Figure 9.
Figure 9. Changes of structure and composition of AJs upon treatment of monolayer cultures of human hepatocyte-derived (PLC) cells with siRNAs interfering with mRNAs encoding different cadherins. (A–N) Laser-scanning double-label immunofluorescence microscopy of monolayer cultures of PLC cells treated with siRNA interfering with mRNA encoding E-cadherin (B–D) or N-cadherin (E–H) or with both siRNAs (I–M), in comparison with control cultures without siRNA added (A) or treated with siRNA interfering with nuclear lamins A and C (N). Results of treatments for 72 h, with one change of siRNA-containing medium after 24 h, are shown. (A) Densely grown PLC cells immunostained here for N-cadherin (red) and plaque protein ZO-1 (green). Note the extended cell–cell contact regions stained for one of the two markers or in yellow merged color. (B–D) Cells treated with siRNA interfering with E-cadherin show exclusively the N-cadherin reaction in their cell–cell contact regions (B and C, green), whereas only occasionally, some indistinct E-cadherin (red) fluorescence is seen in the perinuclear cytoplasm (C; e.g., cell on the bottom right). All cell–cell contact regions are also positive for all AJ plaque markers as shown, for example, for protein ZO-1 (D, green). (E–H) Upon treatment with siRNA interfering with mRNA encoding N-cadherin, cell–cell contact regions are intensely stained for E-cadherin (green), whereas only very little and faint residual N-cadherin (E–G, red), mostly in endocytotic vesicles, can still be seen in the cytoplasm (F and G). The AJ plaque proteins are continually present (G; e.g., protein ZO-1 is shown in green). The cells are obviously still kept in contact by AJs based on E-cadherin alone (H, red), which is closely associated with the AJ plaque proteins (green; protein ZO-1 in H), as indicated by yellow merge color. (I–M) When the cell cultures are exposed to both, i.e., siRNA interfering with E-cadherin mRNA and siRNA interfering with N-cadherin mRNA, some remaining cell colonies, although mostly smaller, are still recognized that are not demonstrably positive for E- or N-cadherin but in which extended AJ-type contacts are seen that are still positive for AJ plaque proteins, such as protein ZO-1 (green). In contrast, some residual cadherins (E-cadherin is shown in red immunofluorescence) are demonstrable only deep in the cytoplasm, often recognizable as distinct dotlike endocytotic vesicle structures or still in association with the extended AJ plaque-derived cytoplasmic bands positive for protein ZO-1 (green bands in K, L, and M) and other plaque proteins (not depicted; for details of the formation and organization of such zonula adherens–like AJ plaque-derived structures see Kartenbeck et al., 1982). (N) Control cell culture treated in parallel with siRNAs to lamins A/C but reacted with antibodies for E-cadherin (red) and nuclear lamins A/C (green) to demonstrate the stability of the cell monolayer cultures in such experiments. The cells are connected by normal-looking extended AJ structures positive for E-cadherin. Positivity of the latter structures for N-cadherin has also been seen in parallel experiments. In contrast, no nuclear lamina decorated by lamin A/C immunofluorescence is seen. Bars, 10 µm.

Changes of structure and composition of AJs upon treatment of monolayer cultures of human hepatocyte-derived (PLC) cells with siRNAs interfering with mRNAs encoding different cadherins. (A–N) Laser-scanning double-label immunofluorescence microscopy of monolayer cultures of PLC cells treated with siRNA interfering with mRNA encoding E-cadherin (B–D) or N-cadherin (E–H) or with both siRNAs (I–M), in comparison with control cultures without siRNA added (A) or treated with siRNA interfering with nuclear lamins A and C (N). Results of treatments for 72 h, with one change of siRNA-containing medium after 24 h, are shown. (A) Densely grown PLC cells immunostained here for N-cadherin (red) and plaque protein ZO-1 (green). Note the extended cell–cell contact regions stained for one of the two markers or in yellow merged color. (B–D) Cells treated with siRNA interfering with E-cadherin show exclusively the N-cadherin reaction in their cell–cell contact regions (B and C, green), whereas only occasionally, some indistinct E-cadherin (red) fluorescence is seen in the perinuclear cytoplasm (C; e.g., cell on the bottom right). All cell–cell contact regions are also positive for all AJ plaque markers as shown, for example, for protein ZO-1 (D, green). (E–H) Upon treatment with siRNA interfering with mRNA encoding N-cadherin, cell–cell contact regions are intensely stained for E-cadherin (green), whereas only very little and faint residual N-cadherin (E–G, red), mostly in endocytotic vesicles, can still be seen in the cytoplasm (F and G). The AJ plaque proteins are continually present (G; e.g., protein ZO-1 is shown in green). The cells are obviously still kept in contact by AJs based on E-cadherin alone (H, red), which is closely associated with the AJ plaque proteins (green; protein ZO-1 in H), as indicated by yellow merge color. (I–M) When the cell cultures are exposed to both, i.e., siRNA interfering with E-cadherin mRNA and siRNA interfering with N-cadherin mRNA, some remaining cell colonies, although mostly smaller, are still recognized that are not demonstrably positive for E- or N-cadherin but in which extended AJ-type contacts are seen that are still positive for AJ plaque proteins, such as protein ZO-1 (green). In contrast, some residual cadherins (E-cadherin is shown in red immunofluorescence) are demonstrable only deep in the cytoplasm, often recognizable as distinct dotlike endocytotic vesicle structures or still in association with the extended AJ plaque-derived cytoplasmic bands positive for protein ZO-1 (green bands in K, L, and M) and other plaque proteins (not depicted; for details of the formation and organization of such zonula adherens–like AJ plaque-derived structures see Kartenbeck et al., 1982). (N) Control cell culture treated in parallel with siRNAs to lamins A/C but reacted with antibodies for E-cadherin (red) and nuclear lamins A/C (green) to demonstrate the stability of the cell monolayer cultures in such experiments. The cells are connected by normal-looking extended AJ structures positive for E-cadherin. Positivity of the latter structures for N-cadherin has also been seen in parallel experiments. In contrast, no nuclear lamina decorated by lamin A/C immunofluorescence is seen. Bars, 10 µm.

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