Journal: The Prostate

Article Title: A CD24-p53 Axis Contributes to African-American Prostate Cancer Disparities

doi: 10.1002/pros.23973

Figure Lengend Snippet: (A) Representative IHC data showed no CD24 staining in normal prostate (left panel) or benign prostatic hyperplasia (BPH, right panel). (B) Representative IHC data showed CD24 staining of prostate cancer cells with no staining of adjacent normal prostate epithelial cells. The red arrows indicate adjacent normal prostate epithelial cells. (C) CD24, mutp53, MDM2, and ARF staining of representative UAB prostate cancer samples. Representative IHC data showed co-expression of CD24 and mutant p53 from the same case. (D) CD24 and mutp53 IHC staining of two representative PCaP TMA prostate cancer samples. mutp53, mutant p53 protein. All experiments were repeated three times.

Article Snippet: Antibodies were human CD24 (ML5, BD Biosciences), p53 (DO-1, Santa Cruz Biotechnology), MDM2 (SMP14, BD Biosciences), and ARF (also p14-ARF, E3X6D, Cell Signaling).

Techniques: Staining, Expressing, Mutagenesis, Immunohistochemistry

Journal: The Prostate

Article Title: A CD24-p53 Axis Contributes to African-American Prostate Cancer Disparities

doi: 10.1002/pros.23973

Figure Lengend Snippet: Ethnic/racial distribution comparisons in gene expressions

Article Snippet: Antibodies were human CD24 (ML5, BD Biosciences), p53 (DO-1, Santa Cruz Biotechnology), MDM2 (SMP14, BD Biosciences), and ARF (also p14-ARF, E3X6D, Cell Signaling).

Techniques: Significance Assay

Journal: The Prostate

Article Title: A CD24-p53 Axis Contributes to African-American Prostate Cancer Disparities

doi: 10.1002/pros.23973

Figure Lengend Snippet: (A-C) Pearson correlations of the H-scores of CD24 relative to mutp53, MDM2, and ARF staining in UAB prostate cancer samples. (D-G) Protein expression levels of CD24, mutp53, MDM2, and ARF for AAs and EAs in the UAB prostate cancers. mutp53, mutant p53 protein; AA, African-American; EA, European-American.

Article Snippet: Antibodies were human CD24 (ML5, BD Biosciences), p53 (DO-1, Santa Cruz Biotechnology), MDM2 (SMP14, BD Biosciences), and ARF (also p14-ARF, E3X6D, Cell Signaling).

Techniques: Staining, Expressing, Mutagenesis

Journal: Cancers

Article Title: CDKN2A -Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome

doi: 10.3390/cancers13205139

Figure Lengend Snippet: IHC/IF staining conditions for antibodies used in this study.

Article Snippet: P14 , mouse , Cell Signaling , 2407S , ST Citrate , 1:50.

Techniques: Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma

doi: 10.1186/s13046-018-0873-5

Figure Lengend Snippet: Genetic alterations in NPC-PDX tumors. ( a ) Copy number variations (CNV) of NPC-PDX tumors versus corresponding patient’s peripheral blood mononuclear cells (PBMC). Genome-wide CNV alterations in four paired PDX tumor samples (ST, LN, LG and LV). CCND1 CNV gain (red arrow) and CDKN2A CNV loss (green arrow) are indicated. ( b ) HE and EB virus-encoded small RNA (EBER) staining of parental NPC tumor with bone metastasis and its derived NPC-PDX. ( c ) CNV profile comparisons of NPC FFPE-Bone and PDX-Bone based on WES and ( d ) ultra-deep sequencing of cancer panel-409 are shown (genes associated with or without copy number alteration are indicated in different colors or in grey, respectively). Observed copy number for each evaluated position is shown on the y-axis as a log 2 scale. Correlation plots with Pearson’s correlation coefficient, r, indicating similarities between two CNV profiles

Article Snippet: Antibodies used in this study: RB1 (CusaBio PA003948), RB-P (Cell Signaling 9307), E2F1 (Santa Cruz SC-193), CDK2 (CusaBio PA001533), CDK4 (Santa Cruz SC-23896), CDK6 (Santa Cruz SC-53638), CCND1 (Santa Cruz SC-8396), CCNE2 (Proteintech 11,935–1-AP), CDKN2A (Prosci 4211), CDKN1A (Santa Cruz SC-6246), PCNA (Proteintech 10,205–2-AP) and GAPDH (Santa Cruz FL-335).

