Journal: Oncogene

Article Title: p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

doi: 10.1038/sj.onc.1200897

Figure Lengend Snippet: Figure 1 p53 overexpression induces apoptosis of SK-MEL-110 (MEL) cells. (a) Eect of Ad.null or AdCMV.p53 infections in SK-MEL-110 cells and VSMC. Infected cells (100 p.f.u./cell in all experiments) were analysed for relative changes in cell number (counted using a hemocytometer) 48 h after infection with the indicated viruses (solid bars, Ad.null; hatched bars, AdCMV.p53). Values represent mean+SEM of three independent experiments. (b) Apoptotic cell death of SK-MEL-110 cells infected with AdCMV.p53. Thirty-six hours after infection of VSMC and SK- MEL-110 cells with 100 p.f.u./cell Ad.null (null) or AdCMV.p53 (p53), genomic DNA was extracted and assayed from fragmenta- tion. MW: Molecular weight marker

Article Snippet: Fifty-mg samples of total cell lysates were size-fractionated by SDS ± PAGE and transferred onto PVDF membranes using standard techniques (Harlow and Lane, 1988). p53 and p21Waf1/Cip1 proteins were detected with the ECL system (Amersham, Arlington Heights, IL) following incubation with the monoclonal anti-human p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal anti-p21 antibody (Oncogene Science).

Techniques: Over Expression, Infection, Molecular Weight, Marker

Journal: Oncogene

Article Title: p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

doi: 10.1038/sj.onc.1200897

Figure Lengend Snippet: Figure 2 (a) p53 expression in VSMC and SK-MEL-110 cells. Cells were infected with 100 p.f.u./cell AdCMV.p53 and p53 expression was monitored 24 h later by Western blot analysis. (b) Northern blot analysis of p21Waf1/Cip1, GADD45 and p53 mRNA expression in Ad.null- or AdCMV.p53-infected VSMC and SK-MEL-110 cells. Expression was monitored at the times indicated following infection. Loading and transfer dierences among samples were normalized using an oligomer recognizing 18S rRNA

Article Snippet: Fifty-mg samples of total cell lysates were size-fractionated by SDS ± PAGE and transferred onto PVDF membranes using standard techniques (Harlow and Lane, 1988). p53 and p21Waf1/Cip1 proteins were detected with the ECL system (Amersham, Arlington Heights, IL) following incubation with the monoclonal anti-human p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal anti-p21 antibody (Oncogene Science).

Techniques: Expressing, Infection, Western Blot, Northern Blot

Journal: Oncogene

Article Title: p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

doi: 10.1038/sj.onc.1200897

Figure Lengend Snippet: Figure 3 (a) p21Waf1/Cip1 protein expression in SK-MEL-110 cells 24 h after infection with the indicated viruses. (b) Loss in cell viability of control SK-MEL-110 cultures infected with AdCMV.p53 relative to that of p21Waf1/Cip1-overexpressing cells infected with AdCMV.p53. Abscissa, ®rst infection with Ad.null (left), or AdWAF1 (right); second infection: solid bars (Ad.null), hatched bars (AdCMV.p53). Values represent mean+SEM of at least three independent experiments. (c) Time-dependent changes in cell number of SK-MEL-110 cultures that had been infected with Ad.null (null) or AdWAF1 (p21) 48 h prior to a subsequent infection with either Ad.null or AdCMV.p53 (p53). Infection groups: null/null (- -&- -), null/p53 (±&±), p21/null (- -&- -) and p21/p53 (±&±). Values represent mean+SEM of at least three independent experiments. (d) FACS analysis of SK-MEL-110 cultures after sequential infections with Ad.null (null) or AdWAF1 (p21) for 48 h (®rst infection) followed by Ad.null (null) or AdCMV.p53 (p53) for the following 48 h (second infection). Note the apoptotic peak (arrowhead), readily visible in the null/p53 treatment group

Article Snippet: Fifty-mg samples of total cell lysates were size-fractionated by SDS ± PAGE and transferred onto PVDF membranes using standard techniques (Harlow and Lane, 1988). p53 and p21Waf1/Cip1 proteins were detected with the ECL system (Amersham, Arlington Heights, IL) following incubation with the monoclonal anti-human p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal anti-p21 antibody (Oncogene Science).

