Journal: Molecular and Cellular Biology

Article Title: Selection against PUMA Gene Expression in Myc-Driven B-Cell Lymphomagenesis

doi: 10.1128/mcb.00907-07

Figure Lengend Snippet: FIG. 3. The p53 tumor suppressor pathway is bypassed in Puma-deficient lymphomas. E-Myc transgenic lymphomas from Puma/ (n 5), Puma/ (n 9), and Puma/ (n 8) mice were analyzed for changes in p53 and p19Arf gene expression. (A) Southern blot analysis. (B) Western blot analysis. Lymphoma samples with chromosomal alterations in Arf (no. 4, 12, 15, and 19) or mutations in p53 (no. 5, 8, and 22) are highlighted by asterisks.

Article Snippet: Blots were probed with antibodies to the following diluted in a 1% milk TBS-T solution: Puma N terminus (Sigma), p53 (pAb122; Becton Dickinson [BD]), AB7 (Calbiochem), -actin (AC-15; Sigma), Bax (N20; Santa Cruz), Bcl-XL (clone 44; BD), Bcl-2 (clone 7; BD), Bim-EL (22-40; Calbiochem), Mcl-1 (Rockland Labs), and p19Arf (SC32748; Santa Cruz).

Techniques: Transgenic Assay, Gene Expression, Southern Blot, Western Blot

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: ADMET & DMPK

Article Title: Anti-inflammatory potential of plant-derived extracellular vesicles from Solanum nigrum L. integrated in gelatine-dopamine hydrogel on RAW 264.7 and MC3T3 cells

doi: 10.5599/admet.3149

Figure Lengend Snippet: Interleukin-6 (IL-6) concentration by (A) RAW 264.7 and (B) MC3T3 cells LPS induction and PDEV (2.5, 5 and 10 μg mL -1 ) treatment released from Gel-Dop hydrogel. Data presented as mean ± standard deviation (*** p < 0.001, **** p <0,0001)

Article Snippet: IL-6 concentration was measured using a mouse IL-6 ELISA kit (BT Lab) according to the kit protocol.

Techniques: Concentration Assay, Standard Deviation

Journal: JCI Insight

Article Title: BPDCN MYB fusions regulate cell cycle genes, impair differentiation, and induce myeloid–dendritic cell leukemia

doi: 10.1172/jci.insight.183889

Figure Lengend Snippet: ( A ) V5 and IgG CUT&RUN tracks at the Il3ra , Bcl2 , and Myc loci in Cdkn2a -KO MYB::PLEKHO1 leukemia cells. ( B ) TPM of Il3ra (CD123), Bcl2 , Myc , Ncam1 (CD56), Nrp1 (BDCA4), and Irf4 in Hoxb8-FL cell–derived pDCs ( n = 8) and Hoxb8-FL cell–derived leukemias of the indicated genotypes ( n = 4 per genotype). ( C ) Growth curves of Hoxb8-FL.MS5 cells transduced with V5-MYB constructs of the indicated genotypes. ( D ) Western blots showing p16INK4A and p19ARF expression in Hoxb8-FL.MS5 cells of the indicated genotypes. ( E ) Cell cycle analysis of Hoxb8-FL.MS5 cells. (Left) Representative flow plots of the indicated genotypes. (Right) Quantification of cells in each cell cycle phase as a proportion of total cells ( n = 4 per genotype). Significance tests compare proportion of cells in G1. ( A and E ) Data represent mean ± SEM. Significance determined by 1-way ANOVA with Tukey correction for multiple comparisons. * P <0.05, ** P <0.01, *** P < 0.001.

Article Snippet: Antibodies used for Western blots were: MYB (Abcam, ab45150), V5 (Cell Signaling Technology, 13202), ACTB (Santa Cruz Biotechnology Inc., sc-47778), GAPDH (Cell Signaling Technology, 2118), p16INK4A (Cell Signaling Technology, 29271), and p19ARF (Cell Signaling Technology, 77184).

Techniques: Derivative Assay, Transduction, Construct, Western Blot, Expressing, Cell Cycle 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: 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: Gene Expression, Control, Knockdown, 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