Techniques: Genome Wide, Virus, Staining, Derivative Assay, Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma

doi: 10.1186/s13046-018-0873-5

Figure Lengend Snippet: CCND1 mRNA expression and IHC staining in NPC patients and PDX tumors. ( a ) The expression fold change of candidate genes ( CCND1, CDKN2A and CDKN2B ) are indicated based on the cDNA microarray data of five PDX tissues, and C666–1 (EBV-positive NPC cells) and NP69 (immortalized normal nasopharyngeal cells, as control) cell lines. ( b ) Agarose gel electrophoresis of RT-PCR products of CCND1 in PBMC, two NPC cell lines and five PDXs (GAPDH serves as an internal control). Cyclin D1 IHC staining in ( c ) NPC no.13 patient, with NPC primary site, NPC metastatic to bone, and PDX-Bone tumor and ( d ) NPC no.2 patient, with NPC metastatic to lymph node, and PDX-LN tumor

Article Snippet: Antibodies used in this study: RB1 (CusaBio PA003948), RB-P (Cell Signaling 9307), E2F1 (Santa Cruz SC-193), CDK2 (CusaBio PA001533), CDK4 (Santa Cruz SC-23896), CDK6 (Santa Cruz SC-53638), CCND1 (Santa Cruz SC-8396), CCNE2 (Proteintech 11,935–1-AP), CDKN2A (Prosci 4211), CDKN1A (Santa Cruz SC-6246), PCNA (Proteintech 10,205–2-AP) and GAPDH (Santa Cruz FL-335).

Techniques: Expressing, Immunohistochemistry, Microarray, Control, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma

doi: 10.1186/s13046-018-0873-5

Figure Lengend Snippet: Correlation of CNV of CCND1, CDKN2A and RAD52 with high EBV copy number in 22 NPC plasma. ( a ) CNV of CCND1 , CDKN2A and RAD52 in 22 NPC plasma with high EBV DNA load (> 5000 copies/ml) based on Q-PCR results. ( b ) Correlation plot between CNV of CCND1 , CDKN2A and RAD52 and log EBV DNA load in 22 NPC plasma samples ( c ) Correlation between CNV of the CCND1 / CDKN2A ratio and log EBV load in 22 NPC plasma. Pearson’s correlation coefficient, r, and equations of regression are indicated

Article Snippet: Antibodies used in this study: RB1 (CusaBio PA003948), RB-P (Cell Signaling 9307), E2F1 (Santa Cruz SC-193), CDK2 (CusaBio PA001533), CDK4 (Santa Cruz SC-23896), CDK6 (Santa Cruz SC-53638), CCND1 (Santa Cruz SC-8396), CCNE2 (Proteintech 11,935–1-AP), CDKN2A (Prosci 4211), CDKN1A (Santa Cruz SC-6246), PCNA (Proteintech 10,205–2-AP) and GAPDH (Santa Cruz FL-335).

