Journal: American Journal of Translational Research

Article Title: S-phase kinase-associated protein 2 impairs the inhibitory effects of miR-1236-3p on bladder tumors

doi:

Figure Lengend Snippet: Primers used in this study

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) and then incubated with primary antibodies, including those against Skp2 (1/1000) (Affinity, USA), P21 (1/1000) (BD Biosciences), P27 (1/1000) (Affinity, USA), CDK1 (1/1000) (Boster, China), GAPDH (1/500) (Boster, China) and β-actin (1/500) (Boster, China), overnight at 4°C.

Techniques: Control

Journal: American Journal of Translational Research

Article Title: S-phase kinase-associated protein 2 impairs the inhibitory effects of miR-1236-3p on bladder tumors

doi:

Figure Lengend Snippet: miR-1236 induced Skp2 expression independent of p21 activation and influenced downstream Skp2 gene expression. A. Skp2 mRNA levels were evaluated by qRT-PCR. **P<0.01 compared to control miRNA group. B. Expression of Skp2 protein was further detected by Western blot analysis. Only miR-1236 up-regulated Skp2 expression. C. Expression of p21 and Skp2 mRNA in BCa cells was detected by qRT-PCR. *P<0.05, **P<0.01 compared to control miRNA group. D. Western blotting was conducted to detect the expression of p21 and Skp2 protein in BCa cells. Knockdown with sip21 had no influence on the up-regulation of Skp2 by miR-1236. E. mRNA expression levels of p27 and CDK1 were assessed by qRT-PCR. *P<0.05 compared to the control miRNA group. F. P27 and CDK1 expression levels were detected by Western blot in 5637 and T24 cells. miR-1236 decreased p27 expression and up-regulated CDK1 expression. dsRNA-245 had no significant effect on p27 and CDK1. G. Expression of Skp2, p27 and CDK1 was detected by qRT-PCR. GAPDH served as an internal control. **P<0.01, ***P<0.001 compared with the miR-1236 group. H. Skp2, p27 and CDK1 protein expression levels were detected by Western blot. β-actin levels were used as an internal control. miR-1236 influenced p27 and CDK1 expression through up-regulation of Skp2.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) and then incubated with primary antibodies, including those against Skp2 (1/1000) (Affinity, USA), P21 (1/1000) (BD Biosciences), P27 (1/1000) (Affinity, USA), CDK1 (1/1000) (Boster, China), GAPDH (1/500) (Boster, China) and β-actin (1/500) (Boster, China), overnight at 4°C.

Techniques: Expressing, Activation Assay, Gene Expression, Quantitative RT-PCR, Control, Western Blot, Knockdown

Journal: PeerJ

Article Title: Dieting alleviates hyperuricemia and organ injuries in uricase-deficient rats via down-regulating cell cycle pathway

doi: 10.7717/peerj.15999

Figure Lengend Snippet: (A) Casp3, Cdk1 and Ki67 in the liver was evaluated at mRNA level, and a high value of FPKM means a high expression; (B) Casp3, Cdk1 and Ki67 in the liver was evaluated by Western blot; C, Results of B were quantified by relative brightness. FPKM, Fragments Per Kilobase of exon model per Million mapped fragments. Control, control group, KDY rats were given free approach to food and water; 75% dieting, 75% dieting group, rats were given 75% food of control group at the same age in days and given free access to water. * P < 0.05 vs control group, Student’s t -test, two-tailed.

Article Snippet: Hematoxylin-eosin (HE) staining kits, mouse anti- β -actin antibody (BM0627), rabbit anti-CASP3 antibody (BA2142), rabbit anti-CDK1 antibody (PB9533), rabbit anit-Ki67 antibody (M00254-4), and horseradish peroxidase (HRP) conjugated goat anti-rabbit/mouse IgG (H+L) (BA1056) were all purchased from Boster Biological Engineering Co., Ltd (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Cyclin A–CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry

doi: 10.1016/j.jbc.2023.102957

Figure Lengend Snippet: Depletion of cyclin A but not cyclin B1, CDK1, or CDK2 results in CDC25A accumulation. Specificity of cyclin A depletion–mediated CDC25A accumulation. Cells from AID Cyclin A KO , mAID Cyclin B1 KO , AID CDK1 KO , and AID CDK2 KO were synchronized with a double thymidine block procedure. The cells were then cultured in fresh or DI-containing medium. A and B , after 6 h, the cells were harvested and analyzed with ( A ) immunoblotting and ( B ) flow cytometry. AID, auxin-induced degron; DI, Dox and IAA.

