Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: Caudal discs of SM/J mice evidence early cellular senescence and a senescence signature during degeneration. At four weeks of age, SM/J mice have increase abundance of senescence markers in their caudal discs, evidenced by ( a–a” ) p19 and ( b–b” ) p21 abundance relative to age-matched B6J discs. c Microarray analysis of 4-week-old and 17-week-old SM/J NP and AF shows distinct clustering in both tissues between the two timepoints. Thematic analysis in CompBio of enriched concepts in 17-week-old NP tissues, compared to 4-week-old tissues shows: ( d’ ) Beta-galactoside Alpha-2,3-sialytransferase Activity is an upregulated theme in the NP; ( e ) VEGF-A Complex is an upregulated theme in the AF; ( f ) CDK1 Phosphorylates Condensin is a downregulated theme in the NP; and ( g ) RUNX2 Regulates Osteoblast Differentiation is a downregulated theme in the AF. h Venn Diagram showing the gene-level overlap between SM/J tissue profiles and the SenMayo geneset, with the overlapping genes shown in ( i ). Data are shown as mean ± SD. Significance was determined using an unpaired t -test or Mann-Whitney test, as appropriate. n C57BL/6J 4w = 8; n SM/J 4w = 5; n SM/J 17w = 6

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Microarray, Activity Assay, MANN-WHITNEY

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: DQ reduces caudal disc degeneration and senescence in SM/J mice. a , b Schematic showing study design: intraperitoneal injections of DQ, Nav., or a Vehicle control were administered once every week to mice starting at 4 weeks of age and ending at 17 weeks of age. a’ –a’” SafraninO/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows DQ improves disc degeneration in SM/J mice. Images reflect the range of degenerative outcomes across treatment cohorts. b’ –b” Safranin/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows Nav. does not improve disc degeneration in SM/J mice. Quantitative immunohistochemistry shows reduced ( c–c” ) p19 (NP and AF) and ( d–d” ) p21 (AF only) in DQ-treated SM/J discs. SASP markers of ( e–e” ) TGF β, ( f–f” ) IL-6, ( g–g” ) MMP13, and ( h–h” ) IL-1β indicate DQ mediates SASP in SM/J discs. Data are shown as mean ± SD. Significance was determined using an unpaired t -test or Mann-Whitney test, as appropriate. Distribution statistics were determined using a χ 2 test. 17 weeks old ( n DQ = 11, 6 females + 5 males; n CT = 13, 6 females + 7 males; n Nav. = 7, n Veh. = 7)

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Control, Staining, Modification, Immunohistochemistry, MANN-WHITNEY

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: Caudal discs of SM/J mice evidence early cellular senescence and a senescence signature during degeneration. At four weeks of age, SM/J mice have increase abundance of senescence markers in their caudal discs, evidenced by ( a–a” ) p19 and ( b–b” ) p21 abundance relative to age-matched B6J discs. c Microarray analysis of 4-week-old and 17-week-old SM/J NP and AF shows distinct clustering in both tissues between the two timepoints. Thematic analysis in CompBio of enriched concepts in 17-week-old NP tissues, compared to 4-week-old tissues shows: ( d’ ) Beta-galactoside Alpha-2,3-sialytransferase Activity is an upregulated theme in the NP; ( e ) VEGF-A Complex is an upregulated theme in the AF; ( f ) CDK1 Phosphorylates Condensin is a downregulated theme in the NP; and ( g ) RUNX2 Regulates Osteoblast Differentiation is a downregulated theme in the AF. h Venn Diagram showing the gene-level overlap between SM/J tissue profiles and the SenMayo geneset, with the overlapping genes shown in ( i ). Data are shown as mean ± SD. Significance was determined using an unpaired t -test or Mann-Whitney test, as appropriate. n C57BL/6J 4w = 8; n SM/J 4w = 5; n SM/J 17w = 6

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Microarray, Activity Assay, MANN-WHITNEY