Techniques: Clinical Proteomics

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: FOXO3 and p16 INK4a oppositely modulate the vulnerability of human HD NSCs. Significance was tested using two‐way ANOVA (panels a–c), paired t test (panels d) and Mann‐Whitney test (panel g). ns: not significant. (A) Human HD NSCs show reduced rates of cell growth. Data are mean ± SEM . (b) Reducing FOXO3 does not alter the growth of C116 NSCs (left panel) and strongly reduces the growth of HD NSCs (right panel), with no change detected in HTT mRNA levels (see Figure , left panel). Data are mean ± SEM . (c) Reducing p16 INK4a slightly increases the growth of C116 (left panel) and HD (right panel) NSCs. Reducing p16 INK4a does not alter HTT mRNA levels in HD NSCs (see Figure , right panel). Data are mean ± SEM . (d) Reducing FOXO3 increases the mortality of HD NSCs with no effect detected in C116 NSCs (left: * p < .05). Reducing p16 INK4a decreases the mortality of HD NSCs with no effect detected in C116 NSCs (right: * p < .05). (e) Lenti‐myc‐ p16 INK4a transduction promotes nuclear release of HMGB1 in cytoplasm of HD and corrected (C116) MSNs. HD and C116 MSNs transduced for 4 days with lenti‐myc‐ p16 INK4a (red) were immunostained with HMGB1 (green). NT: transduction without myc‐ p16 INK4a . HMGB1 co‐localizes with the nucleus (DAPI), with low level in cytoplasm. The transduction with lenti‐myc‐ p16 INK4a significantly relocates HMGB1 into cytoplasm of HD and C116 MSNs (arrowhead). Scale bars: 100 µm. (f) Upper panel: The quantification of cytoplasmic HMGB1 pixel intensity shows a significant increase of nuclear HMGB1 release in HD vs. C116 MSNs and in HD vs. C116 MSNs following p16 INK4a overexpression (Wilcoxon ranked‐sum test: C116‐ p16 INK4a vs. HD‐ p16 INK4a , p = 6.1e‐22; C116‐ p16 INK4a vs. HD‐NT, p = 3.4e‐06; C116‐ p16 INK4a vs. C116‐NT, p = 1.8e‐23; HD‐ p16 INK4a vs. HD‐NT, p = 2.3e‐7; HD‐NT vs. C116‐NT, p = 5.6e‐37). Lower panel: data normalized against Myc‐p16 INK4a levels using the ratio (sum of HMGB1 intensity in ‘cells’ MOI 1/number of cells detected in cells MOI 1)/(sum of myc‐tag intensity in ‘cells’ MOI 1/number of cells detected in ‘cells’ MOI 1). The ratios show that HMGB1 relocalization is CAG‐repeat‐length‐dependent. C116‐p16 INK4a : 1604 cells; C116‐NT: 1403 cells; HD‐p16 INK4a : 879 cells; HD‐NT: 1792 cells

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: MANN-WHITNEY, Transduction, Over Expression

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: Gene expression analyses in human NSCs. The mRNA levels are normalized to cells treated with nontargeting control (NTC) siRNAs (siRNA tests) or to C116 cells or cells without growth factor (GF) deprivation (other experiments). ns, not significant. (a) ETS2 mRNA levels are increased by FOXO3 reduction in HD NSCs subjected to GF deprivation with no effect detected in basal conditions nor in normal HTT cells (left panel: * p < .05). ETS2 mRNA levels are decreased in HD NSCs (middle panel: ** p < .01). GF deprivation does not change ETS2 mRNA levels in C116 and decreases ETS2 mRNA levels in HD NSCs (right panel: * p < .05). (b) p16 INK4a mRNA levels are decreased by ETS2 reduction in HD NSCs in basal conditions and in cells subjected to stress with no effect detected in normal HTT cells (left panel: * p < .05, ** p < .01). p16 INK4a mRNA levels are increased in HD NSCs (middle left panel: *** p < .001). GF deprivation does not change p16 INK4a mRNA levels in C116 NSCs and decrease p16 INK4a mRNA levels in HD NSCs (middle right panel: * p < .05). p16 INK4a mRNA levels tend to be increased by FOXO3 knockdown in HD NSCs subjected to GF deprivation (right panel: not significant with p = .0736). (c) ETS2 and p16 INK4a mRNA levels are decreased by overexpression of FOXO3, but not that of FOXO3‐TM, in human HD NSCs subjected to GF deprivation. The mRNA levels are normalized to cells treated with empty vector. * p < .05 and ** p < .01. (d) p16 INK4a mRNA levels are decreased by ETS1 reduction in C116 NSCs in basal conditions and in HD NSCs in both basal and stress conditions (upper panel: * p < .05, ** p < .01). ETS1 mRNA levels are unchanged in HD compared with C116 NSCs (lower left panel). GF deprivation does not change ETS1 mRNA levels in C116 NSCs and decreases ETS1 mRNA levels in HD NSCs (lower right panel: * p < .05). (d) Working model for effect of FOXO3 target reprogramming on the ETS2‐p16 INK4a pathway

