siRNA for Jagged-2 inhibited proliferation of IEC-6 cells.

<p>Cells were plated onto 6-well plates. Inhibition of Jagged-2 with siRNA was carried out as mentioned above (see the Materials and Methods). <i>A</i>: After 48 h of culture, protein was extracted from IEC-6 cells plated on 6-well plates. Western blot analysis showed that siRNA for Jagged-2 downregulated protein expression of Jagged-2, NICD-1, and Hes-1 of IEC-6 cells compared with the si-NC or control group. GAPDH was used as loading control. <i>B</i>: Graphic representation of relative expression of Jagged-2, NICD-1, and Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 5). **<i>p</i><0.01 versus control group. <i>C</i>: Cell count of IEC-6 cells was taken for control group, si-NC, and siRNA for Jagged-2 group (magnification×400). <i>D</i>: IEC-6 cells were plated onto a 96-well plate. Inhibition of Jagged-2 with siRNA was carried out as mentioned above (see the Materials and Methods). MTT assay showed that siRNA for Jagged-2 decreased the cell number of IEC-6 cells significantly. Data are given as the means ± SDs (<i>n</i> = 10) **<i>p</i><0.01 versus control group. Control: IEC-6 cells were cultured normally. si-NC: IEC-6 cells were transfected with unrelated control siRNA. siRNA: IEC-6 cells were transfected with siRNA for Jagged-2.</p

siRNA for Hes-1 inhibited proliferation of IEC-6 cells.

<p>Cells were plated onto 6-well plates. Inhibition of Jagged-2 with siRNA was carried out as mentioned above (see the Materials and Methods). <i>A</i>: After 48 h of culture, protein was extracted from IEC-6 cells. Western blot analysis showed that siRNA for Hes-1 downregulated protein expression of Hes-1 in IEC-6 cells compared with the si-NC or control group. GAPDH was used as loading control. <i>B</i>: Graphic representation of relative expression of Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 5). **<i>p</i><0.01 versus control group. <i>C</i>: Cell count of IEC-6 cells was taken for control group, si-NC, and siRNA for Hes-1 group (magnification×400). <i>D</i>: IEC-6 cells were plated onto a 96-well plate. Inhibition of Hes-1 with siRNA was carried out as mentioned above (see the Materials and Methods). MTT assay showed that siRNA for Hes-1 decreased the cell number of IEC-6 cells significantly. Data are given as the means ± SDs (<i>n</i> = 10) **<i>p</i><0.01 versus control group. Control: IEC-6 cells were cultured normally. si-NC: IEC-6 cells were transfected with unrelated control siRNA. siRNA: IEC-6 cells were transfected with siRNA for Hes-1.</p

PCNA expression is increased in intestinal crypt epithelial cells after I/R (2 h).

<p><i>A</i>: Immunostaining of rat intestinal tissues showing expression of PCNA. Brown staining showed positive results for PCNA (original magnification ×200). Magnified view of the squared area is shown in the right of the original picture (original magnification ×400). <i>B</i>: Graphic representation of relative number of PCNA positive cells. Data are given as the means ± SDs (<i>n</i> = 7). **<i>p</i><0.01 versus sham group.</p

Immunofluorescence evidence for increased Notch signaling activation in intestine after I/R injury.

<p>Frozen sections of intestine from sham operated rats or rats after I/R injury were stained with antibodies against Jagged-2 (A), NICD-1 (B), and Hes-1 (C). Nuclei were stained with DAPI (blue). Images (A: magnification×200; B, C: magnification×400) were taken by confocal microscopy.</p

Inhibition of Notch signaling by DAPT suppressed cell proliferation of IEC-6 cells.

<p><i>A</i>: IEC-6 cells with DMSO (Control) or DAPT (20 µM) for indicated times (magnification×100). <i>B</i>: IEC-6 cells were plated onto a 96-well culture plate, and DAPT was added. MTT assay was performed to examine the proliferation of IEC-6 cells. Data are given as the means ± SDs (<i>n</i> = 10). <i>C</i>: Protein was extracted from IEC-6 cells plated onto a 6-well plate, and protein expression of NICD-1, and Hes-1 was examined by Western blot. GAPDH was used as the loading control. <i>D</i>: Graphic representation of relative expression of NICD-1, and Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 5). **<i>p</i><0.01 versus control group. *<i>p</i><0.05 versus control group.</p

Transcripts of Notch signaling components were increased in intestine after I/R injury.

<p><i>A</i>: The I/R and sham-operated rats were sacrificed at indicated times, mRNA of intestinal mucosa was collected, and RT-PCR was performed to detect mRNA levels of Jagged-2, Notch-1, and Hes-1. GAPDH was used to verify equivalent loading. <i>B</i>: mRNA expression of Jagged-2, Notch-1, and Hes-1 detected by Real-time PCR. Data are given as the means ± SDs (<i>n</i> = 7). **<i>p</i><0.01 versus sham group.</p

The protein expressions of Notch signaling components were increased in intestine after I/R injury.

<p><i>A</i>: The I/R and sham-operated rats were sacrificed at indicated times, and Western blot was performed to detect protein expression of Jagged-2, NICD-1, and Hes-1. GAPDH was used to verify equivalent loading. <i>B</i>: Graphic representation of relative expression of Jagged-2, NICD-1, and Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 7). **<i>p</i><0.01 versus sham group. *<i>p</i><0.05 versus sham group.</p