The primers used.

<p>The primers used.</p

Inhibition of ICAM-1 <i>N</i>-glycan elongation or processing by ATRA suppresses cell adhesion.

<p>A, Schematic presentation of the protocol used to determine the effect of ATRA on cell adhesion. Briefly, SW480 cells were transfected with the GnT-III specific siRNA, treated with ATRA and then co-incubated with the HUVEC monolayer. The cell adhesion was assessed by counting the cells attached to the HUVEC monolayer. B, The cells attached to the HUVEC monolayer were observed under a confocal microscope. C, The adherent cells were analyzed by cell counting. D, SW480 cells were pretreated with 10 µM U0126 and then with 25 µM ATRA. The cells attached to the HUVEC monolayer were observed under a confocal microscope. E, The adherent cells were analyzed by cell counting.</p

ATRA-induced GnT-III expression is involved in the modulation of ICAM-1 <i>N</i>-glycan composition.

<p>A, SW480 cells were treated with 25 µM ATRA for 0, 18 and 36 h. The expression of GnT-III and GnT-V at the mRNA levels was detected by real-time RT-PCR (n = 3). B and C, SW480 cells that were transiently transfected with 50 nM of the siRNA specifically targeting GnT-III were treated with 25 µM ATRA. The efficiency of transfection was analyzed by real-time RT-PCR (n = 3, B) and the expression of ICAM-1 by Western blot (C). D, SW480 cells were treated with 25 µM ATRA. Then immunoprecipitation by the antibody against ICAM-1 (1.5 µg per 500 µg of total protein) was performed. The immunoprecipitated products were subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane and consecutively incubated with biotinylated L-PHA or E-PHA lectin, streptavidin-labled rabbit IgG and HRP-labeled goat anti-rabbit IgG. Bound HRP on the membranes was detected by ECL. E, SW480 cells were pretreated with 0.7 nM stauroporine, 10 µM H-89, 10 µM SB203580, 10 µM U0126, 50 µM PD98059, 20 µM SP600125 or 50 µM LY294002 for 2 h and then exposed to 25 µM ATRA for 36 h. The expression of ICAM-1 and phosphorylation of ERK were analyzed by Western blot. F, SW480 cells were pretreated with 10 µM U0126 and then with 25 µM ATRA. The expression of GnT-III at the mRNA level was analyzed by real-time RT-PCR (n = 3).</p

Inhibition of ICAM-1 <i>N</i>-glycan elongation or processing by ATRA suppresses trans-endothelial migration of U937 cells.

<p>A, Schematic presentation of the protocol used to determine the effect of ATRA on trans-endothelial migration of U937 cells. Briefly, U937 cells were treated with ATRA for 36 h and then co-incubated with the confluent monolayer of HUVEC cells, which were treated with 10 ng/ml TNFα and cultured on matrigel-coated transwell filters. 50 ng/ml IL-8 were added to the lower chamber. B to D, U937 cells attached to the HUVEC monolayer and trans-migrated through the matrix-membrane unit were estimated by confocal microscopy (B), cell counting (C) and flow cytometry (D).</p

ER stress is not the main cause for alteration of ICAM-1 <i>N</i>-glycan composition.

<p>A, Total RNA was isolated from SW480 cells that treated with 0, 5, 10, 25 and 50 µM ATRA or 5 µg/ml Tunicamycin. The expression of XBP-1 at the mRNA level was assessed by RT-PCR. B, SW480 cells were treated with 25 µM ATRA for 0, 1, 3, 6, 12, 18, 24 and 36 h. The expression of XBP-1 at the mRNA level was assessed by RT-PCR. C and D, SW480 cells were treated with 0, 5, 10 and 25 µM ATRA or with 5 µg/ml tunicamycin for 36 h. The expression of BiP (C) and EDEM (D) at the mRNA levels was evaluated by real-time RT-PCR (n = 3). E, SW480 cells were treated with 25 µM ATRA for 36 h or with 5 µg/ml tunicamycin for 12, 24 and 36 h. The expression of ICAM-1 was analyzed by Western blot. F, SW480 cells were grown on coverslips for 12 h and then incubated with 25 µM ATRA for 36 h. Then the cells were labeled with the anti-ICAM-1 antibody and FITC-conjugated goat anti-mouse IgG and examined under a confocal microscope. G, SW480 cells were treated with 25 µM ATRA for 48 h. The expression of ICAM-1 on the cell membranes was analyzed by flow cytometry. For the analysis of total ICAM-1 expression in the cells, the cells were fixed, permealized and stained. The stained cells were analyzed by flow cytometry.</p

ATRA induces the expression and <i>N</i>-glycan modification of ICAM-1.