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Expressing, Over Expression, Plasmid Preparation

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: Human HD prepatterned NSCs show increase of p16 INK4a and of SA‐β‐gal activity. (a) p16 INK4a mRNA levels are increased in HD prepatterned NSCs. Data are mean ± SD (** p < .01), N = 3. (b) Representative images for modest p16 INK4a increase in HD NSCs. Scale bar in all panels: 100 µm. (c) Quantification of nuclear p16 INK4a pixel intensity for 532 C116‐NSCs and 1000 HD NSCs. Data are mean ± SD (** p < .01). (d) Representative images for increase of SA‐ß‐gal activity in HD NSCs. Scale bar in all panels: 200 µm. (e) Quantification of SA‐ß‐gal activity for 547 C116‐NSCs and 645 HD NSCs. Data are mean ± SD (**** p < .0001). (f) Frequency distribution of SA‐ß‐gal signals for data shown in panel (e)

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Activity Assay

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: p16 INK4a expression is elevated in human HD MSNs. (a) Representative images of human NSC‐derived MSNs using defined enhanced media (Synaptojuice medium). (b) RT‐PCR analysis of p16 INK4a , FOXO3, and Ryk in C116 and HD MSNs reveals modest increase of FOXO3 mRNA levels and robust increase of p16 INK4a and Ryk mRNA levels in HD MSNs. Data are mean ± SD (* p < .05, *** p < .001). N = 3. (c) Immunofluorescence analysis reveals dramatic increase of p16 INK4a in HD MSNs. Scale bar in all panels: 100 µm. (d) Quantification of nuclear p16 INK4a pixel intensity for N = 596 C116 NSCs and N = 609 HD NSCs. Data are mean ± SD (**** p < .0001). (e) Frequency distribution of nuclear p16 INK4a signals for data shown in Panel (d). (e) RT‐PCR analysis of CDKN2AIP, MMP3, SELL, IGFBP7, EST1, and EST2 show increased mRNA levels in HD MSNs compared with C116 MSNs. Data are mean ± SD (* p < .05, ** p < .01, *** p < .001). N = 3

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

doi: 10.1172/JCI77321

Figure Lengend Snippet: (A) Representative immunofluorescence image of EdU incorporation in human fibroblasts infected with WT or mutant p16 jTALE-DNMT lentivirus. After 72 hours of infection, cells were incubated with EdU for 1 hour and stained for EdU. Cell nuclei are stained blue (DAPI), and EdU-positive nuclei are stained red. Images were taken at ×10 magnification (n = 7). (B) Percent EdU incorporation of cells infected with p16 jTALE-DNMT WT or mutant lentivirus, with (n = 4) or without (n = 7) coinfection of CMV-p16 lentivirus. Three random images were counted for each biological replicate (mean ± SEM). ***P < 0.001; NS, not significant; 2-tailed t test. (C) Population doubling time of human fibroblasts infected with p16 jTALE-DNMT WT or mutant lentivirus (mean ± SEM; n = 4). *P < 0.05, 2-tailed t test. (D) Transcript levels of cell cycle regulators in human fibroblasts transduced with WT p16 jTALE-DNMT lentivirus relative to mutant lentivirus. Expression was normalized to HPRT1 mRNA levels (mean ± SEM; n = 3). Two-tailed t test. (E) Average percent DNA methylation of CpGs at the nearest CpG island of cell cycle regulators evaluated in D. Average DNA methylation was measured by PCR amplification of bisulfite-converted genomic DNA followed by high-throughput sequencing (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001, 2-tailed t test. CKI, cyclin-dependent kinase inhibitor.

Article Snippet: For p16 re-expression experiments, a lentiviral vector incorporating the human p16 cDNA clone under a CMV promoter was purchased from OriGene Technologies Inc. (catalog RC220937L1).

