Figure 6
Number of autophagosomes and autolysosomes do not change by hyperosmolarity in NP cells. (a) Schematic diagram of tandem mCherry-EGFP-LC3B plasmid. Autophagosomes in the NP cells expressing mCherry-EGFP-LC3B are tagged with both fluorophores and therefore appear yellow/green. When these autophagosomes fuse with lysosomes, acid labile EGFP signal is lost, leaving mCherry signal (red), which measures autophagic flux. (b) NP cells transduced with retrovirus expressing a tandem mCherry-EGFP-LC3B construct cultured under either iso- or hyperosmotic condition showed that the numbers of autophagosome (yellow/green puncta) and autolysosome (red puncta) did not change by hyperosmolarity. Scale bar: 25 μm. (c) Quantification of puncta area per cell using Colocalization Plugin of ImageJ software confirmed the insensitivity of autophaigc flux to hyperosmotic stimulus. At least 27 cells per group imaged at 126x magnification from 10 random microscopic fields were used for quantification analysis. Bars represent mean ± SEM (n = 3). Student t test was used to determine statistical significance. NS, non-significant.