Article Snippet: The following antibodies were obtained from the indicated sources: monoclonal antibodies against beta-actin (Sigma-Aldrich); cyclin A2 (AT10, a gift from Tim Hunt, Cancer Research UK); cyclin B1 (sc-245, Santa Cruz Biotechnology), cyclin E1 (sc-247, Santa Cruz Biotechnology), CDK1 (sc-54, Santa Cruz Biotechnology), CDK2 (sc-6248, Santa Cruz Biotechnology), CDK1(Y15p) (612307, BD Biosciences), cleaved PARP1 (552597, BD Biosciences), CDC25A (sc-7389, Santa Cruz Biotechnology), CDC25B (ab124819, Abcam), CDC25C (sc-13138, Santa Cruz Biotechnology), WEE1 (sc-5285, Santa Cruz Biotechnology), CDC27 (610455, BD Biosciences), EMI1 (37-6600, Zymed Laboratories), PLK1 (sc-17783, Santa Cruz Biotechnology), polyclonal antibodies against phosphorylated histone H3 S10 (sc-8656R, Santa Cruz Biotechnology), MYT1 (sc-13081, Santa Cruz Biotechnology), γ-H2AX (A300-081A, Bethyl Laboratories), and HA (A190-208A, Bethyl Laboratories).

Techniques: Blocking Assay, Cell Culture, Western Blot, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Cyclin A–CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry

doi: 10.1016/j.jbc.2023.102957

Figure Lengend Snippet: Cyclin A regulates CDC25A throughout the cell cycle. A , CDC25A expression starts at late S, accumulates in G 2 , and degrades at the end of mitosis. HeLa cells were synchronized using a double thymidine block procedure. Cells at different cell phases of the cell cycle were harvested at the indicated time points after release from the block. NOC was added to the mitotic sample (M) for 6 h before cells were isolated with mechanical shake-off. A portion of the mitotic cells were released from the block by washing and re-plating in NOC-free medium for 3 h before harvested as G 1 cells. Lysates were prepared and analyzed with immunoblotting. Histone H3 S10 phosphorylation and CDC27 mobility shifts are mitotic markers. Dephosphorylation of CDK1 Y15 occurred as cells entered mitosis. Note that CDC25A was phosphorylated and displayed a gel mobility shift during mitosis. The DNA contents of the cells were also examined with flow cytometry to validate the synchronization ( Fig. S3 A ). B , preventing cyclin A accumulation during late G 1 promotes CDC25A accumulation. AID Cyclin A KO cells were synchronized in mitosis using a NOC block procedure as described in . The cells were then released into NOC-free medium for 3 h and 6 h to obtain cells in early and late G 1 , respectively. The cells were treated with or without DI for 3 h before harvested. Asynchronized cells were included for comparison. Cell-free extracts were prepared and analyzed with immunoblotting. The synchronization was also assessed with flow cytometry ( Fig. S3 B ). C , destruction of cyclin A promotes CDC25A accumulation during S phase. AID Cyclin A KO cells were synchronized at early S phase with a double thymidine block procedure. The cells were then mock- or DI-treated, either after released from the block or continually incubated with thymidine. After 6 h, the cells were harvested and analyzed with immunoblotting. Flow cytometry analysis of the samples are shown in Fig. S3 C . D , loss of cyclin A specifically increases CDC25A but not CDC25B or CDC25C in G 2 cells. After synchronization with a double thymidine block, AID Cyclin A KO cells were released into drug-free or DI-containing medium. After 9 h, G 2 cells were obtained for immunoblotting analysis. Flow cytometry analysis of the samples are shown in Fig. S3 D . E , destruction of cyclin A promotes CDC25A accumulation during both G 2 and mitosis. AID Cyclin A KO cells were synchronized with a double thymidine block and released into drug-free or DI-containing medium. NOC was added after 4 h. G 2 and mitotic cells were isolated as described in panel A . Flow cytometry analysis of the samples are shown in Fig. S3 E . AID, auxin-induced degron; DI, Dox and IAA; NOC, nocodazole.