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: DQ reduces caudal disc degeneration and senescence in SM/J mice. a , b Schematic showing study design: intraperitoneal injections of DQ, Nav., or a Vehicle control were administered once every week to mice starting at 4 weeks of age and ending at 17 weeks of age. a’ –a’” SafraninO/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows DQ improves disc degeneration in SM/J mice. Images reflect the range of degenerative outcomes across treatment cohorts. b’ –b” Safranin/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows Nav. does not improve disc degeneration in SM/J mice. Quantitative immunohistochemistry shows reduced ( c–c” ) p19 (NP and AF) and ( d–d” ) p21 (AF only) in DQ-treated SM/J discs. SASP markers of ( e–e” ) TGF β, ( f–f” ) IL-6, ( g–g” ) MMP13, and ( h–h” ) IL-1β indicate DQ mediates SASP in SM/J discs. Data are shown as mean ± SD. Significance was determined using an unpaired t -test or Mann-Whitney test, as appropriate. Distribution statistics were determined using a χ 2 test. 17 weeks old ( n DQ = 11, 6 females + 5 males; n CT = 13, 6 females + 7 males; n Nav. = 7, n Veh. = 7)

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Control, Staining, Modification, Immunohistochemistry, MANN-WHITNEY

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: JUN pathway inhibition mimics the positive effect of DQ in decreasing senescence and SASP in human degenerated NP cells. a Grade IV and V human degenerated NP cells show a lower percentage of β-Gal-staining after treatment with DQ and JUN inhibitor, or only DQ, respectively. b Grade IV Human NP cells exhibit lower expression of IL-6 , MMP2 , and MMP13 compared to the stimulus group. Additionally, DQ treatment resulted in a decrease in CDKN1A and IL-6 , while T5224 enhanced MMP13 expression. c Grade V human NP cells exhibit lower expression of CDKN1A, CDKN2A, IL-6 , CCL2 , MMP2 , and MMP13 relative to the stimulus group. DQ treatment attenuated the expression of IL-6 and CCL2 , and MMP2 . T5224 ameliorated CDKN1A , IL-6 , CCL2 , and MMP2 expression. Data are shown as mean ± SD. Significance was determined using Dunnett’s multiple comparisons test ( n = 3 independent experiments, performed in triplicate)

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Inhibition, Staining, Expressing

Journal: eBioMedicine

Article Title: Renal podocyte senescence in human obesity detected by urinary extracellular vesicles

doi: 10.1016/j.ebiom.2025.106116

Figure Lengend Snippet: Increased proportion of senescence- and inflammation-associated uEVs in obesity . (A) The proportion of P16 + uEVs was significantly higher in patients with obesity (OB) (n = 21) compared to healthy volunteers (HV, n = 10), suggesting magnified cellular senescence in obesity. (B) The proportions of P16 + uEVs co-expressing the podocyte marker PODXL was higher in OB, but not those expressing URAT1, UROMODULIN, or PROMININ; (C) Representative flow cytometry dot plots depicting P16 + PODXL + uEVs in HV and OB; (D) The percentage of MCP-1 + uEVs was elevated in OB, indicating an inflammatory renal phenotype; (E) The proportion of MCP-1 + PODXL + uEVs was higher in OB, while no significant differences are observed in MCP-1 + uEVs expressing renal tubular cell markers; (F) Representative flow cytometry plots showing MCP-1 + PODXL + uEVs in HV and OB; (G) The proportion of P16 + MCP-1 + uEVs was higher in OB, indicating the coexistence of senescence and inflammation in renal-derived uEVs; (H) P16 + MCP-1 + PODXL + uEVs were significantly increased in OB, while other triple-positive renal tubular cells were not; (I) Representative flow cytometry plots for P16 + MCP-1 + PODXL + uEVs in HV and OB; ∗P < 0.05, ∗∗∗∗P < 0.0001; uEVs, urinary extracellular vesicles; HV, healthy volunteers; OB, obesity; PODXL, podocalyxin; MCP-1, monocyte chemoattractant protein-1; URAT1, urate transporter-1; UROMODULIN, uromodulin; PROMININ, prominin-1.

Article Snippet: Flow cytometry analysis of isolated EVs was conducted following staining with 0.5 mM Tag-it Violet (TIV) cell-labelling solution (BioLegend, San Diego, CA) at 37 °C for 2 h. To confirm EVs identity, samples were stained with tetraspanin markers CD9 (BioLegend, Cat#312105), CD63 (BioLegend, Cat#353007), and CD81 (BioLegend, Cat#349503), in addition to cell-specific antibodies, including p16 (Novus, Cat#NB200-174), MCP-1 (ThermoFisher, Cat#MA5-17040), podocalyxin (PODXL; ThermoFisher, Cat#MA5-15846), nephrin (ThermoFisher, Cat#MA5-41644), urate transporter-1 (URAT1; Creative Diagnostics, Cat#DCABH-7215), uromodulin (Novus, Cat#NBP1-50321), and prominin-1 (R&D, Cat#MAB2024).

