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

Journal: The Journal of pathology

Article Title: Machine learning models predict the primary sites of head and neck squamous cell carcinoma metastases based on DNA methylation.

doi: 10.1002/path.5845

Figure Lengend Snippet: Figure 1. Histopathology and anatomy of HNSC. (A) H&E and (B) p16 immunohistochemistry images of an HNSC lymph node metastasis from the validation set. (C) H&E and (D) p16 immunohistochemistry images of the corresponding primary T1 tumor located in the tonsil. Scale bars, 50 μm. (E) Illustration of the workflow for the developed classifiers, including the relevant anatomical subregions of the head and neck.

Article Snippet: The D7C1M p16 antibody (Cell Signaling Technology, Danvers, MA, USA) was used diluted 1:1000.

Techniques: Histopathology, Immunohistochemistry, Biomarker Discovery

Journal: The Journal of pathology

Article Title: Machine learning models predict the primary sites of head and neck squamous cell carcinoma metastases based on DNA methylation.

doi: 10.1002/path.5845

Figure Lengend Snippet: Figure 3. Classification results on the validation cohort of HNSC lung and lymph node metastases. (A) Two-dimensional representation of the full dataset (n = 469) based on a t-SNE computed from the DNA methylation profiles of its samples. The full dataset contains the reference cohort (n = 405) of primary HNSC tumors and the validation cohort (n = 64) of HNSC metastases in lung and lymph nodes. Samples from the reference cohort are displayed in transparent colors, and samples from the validation cohort in opaque colors. Individual samples are color-coded according to the region of origin as used in the classification. (B) Heatmap of the validation cohort (n = 64) showing the anno- tated region of origin, the predictions of the four classifiers, the total number of wrong predictions across the classifiers, the p16 status, the location of the metastases, and an estimation of tumor purity for every sample. (C) Confusion matrices for the result of the four classifiers on the validation cohort (n = 64) showing the relationship between the annotated and the predicted regions of origin. The main numbers show absolute counts of the respective cases; row and column percentages are displayed on the right and at the bottom of each square. The row and column percentages of the diagonal entries are the sensitivity and positive predictive value of the corresponding class, respectively. The overall accuracy of each classifier on the validation cohort is given below the confusion matrix.

Article Snippet: The D7C1M p16 antibody (Cell Signaling Technology, Danvers, MA, USA) was used diluted 1:1000.

Techniques: Biomarker Discovery, DNA Methylation Assay

Journal: bioRxiv

Article Title: Progressive Senescence Programs Induce Intrinsic Vulnerability to Aging-related Breast Cancer

doi: 10.1101/2023.05.08.539818

Figure Lengend Snippet: (A) Diagram showing the workflow of the transcription factors enrichment analysis for each cell state using the ENCODE, ChEA, and TRRUST database. Reliable candidates of transcription factors regulating each state were supported by at least two databases. (B) Table showing enriched transcription factors based on differentially expressed genes in each cell state. Black lines indicate TFs enrichment in two databases. (C) Activity score of transcription factors enriched in e-Sen state presented by target gene index over the pseudotime. We used the differentially expressed genes in each cell state, and inferred the transcription factors with the CHIP-seq target database. If the targets of certain TF factor are enriched in specific cell state, the TF factor will be popped out as a signature factor. (D) Representative β-gal staining images of mammary gland from young (3 month), aged (17 month) and K14 - Cre Bcl11b fl/fl (3 month) mice showing senescent cells. Scale bar, 20μm. (E) Percentage of p16 Ink4a (scale bar, 20μm) positive cells in mammary epithelial cells from young (4 month), old (22 month), control K14 - Cre Bcl11b wt/wt (4 month) and K14 - Cre Bcl11b fl/fl (4 month) mice. Statistical analysis was performed using two-tailed unpaired t-tests; data are presented as mean ± SD; ***P<0.001, ****P<0.0001. (F) Relative basal luminal proportion of mammary gland epithelia in young (n=54) and old (n=13) mice. Statistical significance was determined by two-tailed unpaired t-tests; data are presented as mean ± SD; ****P<0.0001. (G) Quantification of relative basal/luminal proportion in mammary epithelia in K14 - Cre Bcl11b fl/fl mTmG reporter mice (n=9). GFP+ cells were regarded as Bcl11b KO cells while tdTomato+ cells were regarded as WT cell control in the same gland. Statistical significance was determined by two-tailed unpaired t-tests; data are presented as mean ± SD; ****P<0.0001. (H-I) Density map (H) and percentage (I) of mammary cell states in each age group and K14 - Cre Bcl11b fl/fl group. (J) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showing pathways significantly enriched in K14 - Cre Bcl11b fl/fl CD49f high EpCAM low cells. (K) Boxplots showing the scEntropy score of K14 - Cre Bcl11b fl/fl CD49f high EpCAM low cells increased to a level similar to l-Sen cells. The interquartile (box limits) and median (center blank line). (L) Comparison of SASP gene score in WT and K14 - Cre Bcl11b fl/fl CD49f high EpCAM low cells. The box plots show the interquartile (box limits) and median (center blank line). Statistical analysis was performed using wilcoxon test.