Article Snippet: The following antibodies were obtained from the indicated sources: monoclonal antibodies against beta-actin (Sigma-Aldrich); cyclin A2 (AT10, a gift from Tim Hunt, Cancer Research UK); cyclin B1 (sc-245, Santa Cruz Biotechnology), cyclin E1 (sc-247, Santa Cruz Biotechnology), CDK1 (sc-54, Santa Cruz Biotechnology), CDK2 (sc-6248, Santa Cruz Biotechnology), CDK1(Y15p) (612307, BD Biosciences), cleaved PARP1 (552597, BD Biosciences), CDC25A (sc-7389, Santa Cruz Biotechnology), CDC25B (ab124819, Abcam), CDC25C (sc-13138, Santa Cruz Biotechnology), WEE1 (sc-5285, Santa Cruz Biotechnology), CDC27 (610455, BD Biosciences), EMI1 (37-6600, Zymed Laboratories), PLK1 (sc-17783, Santa Cruz Biotechnology), polyclonal antibodies against phosphorylated histone H3 S10 (sc-8656R, Santa Cruz Biotechnology), MYT1 (sc-13081, Santa Cruz Biotechnology), γ-H2AX (A300-081A, Bethyl Laboratories), and HA (A190-208A, Bethyl Laboratories).

Techniques: Expressing, Blocking Assay, Isolation, Western Blot, De-Phosphorylation Assay, Mobility Shift, Flow Cytometry, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Cyclin A–CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry

doi: 10.1016/j.jbc.2023.102957

Figure Lengend Snippet: A model of the role of cyclin A–CDK in modulating CDC25A level during G 2 -M. During the G 2 -M transition, members of the CDC25 family antagonize WEE1 (and MYT1) by removing the inhibitory phosphorylation on cyclin B–CDK1 complexes. Cyclin A–CDK complexes are involved in the activation of CDC25B and CDC25C through the PLK1 pathway. We showed that cyclin A–CDK complexes contribute to transcriptional repression of CDC25A during interphase, preventing premature activation of cyclin B–CDK1. Downregulation of cyclin A removes this repression and promotes CDC25A accumulation. We postulate that the excess CDC25A acts as a compensatory mechanism for cyclin A-depleted cells to overcome the G 2 -M barrier by promoting cyclin B–CDK1 activation.

Article Snippet: The following antibodies were obtained from the indicated sources: monoclonal antibodies against beta-actin (Sigma-Aldrich); cyclin A2 (AT10, a gift from Tim Hunt, Cancer Research UK); cyclin B1 (sc-245, Santa Cruz Biotechnology), cyclin E1 (sc-247, Santa Cruz Biotechnology), CDK1 (sc-54, Santa Cruz Biotechnology), CDK2 (sc-6248, Santa Cruz Biotechnology), CDK1(Y15p) (612307, BD Biosciences), cleaved PARP1 (552597, BD Biosciences), CDC25A (sc-7389, Santa Cruz Biotechnology), CDC25B (ab124819, Abcam), CDC25C (sc-13138, Santa Cruz Biotechnology), WEE1 (sc-5285, Santa Cruz Biotechnology), CDC27 (610455, BD Biosciences), EMI1 (37-6600, Zymed Laboratories), PLK1 (sc-17783, Santa Cruz Biotechnology), polyclonal antibodies against phosphorylated histone H3 S10 (sc-8656R, Santa Cruz Biotechnology), MYT1 (sc-13081, Santa Cruz Biotechnology), γ-H2AX (A300-081A, Bethyl Laboratories), and HA (A190-208A, Bethyl Laboratories).

Techniques: Activation Assay

Journal: Cancers

Article Title: Anticancer Effects and Molecular Mechanisms of Apigenin in Cervical Cancer Cells

doi: 10.3390/cancers14071824

Figure Lengend Snippet: Proposed mechanism and signaling pathways of the apoptosis and cell cycle arrest induced by apigenin in cervical cancer cells. Cell cycle G2/M phase-related proteins CDK1, CDC25C, cyclin B1, and p21, and apoptosis-related proteins, Bcl-2 and Bax, were detected in HeLa ( A ) and C33A ( B ) cells with or without 50 μM apigenin treatment for 24 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * p < 0.05 indicates a significant difference as compared with the corresponding control. CON, 0.1% DMSO; Api 50, 50 μM apigenin.