Techniques: Expressing, Marker, Flow Cytometry, Derivative Assay

Journal: eBioMedicine

Article Title: Renal podocyte senescence in human obesity detected by urinary extracellular vesicles

doi: 10.1016/j.ebiom.2025.106116

Figure Lengend Snippet: Correlation between podocyte-derived P16 + uEVs and clinical parameters . Spearman correlation analysis showed that the proportions of both P16 + PODXL + (red) and P16 + PODXL + NEPHRIN + (black) uEVs correlated directly with (A) BMI, (B) HOMA-IR, (C) insulin, and (D) urinary MCP-1; BMI, body mass index; HOMA-IR, homoeostatic model assessment for insulin resistance; A trend line is shown to illustrate the overall directionality of the association. MCP-1, monocyte chemoattractant protein-1; PODXL, podocalyxin; NEPHRIN, nephrin.

Article Snippet: Flow cytometry analysis of isolated EVs was conducted following staining with 0.5 mM Tag-it Violet (TIV) cell-labelling solution (BioLegend, San Diego, CA) at 37 °C for 2 h. To confirm EVs identity, samples were stained with tetraspanin markers CD9 (BioLegend, Cat#312105), CD63 (BioLegend, Cat#353007), and CD81 (BioLegend, Cat#349503), in addition to cell-specific antibodies, including p16 (Novus, Cat#NB200-174), MCP-1 (ThermoFisher, Cat#MA5-17040), podocalyxin (PODXL; ThermoFisher, Cat#MA5-15846), nephrin (ThermoFisher, Cat#MA5-41644), urate transporter-1 (URAT1; Creative Diagnostics, Cat#DCABH-7215), uromodulin (Novus, Cat#NBP1-50321), and prominin-1 (R&D, Cat#MAB2024).

Techniques: Derivative Assay

Journal: eBioMedicine

Article Title: Renal podocyte senescence in human obesity detected by urinary extracellular vesicles

doi: 10.1016/j.ebiom.2025.106116

Figure Lengend Snippet: Correlation between P16 + MCP-1 + PODXL + podocyte-derived uEVs and clinical parameters . The proportion of P16 + MCP-1 + PODXL + uEVs in HV (blue, n = 10) and OB (red, n = 21) was significantly positively correlated with (A) BMI, (B) HOMA-IR, (C) insulin, (D) and urinary (U) levels of KIM-1, (E) NGAL, (F) protein, and (G) TNF-α; BMI, body mass index; HOMA-IR, homoeostatic model assessment for insulin resistance; A trend line is shown to illustrate the overall directionality of the association. KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; TNF-α, tumour necrosis factor-alpha.

Article Snippet: Flow cytometry analysis of isolated EVs was conducted following staining with 0.5 mM Tag-it Violet (TIV) cell-labelling solution (BioLegend, San Diego, CA) at 37 °C for 2 h. To confirm EVs identity, samples were stained with tetraspanin markers CD9 (BioLegend, Cat#312105), CD63 (BioLegend, Cat#353007), and CD81 (BioLegend, Cat#349503), in addition to cell-specific antibodies, including p16 (Novus, Cat#NB200-174), MCP-1 (ThermoFisher, Cat#MA5-17040), podocalyxin (PODXL; ThermoFisher, Cat#MA5-15846), nephrin (ThermoFisher, Cat#MA5-41644), urate transporter-1 (URAT1; Creative Diagnostics, Cat#DCABH-7215), uromodulin (Novus, Cat#NBP1-50321), and prominin-1 (R&D, Cat#MAB2024).

Techniques: Derivative Assay

Journal: eBioMedicine

Article Title: Renal podocyte senescence in human obesity detected by urinary extracellular vesicles

