KFERQ-PA-mCherry1 puncta number and distribution in MCF-10A-NC and MCF-10A-6SA cells. Right, quantitative analysis of the number of puncta per cell in >20 cells in at least eight different fields. Nuclei are labeled with DAPI. Scale bar represents 20 μm. **P<0.01 as compared with puncta number in MCF-10A-NC cells.
