Journal: Cell Cycle

Article Title: Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent

doi: 10.1080/15384101.2017.1339850

Figure Lengend Snippet: CD45NEG cells isolated from alginate capsules are highly p16-luciferase positive. (A) Representative bioluminescent in vivo imaging of 20-week old p16Ink4a/Luc mice pre-implantation (top panel) and post-implantation (lower panel) of alginate-embedded NDFs. The scale depicts relative luminescent signal intensity of minimum and maximum thresholds, displayed in terms of radiance. (B) Representative brightfield images of alginate capsules containing embedded irradiation-induced senescent NDFs before implantation (left panel) and post-implantation at the indicated time points. Bar, 0.1mm. (C) Representative brightfield images of alginate beads containing embedded with NDFs (as in A) subjected to TrypLE and shaking on a Thermoshaker for 10, 20 and 30 minutes at 37°C. Bar, 0.1mm. (D) Cell composition analysis of isolated capsule-bound cells (CBCs) from p16Ink4a/Luc mice by flow cytometry on live cells immunostained for surface markers. The percent contribution to major cell types is depicted: eosinophils (Eos), macrophages (Mac) and remaining cell populations, including B lymphocytes (Other) and CD45NEG cells. Analysis depicts a representative experiment. (E) Cell lysates from whole lavage or CBCs (isolated as in A) were normalized to cell number and assayed for luciferase activity. Values were and normalized to cell number. Standard deviations were calculated from triplicates, and an unpaired Student's t-test was used for statistical analysis. ** indicates p-value of <0.001. (F) Cell sorted populations from p16Ink4a/Luc mice were assayed for luciferase activity. Values were normalized to cell number. Standard deviations were calculated from triplicates, and an unpaired Student's t-test was used for statistical analysis. ** indicates p-value of <0.001. (G) CD45NEG and CD45POS cell sorted populations from p16Ink4a/Luc mice were assayed for luciferase activity. Values were normalized to cell number. Standard deviations were calculated from triplicates, and an unpaired Student's t-test was used for statistical analysis. ** indicates p-value of <0.001. (H) Adipose-derived mesenchymal stromal cells (MSCs) isolated from p16Ink4a/Luc mice were assayed for luciferase activity following 10-days post treatment with IRR (20 Gy) or in the absence of IRR treatment, respectively. Values were normalized to cell number. Standard deviations were calculated from triplicates, and an unpaired Student's t-test was used for statistical analysis. ** indicates p-value of <0.001. (I) Quantification of the percentage of SA-βGal-positive MSCs treated with or without IRR (as in A). Standard deviations were calculated from triplicates, and an unpaired Student's t-test was used for statistical analysis. ** indicates p-value of <0.001. (J) Quantification of the percentage of EdU-positive MSCs treated with or without IRR (as in A). Standard deviations were calculated from triplicates, and an unpaired Student's t-test was used for statistical analysis. ** indicates p-value of <0.001.

Article Snippet: After blocking for 30 min at room temperature with 5% (wt/vol) nonfat dry milk in TBS-T, membranes were incubated in TBS-T for 1 hour at room temperature with the following primary antibodies: rabbit polyclonal anti-p16(Ink4a) antibody (ProteinTech, 10883–1-AP), mouse monoclonal anti-p16(Ink4a) antibody (Cell Applications, CP10342) and GAPDH (Cell Signaling, D16H11).

Techniques: Isolation, Capsules, Luciferase, In Vivo Imaging, Irradiation, Flow Cytometry, Activity Assay, Derivative Assay

Journal: Oncology letters

Article Title: P16 INK4A is required for cisplatin resistance in cervical carcinoma SiHa cells.

doi: 10.3892/ol.2014.2814

Figure Lengend Snippet: Figure 2. P16INK4A‑CDK4 interaction is upregulated in SiHa‑DDP cells with pRb inactivation. (A) Western blot analysis revealed the changes in P16, cyclin D1 and pRb protein expression, indicating an inverse cor relation with P16 and cyclin D1, pRb in DDP‑resistant SiHa‑DPP cells. (B) Co‑immunoprecipitation was conducted to evaluate the enhanced interaction between P16 and CDK4 in SiHa‑DDP cells when compared with SiHa cells. DDP, cisplatin; Rb, retinoblastoma protein; pRb, phosphorylated retinoblastoma protein; CDK, cyclin‑dependent kinase.

Article Snippet: The membranes were then incubated with human anti-mouse monoclonal p16 antibody (Sigma-Aldrich, St. Louis, MO, USA), human anti-mouse polyclonal pRb and β-actin antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), human anti-rabbit monoclonal CDK4 antibody (Cell Signaling Technology, Inc.) overnight at 4 ̊C.