doi: 10.1016/j.ebiom.2025.106116

Figure Lengend Snippet: Histological evidence of increased podocyte senescence in obesity . (A) Representative immunofluorescence images of kidney biopsy samples from HV (n = 6) and OB (n = 7) triple-stained for p16 (green), PODXL (red), and DAPI (blue). Scale bar represents 100 μm. (B–C) Line profile analysis of fluorescence intensity across glomeruli shows increased p16 signal in obese glomeruli, overlapping with DAPI and confined to PODXL + regions. (D) Quantification of senescent podocytes (P16 + PODXL + ) shows an increased proportion in OB glomeruli. Each dot represents an analysed glomerulus, and different colours indicate different individuals, for a total of 45 glomeruli from six HV and 55 glomeruli from seven OB participants. (E) Spearman correlation analysis showed a strong direct correlation between P16 + PODXL + uEVs and BMI, consistent with an obesity-driven increase in podocyte senescence, and a weaker but significant direct correlation with plasma glucose levels (F). PODXL, podocalyxin; OB, obesity; HV, healthy volunteers; BMI, body mass index; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Flow cytometry analysis of isolated EVs was conducted following staining with 0.5 mM Tag-it Violet (TIV) cell-labelling solution (BioLegend, San Diego, CA) at 37 °C for 2 h. To confirm EVs identity, samples were stained with tetraspanin markers CD9 (BioLegend, Cat#312105), CD63 (BioLegend, Cat#353007), and CD81 (BioLegend, Cat#349503), in addition to cell-specific antibodies, including p16 (Novus, Cat#NB200-174), MCP-1 (ThermoFisher, Cat#MA5-17040), podocalyxin (PODXL; ThermoFisher, Cat#MA5-15846), nephrin (ThermoFisher, Cat#MA5-41644), urate transporter-1 (URAT1; Creative Diagnostics, Cat#DCABH-7215), uromodulin (Novus, Cat#NBP1-50321), and prominin-1 (R&D, Cat#MAB2024).

Techniques: Immunofluorescence, Staining, Fluorescence, Clinical Proteomics

Journal: eBioMedicine

Article Title: Renal podocyte senescence in human obesity detected by urinary extracellular vesicles

doi: 10.1016/j.ebiom.2025.106116

Figure Lengend Snippet: Schematic model of the association among obesity, podocyte senescence, and uEVs . Obesity-related metabolic and inflammatory stress is associated with podocyte senescence, reflected by p16 expression and SASP activity. Senescent podocytes release uEVs enriched in p16 and MCP-1, detectable in urine as indicators of renal stress. These changes may precede overt structural or functional impairment. [Graphical elements were created with BioRender.com ].

Article Snippet: Flow cytometry analysis of isolated EVs was conducted following staining with 0.5 mM Tag-it Violet (TIV) cell-labelling solution (BioLegend, San Diego, CA) at 37 °C for 2 h. To confirm EVs identity, samples were stained with tetraspanin markers CD9 (BioLegend, Cat#312105), CD63 (BioLegend, Cat#353007), and CD81 (BioLegend, Cat#349503), in addition to cell-specific antibodies, including p16 (Novus, Cat#NB200-174), MCP-1 (ThermoFisher, Cat#MA5-17040), podocalyxin (PODXL; ThermoFisher, Cat#MA5-15846), nephrin (ThermoFisher, Cat#MA5-41644), urate transporter-1 (URAT1; Creative Diagnostics, Cat#DCABH-7215), uromodulin (Novus, Cat#NBP1-50321), and prominin-1 (R&D, Cat#MAB2024).

Techniques: Expressing, Activity Assay, Functional Assay

Journal: Cancers

Article Title: Derivation of Genetically Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

doi: 10.3390/cancers17183002

Figure Lengend Snippet: Properties of BN and YN HBs and their progeny immortalized cell lines. ( A ) Gross appearance of typical tumors generated by the indicated combinations of oncogenic drivers. Images of previously generated BY and BYN tumors are included for comparison. To allow for the establishment of immortalized BN and YN cell lines, all tumors were generated with the inclusion of 2 Crispr/Cas9 vectors encoding 4 different gRNAs directed against exon 2 of the Cdkn2a locus . ( B ) H&E-stained sections of tumors from ( A ). Note the more prominent blood vessels in BN tumors (black arrow) and the previously reported fluid-filled cysts adjacent to regions of necrosis in BYN tumors (black and green arrows, respectively) [ , ]. Scale bars = 500 μm. ( C ) H&E-stained cells from the indicated immortalized cell lines propagated on coverslips in vitro. BY1 and BYN2 cell lines were derived and characterized previously and are included here for comparative purposes . Scale bars = 50 μm. ( D ) Growth curves of the indicated cell lines and their suppression via the enforced expression of WT p16 INK4A and p19 ARF . The indicated cell lines were transfected with a control pSBbi-RP SB vector or with vectors encoding WT p16 INK4A or p19 ARF . Two days later, the cells were seeded into 12-well plates and maintained in 2 μg/mL of puromycin while monitoring dTomato expression. Subsequent growth was monitored using an Incucyte S3 Live-Cell imaging and Analysis System. Each point represents the mean of 4 replicas +/− 1 S.E. Note that BN2 cells were resistant to transfection on multiple occasions.

