Supplemental Material - Karbanová et al. - Vol. 56, pp 977-993

Online Supplemental data for Karbanová et al.: Supplemental Figures and Table

Files in this Data Supplement:

• Supplemental Table ST1 - Expression of PROMININ-1 in various human glands
• Supplemental Figure SF1 - The 80B258 mAb does not recognize the human prominin-1 under native labeling conditions. (A) 80B258 mAb recognizes the human PROMININ-1 by indirect immunofluorescence only under the denaturing condition. CHO cells transiently transfected with human PROMININ-1 were paraformaldehyde-fixed, SDS-treated, double-labeled with 80B258 mAb (left panel) and αhE2 antiserum (right panel), and analysed by double immunofluorescence. Note the co-localization of both immunoreactivities at the cell surface (arrows), consistent with both antibodies recognized the same protein (insets high magnification) upon denaturing condition. No immunoreactivity was observed without the SDS pre-treatment (not shown). Scale bar 10 μm. (B) Detection of 80B258 epitope by flow cytometry only under the denaturing condition. A suspension of living (grey or red or blue area) or paraformaldehyde-fixed, SDS-treated (green area) human Caco-2 cells were incubated either with 80B258 (red or green area) or AC133 (blue area) mAb or without primary antibody (grey area), as negative control, followed by the goat anti-mouse Alexa Fluor 488-conjugated secondary antibody, and analysed by flow cytometry. Note the lack of detection of prominin-1 by 80B258 (red), but not AC133 (blue), mAb under the native labeling condition.
• Supplemental Figure SF2 - Immunohistochemistry of PROMININ-1 in human salivary glands and sweat glands of axilla using mAb AC133 and isotype IgG1 control. Paraffin-embedded sections containing salivary submandibular glands (A-G), eccrine (H-J) and apocrine (K-M) sweat glands were immunolabeled with either AC133 mAb (A-E, H, I, K, L) or mouse isotype IgG1 (MOPC-21) control (F, G, J, M) and counterstained with light green. Samples were either heated in microwave (B-E, G, I, J, L, M) or incubated with SDS solution (A, F, H, K) prior the labeling. In submandibular glands, serous acini, serous demilunes and mucous tubuli are indicated with black, orange and blue dotted lines, respectively. The intercalated ducts where an occasional AC133 staining where observed (C, D, blue arrowheads) are indicated with black lines. Very rare cells located in veins were also AC133-positive (E, black arrowheads). In eccrine (H-J) and apocrine (K-M) sweat glands, secretion (white asterisks) found in the lumen of the duct (I, red dotted line) and secretory (L) portion, respectively, were weakly positive for AC133 mAb. A weak AC133 staining was detected as well at the luminal surface of cells (L, black arrowheads) located in the secretory portion of apocrine, but not eccrine (black dotted lines), sweat glands. The insets in panels I and L demarcate regions displayed at higher magnification in I1 and L1, respectively. Note that as for salivary and sweat glands, the AC133 staining was observed only upon microwave pre-treatment. No immunoreactivity was detected using isotype IgG1 control (F, G, J, M). Scale bar 50 μm.
• Supplemental Figure SF3 - Immunohistochemistry of PROMININ-1 in human lacrimal gland and the gland of Moll of eyelid using mAb AC133 and isotype IgG1 control. Paraffin-embedded sections containing accessory lacrimal gland of Wolfring (A, B), the gland of Moll (C-E) and conjunctival epithelium (F, G) were immunolabeled with either AC133 (A, C, F) or 80B258 (E) mAb or mouse isotype IgG1 (MOPC-21) control (B, D, G) and counterstained with light green. Samples were either heated in microwave (A, C, D, F, G) or incubated with SDS solution (B, E) prior the labeling. Neither mAb AC133 nor isotype control exhibited any immunoreactivity at the apical surface of various epithelia (white arrowheads), in contrast to the mAb 80B258 (black arrowheads). In the lumen of the gland of Moll, secreted materials were labeled strongly with the mAb 80B258, and weakly for mAb AC133 (white asterisks) whereas no labeling was detected with isotype control (black asterisks). Note that the AC133 immunoreactivity associated with the secretion was not detected when SDS pre-treatment was used instead the microwave (data not shown). The insets in panels C and D demarcate two regions displayed at higher magnification in C1/C2 and D1/D2, respectively. Scale bar 50 μm.
• Supplemental Figure SF4 - Immunohistochemistry of PROMININ-1 in human liver and pancreas using mAb AC133 and isotype IgG1 control. Paraffin-embedded sections of liver (A-C) and pancreas (D-J) were immunolabeled with either mAb AC133 (A, B, D-H,) or mouse isotype IgG1 (MOPC-21) control (C, I, J) and counterstained with light green (C, D, I, J) or hematoxylin-eosin (A, B, E-H). Samples were either heated in microwave (A-C, E-H, J) or incubated with SDS solution (D, I) prior the labeling. In liver, no immunorectivity was detected using either mAb AC133 or isotype IgG1 control. The portal space is outlined by a red dotted line and the intraportal bile ductules lined by a simple cuboidal epithelium are indicated with black dotted lines. In pancreas, a weak and rare AC133 immunoreactivity was detected at the luminal surface of cells located in intercalated ducts (blue arrowheads). Very rare cells located in the interstitium were also AC133-positive (black arrowhead). Note that the AC133 staining was observed only upon microwave pre-treatment. Secretory portions are outlined by a dotted line. The inset in panel E demarcates a region displayed at higher magnification in E1. Scale bar 50 μm.