<p>A, SW480 cells were treated with 0, 5, 10, 25 and 50 µM ATRA for 36 h or with 25 µM ATRA for 0, 12, 24 and 36 h. The expression of ICAM-1 was analyzed by real-time RT-PCR (n = 3) and Western blot. Equal loading was verified by detection of GAPDH. B, HUVEC cells were treated with 25 or 50 µM ATRA for 0, 24 and 36 h. The expression of ICAM-1 was analyzed by real-time RT-PCR (n = 3) and Western blot. C, U937 and WISH cells were treated with either 10 ng/ml IFN-γ or 25 µM ATRA or both. The expression of ICAM-1 was analyzed by Western blot. D, WISH cells were treated with 25 µM ATRA. The cell lysates were immunoprecipitated by the antibody against ICAM-1 and then treated with PNGase F. The digested samples were blotted and detected by Western blot.</p

Schematic presentation of the mechanisms by which ATRA modulates the structure of <i>N</i>-glycans linked to ICAM-1.

<p>Schematic presentation of the mechanisms by which ATRA modulates the structure of <i>N</i>-glycans linked to ICAM-1.</p

The transcription of <i>Erbin</i> is controlled in a cell cycle-dependent manner.

<p>A, The cells were arrested by a double-thymidine block or nocodazole treatment. Total RNA was extracted and <i>Erbin</i> mRNA expression analyzed by real-time RT-PCR. B, Schematic maps of the plasmids that carry the various <i>Erbin</i> promoter fragments driving the expression of luciferase (left). HeLa cells were transiently co-transfected with these plasmids and pRL-TK, then synchronized by a double-thymidine block and nocodazole treatment, respectively. The luciferase activities were assayed (right). C, HeLa cells were transiently transfected with pLuc-483, synchronized by double-thymidine block or nocodazole treatment, respectively, and then harvested at the different stages of the cell cycle. The luciferase assays were performed. (T), double-thymidine block; (N), nocodazole treatment.</p

Erbin protein expression is cell cycle-dependent.

<p>A, SKBR3 cells were stained by immunofluorescent method using anti-Erbin antibody. DAPI staining was utilized to display the nuclei. The stained cells were observed under a laser scanning confocal microscope. Bar = 10 µm. B, celllular DNA content was analyzed by flow cytometry. C, MCF-7 cells were arrested at G2/M phase using nocodazole followed by their release into the cell cycle. Cells at different stages of cell cycle were lysed and whole-cell lysates subjected to SDS-PAGE and Western blot analysis with anti-Erbin antibody. Analysis of cyclin A, cyclin B1 and cyclin D1 were utilized to dissect cell cycle progression. D, HeLa, 293T, LO2 and HL-7702 cells were synchronized by a double-thymidine block or nocodazole treatment and cellular DNA content was analyzed by flow cytometric analysis and Erbin expression detected by Western blot.</p

c-Myb binds to the c-Myb consensus sequences in the <i>Erbin</i> promoter.

<p>A, The association of c-Myb with the <i>Erbin</i> promoter in vivo was demonstrated by ChIP assays. B, The biotinylated double-stranded oligonucleotides harboring the consensus motif of c-Myb were incubated with 200 µg of the nuclear extracts and the binding of the nuclear proteins to the biotinylated oligonucleotides was analyzed in the presence or absence of five- and fifteen-fold amounts of double-stranded oligonucleotide competitors containing the c-Myb motif by DNA affinity precipitation assays. C and D, The binding activities of exogenous and endogenous c-Myb to c-Myb site in the <i>Erbin</i> promoter during cell cycle was analyzed by ChIP assays with the anti-HA (C) and anti-c-Myb antibodies (D) and real-time PCR.</p