Article Snippet: Antibodies used included those directed against p16 INK4A (# Ab211542 , 1:2000, Abcam, Cambridge, UK), p19 ARF (#NB200-174, 1:1000, Novus Biologicals, Centennial, CO), GAPDH (#G8795, 1:10,000, Sigma-Aldrich, Inc. St Louis, MO, USA), YAP (#4912, 1:1000, Cell Signaling Technologies [CST], Inc., Danvers, MA, USA).

Techniques: Generated, Comparison, CRISPR, Staining, In Vitro, Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Live Cell Imaging

Journal: Cancers

Article Title: Derivation of Genetically Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

doi: 10.3390/cancers17183002

Figure Lengend Snippet: YN and BN cell lines express small, unique subsets of p16 INK4A and p19 ARF mutants. ( A ) Depictions of the proteins encoded by the 5 most abundant Cdkn2a mutations identified in each of the indicated cell lines. The frequencies with which they were detected based on deep sequencing of Cdkn2a exon 2 PCR products obtained from cell lines are indicated to the right of each cartoon. Asterisks indicate the mutants that were deemed the most likely to be expressed as proteins, based upon in silico translation of the open reading frames and the sizes of the actual proteins observed by immunoblotting (panel B ). contains a more comprehensive list of the mutations identified and their abundance. *: Proteins predicted to be expressed. ( B ) Immunoblots of mutant p16 INK4A and p19 ARF proteins expressed by the indicated cell lines. Included as controls were a sample of normal liver (L) and a primary BY HB (T) with intact Cdkn2a loci. The latter expressed WT p16 INK4A and p19 ARF as previously described . Expression of each protein relative to that of GAPDH, as determined by densitometric scanning, is shown beneath the blot. ( C ) BY-derived mutant and/or fusion p16 INK4A and p19 ARF proteins were used for expression in BN and YN cell lines. All encoded proteins were V5 epitope-tagged to allow expression levels to be directly compared. See ref. for previous characterization. ( D ) Approach to evaluating the growth suppressive effects of the mutants depicted in C on BN1 and YN2 cells. ( E ) Differential selection of p16 INK4A /p19 ARF mutants. The indicated cell lines were individually co-transfected with pSBbi SB vectors encoding the mutant proteins depicted in panel ( C ) plus an equal amount of the empty pSBbi vector. Two days later, half the cells were used to assess the transient expression of each protein as described in ( D ). The remaining cells were selected in puromycin for 14 and 21 days and assessed for the expression of their respective protein at these times using anti-V5 or anti-p16 INK4A antibodies. Expression of each protein relative to that of GAPDH, as determined by densitometric scanning, is shown to the right of the blot. ( F ) Selective retention of the non-resolvable mutants shown in ( E ). BN1 cells were separately co-transfected with the empty pSBbi vector alone plus one encoding each of the four indicated mutants. Puromycin-resistant clones were then selected and expanded for 3 weeks as in ( E ), followed by immunoblotting to detect each of the V5-tagged mutants. Expression of each protein relative to that of GAPDH, as determined by densitometric scanning, is shown beneath the blot. ( G ). The indicated vectors were transfected into YN2 or BN1 cells, which were then seeded into 12-well plates 2 days later in the presence of puromycin and enumerated over the course of the next 10 days. Each point represents the mean of 4 replicas +/− 1 S.E. Original Western Blot images in .

Article Snippet: Antibodies used included those directed against p16 INK4A (# Ab211542 , 1:2000, Abcam, Cambridge, UK), p19 ARF (#NB200-174, 1:1000, Novus Biologicals, Centennial, CO), GAPDH (#G8795, 1:10,000, Sigma-Aldrich, Inc. St Louis, MO, USA), YAP (#4912, 1:1000, Cell Signaling Technologies [CST], Inc., Danvers, MA, USA).

Techniques: Sequencing, In Silico, Western Blot, Mutagenesis, Expressing, Derivative Assay, Selection, Transfection, Plasmid Preparation, Clone Assay