Article Snippet: The membranes were then blocked with 0.5% bovine serum albumin (BSA, A3294, Sigma-Aldrich) in PBS/0.5% Tween 20 (#9005-64-5, Sigma-Aldrich) for 1 h, followed by incubation with primary antibodies (1/800-1/1000; diluted in blocking buffer) overnight at 4 °C: CDK1 (E-AB-64159), CDC25c (E-AB-63512), cyclin B1 (E-AB-70114), p21 cip1 (E-AB-65412), Bcl-2 (E-AB-15522), and Bax (E-AB-30629e) purchased from Elabscience Technology (Houston, TX, USA); phospho-AKT (#4060S), AKT (#9272S), phospho-mTOR (#2971S), and mTOR (#2972S) (Cell Signaling Technology; Danvers, MA, USA); phospho-FAK (GTX129840), FAK (GTX100764), paxillin (GTX129840), and integrin β1 (GTX128839) (GeneTexT Biotechnology; Hsinchu city, Taiwan); PI3K(p85) (ARG55392), fibronectin (ARG66162), N-cadherin (ARG22587), vimentin (ARG66199), laminin (ARG59198), and E-cadherin (ARG66195) (Arigo Biolaboratories Biotechnology; Hsinchu city, Taiwan); phospho-PI3K(p85) (AB182651; Abcam Technology; Cambridge, UK) and GAPDH (MA5-15738; Thermo Fisher Scientific Inc.).

Techniques: Protein-Protein interactions, Western Blot, Control

Journal: Cancers

Article Title: Anticancer Effects and Molecular Mechanisms of Apigenin in Cervical Cancer Cells

doi: 10.3390/cancers14071824

Figure Lengend Snippet: Schematic representation of the anticancer molecular mechanism of apigenin in cervical cancer. Apigenin down-regulated FAK signaling (FAK, paxillin, and integrin β1) and PI3K/AKT signaling (PI3K, AKT, and mTOR), which inactivated or activated various signaling targets, such as Bcl2, Bax, p21 cip1 , CDK1, CDC25c, cyclin B1, fibronectin, N-cadherin, vimentin, laminin, and E-cadherin, leading to mitochondrial-mediated apoptosis, G2/M-phase arrest, and reduced EMT to induce anticancer effects in cervical cancer.

Article Snippet: The membranes were then blocked with 0.5% bovine serum albumin (BSA, A3294, Sigma-Aldrich) in PBS/0.5% Tween 20 (#9005-64-5, Sigma-Aldrich) for 1 h, followed by incubation with primary antibodies (1/800-1/1000; diluted in blocking buffer) overnight at 4 °C: CDK1 (E-AB-64159), CDC25c (E-AB-63512), cyclin B1 (E-AB-70114), p21 cip1 (E-AB-65412), Bcl-2 (E-AB-15522), and Bax (E-AB-30629e) purchased from Elabscience Technology (Houston, TX, USA); phospho-AKT (#4060S), AKT (#9272S), phospho-mTOR (#2971S), and mTOR (#2972S) (Cell Signaling Technology; Danvers, MA, USA); phospho-FAK (GTX129840), FAK (GTX100764), paxillin (GTX129840), and integrin β1 (GTX128839) (GeneTexT Biotechnology; Hsinchu city, Taiwan); PI3K(p85) (ARG55392), fibronectin (ARG66162), N-cadherin (ARG22587), vimentin (ARG66199), laminin (ARG59198), and E-cadherin (ARG66195) (Arigo Biolaboratories Biotechnology; Hsinchu city, Taiwan); phospho-PI3K(p85) (AB182651; Abcam Technology; Cambridge, UK) and GAPDH (MA5-15738; Thermo Fisher Scientific Inc.).

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Ethanol Extract of Root of Prunus persica Inhibited the Growth of Liver Cancer Cell HepG2 by Inducing Cell Cycle Arrest and Migration Suppression

doi: 10.1155/2017/8231936

Figure Lengend Snippet: TSG treatment induced G2/M phase arrest of HepG2 cells. (a) Flow cytometry analysis of DNA content of HepG2 cells subjected to indicated treatment. ((b), (c)) Statistical analysis of cell subpopulation ratios for each phase of cell cycle. ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001. (d) Western blotting analysis to detect the expression of CDK1 and Cdc25c.

Article Snippet: Antibodies used in this study included rabbit anti-Cdc25c polyclonal antibody (Cat# D154112, Sangon Bio-tech, China), rabbit anti-CDK1 polyclonal antibody (Cat# D160158, Sangon Bio-tech, China), rabbit anti-MMP9 polyclonal antibody (Cat# 10375-2-AP, Proteintech, USA), rabbit anti-MMP3 polyclonal antibody (Cat# 17873-1-AP, Proteintech, USA), mouse anti- β -Actin monoclonal antibody (Cat# TA-09, ZSGB-Bio, China), HRP-conjugated goat anti-mouse polyclonal antibody (Cat# ZB2305, ZSGB-Bio, China), and HRP-conjugated goat anti-rabbit polyclonal antibody (Cat# ZB2301, ZSGB-Bio, China).

Techniques: Flow Cytometry, Western Blot, Expressing