Techniques: Expressing, Infection, Control

Journal: Oncogene

Article Title: p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

doi: 10.1038/sj.onc.1200897

Figure Lengend Snippet: Figure 4 p53-mediated cytotoxicity in mouse embryonal ®broblasts lacking the p21Waf1/Cip1 gene (p217/7 MEFs) is prevented by prior expression of p21Waf1/Cip1. (a) Western blot analysis of p53 expression in p217/7 MEFs following infection with either Ad.null AdCMV.NLSbgal or AdCMV.p53 adenoviruses at the indicated p.f.u./cell. (b) Cytotoxic eect of p53 overexpression in p217/7

Article Snippet: Fifty-mg samples of total cell lysates were size-fractionated by SDS ± PAGE and transferred onto PVDF membranes using standard techniques (Harlow and Lane, 1988). p53 and p21Waf1/Cip1 proteins were detected with the ECL system (Amersham, Arlington Heights, IL) following incubation with the monoclonal anti-human p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal anti-p21 antibody (Oncogene Science).

Techniques: Expressing, Western Blot, Infection, Over Expression

Journal: Nature communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function.

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: Fig. 1 ADH-6 abrogates amyloid formation of aggregation-prone region of p53 DBD. a Schematic representation of the different domains of p53. The DBD (residues 102–292) contains an aggregation-nucleating subdomain (residues 251–258) that is necessary and sufficient to drive p53 aggregation14,17,28. Another segment of interest comprises residues 213–217, which is the antigen recognized by the PAb 240 antibody that binds to partially unfolded p53. Also highlighted in the DBD is R248, one of the most common mutation hotspots in p53 (IARC TP53 database; https://p53.iarc.fr)9. b Structure of p53 DBD. Highlighted are the aggregation-nucleating subdomain (green) and the epitope recognized by PAb 240 (red). Both segments are buried in the fully folded p53 structure. The 3D image was generated using PyMOL 2.3.5 (Schrödinger, New York, NY). c Primary sequences of the studied WT and mutant R248W p53 DBD-derived peptides, denoted pWT and pR248W, respectively, which span residues 248–273. The peptides include the aggregation-prone 252–258 sequence, as well as R248 and another of the most common mutation hotspots in p53 and R273 (IARC TP53 database; https://p53.iarc.fr)9. d Chemical structures of the oligopyridylamides ADH-1 and ADH-6. e, f Effects of the oligopyridylamides on pR248W amyloid formation. Kinetic profiles (left panel) and representative transmission electron microscopy (TEM) images (right panel) for aggregation of 25 μM pR248W in the absence or presence of an equimolar amount of ADH-1 or ADH-6 co-mixed at the start of the reaction (e) or added during the growth phase (i.e. 5 h after the start of the reaction) (f). Kinetic aggregation profiles were acquired by measuring the fluorescence of the thioflavin T (ThT) reporter (λex/em = 440/480 nm) at 5-min intervals at 37 °C (n = 4). TEM images were acquired at 10 h after the start of the aggregation reaction. Scale bar = 100 nm. g Characterization of the binding interaction of the oligopyridylamides and pR248W measured using steady-state intrinsic tryptophan fluorescence quenching. A 5 µM solution of pR248W was titrated with increasing concentrations of ADH-1 (left panel) or ADH-6 (right panel) and the tryptophan fluorescence after each addition was normalized to account for the dilution (total dilution during the titration was <1%) and plotted against the ligand concentration. The equilibrium dissociation constants (Kd) were then determined using a one-site-specific binding equation (Eq. 1). h Effects of the oligopyridylamides on pR248W oligomerization monitored using the dot blot assay. Samples of 10 μM pR248W were incubated with or without an equimolar amount of ADH-1 or ADH-6 for 0–24 h, and the presence of oligomers was detected using an amyloid oligomer-specific polyclonal antibody (A11)35. i Effects of the oligopyridylamides on the self-assembly driven structural transition of pR248W. Time-dependent circular dichroism (CD) spectra of 10 µM pR248W alone (left panel) or in the presence of an equimolar amount of ADH-1 (middle panel) or ADH-6 (right panel).

Article Snippet: Genes for WT and R248W p53 DBDs were respectively amplified from the vector pCMV-Neo-Bam carrying WT and R248W p53 constructs (plasmids #16434 and #16437, respectively; Addgene, Watertown, MA).

