Fig 1: AQP5 is expressed in L635- and H felis infection-induced SPEM in the gastric corpus of mice. (A) Immunofluorescence staining for AQP5, mucus marker GSII-lectin, SPEM marker CD44 variant 9 (CD44v9), and epithelial membrane marker p120 in gastric corpus tissues from mice with or without L635 treatment for 1, 2 or 3 days. Arrows indicate AQP5-expressing cells. Scale bar = 100 μm or 50 μm (expanded images). (B) Quantification of AQP5 single or AQP5 and GSII-lectin co-positive gland per ×20 field (n = 6). AQP5 expression was first observed at the base of a few glands of mice treated with L635 for 1 day without GSII expression. Over time, the numbers of AQP5 single positive or AQP5 and GSII co-positive glands were significantly increased in cells at the bases of glands. ∗P < .05, ∗∗P < .01. (C) Quantification of CD44v9 single or AQP5 and CD44v9 co-positive gland per ×20 field (n = 6). CD44v9-expressing glands were prominently observed after L635 treatment for 3 days. Most of these glands co-expressed AQP5. ∗P < .05, ∗∗∗∗P < .0001.
Fig 2: Niclosamide Effect on AQP5 abundance in response to tonicity changes. (A) Normalized AQP5-HiBiT luminescence in response to 6-h treatment with hypotonic media (∼150 mOsm), and hypertonic media (∼400 mOsm). N = 48 wells/condition. Graph is representative of repeated independent experiments. (B) Normalized AQP5 HiBiT luminescence response to 6-h treatment with hypotonic media, Niclosamide, or combination. N = 48 wells/condition. Graph is representative of repeated independent experiments. *p <0.05, **p <0.01, *** p <.001, ****p <0.0001 by 1-way ANOVA with correction for multiple comparisons by Tukey’s method. (C) Calcein in A549 cells in response hypertonic stimuli of 400, 420, and 450 mOsm. (D) Calcein fluorescence in vehicle or Niclosamide-treated (2 μM, 6 h) A549 cells. Dots represent average values of n = 3 wells, with error bars displaying SEM values at a given time-point.
Fig 3: Development of an assay to measure AQP5 Abundance. (A) Overview of AQP5-HiBiT stable cell line generation. HiBiT blotting shows AQP5-HiBit construct response to cycloheximide (CHX) and MG-132 in pooled cells. (B) AQP5-HiBiT signal in cells were treated with vehicle or MG-132 (n = 128 wells/condition) used for Z′ score calculation. (C) Overview of screening procedure. AQP5-HiBit cells were plated in 384-well plates and treated with 1153 compounds from the Selleck FDA drug library. AQP5-HiBiT luminescence values were recorded. (D) Drug screen results sorted by ranked AQP5-HibiT luminescence, with compounds of interest colorized as indicated.
Fig 4: Cell viability after treatment with Niclosamide, candesartan, and Panobinostat. (A,B) Viability of AQP5-HiBiT cells in response to 6 h (A) and 24 h (B) Niclosamide at various doses. (C,D). Viability of AQP5-HiBiT cells in response to 6 h (C) and 24 h (D) Panobinostat at various doses. (E,F) Viability of AQP5-HiBiT cells in response to 6 h (E) and 24 h (F) candesartan at various doses.
Fig 5: AQP5 is expressed in DMP777-induced SPEM in gastric corpus of mice. (A) Immunofluorescence staining for AQP5, mucus marker GSII-lectin, SPEM marker CD44v9, and epithelial membrane marker p120 in gastric corpus tissues from mice without or with DMP777 treatment for 1, 3, 7 or 14 days. Scale bar = 100 μm or 50 μm (expanded images). (B) Quantification of AQP5 single or AQP5 and GSII-lectin co-positive gland per ×20 field (n = 4–6). AQP5 expression was first observed at the base of a few glands in mice treated with DMP-777 for 3 days with small amount of GSII co-expression. Over time, the numbers of AQP5 and GSII-lectin co-positive glands were significantly increased at the bases of glands. ∗P < .05, ∗∗P < .01. (C) Quantification of CD44v9 single or AQP5 and CD44v9 co-positive glands per ×20 field (n = 4–6). CD44v9 expression was observed at the base of few glands after DMP-777 treatment for 7 days, but it was markedly increased at 14 days of treatment with AQP5 co-expression. ∗∗∗P < .001.
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