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. 2022 Jul 1;14(4):925–944. doi: 10.1016/j.jcmgh.2022.06.008

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Mechanism of lysosomal disintegrity of cultured cells by HNE.A, The expression of GPR40, GPR109A, and GPR120 in Huh-7 cells, HepG2 cells, the human liver, monkey liver, and monkey pancreas was evaluated by a Western blotting analysis. B, The relative fold expressions by real-time PCR indicates that GPR120 in HepG2 cell is suppressed by siRNA (#1 and #3). C, Viability of HNE-treated HepG2 cells with (#1 and #3) and without (NC) the down-regulation of GPR120 by siRNA. D, Lysosomes of HepG2 cell, with 25 μM HNE treatment, in which GPR120 was suppressed by siRNA (#1 and #3) were stained with LysoTracker. Red, LysoTracker; blue, DAPI. E, GPR120 in HepG2 cells was down-regulated by siRNA (#1 and #3), and cells were treated with 100 μM HNE. Lysosomes were stained with LysoTracker 6 hours after the addition of HNE. Flow cytometry was used in the analysis, and results are shown in the histogram. NC means HNE-treated HepG2 cells without the down-regulation of GPR120. F, Flow cytometry was used in the analysis, and results are shown in the mean fluorescence intensity (MFI) of LysoTracker. G, A Western blotting analysis of activated μ-calpain in HepG2 cells is shown. H, Bands of panel G are quantified and shown as relative fold ratios. I, The relative fold expressions by real-time PCR indicates that μ-calpain in HepG2 cell is suppressed by siRNA (#4 and #5). J, μ-calpain in HepG2 cells was down-regulated by siRNA (#4 and #5) and cells were treated with 100 μM HNE. Lysosomes were stained with LysoTracker, and changes in the cell, and lysosomes were observed using time-lapse imaging. Blue, Hoechst; red, LysoTracker. Bright field images are shown in the square on the bottom right corner of each image. In the negative control without μ-calpain down-regulation (NC), lysosomes were no longer present, and HepG2 cells died 5 hours after the HNE treatment. In contrast, lysosomes were retained in cells in which μ-calpain was down-regulated by siRNA (#4 and #5), and many cells survived even after the HNE treatment. K, Viability of HNE-treated HepG2 cells with (#4 and #5) and without (NC) the down-regulation of μ-calpain by siRNA. The graph shows results of cell viability obtained 12 hours after the treatment with 40 μM HNE. L, A Western blotting analysis of activated μ-calpain in HepG2 cells with (#1-#3) and without (NC) the down-regulation of GPR120 by siRNA is shown. M, Bands of panel L are quantified and shown as relative fold ratios. NC, HNE-treated HepG2 cells without the down-regulation of μ-calpain.