Fig. 5: Hyperosmotic stress-induced p62 bodies primarily contain NBR1 and TAX1BP1.
From: Rapid activation of p62 body-mediated autophagy in human cells under hyperosmotic stress
A WB analysis of T24 and U2OS cells exposed to hyperosmotic stress using the indicated antibodies. Cells were cultured under normosmotic conditions without medium exchange (No Me), or shifted to DMEM containing indicated concentrations of sucrose for 1 h followed by cell lysis. B Quantification of WB data in panel A. Band intensities were normalized to GAPDH, and expressed relative to “0 M” condition. Data are presented as mean ± SD from three independent experiments. Each value was statistically compared to “0 M” condition using one-way ANOVA followed by Tukey’s test. * P < 0.05, ** P < 0.01, *** P < 0.001. C WB analysis of T24 and U2OS cells exposed to hyperosmotic stress with lysosomal or proteasomal inhibitor. Cells were cultured under normosmotic conditions without medium exchange (No Me), or shifted to DMEM containing 0.2 M sucrose and DMSO (vehicle), BafA1 (100 nM), or Epoxomicin (3.6 µM) for 1 h, before cell lysis. Indicated antibodies were used. D Quantification of WB data in panel C. Band intensities were normalized to GAPDH, and expressed relative to “No Me” condition. Data are presented as mean ± SD from three independent experiments. Each value was statistically compared to “No Me” condition using one-way ANOVA followed by Tukey’s test. * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant. E Immunostaining of T24 cells exposed to hyperosmotic stress with lysosomal inhibitor. Cells were cultured in DMEM containing 0.2 M sucrose and DMSO (vehicle) or BafA1 (100 nM) for 1 h. They were fixed for immunostaining with antibodies against p62 (H00008878-MO1) and NBR1 or TAX1BP1. Boxed regions are enlarged and shown as insets. Scale bars = 20 µm and 2 µm (insets). F Quantification of colocalization in panel E using Manders’ coefficients. The fraction of p62-positive area overlapping with NBR1, or TAX1BP1 was quantified in 20 cells from a representative experiment. Data are presented as mean ± SD. Statistical comparisons were carried out using Welch’s t-test.
