Journal: International Journal of Molecular Sciences

Article Title: Evodiamine Induces Apoptosis and Inhibits Migration of HCT-116 Human Colorectal Cancer Cells

doi: 10.3390/ijms161126031

Figure Lengend Snippet: Effects of AG490 and EVO on PGI, the JAK2/STAT3 signaling pathway, and MMP3 in HCT-116 cells. ( A , C ) HCT-116 cells were treated with various concentrations of AG490 for 48 h and then stimulated with 25 ng/mL IL-6 for 15 min, after which whole-cell extracts of PGI, MMP3, JAK2, p-JAK2 (data not shown), STAT3, and p-STAT3 were analyzed by Western blotting; ( E ) HCT-116 cells were treated with 50 μmol/L AG490, 6 μmol/L EVO, and combined 50 μmol/L AG490 with 6 μmol/L EVO for 6 h, then stimulated with 25 ng/mL IL-6 for 15 min. Protein expression was analyzed by Western blotting; ( B , D , F ) The graph represents densitometry of the results of three independent experiments (mean ± SD). * p < 0.05, ** p < 0.01 compared to the control. The control group of A–C with 25 ng/mL IL-6 + 0 μmol/L AG490, while E with 25 ng/mL IL-6 + 0 μmol/L AG490 + 0 μmol/L EVO.

Article Snippet: AG490 was obtained from Tocris Bioscience (Bristol, UK).

Techniques: Western Blot, Expressing, Control

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 1. Identification of Piezo1∆GC mice. (A) Immunofluorescence co-localization of Piezo1 and Agr2 (label goblet cells) in WT and Piezo1∆GC mouse colons. The yellow box shows a magnified localized image. Scale bar: 100 µm. (B) mRNA level of Piezo1 in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). At least three independent experiments were conducted. Data are expressed as the mean ± SEM. (n = 5 mice). ** p < 0.01.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques:

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 2. Decreased GC numbers and thinner mucus layer in Piezo1∆GC mouse colons. (A) Im- munofluorescence staining of Agr2 in WT and Piezo1∆GC mouse colons. Scale bar: 100 µm. The crypt was divided equally into three parts: upper, middle, and base. The white arrow indicates a goblet cell. (B) Statistical analysis of goblet cells/epithelial cells in (A). (C) Statistical analysis of goblet cells in different parts/epithelial cells in (A). (D) RNA level of Mucin2 in colon tissues from WT and Piezo1∆GC mice (normalized to GAPDH). (E) AB-PAS staining of mucus in WT and Piezo1∆GC mouse colons. The red arrows indicate the mucus layer. Scale bar: 50 µm. (F) Statistical analysis of mucus layer thickness in (D). At least three independent experiments were conducted. Data are expressed as the mean ± SEM. (n = 5 mice). ns, not significant; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Staining

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 6. Abnormal intestinal epithelial cell composition and impaired colon stem cell niche in Piezo1∆GC mice. (A) RNA levels of Ki67 and stem cell markers (Lgr5, Sox9, and EphB2) in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). (B) Immunohistochemistry of Ki67 in WT and Piezo1∆GC mouse colons. Scale bar: 100 µm. (C) Statistical analysis of Ki67 positive cell ratio in (B). (D) RNA levels of stem cell niche marker (cKit), differentiated colonocyte marker (Alpi), goblet cell marker (Agr2), and enteroendocrine cell marker (Chga) in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). (E) Immunohistochemistry of cKit in WT and Piezo1∆GC mouse colons. The white dashed lines mark the crypt borders, and the red arrow indicates a cKit-positive cell. Scale bar: 50 µm. (F) Statistical analysis of cKit-positive cell ratio in (E). (G,H) Immunofluorescence staining of Alpi (G) and Chga (H) in WT and Piezo1∆GC mouse colons. The white dashed lines mark crypt borders, and the white arrow indicates a differentiated colonocyte in (G) and an enteroendocrine cell in (H). Scale bar: 25 µm. (I,J) Statistical analysis of differentiated colonocyte ratio in (G) and enteroendocrine cell ratio in (H). At least three independent experiments were conducted. Data are presented as the mean ± SEM. (n = 5 mice). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Immunohistochemistry, Marker, Staining

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 7. Decreased self-renewal capacity of colon stem cells from Piezo1∆GC mice. (A) Representative images of colonoids from WT and Piezo1∆GC mice at day 5. Images of one colonoid on day 1, 3, and 5 are below, reflecting the growth process of a colonoid. Scale bar: 100 µm. (B–D) Indicators related to colonoids growth: number of buds per colonoid (B), surface area per colonoid (C), and percentage of colonoids with buds per well (D). (E) Immunofluorescence staining of EDU and Ki67 in colonoids from WT and Piezo1∆GC mice. Scale bar: 100 µm. (F) Statistical analysis of EDU and Ki67 positive cell ratio in (E). (G) Immunofluorescence staining of differentiated colonocyte (Alpi), goblet cell (Agr2), and enteroendocrine cell (Chga) in colonoids from WT and Piezo1∆GC mice. Scale bar: 100 µm. (H) Statistical analysis of Alpi, Agr2, and Chga positive cell ratio in (G). (I) RNA levels of Ki67 from WT and Piezo1∆GC mouse colonoids (normalized to GAPDH). (J) AB-PAS staining of colonoids from WT and Piezo1∆GC mice. The red arrows indicate mucus secreted by goblet cells in colonoids. Scale bar: 100 µm. At least three independent experiments were conducted. Data are presented as the mean ± SEM. (n = 5 mice). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Staining