Techniques: Mutagenesis, Generated, Derivative Assay, Sequencing, Transmission Assay, Electron Microscopy, Binding Assay, Titration, Concentration Assay, Dot Blot, Incubation, Circular Dichroism

Journal: Nature communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function.

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: Fig. 2 NMR-based determination of p53 DBD–ADH-6 interaction interface. a, b Overlay of 15N-1H HSQC maps of 19 μM WT (a) and 24 μM R248W (b) p53 DBD in H2O/D2O (96/4) with 16.7 mM DTT, without (green contours) or with (red contours) ADH-6 addition (protein:ligand 1:11 in a and 1:15 in b). The assignments are reported only outside the rightmost regions. These regions are crowded because of the presence of partially unfolded species that also interact with ADH-6 as highlighted by the boxed peak in each panel. c HSQC contour maps overlay of mutant R248W p53 DBD at different protein: ADH-6 ratios (1:0 green, 1:8 cyan, and 1:15 red) showing the increment of cumulated chemical shift perturbation (CSP) with ligand concentration (Eq. 2). d The five clusters of the two p53 DBD variants (WT and mutant R248W) that show high (>0.025) or medium (>0.015) CSP values137. Cluster 1 (highlighted in blue) includes residues T118, Y126, E271, C275, and G279; cluster 2 (highlighted in magenta) includes residues R196, E198, G199, L201, Y220, and E221; cluster 3 (green) includes T102, Y103, Q104, G105, L257, L264, and R267; cluster 4 (orange) includes E171, R174, H179, R209, and G244; and cluster 5 (cyan) includes S94, A161, I162, L206, and S215. Clusters 1 and 2 are at the front in the cartoon on the left; clusters 3–5 are at the front in the cartoon on the right. The 3D image was generated using PyMOL 2.3.5 (Schrödinger, New York, NY).

Article Snippet: Genes for WT and R248W p53 DBDs were respectively amplified from the vector pCMV-Neo-Bam carrying WT and R248W p53 constructs (plasmids #16434 and #16437, respectively; Addgene, Watertown, MA).

Techniques: Mutagenesis, Concentration Assay, Generated

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 4 PARIS expression leads to p53 activation via PARIS Y137 phosphorylation-dependent epigenetic repression of MDM4. (A) Representative immunoblots examining the expression of MDM4, pS15-p53, p53 and FLAG (PARIS) in SH-SY5Y cells transfected with FLAG-PARIS wild-type or a Y137F mutant (48 h) using the indicated antibodies. b-Actin serves as an internal loading control. (B–D) Relative expression levels of MDM4 (B), p53 (C) and pS15-p53 (D) in the indicated experimental groups from A normalized to the internal loading control (b-actin; n = 3 per group). (E) Quantification of the relative expression of MDM4 mRNA in SH-SY5Y cells transfected (48 h) with mock or FLAG-PARIS and treated with TSA (300 nM, 42 h) deter- mined by RT-qPCR (normalized to internal GAPDH loading control; n = 3 per group). (F) Representative immunoblots of FLAG (PARIS) and MDM4 ex- pression in SH-SY5Y cells transfected (48 h) with mock or FLAG-PARIS and treated with TSA (300 nM, 42 h). (G) Quantification of the relative expression of MDM4 protein in the experimental groups in F normalized to b-actin (n = 3 per group). (H) A schematic diagram depicting the promoter structures of human MDM4 (hMDM4). IRS1, IRS2 and IRS3 motifs are indicated (top). Anti-acetyl-histone and anti-FLAG ChIP analysis of putative IRS (motif 1, 2 and 3) within the MDM4 promoter region in SH-SY5Y cells transfected with mock, or FLAG-PARIS wild-type (48 h, bottom) with or without the HDAC inhibitor TSA (300 nM, 42 h). The non-IRS region within the MDM4 promoter (Ctrl motif) was used as a negative control. Samples immuno- precipitated using either anti-histone antibodies or rabbit IgG were included as experimental controls in ChIP assays. (I) Quantification of relative ace- tylated histone enrichment on the indicated motifs located within MDM4 promoter determined by PCR amplification of ChIPed DNA in H (n = 3 per group). Data are expressed as mean SEM. *P 5 0.05, **P 5 0.01 and ***P 5 0.001 and statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Western Blot, Transfection, Mutagenesis, Control, Quantitative RT-PCR, Negative Control