Article Snippet: Sections were blocked with TBS+0.1% Triton X-100+2% BSA+10% Donkey serum for 1 hr at RT, and then stained with primary antibody mouse anti-p16 Ink4a (Santa Cruz, sc-1661), Rb anti-p65 (Cell signaling technology, 8242), Rat anti-Bcl11b (Abcam, ab18465) 1:200 overnight at 4°C.

Techniques: Activity Assay, ChIP-sequencing, Staining, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Progressive Senescence Programs Induce Intrinsic Vulnerability to Aging-related Breast Cancer

doi: 10.1101/2023.05.08.539818

Figure Lengend Snippet: (A) Representative immunofluorescence staining of p16 Ink4a (scale bar, 20μm) in mammary epithelial cells from young (4 month), old (22 month), control K14 - Cre Bcl11b wt/wt (4 month) and K14 - Cre Bcl11b fl/fl (4 month) mice. (B) Representative FACS plots of basal cells and luminal cells in young (2m) and old (20m) mammary gland lineage-population. (C) FASC analysis of mammary epithelia in K14 - Cre Bcl11b fl/fl mTmG mice showing the relative proportion of basal cells and luminal cells. (D) Representative wholemount staining and duct width quantifications of young (2m-5m) and aged (12m-29m) mammary glands. Scale bar, 500μm; statistical analysis was performed using two-tailed unpaired t-tests; data are presented as mean ± SD; ****P<0.0001. (E) Schematic diagram for the workflow of the ageing phenotype quantification for WT and K14 - Cre Bcl11b fl/fl mammary cells after multiple rounds of reproduction cycles. Basically, WT and K14 - Cre Bcl11b fl/fl mammary cells were transplanted onto cleared fat pads. Recipient mice were mated with male mice for 3 rounds. Whole mount and colony formation assays were performed after the last round of pregnancy. (F-H) Representative pictures of mammary wholemounts (F; Scale bar, 500μm), HE staining (G; Scale bar, 200μm) and colony formation (H; Scale bar, 1000μm) of WT and K14 - Cre Bcl11b fl/fl transplanted mammary glands after multiple rounds of pregnancy. Statistical analysis was performed using two-tailed unpaired t-tests; data are presented as mean ± SD; *P<0.05. (I) ELDA plot of limiting dilution transplant of young (3m) and old (18m) mammary gland lineage-cells. P<0.0001. (J) Table for limiting dilution transplant of young (3month) and old (18 month) mammary gland lineage-cells.

Article Snippet: Sections were blocked with TBS+0.1% Triton X-100+2% BSA+10% Donkey serum for 1 hr at RT, and then stained with primary antibody mouse anti-p16 Ink4a (Santa Cruz, sc-1661), Rb anti-p65 (Cell signaling technology, 8242), Rat anti-Bcl11b (Abcam, ab18465) 1:200 overnight at 4°C.

