Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: Cdkn2a mediates Usp16 response to Wnt3a. ( a ) Basal cell expansion is observed in the mammary gland of Cdkn2a +/− mice. A Graph quantifying the basal/luminal ratio is shown. Each dot represents an individual mouse. ( b ) Cdkn2a +/− sorted basal cells show an increased induction of Axin2 mRNA levels after 16 hours of Wnt3A stimulation (50 ng/ml). Two independent experiments were performed for a total of 4 animals per group. ( c ) Terminal Tip Fibroblasts (TTFs) derived from Cdkn2a +/− animals show an increase response to two different doses of Wnt3a (20 ng/ml and 50 ng/ml). The graph shows activation of a Top/Flash reporter for Wnt activity normalized by Renilla expression, co-transfected along with the reporter. ( d ) TTFs derived from Cdkn2a −/− animals and transfected with a vector expressing a Usp16 transgene or a control vector were analyzed for their ability to activate the Top-flash reporter. No differences were observed with two different doses of Wnt3a (20 ng/ml and 100 ng/ml). ( e ) MEFs cells were transiently transfected with two individual siRNAs targeting Bmi1. Activation of 6KD Wnt reporter is shown, normalized by Renilla expression. Cells were treated with 100 ng/ml of Wnt3a for 16 hours. ( f ) Two different human foreskin cultures were transiently transfected with siRNA targeting USP16, p16 Ink4a or a scramble sequence. USP16 and p16 Ink4a downregulation promotes the induction of AXIN2 mRNA expression 24 hours after treatment with 100 ng/ml of Wnt3a.

Article Snippet: Human fibroblasts (wild type: CRL-2088, CRL-2076, CTRL-1634 and PCS-201-010; Down’s syndrome: CCL-54, CRL-7090 and CRL-7031) were purchased from ATCC.

Techniques: Derivative Assay, Activation Assay, Activity Assay, Expressing, Transfection, Plasmid Preparation, Control, Sequencing

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: Usp16 trisomy affects the Wnt pathway in Down’s Syndrome. ( a ) SYBR Green analyses revealed a marked decrease in the expression of a Top-Gal reporter of Wnt activity in mammary epithelial cells FACS-sorted from Ts65Dn mice compared to wt animals (n = 3 per group). ( b ) TTFs derived from animals with the indicated genotypes were analyzed for their ability to activate the Top-flash reporter in response to 20 ng/ml of Wnt3a. Data were normalized based on induction in wt animals. Wt and Ts65Dn/Usp16 +/− cells activate Top-Flash more efficiently than Ts65Dn-derived cells. (wt, n = 5; Ts65Dn, n = 5; Ts65Dn/Usp16 +/− , n = 2). ( c ) Microarray analysis shows clustering of wt, Ts65Dn and Ts65Dn/Usp16 +/− RNA expression based on a Wnt signature of genes differentially expressed between wt and Ts65Dn cells (p < 0.05). (n = 2 per group). Ts65Dn/Usp16 +/− RNA expression (samples in the middle) clusters with wt expression (samples on the right). ( d ) FACS-sorted epithelial cells from Ts65Dn mammary glands form less colonies in vitro compared to their wt counterpart. Axin2 heterozygosis increases the ability of Ts65Dn cells to form colonies (n = 3 animals per group; n = 2 for wt animals). Shown is passage P0. ( e , f ) Real time PCR analyses showed that human DS foreskin fibroblasts are less responsive to Wnt3a than cells derived from healthy patients in the activation of AXIN2, RSPO2 and RSPO3 (healthy, n = 4: DS, n = 3).

Article Snippet: Human fibroblasts (wild type: CRL-2088, CRL-2076, CTRL-1634 and PCS-201-010; Down’s syndrome: CCL-54, CRL-7090 and CRL-7031) were purchased from ATCC.

Techniques: SYBR Green Assay, Expressing, Activity Assay, Derivative Assay, Microarray, RNA Expression, In Vitro, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Aging Cell

Article Title: Single‐Cell Fluorescence Imaging Reveals Heterogeneity in Senescence Biomarkers and Identifies Rapamycin‐Responsive Sub‐Populations

