Article Figures & Data
Figures
- Fig. 1.
Indomethacin (IM) disrupts autophagy flux in IEC6 cells. (A) IEC6 cells were exposed to 200 μM IM for 4 hours, either alone or in combination with 10 μM CQ or 10 μM Baf A1. Subsequently, LC3 and p62 expression levels were assessed using western blotting. (B) Quantitative analysis of the LC3-II/LC3-I ratio as shown in (A). Values are presented as the mean ± S.D. (n = 3) (*P < 0.05). (C) Quantitative assessment of p62 protein expression from (A). Values are expressed as the mean ± S.D. (n = 3) (*P < 0.05).
- Fig. 2.
IM does not inhibit autophagosome-lysosome fusion in IEC6 cells. (A) IEC6 cells were stained for Lamp2 (red) and LC3 (green) to examine the effects of IM. White arrows highlight the fusion of autophagosomes with lysosomes. Scale bar, 50 μm. (B) Quantification of confocal images (Mander’s colocalization) between Lamp2 and LC3 as depicted in (A). Values are expressed as the mean ± S.D. (n = 5) (*P < 0.05).
- Fig. 3.
IM reduces lysosomal activity and acidification, but these effects are mitigated by Rapa treatment or low-glucose (glu) conditions in IEC6 cells. (A) IEC6 cells were exposed to 200 μM IM and subsequently stained with Magic Red dye to assess lysosomal activity. Scale bar, 50 μm. (B) Quantitative analysis of the Magic Red fluorescence intensity per cell shown in (A). (C) IEC6 cells were treated with 200 μM IM and then stained with LysoSensor dye to measure lysosomal pH. Scale bar, 50 μm. (D) Quantitative evaluation of LysoSensor fluorescence intensity per cell as depicted in (C). (E) Lysates from IEC6 cells treated with IM, either alone or in combination with low-glucose/0.5% serum or Rapa, were subjected to coimmunoprecipitation using anti-V0D antibody. This was followed by western blotting using the anti-V1A antibody. (F) Lysates from IM-treated IEC6 cells, either alone or combined with low-glucose/0.5% serum or Rapa, were subjected to coimmunoprecipitation using the anti-V1A antibody. This was followed by western blotting using the anti-V0D antibody. FCS, fetal calf serum; IP, immunoprecipitation; n.s., not significant.
- Fig. 4.
IM induces ulcers and inflammation in the mouse small intestine. (A) Histologic evaluation of small intestinal lesions caused by IM as visualized using Evans blue staining. The red circle highlights an ulcerated area. Scale bar, 5 mm. (B) Quantification of the ulcer ratio (ulcer area:total area) as depicted in (A). Values are presented as the mean ± S.D. (n = 4) (*P < 0.05). (C) Histologic examination of small intestinal lesions due to IM using H&E staining. Scale bar, 5 mm. (D) Relative mRNA expression levels of interleukin 6 were determined in mouse hearts following IM treatment. Values are expressed as mean ± S.D. (n = 3) (*P < 0.05).
- Fig. 5.
Fasting or Rapa mitigates IM-induced small intestinal ulceration in mice. (A) Histologic examination of small intestinal lesions caused by IM administration, with or without Rapa, using Evans blue staining. Scale bar, 5 mm. (B) Histologic examination of small intestinal lesions caused by IM administration, with or without fasting, using Evans blue staining. Scale bar, 5 mm. (C) Quantification of the ulcer ratio (ulcer area:total area) as depicted in (A). Values are expressed as the mean ± S.D. (n = 4) (*P < 0.05). (D) Quantification of the ulcer ratio (ulcer area:total area) as depicted in (B). Values are expressed as the mean ± S.D. (n = 4) (*P < 0.05). (E) Histologic examination of small intestinal lesions caused by IM administration, with or without Rapa, using H&E staining. Scale bar, 100 μm. (F) Histologic examination of small intestinal lesions caused by IM administration, with or without fasting, using H&E staining. Scale bar, 100 μm. (G) Homogenates of the small intestine from IM-treated mice, with or without fasting or Rapa, were subjected to coimmunoprecipitation using the anti-V0d antibody. This was followed by western blotting with the anti-V1A antibody. (H) Homogenates of the small intestine from IM-treated mice, with or without fasting or Rapa, were subjected to coimmunoprecipitation using the anti-V1A antibody, followed by western blot analysis with the anti-V0d antibody. IP, immunoprecipitation.
- Fig. 6.
IM binds to V-ATPase in a concentration-dependent manner. (A) Beads immobilized with IM (+) or empty (-) were incubated with cytoplasmic extracts from IEC6 cells. Bound proteins were eluted using Laemmli dye, then subjected to SDS-PAGE and subsequent western blotting using anti-V1A, anti-V1B2, and anti-V0d antibodies. (B) Beads immobilized with 2, 5, or 10 mM of IM (+) or empty (-) were incubated with cytoplasmic extracts from IEC6 cells. After eluting bound proteins with Laemmli dye, they were analyzed using SDS-PAGE and western blotting using anti-V1A, anti-V1B2, and anti-V0d antibodies. (C) Beads immobilized with IM (+) or empty (-) were incubated with homogenates from mouse small intestine. Bound proteins were eluted using Laemmli dye and subsequently analyzed using SDS-PAGE and western blotting using anti-V1A, anti-V1B, and anti-V0d antibodies.
Additional Files
Data Supplement
- Supplemental Figures -
Supplemental Figure 1. IM-immobilized beads disrupt autophagy flux in IEC6 cells without altering the bioactivity of IM.
Supplemental Figure 2. IM-immobilized beads decrease both the activity and acidification of lysosomes in IEC6 cells without altering the bioactivity of IM.
Supplemental Figure 3. IM reduces lysosomal activity and acidification, but these effects are mitigated by rapamycin (Rapa) treatment, low glucose conditions, and with a combination of both Rapa and low glucose conditions in IEC6 cells.
- Supplemental Figures -
