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: 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: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry