Release of ALCAM is sensitive to ADAM17/TACE silencing.

<p>(A) Analysis of lysates from TPC-1 and A2774 cells, resolved on 4–12% SDS-PAGE and immunoblotted with anti-ADAM17/TACE antibodies. Arrows indicate inactive (130 kDa) and active (80 kDa) forms of ADAM17/TACE enzyme. Normalization of results was obtained with immunoblotting analysis of beta-actin. (B) Expression of ADAM17/TACE protein by TPC-1 cells transfected with an ADAM17/TACE-specific small interfering RNA (siRNA) (OTP17), or with non-targeting siRNA (NT) as detected by western blot. The amount of protein was calculated by comparative densitometric scanning with beta-actin. (C) ELISA detection of ALCAM release by TPC-1 cells after transfection with siRNA specifically inhibiting ADAM-17/TACE (OTP17, black column) or with non-targeting siRNA (NT, white column). (D) Conditioned medium (CM) from TPC-1 cells, cultured with pervanadate (PV) (60 min), epidermal growth factor (EGF) (24 h), or medium alone (ctr, 24 h) in the presence (black columns) or in absence (white columns) of CGS27023A (CGS) was assessed by ELISA for ALCAM. Columns, means of three experiments (cells cultured in presence of 10, 1, or 0.1 µM CGS); bars, SD. *, P<0.05. Grey bar: in absence of CGS, but in presence of orthovanadate (OV).</p

Immunohistochemical analysis of ALCAM.

<p>Membranous positive staining in surrounding non-tumor tissue (A and C) and in medullary thyroid carcinomas (MTCs) (B). Cytoplasmic and membranous positive staining in papillary thyroid carcinomas (PTCs) (D). 1, low-power field; 2, high-power field; N (normal tissue), T (tumor tissue).</p

Changes in ALCAM expression induced by ionomycin or phorbol myristate acetate (PMA).

<p>Total lysates (Lys) and conditioned medium (CM) from TPC-1 cells, treated or not with 1 µM ionomycin (iono) or 50 ng/mL PMA for 2 h, were resolved by 3–8% SDS-PAGE and immunoblotted with ALCAM antibody. Both treatments led to an increase of secreted ALCAM isoforms; especially PMA induced a gain of 60-kDa isoform expression. Arrow indicates the membrane-localized ALCAM isoform in lysate, and arrowheads indicate shed-ALCAM isoforms in CM, respectively. Beta-actin was used as a loading control.</p

Immunohistochemical reactivity of thyroid tumors.

<p>ALCAM, activated leukocyte cell adhesion molecule; PTC, papillary thyroid carcinoma;</p><p>MTC, medullary thyroid carcinoma.</p><p>CD56 and TTF-1 were negative in three out of four PTC cases. MTCs showed comparable positive immunostaining results for CD56 but were completely negative for TTF-1.</p

CGS decreases TPC-1 cell motility.

<p>Cells were seeded onto cell culture plates, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017141#s4" target="_blank">materials and methods</a>. TPC-1 cells were exposed to CGS treatment or vehicle. At cell confluence, a scratch was performed using a 10 µl tip and the wound measured. After a 15 h treatment, the plate area remaining free of cells was measured. The cell-free area accounted for 44.5% of the image when the scratch was applied, P<0.05 versus DMSO control.</p

ALCAM expression in thyroid cell lines.

<p>Analysis of TT and MZ-CRC1 cell lysates showing ALCAM protein expression in these thyroid cell lines. SKOV-3 and TPC-1 cell lysates were included as controls. Total lysates from each cell line were resolved by 4–12% SDS-PAGE and immunoblotted with anti-ALCAM antibodies. Arrow indicates the fully glycosylated ALCAM isoform. Beta-actin was used as a loading control.</p

ALCAM expression in normal and tumor human thyroid tissue samples.

<p>(A) Total protein extracts (30 µg) from 11 papillary thyroid carcinomas (Ca Pap), 5 medullary thyroid carcinomas (Ca Mid), and 5 normal thyroid tissues (Thyroid) were resolved by 4–12% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with anti-ALCAM antibody. (*) indicates a non-specific background band. Beta-actin was used as a loading control. (B) Western blot analysis of ALCAM expression of total protein extracts (30 µg) from three PTCs, two MTCs, and three CTRLs, treated (+) or not (−) with N-glycosidase F. Beta-actin was used as a loading control.</p

Western blot analysis of ALCAM expression in lysate and conditioned medium of TPC-1 cell line.

<p>(A) Analysis of TPC-1 cell lysate (Lys) and conditioned medium (CM) using anti-ALCAM antibody. Arrow indicates membrane-localized ALCAM isoform in lysate, and arrowheads indicate shed-ALCAM isoforms in CM, respectively. The SKOV-3 cell line was used as positive control. (B) TPC-1 cell lysate and CM were treated or not with a mixture of O-glycosidase (O) and sialidase (S) and analyzed by western blot using anti-ALCAM antibody. Continuous arrows indicate membrane and shed ALCAM forms before digestion, and dashed arrows indicate digestion products. (*) indicates less evident bands of ALCAM in CM. (C) TPC-1 cell lysate and CM were treated or not with O-glycosidase (O), sialidase (S), or PNGaseF (F) and analyzed by western blot using anti-ALCAM antibodies. Continuous arrows indicate membrane and shed ALCAM forms before digestion, and dashed arrows indicate digestion products.</p

Additional file 2: of MIF/CD74 axis is a target for novel therapies in colon carcinomatosis

Figure 1. Phase contrast microscope images of C1 and C2 organoids. Figure 2. Ingenuity Pathway Analysis of C2 secretome. Figure 3. Western blot analysis of C2 organoids after 4-IPP treatments. (PDF 282 kb