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 5 Pharmacological inhibition of c-Abl activation restores behaviour/motor deficits and dopaminergic degeneration and prevents MDM4 repres- sion and p53 activation in mice with AAV-PARIS injection. (A) Representative exploratory paths from an open field test of mice that underwent AAV- Con or AAV-PARIS stereotaxic nigral injections (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). (B) Anxiety assessment of each experimental mouse group by examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 9 AAV-Con-injected mice, n = 10 AAV-Con-injected mice + nilotinib, and n = 8 AAV-PARIS-injected mice). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of vertical pole. (D) Motor coordination of each experimental mouse group used in B determined by the latency to fall in an accelerating rotarod test. (E) Representative TH immunohistochem- ical staining with Nissl counterstain of substantia nigra from mice that underwent AAV-Con or AAV-PARIS stereotaxic nigral injections (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). The substantia nigra and ventral tegmental area regions are indicated by dot- ted yellow and white lines, respectively. Scale bar = 500 mm. (F) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) in the indicated mouse groups (n = 4 AAV-Con-injected mice + DMSO, n = 5 AAV-Con-injected mice + nilotinib and n = 4 AAV-PARIS-injected mice). (G) Representative TUNEL assay images of ventral midbrain from mice that underwent stereotaxic nigral injection of AAV-Con or AAV-PARIS (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). The coronal brain sections were counterstained with DAPI. (H) Quantification of the percentage of TUNEL-labelled cells in AAV-Con- or AAV-PARIS-injected ventral midbrain regions from mice with or without nilotinib treatment (n = 16 sections from four mice per group). (I) Representative immunoblots examining pY137-PARIS, PARIS, c-Abl, pY245-c-Abl, MDM4, pS15-p53 and p53 expression in the ventral midbrain of AAV-Con- or AAV-PARIS-injected mice with or without nilotinib treatment using the indicated antibodies. (J) Quantification of the relative expression of pY137-PARIS, PARIS, c-Abl, pY245-c-Abl MDM4, pS15-p53 and p53 proteins nor- malized to b-actin (n = 5 AAV-Con-injected mice and n = 4 AAV-PARIS-injected mice). Data are expressed as mean SEM. Statistical analyses was per- formed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. **P 5 0.01 and ***P 5 0.001. DMSO = dimethyl sulphoxide; WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Inhibition, Activation Assay, Injection, Staining, TUNEL Assay, Western Blot, Expressing, Two Tailed Test

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 6 Pharmacological inhibition of c-Abl activity in in vivo adult parkin knockout mice prevents motor dysfunction and dopaminergic neurode- generation with concomitant blocking of PARIS phosphorylation and p53 activation. (A) Representative exploratory paths from an open-field test of 6-month-old wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 m) and treated with the c-Abl in- hibitor nilotinib (200 mg nilotinib per 1 kg diet, p.o. for 2 months) or standard chow diet (chow). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 4 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 4 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre with or without nilotinib treatment (200 mg nilotinib per 1 kg diet, p.o. for 2 months). Scale bar = 500 mm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) that expe- rienced stereotaxic nigral injection of AAV-GFPCre with or without nilotinib treatment (200 mg nilotinib per 1 kg diet, p.o. for 2 months). The coronal brain sections were counterstained with DAPI. Magnified images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL- labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice with or without nilotinib treatment (n = 16 sections from four mice per group). (H) Representative immunofluorescence images examining the expression of pY245-c-Abl in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treat- ment. (I) Quantification of the relative pY245-c-Abl fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). (J) Representative immunofluorescence images examining the expression of pY137-PARIS in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treatment. (K) Quantification of the relative pY137-PARIS fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). (L) Representative immunofluorescence images examining the expression of pS15-p53 in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treatment. (M) Quantification of the relative pS15-p53 fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). Data are expressed as mean SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P 5 0.001. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Inhibition, Activity Assay, In Vivo, Knock-Out, Blocking Assay, Phospho-proteomics, Activation Assay, Injection, Immunohistochemical staining, Staining, TUNEL Assay, Expressing, Two Tailed Test