Techniques: Immunofluorescence, Infection, Mutagenesis, Incubation, Staining, Transduction, Expressing, Two Tailed Test, DNA Methylation Assay, Amplification, Next-Generation Sequencing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: Senescence markers in primary human and mouse VSMCs undergoing senescence. ( A ) % EdU + in cultured human VSMCs (Control), after 24h treatment with 500 nM doxorubicin (Dox 1d), after an additional 21 days recovery in control conditions (control 21d) or after doxorubicin (Dox 1d+ 21d), or at replicative senescence (RS). ( B – D ) mRNA levels of Lamin B1, p16, and p21 in cell populations described in ( A ) relative to control (1d) cells. ( E ) Western blot for Lamin B1, p16, p21, and p53 for cells treated in ( A ). n = 6–8 human VSMC isolates. ( F and G ) EdU + % ( F ) or SAβG + % ( G ) of mouse p16-3MR VSMCs treated increasing concentrations of Doxorubicin for 1 day followed by 7 days recovery vs. vehicle control. ( I – K ) qPCR for Lamin B1, IL6, p16, or p21 mRNA expression for cells treated in ( F ). ( L ) Western blot of mouse cells as treated in ( F ) for Lamin B1, p16, or p21. n = 3–8 mouse VSMC isolates. Data are means (SD), one-way ANOVA with correction for multiple comparisons ( A ) or unpaired Student’s t -test vs. Control 1d ( B – D ) or vs. Vehicle (Dox 0 nM) ( F – K ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Cell Culture, Control, Western Blot, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: p16/Cdkn2a is detected in VSMCs in mouse atherosclerotic plaques. ( A and B ) UMAP plots showing scRNA-seq profiles of unsorted aortic cells from Myh11Cre ERt2+ /Confetti + mice ( A ), or sorted Confetti + VSMCs from atherosclerotic plaque and media of fat-fed Myh11Cre ERt2 /Confetti + /ApoE −/− mice ( B ). Log-transformed expression levels of Myh11 and p16/Cdkn2a are shown alongside e-Cadherin/Cdh5 and Pdgfrα ( A ) or Cd68 and Ly6a/Sca1 ( B ) using a scale from white to dark red. Insets show high power regions of clusters 6, 8, and 9 and expression of p16 in ( B ). Feature plots show log-normalized expression levels. ( C ) % cells in each cluster with detectable expression of p16/cdkn2a after 14 weeks or 18 weeks or high-fat feeding, or combined.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Transformation Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: GCV treatment of p16-3MR mice does not affect atherosclerosis, but induces inflammation. ( A ) Aortic root plaques in ApoE→ApoE, p16→ApoE, ApoE→p16, or p16→p16 mice + GCV, or p16→p16 mice + saline, stained with Masson’s trichrome, TUNEL, or Mac3. Scale bar = 300 µm. High power inset shows apoptotic cell and nuclear debris from outlined area. ( B ) Plaque area for mice in ( A ). ( C and D ) Number of TUNEL + cells/aortic root plaque ( C ) or %Mac3 + cells ( D ) for mice in ( A ). ( E – G ) Relative mRNA expression for p16, IL18, or TNFα in experimental mice. Data are means (SD) n = 5–10 mice. One-way ANOVA with correction for multiple comparisons ( B – D ) or Kruskal–Wallis H test followed by Dunn’s multiple comparisons test ( E – G ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Saline, Staining, TUNEL Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 (Navitoclax) selectively reduces senescent VSMCs. ( A and B ) Photomicrographs ( A ) or quantification ( B ) of mouse VSMCs stained for SAβG, as replicating control cells or after dox1 + 7 days treatment, or each group ±1 µM ABT-263 treatment for 48 h. ( C ) Western blot and quantification for p16 in cells treated in ( A and B ). ( D ) Fold change in mRNA expression compared with control replicating cells for p16 and a range of SASP cytokines against the housekeeping gene HMBS. Data are means (SD), n = 4–5. Unpaired Student’s t -test. ( E ) Mouse macrophages cultured for 28 days, then treated with 1 µM ABT-263 for 48 h and stained for SAβG. Data are means (SD), n = 3. Unpaired Student’s t -test.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Staining, Control, Western Blot, Expressing, Cell Culture

