Figure 1.
Human intestinal epithelial cells express both BEST2 and BEST4 in vivo.
Representative data of three independent immunohistochemical analyses using human intestinal tissues are shown. (A) A single stain of BEST2 and BEST4 in the human small intestine and colon (Original magnification 200x). Goblet-shaped cells that are positive for BEST2 (red) are present in the colon but not in the small intestine. Columnar-shaped cells that are positive for BEST4 (green) are present both in the colon and in the small intestine. A magnified view of the squared area is shown in the lower column (Original magnification 400x). (B) Double-immunostaining of BEST2 with CDH1 (Original magnification 400x). In the human colon tissue, BEST2 (red) was expressed in CDH1-positive IECs (green). A magnified view of the squared area is shown in the right column (Original magnification 800x). (C) Double-immunostaining of BEST4 with CDH1 (Original magnification 400x). Both in the small intestine and in the colon, BEST4 (green) was expressed in CDH1-positive IECs (red). A magnified view of the squared area is shown in the right column (Original magnification 800x).
Figure 2.
BEST2 is expressed specifically in human colonic goblet cells.
(A) Double-immunostaining of BEST2 with MUC2 (Original magnification 400x). In the human colon tissue, BEST2 (red) was expressed exclusively in MUC2-positive IECs (green). A magnified view of the squared area is shown in the right column (Original magnification 800x). (B) Double-immunostaining of BEST4 with MUC2 (Original magnification 400x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express MUC2 (red). A magnified view of the squared area is shown in the right column (Original magnification 800x). Representative data of three independent analyses are shown.
Figure 3.
BEST4 is expressed in post-mitotic IECs other than secretory lineage cells.
(A) Double-immunostaining of BEST4 with Ki-67 (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express Ki-67 (red). (B) Double-immunostaining of BEST4 with Lysozyme (Original magnification 200x). In the small intestine, BEST4-positive cells (green) did not co-express with Lysozyme (red). (C) Double immunostaining of BEST4 with Chromogranin-A (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with Chromogranin-A (red). A magnified view is shown in the lower column (Original magnification 400x). (D) Double-immunostaining of BEST4 with HPGDS (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with HPGDS (red). A magnified view is shown in the lower column (Original magnification 400x). Representative data of three independent analyses are shown.
Figure 4.
BEST4 is expressed in the absorptive cells of the human intestine in vivo.
(A) Double-immunostaining of BEST4 with CD10 in the human small intestine (Original magnification 200x). In the human small intestinal tissue, BEST4 (green) was expressed in CD10-positive IECs (red). A magnified view of the squared area is shown in the lower column (Original magnification 400x). (B) Double-immunostaining of BEST4 with Villin in the human small intestine (Original magnification 400x). In the human small intestinal tissue, BEST4 (green) was expressed in Villin-positive IECs (red). A magnified view of the squared area is shown in the lower column (Original magnification 400x). (C) Double-immunostaining of BEST4 with Villin in the human colon (Original magnification 800x). In the colon, BEST4 (green) was expressed in Villin-positive IECs (red). A magnified view of the squared area is shown in the lower column (Original magnification 800x). Representative data of three independent analyses are shown.
Figure 5.
Expression of BEST2 is markedly down-regulated in active lesions of UC.
The immunohistochemical analysis of BEST2 and BEST4 expression using colonic tissues of UC patients is shown. Representative data obtained from three subjects in each group are presented. (A) Expression of BEST2 is down-regulated in the active lesions of UC patients, where goblet cell mucins are depleted. The double staining of BEST2 (green) and WGA-lectin (red) using tissues from the normal human colon or from active and inactive lesions of UC patients is shown (Original magnification 400x). The red staining of WGA-lectin represents goblet cell mucins. Each series represents staining results obtained from a different patient (Original magnification 400x). (B) The expression of BEST4 is maintained at the surface epithelium of active, as well as inactive, lesions in UC patients. Single staining of BEST4 (green) using tissues from the normal human colon or from active and inactive lesions of UC patients is shown (Original magnification 400x). Each figure represents a staining result obtained from a different patient.
Figure 6.
BEST2 expression is significantly up-regulated in HT-29 cells by the induction of goblet cell differentiation.
(A) LY411575 induces the expression of BEST2 in HT-29 cells. The HT-29 cells were treated with either LY411575 (1μM) or DMSO for the indicated period of time and then subjected to quantitative RT-PCR analysis or immunostaining. The quantitative data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO of the corresponding time period, as determined by Student’s t-test. The immunostaining of MUC2 (red, left panel) and BEST2 (green, left panel) after 120 h of treatment showed a clear increase in the number of MUC2- or BEST2- positive cells by LY411575 (Original magnification 200x). (B) The expression of absorptive cell marker genes, as well as BEST4, was not induced in HT-29 cells by LY411575. HT-29 cells were treated with either LY411575 or DMSO for 72 h and subjected for quantitative RT-PCR analysis. Data were normalized to expression levels of β-actin. Error bars represent S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO, determined by Student’s t-test. (C) LY411575 induces BEST2 expression in MUC2-positive cells. HT-29 cells were treated with either LY411575 or DMSO for 120 h and subjected to double-immunostaining. The analysis of LY411575-treated cells revealed that BEST2-positive (green) cells clearly co-localize within MUC2-positive (red) cells (Original magnification 200x for upper series, 400x for lower series).
Figure 7.
BEST4 expression is significantly up-regulated in Caco-2 cells by the induction of absorptive cell differentiation.
(A) Induction of absorptive cell differentiation induces expression of both BEST4 and Sucrase-Isomaltase, in Caco-2 cells. Caco-2 cells were cultured under full confluency for the indicated period of time and subjected to quantitative RT-PCR analysis. The data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to day 0, as determined by Student’s t-test. (B) The expression of not BEST2, but of absorptive cell marker genes, was induced in Caco-2 cells by the induction of absorptive cell differentiation. Caco-2 cells were cultured in full confluency for 18 days and subjected to quantitative RT-PCR analysis. The data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to day 0, as determined by Student’s t-test. (C) The induction of absorptive cell differentiation generated BEST4 expression in Villin- or Sucrase-Isomaltase- positive cells. Caco-2 cells were cultured in full confluency for 18 days and subjected to immunocytochemistry. Staining of Sucrase-Isomaltase, Villin and BEST4 showed clear increases in the number of BEST4-positive cells (green) at day 18 within the area where the expression of Sucrase-Isomaltase (red) or Villin (red) was uniformly induced (Original magnification 200x). Representative data of three independent analyses are shown.
Figure 8.
Activation level of Notch signaling can alternatively potentiate the expression of BEST2 or BEST4 in LS174T cells.
An analysis of LS174T-NICD cells, a sub-line of LS174T cells in which we can induce expression of activated form of Notch1 (NICD) by doxycycline (DOX) addition, is shown. (A) The up- and down-regulation of the Notch activity level can be achieved by addition of DOX or LY411575 to LS174T-NICD cells. LS174T-NICD cells were pre-cultured with LY411575 (1μM) or DMSO for 48 h and then cultured with LY411575, DOX (100 ng/ml) or both for the following 24 h. Those cells were subjected to immunoblot analysis for NICD, Hes1 and β-actin. A membrane was stripped and re-probed with series of antibodies. (B) The activation level of Notch signaling may potentiate the expression of BEST2 and BEST4 in LS174T cells. LS174T-NICD cells were cultured as described in (A) and subjected to quantitative RT-PCR analysis. Data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05, determined by Student’s t-test.