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 7 Y137F-PARIS-mediated suppression c-Abl-PARIS pathway rescues MDM4 repression, blocks p53 activation and prevents development of motor deficits and dopamine neuron loss in parkin knockout mice. (A) Representative exploratory paths from an open field test of 6-month-old wild- type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 months) AAV-PARIS-Y137F (3 months, phospho- deficient mutant PARIS). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border ver- sus the sum of the centre and periphery zones (n = 8 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 8 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre AAV-PARIS-Y137F. Scale bar = 500 mm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (par- kinfl/fl) that experienced stereotaxic nigral injection of AAV-GFPCre AAV-PARIS-Y137F. The coronal brain sections were counterstained with DAPI. Merged images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL-labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice AAV-PARIS-Y137F (n = 16 sections from four mice per group). (H) Representative immunoblots examining the expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53, and parkin in the AAV-GFPCre AAV-PARIS-Y137F-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice using the indicated antibodies. b-Actin serves as an internal loading control. (I) Quantification of the relative expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53 and parkin proteins normalized to b-actin in the indicated experimental groups (n = 4 mice per group). Data are expressed as mean SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P 5 0.001. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Activation Assay, Knock-Out, Injection, Mutagenesis, Immunohistochemical staining, Staining, TUNEL Assay, Western Blot, Expressing, Control, Two Tailed Test

Journal: IUBMB life

Article Title: Schizandrin A inhibits cellular phenotypes of breast cancer cells by repressing miR-155.

doi: 10.1002/iub.2329

Figure Lengend Snippet: FIGURE 1 Schizandrin A (SchA) suppresses cell proliferation while enhances apoptosis of MDA-MB-231 cells. (a) The molecular structure of SchA. (b) The cells were stimulated with SchA at the showed concentrations, and after 24 h, the viability was determined by CCK-8 assay. (c) Cell proliferation was evaluated based on a BrdU incorporation method. (d,e) p53, CDK4, and cyclin D1 were probed by using the Western blot method. (f) The number of FITC-positive cells was counted using a flow cytometry and was shown in the column chart. (g) Relative expression of Bax, Bcl-2, and caspases was detected by Western blot, and (h) quantitative analysis was illustrated using the column chart. Data from three independent experiments were shown as mean ± SD (*p < .05, **p < .01, ***p < .001). CCK-8, cell counting kit-8; FITC, fluorescein isothiocynate

Article Snippet: The primary antibodies contained antibodies against p53 (9282) (Cell Signaling Technology, Danvers, MA), CDK4 (ab137675) (Abcam, Cambridge, UK), cyclin D1 (ab16663), β-actin (4967), Bax (ab32503), Bcl-2 (ab182858), caspase-3 (14220), caspase-9 (9502), MMP-2 (87809), MMP-9 (ab38898), t-PI3K (3811), p-PI3K (13857), t-AKT (9272), p-AKT (9271), Wnt3a (ab219412), and β-catenin (ab32572).

Techniques: CCK-8 Assay, BrdU Incorporation Assay, Western Blot, Flow Cytometry, Expressing, Cell Counting

Journal: IUBMB life

Article Title: Schizandrin A inhibits cellular phenotypes of breast cancer cells by repressing miR-155.

doi: 10.1002/iub.2329

Figure Lengend Snippet: FIGURE 4 Schizandrin A (SchA) restrains proliferation and migration while induces apoptosis through downregulating miR-155. (a) Exogenous miR-155 mimic was introduced into the cells and qRT-PCR proceeded to examine the level of miR-155. At 24 h after the cells were simulated by 30 μM SchA; (b) BrdU-incorporated cells were counted as the proliferative cells; (c) p53, CDK4, and cyclin D1 were assayed by using the Western blot method; (d) quantitative analysis was illustrated with the column chart. (e) The number of FITC-positive cells were counted by flow cytometry and are shown in the column chart. (f) Relative expression of Bax, Bcl-2, and caspases was detected by Western blot. (g) Quantitative analysis was illustrated with the column chart. (h) Migration ability was measured by 24-Transwell assay; (i) relative expression of MMPs was detected by Western blot; (j) quantitative analysis was illustrated with the column chart. Data from three independent experiments were shown as mean ± SD (*p < .05, **p < .01, ***p < .001)