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 reduces atherosclerosis, but not local SASP cytokine expression. ( A ) ORO staining of mouse descending aorta treated with control (vehicle) or ABT-263, and quantification of %ORO area ( n = 11–14). Scale bar = 3 mm. ( B ) Masson’s trichrome histochemistry of aortic root atherosclerotic plaque from mice treated in ( A ). Panels below show high power view of outlined area. Arrow shows necrotic core. Scale bar = 200 µm. ( C – E ) Aortic root plaque area/total area ( C ) Cap area ( D ), or Core area ( E ) for mice in ( A ). n = 11–13. ( F ) qPCR for relative expression of p16 or SASP cytokines in aortic arches of experimental mice against the housekeeping gene HMBS ( n = 7). Data are means (SD), n = 10. Unpaired Student’s t -test ( A, C – E ) or Mann–Whitney U test ( F ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Expressing, Staining, Control, MANN-WHITNEY

Journal: EBioMedicine

Article Title: p16 INK4A -expressing mesenchymal stromal cells restore the senescence–clearance–regeneration sequence that is impaired in chronic muscle inflammation

doi: 10.1016/j.ebiom.2019.05.012

Figure Lengend Snippet: PDGFRα-positive fibro-adipogenic progenitors (FAPs) express high levels of p16 INK4A in acute muscle injury. (a and b) Flow cytometry analysis of CD11b, F4/80, and p16 INK4A , and percentages of CD11b + F4/80+ p16 INK4A + cells in both AMI and CIM ( n = 3 per group). (c) Representative image of F4/80- and p16 INK4A - immunostained triceps surae in CIM and AMI (1, 2, and 5 days after injury). (d and e) Representative image of hematoxylin and eosin (H&E) staining and PDGFRα- and p16 INK4A - immunostained triceps surae in CIM and AMI (1, 2, and 5 days after injury) and Control (d), and percentage of p16 INK4A + FAPs in randomly chosen fields of view (n = 3–4 per group) (e). Quantitative data are shown as means ± SEM (dot plot). P-values were determined by one-way ANOVA, adjusted by Tukey's method. (*P < .05, **P < .001).

Article Snippet: CDKN2A knockout was performed using the p14 ARF/p16 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA), which includes green fluorescent protein (GFP) and a pool of three plasmids, each encoding Cas9 nuclease and a target-specific 20-nt guide RNA designed for maximum knockout efficiency.

Techniques: Flow Cytometry, Staining, Control

Journal: EBioMedicine

Article Title: p16 INK4A -expressing mesenchymal stromal cells restore the senescence–clearance–regeneration sequence that is impaired in chronic muscle inflammation

doi: 10.1016/j.ebiom.2019.05.012

Figure Lengend Snippet: Placental extract (PE) induces functional senescence in MSCs. (a) Representative image of SA-β-galactosidase in MSCs with or without PE supplementation in vitro . (b–d) Representative confocal images of MSCs immunostained for OCT4 and p16 INK4A with or without PE supplementation in vitro (b), and quantitation of OCT4- and p16 INK4A -positive area ( n = 5 per group) (c and d). (e–g) Relative mRNA expression of DNMT1 , NANOG , OCT4(POU5F1) , SOX2 , p14 ARF ( CDKN2A ), p16 INK4A ( CDKN2A ), p21 ( CDKN1A ), p53 ( TP53 ), and RB1 , and TERT in MSCs+PE ( n = 7 per group). Dashed line indicates mRNA expression in MSCs without PE supplementation. (h) Hierarchical clustering of stemness-, senescence-, and cytokine-related gene expression in MSCs ( n = 8). Red indicates up-regulation and green indicates down-regulation by PE supplementation. (i) Principal component analysis (PCA) of stemness-, senescence-, and cytokine-related gene expression in MSCs (n = 8), MSCs+PE (n = 8), and continuously cultivated (CC) MSCs ( n = 4). (j–o) Representative image of confocal images of F-actin, Yap, phospho-Yap (p-Yap), and LATS1/2 immunostained MSCs with or without PE supplementation in vitro , and quantitation of F-actin–, Yap-, phospho-Yap (p-Yap)–, and LATS1/2-positive areas (n = 5 per group). Quantitative data are shown as means ± SEM (dot plot) or means and medians with interquartile range (IQR) and 1.5× IQR (box-and-whisker plot). P-values were determined by two-tailed Student's t -test. (*P < .05, **P < .001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: CDKN2A knockout was performed using the p14 ARF/p16 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA), which includes green fluorescent protein (GFP) and a pool of three plasmids, each encoding Cas9 nuclease and a target-specific 20-nt guide RNA designed for maximum knockout efficiency.