Techniques: Western Blot, Expressing

Journal: Oncology reports

Article Title: Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene.

doi: 10.3892/or.2016.4935

Figure Lengend Snippet: Figure 3. PD induced cell cycle arrest in the G0/G1 phase in MDA-MB-231 cells. (A) Cells were exposed to 2.5, 5, 10 or 20 µM of PD for 48 h and the cell cycle progression was assessed by flow cytometry. The 5, 10 and 20 µM concentrations of PD induced significant cell cycle arrest in the G0/G1 phase. (B and C) The expression levels of G0/G1 phase-related proteins was analyzed by western blotting after treatment with various concentrations of PD for 24 h. PD down regulated the expression of CDK2, CDK4, CDK6, and Cyclin E. (*p<0.05).

Article Snippet: The antibodies against human CDK2, CDK4, CDK6, and Cyclin E used in the western blot analyses were obtained from Boster Biotechnology (Wuhan, China).

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Oncology reports

Article Title: Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene.

doi: 10.3892/or.2016.4935

Figure Lengend Snippet: Figure 9. The effects of PD on the expression levels of various proteins in MDA-MB-231 xenograft tumors. Western blotting was performed to assess the expression levels of proteins in the MDM2-MDMX-p53 pathway, down- stream proteins and G0/G1 phase-related proteins in mice after treatment with the three concentrations of PD for four weeks. (A) PD downregulated the expression of the MDM2, MDMX, and mutant p53 proteins. (B) PD upregulated the expression of p21 and p27. (C) PD decreased the expression of G0/G1 phase-related proteins (CDK2, CDK4, CDK6 and Cyclin E).

Article Snippet: The antibodies against human CDK2, CDK4, CDK6, and Cyclin E used in the western blot analyses were obtained from Boster Biotechnology (Wuhan, China).

Techniques: Expressing, Western Blot, Mutagenesis

Journal: Translational Cancer Research

Article Title: miR-30b suppresses the progression of breast cancer through inhibition of the PI3K/Akt signaling pathway by targeting Derlin-1

doi: 10.21037/tcr.2019.01.21

Figure Lengend Snippet: Overexpression of miR-30b inhibits cell proliferation and viability of SKBR3 and MDA-MB-231 cells. (A) Relative expression of miR-30b in breast cancer cell lines SKBR3 (left) and MDA-MB-231 (right) cells transfected with miR-30b or miR-NC; (B,C) after transfection for 24 hours, the CCK8 assay showed that overexpression of miR-30b inhibited proliferation of SKBR3 (B) and MDA-MB-231 (C) cells; (D) colony formation assay showed that overexpression of miR-30b inhibited cell growth and viability of SKBR3 and MDA-MB-231 cells; (E) after transfection for 24 hours, overexpression of miR-30b reduced the expression of CDK4 and CDK6 in SKBR3 and MDA-MB-231 cells. *, P<0.05 compared with NC. NC, negative control.

Article Snippet: The primary antibodies anti-CDK4 (Cat # 11026-1-AP, Proteintech Group Inc., Rosemont, IL, USA), anti-CDK6 (Cat # 14052-1-AP, Proteintech Group Inc.), anti-Active Caspase-3 p17-specific (Cat # 25546-1-AP, Proteintech Group Inc.), anti-Notch1 (Cat # 10062-2-AP, Proteintech Group Inc.), anti-Bcl-2 (Cat # ab32124, Abcam, Cambridge, United Kingdom), anti-Bax (Cat # ab32503, Abcam), anti-Caspase-9 (Cat # ab32539, Abcam), anti-p-Akt (Cat # ab81283, Abcam), anti-Akt (Cat # ab32505, Abcam), anti-mTOR (Cat # ab2732, Abcam), anti-p-mTOR (Cat # ab131538, Abcam), anti-P70S6K (Cat # ab32529, Abcam), anti-Cyclin D1 (Cat # ab40754, Abcam), anti-Derlin-1 (Cat # SAB4200148, Sigma) and anti-GAPDH (Cat # ab8245, Abcam) were added to the PVDF membrane in blocking solution and incubated at 4 °C overnight.

Techniques: Over Expression, Expressing, Transfection, CCK-8 Assay, Colony Assay, Negative Control

Journal: Cells

Article Title: Artesunate Inhibits the Cell Growth in Colorectal Cancer by Promoting ROS-Dependent Cell Senescence and Autophagy