Article Snippet: The primary antibodies contained antibodies against p53 (9282) (Cell Signaling Technology, Danvers, MA), CDK4 (ab137675) (Abcam, Cambridge, UK), cyclin D1 (ab16663), β-actin (4967), Bax (ab32503), Bcl-2 (ab182858), caspase-3 (14220), caspase-9 (9502), MMP-2 (87809), MMP-9 (ab38898), t-PI3K (3811), p-PI3K (13857), t-AKT (9272), p-AKT (9271), Wnt3a (ab219412), and β-catenin (ab32572).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Flow Cytometry, Expressing, Transwell Assay

Journal: Mechanisms of ageing and development

Article Title: Cellular senescence in vascular wall mesenchymal stromal cells, a possible contribution to the development of aortic aneurysm.

doi: 10.1016/j.mad.2021.111515

Figure Lengend Snippet: Fig. 5. (A and B) representative images of immunofluorescence labeling of p – H2AX in h – MSCs and AAA – MSCs isolated from two different donors. A FITC conjugated secondary antibody was used to detect the nuclear localization of p – H2AX. AAA – MSCs nuclei showed a stronger green fluorescent signal compared to h – MSCs nuclei (magnification 600X; bar: 100 nm); (C) representative western blot images showing total p53 and P-p53 protein expression in h – MSCs and AAA – MSCs. Results from two different donors were shown. (D) Relative amounts of total p53 and P-p53 protein expression were normalized to the intensity of actin and represented as fold increase relative to h – MSCs of each donor. Western blotting was performed in duplicate and the relative quantification was expressed as mean value ± SD. * represents a significant difference compared to h - MSCs, p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The membranes were blocked with dry milk (blocking reagent) (Invitrogen, Thermo Fisher Scientific, Monza, Italy) for 30 min at room temperature and were then incubated with the following primary antibodies: mouse anti-human p53 antibody (Cell Signaling Technologies, Euroclone, Milan, Pero); mouse anti-human phospho - p53 (Ser 15) (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human p21CIP antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti human p16INK4A antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human Beclin (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human LC3 (Cell Signaling Technologies, Euroclone, Milan, Italy); mouse anti-human tubulin antibody (Sigma- Aldrich, St Louis, Missouri, USA) and mouse anti-human actin antibody (Millipore Merck, Darmstadt, Germany).

Techniques: Immunofluorescence, Labeling, Isolation, Western Blot, Expressing, Quantitative Proteomics

Journal: International journal of molecular sciences

Article Title: Phenolic Profiles of Red Wine Relate to Vascular Endothelial Benefits Mediated by SIRT1 and SIRT6.

doi: 10.3390/ijms22115677

Figure Lengend Snippet: Figure 6. Modulation of p53 by Magliocco. (a,b) Representative confocal images of p53 (red) and vimentin (green). (c) Fluorescence intensity performed by ImageJ software and expressed as arbitrary fluorescence units (AFU) ± SD of n = 3 replicates. (d,e) Western blot analysis of total and (f,g) acetylated (acetyl K382) p53 expression levels. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = Ma, lane 4 = hGlu, lane 5 = PA, lane 6 = Ma+hGlu, and lane 7 = Ma+PA. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU) ± SD of n = 3 replicates. β-Actin was used as the internal control. § p < 0.01 vs. Ctr, * p < 0.05 vs. hGlu or PA, and ** p < 0.01 vs. hGlu or PA.

Article Snippet: Membranes were incubated overnight at 4 ◦C with specific primary antibodies anti-SIRT1 (1:1000, Biorbyt, Cambridge, UK, orb306144), anti-SIRT6 (1:1000, Abcam, Cambridge, UK, ab191385), anti-NF-κB p65 (acetyl K310) (1:1000, Abcam, Cambridge, UK, ab218533), anti-NF-κB (1:1000, Abcam, Cambridge, UK, ab16502), anti- p53 (acetyl K382) (1:700, Abcam, Cambridge, UK, ab75754), anti-p53 (1:1000, Biorbyt, Cambridge, UK, orb323871), and anti-tumor necrosis factor-α (TNF-α) (1:1000, Abcam, Cambridge, UK, ab6671).

Techniques: Fluorescence, Software, Western Blot, Expressing, Molecular Weight, Control