Figure S3. LZTS2 knock-down in RPE cells... | Rockefeller University Press

Figure S3.

$LZTS2 knock-down in RPE cells leads to binucleation. (A) Immunoblots of total cell extracts showing efficient transient knockdown of LZTS2 expression using the mixture of two different LZTS2 siRNAs. Asterisk (*) denotes non-specific band refractory to knock-down and possibly contributing to Golgi localization. (B) Confocal en face view (maximum z-projection; inverted grayscale) showing efficient transient knockdown (48 h) of LZTS2 at cell-cell contacts (denoted by magenta arrows) using the mixture of two different LZTS2 siRNAs. LZTS2-deficient RPE cells exhibit binucleation (denoted by yellow arrows). LZTS2 (magenta), α-cat (green), nuclei (gray). Scale bar, 50 μm. (C) Percentage of binucleated RPE cells upon transient knockdown of LZTS2 expression using the mixture of two different LZTS2 siRNAs. Two-tailed unpaired t test, **P < 0.01. Graph indicates mean ± SD. Data from one experiment. (D) Two confocal z-planes (apical versus basal) corresponding to maximum intensity projection (Max IP) shown in B, to show how binucleated RPE cells (yellow arrows) were distinguished from cell piling (magenta arrows, where apical junction is clearly apparent), which occurs in hTERT-RPE1 cultures. Scale bar, 50 μm. Refer to the image caption for details.$

LZTS2 knock-down in RPE cells leads to binucleation. (A) Immunoblots of total cell extracts showing efficient transient knockdown of LZTS2 expression using the mixture of two different LZTS2 siRNAs. Asterisk (*) denotes non-specific band refractory to knock-down and possibly contributing to Golgi localization. (B) Confocal en face view (maximum z-projection; inverted grayscale) showing efficient transient knockdown (48 h) of LZTS2 at cell-cell contacts (denoted by magenta arrows) using the mixture of two different LZTS2 siRNAs. LZTS2-deficient RPE cells exhibit binucleation (denoted by yellow arrows). LZTS2 (magenta), α-cat (green), nuclei (gray). Scale bar, 50 μm. (C) Percentage of binucleated RPE cells upon transient knockdown of LZTS2 expression using the mixture of two different LZTS2 siRNAs. Two-tailed unpaired t test, **P < 0.01. Graph indicates mean ± SD. Data from one experiment. (D) Two confocal z-planes (apical versus basal) corresponding to maximum intensity projection (Max IP) shown in B, to show how binucleated RPE cells (yellow arrows) were distinguished from cell piling (magenta arrows, where apical junction is clearly apparent), which occurs in hTERT-RPE1 cultures. Scale bar, 50 μm.