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. 2017 Oct 9;6(10):e387. doi: 10.1038/oncsis.2017.85

Figure 1.

Figure 1

Degradation of p65 through lysosome. (a-d) MCF-10A and HeLa cells were treated with a combination of ammonium chloride (40 mM) and leupeptin (100 μM) (referred to NL hereinafter) (a), CQ (50 μM) (b) and Baf (10 nM) (c), or maintained with or without serum (d) for indicated time. Whole-protein lysates were subjected to immunoblots for indicated protein. Upper, at least three independent experiments were done, and representative image of immunoblot images were shown. Below, blot density was analyzed by ImageJ and quantification analysis was shown. ***P<0.001, **P<0.01 and *P<0.05 as compared with the con group. (e) Immunofluorescence co-staining of LAMP2 and p65 in MCF-10A cells treated with NL for 20 h or not. The colocalization between LAMP2 (red) and p65 (green) was analyzed by JAcoP plugin of ImageJ and values were presented as mean±s.e.m. Scale bar represents 20 μm. (f, g) f: MCF-10A cells were treated with MG132 for 12 h at indicated concentration, g: MCF-10A cells were treated with MG132 (10 μM) for indicated time. Whole-cell lysates were analyzed with immunoblots. Representative images were provided in left and quantification analysis of blot density was provided in right. *P<0.05 as compared with the con group. (h) MCF-10A cells were treated with MG132 (10 μM) or CQ (100 μM) alone or with a combination of MG132 and CQ and lysed for immunoblots for indicated proteins. Representative images were provided in left and blot density quantification analysis was shown in right. **P<0.01 and ***P<0.001 as compared with each group. (i, j) The effect of macroautophagy on the expression on P65. (i) MCF-10A and HeLa cells were treated with 3-MA for 24 h with indicated concentrations followed by immunoblotting for indicated proteins. (j) MCF-10A and HeLa cells were stably infected with Siren empty vector or shRNAs against ATG7 followed by immunoblots analysis of indicated proteins. Representative images were provided in left and quantification analysis of blot density of p65 and p62 were shown in right. ns, no significance. *P<0.05, **P<0.01 and ***P<0.001 as compared with con or siren in each group.