Techniques: Functional Assay, In Vitro, Quantitation Assay, Expressing, Gene Expression, Whisker Assay, Two Tailed Test

Journal: EBioMedicine

Article Title: p16 INK4A -expressing mesenchymal stromal cells restore the senescence–clearance–regeneration sequence that is impaired in chronic muscle inflammation

doi: 10.1016/j.ebiom.2019.05.012

Figure Lengend Snippet: MSCs + PE exert therapeutic effects on CIM muscle via immuno-activation. (a) Protocol for treatment of CIM mice using MSCs with or without PE supplementation. (b) Detection of the macrophage phenotype switch in CIM 1, 2, and 5 days after MSC treatment. (c-d) Percentage of CD11b + F4/80+ (total) macrophages (c), CD11b + F4/80+ Ly6Chi pro-inflammatory macrophages (d), and CD11b + F4/80+ Ly6Clow pro-fibrosis macrophages (d) (n = 4 per group). (e) Representative confocal images of F4/80- and p16 INK4A -immunostained triceps surae in CIM 1, 2, and 3 days after injection of PKH-labelled MSCs. (f–j) Flow cytometry analysis of CD11b and Gr-1 (Neutrophil) (f), and CD11b and CD49 (NK cell) (i), and quantification following MSC treatment (g, h, and j). (k) Representative confocal images of CD49b- and p16 INK4A -immunostained triceps surae in CIM 5 days after injection of PKH-labelled MSCs. Quantitative data are shown as means ± SEM (dot plot). P-values were determined by one-way ANOVA adjusted by Tukey's method. (*P < .05, **P < .001).

Article Snippet: CDKN2A knockout was performed using the p14 ARF/p16 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA), which includes green fluorescent protein (GFP) and a pool of three plasmids, each encoding Cas9 nuclease and a target-specific 20-nt guide RNA designed for maximum knockout efficiency.

Techniques: Activation Assay, Injection, Flow Cytometry

Journal: EBioMedicine

Article Title: p16 INK4A -expressing mesenchymal stromal cells restore the senescence–clearance–regeneration sequence that is impaired in chronic muscle inflammation

doi: 10.1016/j.ebiom.2019.05.012

Figure Lengend Snippet: MSCs + PE induce senescence in resident FAPs and improve muscle function. (a) Representative image of PDGFRα and p16 INK4A staining of PKH-labelled MSC–transplanted muscle, 1, 2, and 5 days after MSC transplantation. (b and c) Representative image of H&E staining and quantification of the number of regenerating myofibers with central nuclei. (d-f) Representative image of laminin-immunostained triceps surae in CIM 13 days after MSC transplantation (d), quantification of cross-sectional area of each myofiber (e), and muscle function as evaluated by hindlimb grip strength test (f). Quantitative data are shown as means ± SEM (dot plot). P-values were determined by two-tailed Student's t-test. (*P < .05, **P < .001).

Article Snippet: CDKN2A knockout was performed using the p14 ARF/p16 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA), which includes green fluorescent protein (GFP) and a pool of three plasmids, each encoding Cas9 nuclease and a target-specific 20-nt guide RNA designed for maximum knockout efficiency.

Techniques: Staining, Transplantation Assay, Two Tailed Test