Phase contrast microscope

BV2 cells were seeded into 24-well plates at a density of 2.5 × 10⁵ cells/well and incubated at 37 °C for 24 h. To assess the migration of cells, the cells were equally wounded with a sterile 100-μL pipette tip [26 (link),59 (link),60 (link)]. To evaluate how the tested compounds affected the LPS-induced cell mobility, the cells were treated with 1 µg/mL LPS with and without a series of concentrations of HSR1101 or 10 µM of MAPK inhibitors for 24 h. Simultaneously, control cells were treated with serum-free media containing 1% DMSO instead. The relative cell migration during 24 h were determined by measuring the changes in the area of the wounds at 0 and 24 h under a Nikon phase-contrast microscope (Nikon Instruments Inc., Melville, NY, USA) [60 (link)], using ImageJ version 1.49 software obtained from National Institutes of Health homepage (https://imagej.nih.gov/ij/). The cell migration was expressed as the percentages of the respective vehicle-treated control cells.

Wound closure was performed as previously described^{[17 (link)]}. Mitomycin C (10 μg/mL) was added to the cell culture medium to prevent cell proliferation during migration in the wound healing assay^{[6 (link),17 (link)]}. Wound healing/closure was monitored by assessing the migration of cells for 24 h; photographs were taken at 0 and 24 h with a digital SPOT camera attached to an inverted Nikon phase-contrast microscope. The images were stored in TIF format and processed in Adobe Photoshop (Adobe Systems Inc., Mountain View, CA, USA).

To evaluate the inhibitory effects of NeoDP2, galactose, and AHG on colony formation by human colon cancer cells, HCT-116 cells (Korean Cell Line Bank, Seoul, Republic of Korea) were used. The HCT-116 cells were cultured on a 6-well soft agar plate for two weeks at 37 °C in the presence of various concentrations of NeoDP2, galactose, and AHG. Cell colonies were observed under a Nikon phase-contrast microscope (Nikon, Tokyo, Japan).

Uniform vertical streaks were made in the monolayer culture with 200 μL pipette tips. The cells were immediately washed three times with RPMI medium with 10% FBS to remove detached cells. Mitomycin C (10 μg/mL; Sigma‐Aldrich) was added to the medium to inhibit proliferation, so that migration can be effectively followed.3 Mitomycin C is known to inhibit DNA synthesis using forming covalent crosslinks between complementary strands of DNA. This ultimately prevents separation of complementary strands of DNA, thereby inhibiting DNA replication. Cell migration was monitored for 24 hours, and pictures were taken at 0, 18, and 24 hours time points with a digital SPOT camera attached to an inverted Nikon phase contrast microscope (Nikon Inc., Melville, NY).
For wound closure assay in PC3 cells with KD of SOX2, siRNA KD of SOX2 was first performed on PC3 cells as described above. Twenty‐four hours post‐KD, the SOX2 siRNA transfected cells and nontargeting control siRNA transfected cells were replated in six‐well plates, grown overnight at 37°C, and allowed to reach near‐confluent levels. The wound closure assay was then performed as mentioned above. Parallel cultures were used to confirm SOX2 KD as compared with control siRNA‐treated cells

Morphometric analysis of the ileum (from Meckel’s diverticulum to the ileo-caeco-colic junction) was evaluated according to Giannenas et al [16 ]. One-centimetre-long segments were taken from the centre of each part and fixed in 10% buffered formalin for morphometrical assays under light microscopy. Formalin-fixed intestinal tissues were processed, embedded in paraffin wax, sectioned at 3 μm and stained the haematoxylin-eosin method. Histological sections were examined with a Nikon phase contrast microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 80i; Nikon, Tokyo, Japan). Images were viewed using a 4× Eplan objective (40×) to measure morphometric parameters of intestinal architecture. For this purpose, three favourably orientated sections cut perpendicularly from villus enterocytes to the muscularis mucosae were selected from each animal and measurements were carried as follows: villus height was estimated by measuring the vertical distance from the villus tip to villus-crypt junction level for 10 villi per section; crypt depth (the vertical distance from the villus-crypt junction to the lower limit of the crypt) was estimated for 10 corresponding crypts per section.

The abdominal cavity was opened after slaughter, and the entire small intestine system was removed for histomorphological analysis. Two centimeters of mid-duodenum (from the gizzard exit to the end of the pancreatic loop) were removed, rinsed by physiological saline solution and preserved in 10% buffered formalin [22 (link)]. The tissue samples were later embedded in paraffin, and a 5 µm section of each sample was placed on a glass slide and stained with hematoxylin and eosin according to Baurhoo et al. [23 (link)] The tissue sections were examined by a Nikon phase contrast microscope coupled with a micro-computer integrated digital imaging analysis system (Nikon Eclipse 80i, Nikon Co., Tokyo, Japan). The height and width of 30 villi and the depth of 30 crypts were measured from each group. Villus height was measured from tip (with a lamina propria) of the villus to the base (villus-crypt junction) and villus width was measured at its middle part, while the crypt depth was measured from the villus-crypt junction to the distal limit of the crypt [24 (link)]. Finally, the values of micro-morphological measurements of 10 quail/group were expressed as mean ± SE. The surface area was calculated according to this formula [25 ]:

BV2 cells were plated onto 24-well plates at a density of 5 × 10⁵ cells/well and incubated at 37 °C until the cells reached 80–90% confluence. The cells were then wounded with a sterile scratcher (SPL, Korea) and treated with Aze at the concentrations of 1, 3, and 10 μM in the presence or absence of LPS, as previously reported [34 (link),35 (link)]. The effect of Aze on LPS-induced cell migration was determined by measuring the relative change in the width of the wounds over 24 h using ImageJ software. Images at 0 and 24 h were captured under a Nikon phase-contrast microscope (Nikon Instruments Inc., Melville, NY, USA). The degree of cell migration was presented as a percentage of the vehicle-treated control cells.