doi: 10.1111/acel.70209

Figure Lengend Snippet: Assessment of SA‐βgal and cell proliferation in an MMC‐induced senescence model. (A) Representative images of SA‐βgal staining and EDU incorporation in HDFs treated with vehicle and 200 nM MMC. Images obtained with an IN‐Cell analyser 2200 (SA‐βgal) and Opera Phenix plus (EDU). Images show nuclei stained with Hoechst (blue), SA‐βgal (red)/EDU (Green) and merged image (scale bar = 100 μm). (B) Average fluorescence intensities of SA‐βgal in HDFs treated with vehicle (0.1% DMSO) or different concentrations (50–600 nM) of MMC; Error bars represent mean ± standard deviation from three independent biological replicates. * p < 0.05 (Simple one‐way ANOVA compared with the (0) control group). (C) Percentage of cells positive for EDU incorporation in HDFs treated with MMC; **** p < 0.0001, (Student unpaired t ‐test compared with 0 group). (D) Time course effect of cellular proliferation on HDFs with MMC. (E) Single cell fluorescence intensities of SA‐βgal in HDFs treated with vehicle (0.1% DMSO) or different concentrations (50–600 nM) of MMC. (F) Radar chart of different SA‐βgal fluorescence intensities in HDFs treated with vehicle or different concentrations of MMC. (G) Sub‐population analysis of SA‐βgal fluorescence intensities in HDFs treated with vehicle or 200 nM MMC. (H) Percentage of cells with SA‐βgal intensity of greater than that of the threshold set in the control cells from the respective histograms. Error bars represent mean ± standard deviation from three independent biological replicates. **** p < 0.0001 (Simple one‐way ANOVA compared with the (0) control group).

Article Snippet: Primary human skin fibroblasts (HDFs) (106‐05 N, Sigma‐Aldrich, MO, USA, for Figures and ; or PCS‐201‐010, ATCC, for the remaining figures) were cultured in Dulbecco's Modified Eagle Medium (DMEM, D5523, Sigma‐Aldrich) supplemented with 10% foetal bovine serum (FBS).

Techniques: Staining, Fluorescence, Standard Deviation, Control

Journal: Aging Cell

Article Title: Single‐Cell Fluorescence Imaging Reveals Heterogeneity in Senescence Biomarkers and Identifies Rapamycin‐Responsive Sub‐Populations

doi: 10.1111/acel.70209

Figure Lengend Snippet: Assessment of nuclear area, cell area and P21 expression in an MMC‐induced senescence model in HDFs. Representative images were taken using Opera Phoenix plus at 20× magnification. (A) Representative image of Hoechst‐stained nuclei in HDFs treated with vehicle and 200 nM MMC treated HDFs. Average value of nuclei area (B), cell area (F), P21 (L) in vehicle and MMC treated HDFs; Error bars represent mean ± standard deviation from three independent biological replicates. For Nuclei and cell area (ns, not significant, * p < 0.05 ** p < 0.01) (one‐way ANOVA compared with the (0) control group). For P21 (unpaired t ‐test compared to the control (0) group **** p < 0.0001). Single‐cell data for nuclear area (C) and cell area (G) in HDFs treated with MMC. Individual cell‐derived histogram data categorised into various bin centres for nuclear area (D), cell area (H) and P21 (M) in vehicle‐ and MMC‐treated HDFs. (J) Representative image and quantitation of western blot showing the expression of P21 and β‐Actin, along with the relative quantitiation of expression of P21/β‐Actin expression in MMC‐treated HDFs; * p < 0.05, (Student's unpaired t ‐test compared with 0 control group). (K) Representative images of P21 expression in the nuclei of HDFs treated with vehicle and 200 nM MMC, with nuclei labelled with Hoechst (blue), P21 (red) and merged image (scale bar: 10 μm). Percentage of cells with nuclear area (E), cell area (I) and P21 expression (N) exceeding the threshold set in the control cells, derived from the respective heatmaps in vehicle‐ and MMC‐treated HDFs. Error bars represent the mean ± standard deviation from three independent biological replicates. For nuclear area and cell area (**** p < 0.0001, one‐way ANOVA compared to the control group); For p21 (**** p < 0.0001, unpaired t ‐test compared to the control group).

Article Snippet: Primary human skin fibroblasts (HDFs) (106‐05 N, Sigma‐Aldrich, MO, USA, for Figures and ; or PCS‐201‐010, ATCC, for the remaining figures) were cultured in Dulbecco's Modified Eagle Medium (DMEM, D5523, Sigma‐Aldrich) supplemented with 10% foetal bovine serum (FBS).

Techniques: Expressing, Staining, Standard Deviation, Control, Derivative Assay, Quantitation Assay, Western Blot

Journal: Aging Cell

Article Title: Single‐Cell Fluorescence Imaging Reveals Heterogeneity in Senescence Biomarkers and Identifies Rapamycin‐Responsive Sub‐Populations

doi: 10.1111/acel.70209

Figure Lengend Snippet: Assessment of senescence biomarkers in MMC‐induced senescent HDFs with synchronised cell cycles. Average total fluorescence intensities of SA‐βgal (A), nuclear area (B), cell area (C), nuclear p21 (D) and nuclear p16 (E) in HDFs with synchronised cell cycle treated with vehicle (0.1% DMSO) or different concentrations (50–400 nM) of MMC; Error bars represent mean ± standard deviation from three independent biological replicates. Ns: Not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One‐way ANOVA with Dunnet's post hoc analysis; Mean of all group compared with each other). Single cell fluorescence intensities of SA‐βgal (F), nuclear p21 (I) and nuclear p16 (J) in HDFs treated with vehicle (0.1% DMSO) or different concentrations (50–400 nM) of MMC. Single cell data of nuclear area (G) and cell area (H) in HDFs treated with vehicle (0.1% DMSO) or different concentrations (50–400 nM) of MMC.

