Fig 1.
Wound healing assay in IPEC-J2 cells.
(A) Different growth patterns of IPEC-J2 cells grown in 5% fetal bovine serum (left) or in 5% porcine serum (right) (scale bar = 20 μm). (B) Wound healing assay at timepoint 0h when a scratch is made (left) with wound area A1 and after 6h (right) with wound area A2. The horizontal red line standardises the wound height (a) (scale bar = 200 μm). (C) Illustration of the measurement methodologies for calculating the distance to close the wound over 6 hours, which is the sum of both grey distances (b1 + b2). Method 1 is the methodology used in this paper; method 2 and method 3 are used by other authors. (D) Variances (standard deviation) of different methods measuring the absolute wound closure distance. Method 1 is the methodology used in this paper.
Fig 2.
Wound closure distance over 6 hours.
(A-B) The absolute wound closure distance in different culture conditions. Cells were not starved (5%) or starved (1%) after being exposed to (A) bovine (FBS) or (B) porcine serum (PS). (C) The absolute wound closure distance with different epidermal growth factor (EGF) or spermidine concentrations compared to 1% porcine-starved medium (control). (D) The absolute wound closure distance after exposure to different SCFAs and spermidine compared to 1% porcine-starved medium (control). Experiments were performed in three independent repeats run in duplicate. All results are expressed as minimum, first quartile, sample median, third quartile, and maximum. ‘+’ indicates the mean. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).
Fig 3.
Western blot of EGFR and FFAR2 receptor in different cell lines and intestinal samples.
(Left) EGFR staining, two bands are visible around 130 kD and 70 kD (right), FFAR2 staining (39 kD), and β tubulin staining (52 kD). Samples loaded from left to right: IPEC-J2 cells in 5% bovine serum, IPEC-J2 cells in 5% porcine serum, Caco-2 cells, pig jejunum harvested at day 0 and pig jejunum harvested at day 28.
Fig 4.
Immunohistochemical staining of EGFR and FFAR2 in different intestinal cell lines and pig jejunum.
From left to right: negative control without a primary antibody, EGFR as primary antibody, FFAR2 as primary antibody. From top to bottom: Caco-2 cells, IPEC-J2 cells with 5% bovine serum, IPEC-J2 cells with 5% porcine serum, and below are histological samples of pig jejunum. (scale bar Caco-2 cells and IPEC-J2 cells = 20 μm; scale bar pig jejunum = 50 μm).
Fig 5.
Effect of different growth factors on the proliferation capacity of IPEC-J2 cells in a wound healing assay immunohistochemically stained against Ki67.
All images were taken at T6. (a-b) controls (a) negative control without primary antibody (b) primary antibody without treatment group (c-e) treatment groups (c) EGF 5 ng/mL (d) spermidine 16 μM (e) propionate 0.2 mM. The black arrow shows the proliferation of positive Ki67 cells in the EGF condition. (scale bar = 50 μm).