Table 1.
The nomenclature used for the Phosducin like proteins (PLPs) discussed in this manuscript.
Figure 1.
Transient expression of PhLP3 in CHOK1 adherent cells promotes tubulin redistribution, an imbalance of α and β-tubulin subunits and cell death.
(A) Anti-PhLP3 antibody detects full length PhLP3 (24 kDa) in CHOK1 adherent cells transiently transfected with human PhLP3 (duplicates lane 1 and 2). Endogenous PhLP3 was detected in the plasmid only control (lane 3). β-actin levels are not altered upon PhLP3 overexpression but this promotes an increase of β-tubulin subunits relative to the control and a decrease in α-tubulin subunits (compare lanes 1 and 2 with lane 3). (B) Immunoprecipitation of CCTα in CHOK1 cells transiently transfected with PhLP3. CCTα (top panel) and PhLP3 (bottom panel) were detected in cell lysates transiently transfected with 500 ng–2 µg PhLP3 plasmid and immunoprecipitated with an anti-PhLP3 antibody, but not in plasmid only, untransfected and antibody only controls. (C) Immunofluorescent detection of PhLP3 in CHOK1 cells transfected with PhLP3. An anti-PhLP3 antibody was used to identify cells overexpressing PhLP3 (green) and DAPI (blue). (D). PhLP3 overexpression alters tubulin distribution. Immunofluorescent staining of PhLP3 transfected CHOK1 cells with either anti-α tubulin(red) or anti-βtubulin(green) specific antibodies detects redistribution of both tubulin subunits compared to untransfected controls. (E) PhLP3 overexpression in adherent CHOK1 cells promotes the release of cells into the supernatant and decreases cell viability compared to mock transfected and untransfected controls. Data calculated as mean +/− SEM of n = 2.
Figure 2.
RNAi silencing of PhLP3 in LB01 cells promotes a fibroblast-like morphology and cytoskeletal rearrangement.
(A) Immunoblots using anti-PhLP3 of LB01 cells transfected with mouse siRNA shows a decrease in PhLP3 expression compared to human siRNA and a scrambled negative control siRNA (top panel). Actin is used as an internal loading control (bottom panel). Note that two PhLP3 products (bands) are detected in these samples prepared with phosphatase inhibitors while in figure 1A (where they were absent) only one product is detected: PhLP3 is known to be phosphorylated [45] (see also references in the human PhLP3 Unigene entry UniGene Hs.536122) with the phosphoform displaying higher apparent molecular weight on SDS gels than the unphosphorylated form. (B) LB01 cells were transfected with mouse PhLP3 siRNA for 72 h and the cells were fixed and stained for either actin (rhodamine phalloidin) or (C) tubulin (anti-α-tubulin). The microtubule organising center is indicated by a red arrow. (D) NIH 3T3 mouse fibroblast cells were fixed and stained for F-actin filaments (rhodamine phalloidin, red) and microtubules (anti-α-tubulin, green). (E) Focal adhesions observed with anti-vinculin in cells transfected with the control plasmid or siRNA silencing plasmid 48 h and 72 h post transfection.
Figure 3.
Rescue of PhLP3 silenced CHO LB01 cells promotes aberrant cell division.
LB01 cells were silenced and rescued as described in the methods section and then fixed and co-stained for PhLP3 (green) and with the nuclear DAPI stain (blue). PhLP3 overexpression in LB01 cells leads to more ‘rounded’ cells compared to untransfected controls (A v B) whilst siRNA silencing promotes morphological changes including elongated cells and nuclei and adherent growth (A v C). When LB01 cells were co-transfected with mouse PhLP3 siRNA and human PhLP3 DNA (D and E) the elongated phenotype observed in the PhLP3 silenced cells was no longer present and thus the cells were considered ‘rescued’. Co-transfected cells were either in early anaphase (red arrows), late anaphase (green arrow) or exhibited enlarged nuclei (white arrrow).
Figure 4.
RhoA/p160ROCK pathway regulates actin stress fiber formation in PhLP3 siRNA silenced CHO LB01 cells.
A commercially available RhoA-GTP/Rhotekin pull down assay was carried out on siRNA silenced PhLP3 LB01 cells. Samples were collected 24 h, 48 h and 72 h post transfection and activated RhoA was detected at 72 h post-knockdown with an anti-Rho A antibody but not in plasmid only control cells (A). LB01 cells transfected as in panel A for 72 h were then treated for 24 h with the ROCK inhibitor Y2763 (either 3 µM or 10 µM), fixed and stained for actin (rhodamine phalloidin, red). F-actin disassembly was observed at both Y2763 concentrations and the concentration of peripheral actin was observed with 10 µM Y2763 (B).
Figure 5.
siRNA silencing of PhLP3 promotes MAPK phosphorylation upstream of RhoA activation and PhLP3 overexpression decreases levels of phosphorylated MAPK in suspension CHO LB01 cells.
(A) PhLP3 silenced LB01 cells were fixed and co-stained with either anti-MAPK (second panel, green) and rhodamine phalloidin (for filamentous actin, red) or anti-phosphorylated MAPK (fourth panel, green) and rhodamine phalloidin. DAPI was used to detect DNA. Untransfected cells were used as controls (first and third panel). (B) An increase in phosphorylated MAPK was detected by immunoblotting using an anti-MAPK kinase antibody in cells transfected only with PhLP3 siRNA (lane 2) compared to cells that had been transfected with PhLP3 siRNA in the presence of the ROCK inhibitor Y2763 (lane 3). Cells treated with either Y2763 only, cells transfected with a ‘scrambled’ negative control siRNA and untransfected cells did not show elevated phosphorylated MAPK levels. (C) CHO cells were transiently transfected with PhLP3 for either 24 h or 48 h and levels of phosphorylated MAPK were detected by immunoblotting with an anti-MAPK kinase antibody. Expression levels of phosphorylated MAPK were found to decrease from 24 h to 48 h (compare lanes 1 to 2) and to be lower than in untransfected cells (lanes 3 and 4).