Article Snippet: Primary human skin fibroblasts (HDFs) (106‐05 N, Sigma‐Aldrich, MO, USA, for Figures and ; or PCS‐201‐010, ATCC, for the remaining figures) were cultured in Dulbecco's Modified Eagle Medium (DMEM, D5523, Sigma‐Aldrich) supplemented with 10% foetal bovine serum (FBS).

Techniques: Fluorescence, Standard Deviation

Journal: Aging Cell

Article Title: Single‐Cell Fluorescence Imaging Reveals Heterogeneity in Senescence Biomarkers and Identifies Rapamycin‐Responsive Sub‐Populations

doi: 10.1111/acel.70209

Figure Lengend Snippet: Correlation analysis of senescence‐associated biomarkers and the impact of cellular heterogeneity on the senescence‐associated secretory phenotype (SASP) in MMC‐induced senescence. Representative correlation plot of SA‐βgal total fluorescence intensity versus nuclear area (A) and ell area (B) for the MMC 200 nM group. Spearman correlation coefficient values for SA‐βgal total fluorescence intensity versus nuclear area (C) and cell area (D). Data represent mean ± standard deviation from three independent biological replicates; ns, not significant ( p > 0.05, ordinary one‐way ANOVA with Dunnett's post hoc test compared to the control group). (E) Representative images of IL‐6 expression in HDFs treated with vehicle and 200 nM MMC with nuclei labelled with Hoechst (blue) and IL‐6 labelled (yellow); merged image (Scale bar: 10 μm). Images were captured using the Opera Phoenix plus at 20× magnification. (F) Average IL‐6 expression in HDFs treated with vehicle or 200 nM MMC. Representative correlation graph of nuclear area (100–300 μm 2 ) versus IL‐6 (RFU) (G) and nuclear area (> 300 μm 2 ) versus IL‐6 (RFU) (H). (I) Average Spearman correlation coefficient ( r ) of nuclear area subpopulations versus IL‐6 fluorescence in HDFs treated with 200 nM MMC. Error bars represent the mean ± standard deviation from three independent biological replicates (unpaired t ‐test, * p < 0.05, **** p < 0.001).

Article Snippet: Primary human skin fibroblasts (HDFs) (106‐05 N, Sigma‐Aldrich, MO, USA, for Figures and ; or PCS‐201‐010, ATCC, for the remaining figures) were cultured in Dulbecco's Modified Eagle Medium (DMEM, D5523, Sigma‐Aldrich) supplemented with 10% foetal bovine serum (FBS).

Techniques: Fluorescence, Standard Deviation, Control, Expressing

Journal: Aging Cell

Article Title: Single‐Cell Fluorescence Imaging Reveals Heterogeneity in Senescence Biomarkers and Identifies Rapamycin‐Responsive Sub‐Populations

doi: 10.1111/acel.70209

Figure Lengend Snippet: Assessment of senescence biomarkers in the MMC‐induced senescence model with rapamycin treatment. Average total fluorescence intensity of SA‐βgal (A), nuclear area (B) and P21 (C) in HDFs treated with either MMC or MMC plus rapamycin. Data represent the mean ± SD from n = 3 biological replicates; *** p < 0.001, **** p < 0.0001 (ordinary one‐way ANOVA compared to the MMC 200 nM treated group). Sub‐population analysis of SA‐βgal (D), nuclear area (E) and p21 (F) in HDFs treated with either MMC or MMC plus rapamycin. Percentage of cells having increased expression of SA‐βgal (G), nuclear area (H) and p21 (I) in HDFs treated either with MMC or MMC plus rapamycin using the induction threshold method. Data represents the mean ± SD from n = 3 biological replicates; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (ordinary one‐way ANOVA compared to the MMC 200 nM treated group).

Article Snippet: Primary human skin fibroblasts (HDFs) (106‐05 N, Sigma‐Aldrich, MO, USA, for Figures and ; or PCS‐201‐010, ATCC, for the remaining figures) were cultured in Dulbecco's Modified Eagle Medium (DMEM, D5523, Sigma‐Aldrich) supplemented with 10% foetal bovine serum (FBS).

Techniques: Fluorescence, Expressing