Techniques: Immunofluorescence, Staining, Control, Two Tailed Test

Journal: Biomolecules

Article Title: p19 Arf Exacerbates Cigarette Smoke-Induced Pulmonary Dysfunction

doi: 10.3390/biom10030462

Figure Lengend Snippet: Cigarette smoke (CS) increased Arf/Ink4a levels in ARF-DTR mice. ( a ) Experimental schedule. Four-month-old ARF-DTR mice were exposed to CS for 4 weeks. Diphtheria toxin (DT) or phosphate-buffered saline (PBS) was intraperitoneally administered twice with a 2-week interval. ( b ) An in vivo imaging analysis was performed 4 weeks after the CS exposure. Representative images are shown. ( c , d ) The expression of ( c ) Arf and ( d ) Ink4a mRNA was analyzed by real-time PCR in the lung tissue of ARF-DTR or wild-type mice. Data were normalized to Gapdh in each group. ( e ) Representative images of immunostained lung sections of control (Air), CS and DT/CS ARF-DTR mice using epithelial cell marker EpCAM (green) and ARF-DTR mice using p19 A rf (red) antibodies. Sections were counterstained with DAPI (blue). Bar; 40 μm. Arrowheads indicate p19 Arf -expressing cells. Dotted line (CS, merge) indicates magnified area (CS, bottom). ( f ) The number of p19 Arf -positive epithelial cells in lung tissues of control, CS, DT/CS ARF-DTR mice was counted. ( g ) Representative images of CS lung section immunostained using p16 Ink4a (green) and p19 Arf (red) antibodies. Section was counterstained with DAPI. Arrowheads indicate p19 Arf and p16 Ink4a -expressing cells. Bar; 10 μm. In ( c ), ( d ) and ( f ), bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. Student’s t -test was performed for the analysis of wild-type mice data and no significance was observed. ** p < 0.01 and *** p < 0.001.

Article Snippet: The section were immunostained with rabbit polyclonal antibody against p19 Arf (1:200 dilution, cat# ab80, Abcam, Cambridge, UK), rat monoclonal antibody against p19 Arf (1:100 dilution, cat# sc-32748, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal antibody against p16 Ink4a (1:100 dilution, cat# sc-1207, Santa Cruz Biotechnology, Dallas, TX, USA) and rat monoclonal antibody against EpCAM (1:200 dilution, cat# 552370, BD Biosciences, San Jose, CA, USA) overnight at 4 °C.

Techniques: Saline, In Vivo Imaging, Expressing, Real-time Polymerase Chain Reaction, Control, Marker

Journal: Biomolecules

Article Title: p19 Arf Exacerbates Cigarette Smoke-Induced Pulmonary Dysfunction

doi: 10.3390/biom10030462

Figure Lengend Snippet: Cigarette smoke extract (CSE) inhalation model. ( a ) Experimental schedule. CSE or PBS was intranasally administered 3 times per week for 8 weeks to 4-month-old ARF-DTR or wild-type mice. DT was intraperitoneally injected twice with a 2-week interval. ( b ) Representative images of an in vivo imaging analysis of luciferase expression. ( c , d ) The expression of ( c ) Arf and ( d ) Ink4a mRNA was analyzed by real-time PCR in the lung tissue of ARF-DTR or wild-type mice. Data were normalized to Gapdh in each group. Bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. Student’s t -test was performed for the analysis of wild-type mice data and no significance was observed. * p < 0.05 and ** p < 0.01.

Article Snippet: The section were immunostained with rabbit polyclonal antibody against p19 Arf (1:200 dilution, cat# ab80, Abcam, Cambridge, UK), rat monoclonal antibody against p19 Arf (1:100 dilution, cat# sc-32748, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal antibody against p16 Ink4a (1:100 dilution, cat# sc-1207, Santa Cruz Biotechnology, Dallas, TX, USA) and rat monoclonal antibody against EpCAM (1:200 dilution, cat# 552370, BD Biosciences, San Jose, CA, USA) overnight at 4 °C.

Techniques: Injection, In Vivo Imaging, Luciferase, Expressing, Real-time Polymerase Chain Reaction