Fig 1: Effects of miR-152 and CDK5R1 were examined in mice. SKES-1 cells (1 × 106) were inoculated into the gluteal region of BALB/c nu/nu mice. After 5 weeks, the mouse tumors were collected and analyzed for further experiments. Tumor volumes in implanted mice (a) were compared in each group (b). The subcutaneous tumors are indicated by red dashed lines. The collected mouse tumor tissues were analyzed by protein expression (c) and immunostaining of CDK5R1 and CCNE (d) per quantification unit area in CDK5R1- and CCNE-positive cells was quantified. The densitometric levels of protein bands were quantified and normalized to that of GAPDH. Original magnification, × 400; Scale bars: 50 μm. Data represent mean ± SEM of three independent experiments. **p < 0.01 versus the related control groups. (e) The schema illustrates that low expression of miR-152 causes accumulation of CDK5R1 and contributes to high expression of CCNE.
Fig 2: Changes in the expression of p35/p25 in the cytoplasm and nucleus, and changes in the expression of CDK5R1 after transfection with miR-152. Effects of activation and inhibition of calpain on p35/p25, and subsequent changes in CDK5 activity in the cytoplasm and nuclear fractions were analyzed by immunoblotting (a) and protein expression quantification (b). The cytoplasmic fraction was normalized using GAPDH, and the nuclear fraction was normalized using Lamin B1 as reference. In the nuclear fraction, p25 was increased by calpain activation and p35 was absent, whereas nuclear p25 was decreased by calpain inhibitor. Cytoplasmic p25 was formed from the cleavage of p35 by calpain activation and inhibition. Effects of miR-152 mimic transfection and CDK5R1 knockdown on p35/p25 and CDK5 in the cytoplasm fractions were analyzed by western blot analysis (c) and band concentration quantification (d). Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies. The Input was normalized using GAPDH, and the proteins immunoprecipitated with CDK5 were normalized to CDK5. Western blotting analysis of p35/p25 levels in SKES-1 cells transfected with miR-152 and CDK5R1 siRNA in nucleus fraction (e). Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies. Quantification (f) of changes in the protein expression of co-precipitated p35/p25 in the nuclear fractions were performed. The Input of nucleus fraction was normalized using Lamin B1, and the proteins immunoprecipitated with CDK5 were normalized to CDK5.
Fig 3: Effects and causes of changes in miR-152 and CDK5R1 expression on cell proliferation. (a) Cell proliferation of SKES-1 and RDES cells transfected with miR-152 mimic or CDK5R1 siRNA were compared with those in the negative control group. Data represent mean ± SEM of three independent experiments. SKES-1 cells transfected negative control-miR (20 nM), miR-152 mimic (20 nM), negative control-siRNA (20 nM), CDK5R1-siRNA (20 nM) and untreated cells, followed by western blot analysis with the indicated antibodies. Images (b) from immunoblot analysis of apoptosis induction after transfection with miR-152 mimic and CDK5R1 siRNA. GAPDH level was used as loading control. (c) Ewing’s sarcoma cells were stained with Annexin V-FITC/PI to assess cell apoptosis. Each quadrants represent viable cells (Lower Left quadrant), early apoptotic cells (Lower Right), late or secondary necrotic cells (Upper Right), and primary necrotic cells (Upper Left), respectively. (d) The viability of SKES-1 and RDES cells was determined using Cell Counting Kit‑8 assays. (e) Cell proliferation was assessed by observing the changes in the expression of Ki-67 and PCNA using qPCR. SKES-1 cell-transfected negative control-miR (20 nM), miR-152 mimic (20 nM), negative control-siRNA (20 nM), CDK5R1-siRNA (20 nM) and untreated cells, followed by flow cytometric analysis with 5-bromo-2 deoxyuridine-7-amino-actinomycin D (BrdU-7-AAD) staining. Changes in cell cycle for each condition were analyzed by the BrdU-7-AAD double staining assay (Supplemental Fig.S3c) and the rate of cell-cycle progression (f). (g) Effect of miR-152 mimic and CDK5R1 knockdown on the viability of SKES-1 cells for their ability to grow in soft agar. The results are represented as the mean ± SEM (n.s., no significance). **p < 0.01 versus the related control groups.
Supplier Page from Abcam for Anti-p35 antibody