Figure 1.
Nuclear and nucleolar localization signals in STK35L1.
A) Prediction of nuclear localization signals in STK35L1. NLS was aligned with the known monopartite NLS of ESXR1 and bipartite NLSs of Plk1[22], LIMK2 [22] and HDGF [49]. Basic amino acids are shown in red colors. B) Schematic representation of different EGFP-STK35L1 deletion mutants. The upper axis with coordinates represents the position of amino acids. The predicted bipartite NLS (aa 142–153) is shown as red box. C) Subcellular distribution of different EGFP-STK35L1 deletion mutants. EGFP-STK35L1 was mainly localized in the nucleus and nucleolus (Top left panel; white arrow). The nucleolus appears as a dark spot excluded by the DNA staining with Hoechst-dye (lower left panel, white arrow head). The expression of the various EGFP-STK35L1 deletion mutants in endothelial cells is shown (see text for details).
Figure 2.
EGFP-STK35 intracts with nuclear actin.
A) Nuclear actin coimmunoprecipitates with EGFP-FLAG-STK35. HEK293T cells were transfected with EGFP-FLAG-STK35 and EGFP-FLAG. The nuclei were isolated and EGFP-FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody. Bound proteins were resolved by SDS-PAGE and stained with Coomassie blue. The specific bands were cut, in-gel digested by trypsin and identified by MALDI-TOF and peptide mass fingerprinting. The protein band around 45 kDa (under “*”) was as β-actin (left panel). The other bands were identified as HSP70B1 and EGFP-FLAG-STK35. The same immunoprecipitated samples were blotted with monoclonal anti-actin antibody and the band of 45 kDa was confirmed as β-actin (middle panel). To check the purity of the nuclear preparation, 10 µl of 100 µl nuclear fraction and 10 µl of 4 ml cytoplasmic fraction were subjected to SDS-PAGE and blotted with an anti-actin and an anti-tubulin antibody. The apparent 50∶50 ratio of nuclear to cytoplasmic actin is due to the high amount of actin in the diluted cytoplasmic fraction. Tubulin (55 kDa) could be detected in the cytoplasmic but not in the nuclear fraction, demonstrating the nuclear fraction was free from cytoplasmic proteins (right panel). B) EGFP-STK35 and β-actin colocalized partially in the nucleus. Endothelial cells transfected with EGFP-STK35 (left panel, green) and stained for β-actin (middle panel, red) with anti-actin monoclonal antibody (clone C4) 8 h after transfection. The overlay (right panel) shows colocalization of EGFP-STK35 and β-actin. Arrows indicate some sites of colocalization.
Figure 3.
Identification of Putative class III PDZ-binding domains motif in STK35L1 that mediates its interaction with actin.
A) Class III PDZ binding domains motif (PDZ-BM) was predicted by ELM software and its position within the STK35L1 sequence is indicated as green box; kinase domain: yellow box, NLS: red box. B) GST pull down assay of nuclear lysates from HeLa cells was preformed using recombinant GST or GST-PDM protein (GST coupled to 30 amino acids (aa position 170 to 204) of STK35L1 containing the potential PDZ-BM). Bound proteins were resolved by SDS-PAGE and immunoblotted using a monoclonal anti-β-actin antibody C) Subcellular distribution of different EGFP-STK35L1 deletion mutants in transfected endothelial cells. EGFP-PDM (aa 170–204, contains only the PDZ-BM of STK35L1) was localized to actin stress fibers. EGFP-STK35L1Δ1-169 mutant (lacking NoLS and NLS) was distributed throughout the nucleus and cytoplasm with a partial localization to fiber like structures of the cytoskeleton. EGFP-STK35L1Δ1-96 (lacking NoLS, NLS and PDZ-BM) was diffusely distributed throughout the cytoplasm and the nucleus but no green fiber like structures were visible.
Figure 4.
G1 to S-phase progression is accelerated in STK35L1-silenced endothelial cells.
A) STK35L1 expression level of STK35L1 silenced cells and control cells. B) Cell cycle distribution of control siRNA- and STK35L1 siRNA treated endothelial cells. Cells arrested in Go/G1 phase were stimulated by serum for various times. Values are mean ± S.E. of three independent experiments. Asterisk “*” indicates level of significance p≤0. 05.
Table 1.
mRNA expression of selected cell cycle specific genes in STK35L1-silenced cells.
Figure 5.
STK35L1 regulates endothelial morphogenesis.
A) STK35L1 expression in endothelial cells cultivated in the presence of VEGF on collagen or on Matrigel. The expression level of STK35L1 transcripts at the indicated time points was analyzed by RT-PCR. The relative expression level compared to time 0 hr is shown. B) and C) STK35L1 regulates the formation of cord-like structures on Matrigel. Control siRNA and STK35L1-siRNA transfected cells were grown on Matrigel for 6 h. B) phase contrast micrograph, Scale bar 500 µm. (C) Bar diagram, values are mean ± S.E. of three independent experiments; * p<0.05.
Figure 6.
STK35L1 is required for endothelial cell migration.
A) Migration assay using the IBIDI insert. Endothelial cells transfected with STK35L1 siRNA (red shaded part) or control siRNA (green shaded part) were seeded into different reservoirs of an IBIDI insert. After 8 hours the insert was removed and the closure of the gap was observed by video microscopy. The pictures from one experiment representative of three are shown, Scale bar 100 µm. B) Migration after mechanical injury. Left panel, representative micrographs, cells treated with control siRNA, or cells treated with STK35L1 siRNA are shown immediately 0 hr or 7 hr after mechanical injury; width of the wound is shown in µm. Right panel, Bar diagram, wound closure was quantified as described in the method section. Values are mean ± S.E. of three independent experiments; ‘*’, p<0.05.