doi: 10.3390/cells11162472

Figure Lengend Snippet: Artesunate caused excessive mitochondrial ROS to induce cell senescence and inhibit cell proliferation. ( A ) Artesunate arrested cell cycle at G0/G1 phase in SW480 and HCT116. Cells were seeded in 6-well plates. Once attached, cells were maintained in FBS-free medium for about 16~24 h. Afterwards, cell were treated with artesunate (1, 2, and 4 μM in medium containing 10% FBS) for 72 h and then harvested for PI staining to analyze cell cycle by flow cytometry. ( B ) Artesunate induced cell senescence in SW480 and HCT116. Cells were seeded in 6-well plates and treated with artesunate (1, 2, and 4 μM) for 72 h. Cell senescence was represented via SA-β-gal activity, which was assayed using a SA-β-gal staining kit after artesunate treatment. ( C ) Artesunate treatment downregulated the protein levels of CDK 2/4/6 and upregulated the protein levels of CDKIs, p16, and p21 in SW480 and HCT116. Cells were seeded in 60 mm dishes and treated with artesunate (1, 2, and 4 μM) for 72 h. Then, cells were lysed with RIPA to extract total protein. The protein levels were measured by western blotting. The gray values of protein blots were evaluated by Image J. Relative protein expression was normalized to β-actin. ( D ) NAC attenuated the effect of artesunate on protein expression of p16 in SW480 and HCT116. NAC was used at a concentration of 2 mM and added alone or together with artesunate (4 μM) for 72 h. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ctrl. ### p < 0.001 vs. cells treated with artesunate alone. Data were shown as mean ± SD.

Article Snippet: CDK2, CDK4, CDK6, Cyclin D1, Cyclin E1, p21, Rb, phosphorylated-Rb, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP, Bax, Bcl-2, BIP, PERK, IRE1α, CHOP, DR5, Beclin 1, LC3A/B, Atg3, Atg5, Atg7, and Atg12 primary antibodies for western blotting were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). p16 and phosphor-IRE1α primary antibodies for western blotting were purchased from Abcam (Cambridge, UK). p16, p21, and Cyclin D1 primary antibodies for immunohistochemistry and β-actin, XBP1S, and XBP1U primary antibodies for western blotting were purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Staining, Flow Cytometry, Activity Assay, Western Blot, Expressing, Concentration Assay

Journal: Cells

Article Title: Artesunate Inhibits the Cell Growth in Colorectal Cancer by Promoting ROS-Dependent Cell Senescence and Autophagy

doi: 10.3390/cells11162472

Figure Lengend Snippet: Artesunate inhibited the growth of CT26-derived tumor in vivo. ( A ) Experimental timeline of CT26-derived tumor model in balb/c mice. Balb/c mice were injected CT-26 cells (1 × 10 5 cells for each mouse) subcutaneously to establish the CT26-derived tumor model. Tumor-loaded mice were gavaged with artesunate at 30 mg/kg or 60 mg/kg for 24 days. Body weights and tumor volumes were recorded every three days. ( B ) Curve of tumor volume. ( C ) Tumor weight. Tumor tissues were collected and weighted after treatment. ( D ) Immunohistochemical images of Ki67, Cyclin D1, p16, p21, LC3B, and p-IRE1α. Immunohistochemistry assay was performed using a SABC-POD staining kit. ( E ) Curve of body weight. ( F ) Organ indexes. Lung, heart, spleen, liver, and kidney were collected and weighted to calculate the organ indexes after treatment. * p < 0.05, *** p < 0.001 vs. Model group. Data were shown as mean ± SD.

Article Snippet: CDK2, CDK4, CDK6, Cyclin D1, Cyclin E1, p21, Rb, phosphorylated-Rb, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP, Bax, Bcl-2, BIP, PERK, IRE1α, CHOP, DR5, Beclin 1, LC3A/B, Atg3, Atg5, Atg7, and Atg12 primary antibodies for western blotting were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). p16 and phosphor-IRE1α primary antibodies for western blotting were purchased from Abcam (Cambridge, UK). p16, p21, and Cyclin D1 primary antibodies for immunohistochemistry and β-actin, XBP1S, and XBP1U primary antibodies for western blotting were purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Derivative Assay, In Vivo, Injection, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: Screening of anti-phosphoT172 CDK4 mAbs using immobilized cyclin D3/CDK4 proteic fusions.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques:

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: Sera evaluation by CDK4 peptide ELISA.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques: Peptide ELISA

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: Evaluation of CDK4 T172-phosphospecificity of immune sera.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques:

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: CDK4 T172-phosphospecificity of cloned mAbs.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques: Clone Assay

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: T172-phosphospecific CDK4 sandwich ELISA.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques: Sandwich ELISA

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: S130-phosphorylated p21 is enriched in phospho-CDK4 immunoprecipitation.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation

Journal: Cell Cycle

Article Title: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

doi: 10.1080/15384101.2021.1984663

Figure Lengend Snippet: S10-phosphorylated p27 is enriched in phospho-CDK4 immunoprecipitation.

Article Snippet: Equal amounts of whole cell extract protein were separated according to molecular mass and immunodetected using the following antibodies: monoclonal mouse antibodies against cyclin D3 (DCS-22) (Neomarkers/ThermoFisher); cyclin D1 (DCS-6), p16 (DCS-50), CDK4 (DCS-31) (Santa Cruz Biotechnology); p21, p27, CDK4 antibodies (respectively 12D1, D69C12, D9G3E, Cell Signaling Technology); or rabbit polyclonal (H22) CDK4 and anti-phospho-p27 (S10) antibodies (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation