Journal: Carcinogenesis

Article Title: Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers

doi: 10.1093/carcin/bgab104

Figure Lengend Snippet: Location and functional impact of 28 clinically identified STK11 missense variants. STK11 missense variants represent the most common class of somatic variant and are also the most difficult to assess. We sought to evaluate the functional impact of clinically identified STK11 missense variants by using orthogonal approaches including an STK11 autophosphorylation assay and a luciferase-based p53 transcriptional activation assay. ( A ) The locations of each assessed variant relative to the STK11 polypeptide chain are represented by labeled bubbles where ‘red’ indicates significant loss of functional activity in both assays relative to WT STK11, while ‘green’ represents no significant change in activity relative to WT. ‘Gray’ represents conflicting results. ( B ) Protein from HEK293 cells transfected with Flag-tagged STK11 constructs was isolated and subjected to immunoprecipitation with anti-Flag beads. Purified protein complexes were then subjected to our in vitro kinase assay with (+) and without (−) ATP. Controls were treated separately with lambda phosphatase ( , available at Carcinogenesis Online). All reactions were analyzed by SDS-PAGE and western blot with STK11 antibody. The electrophoretic mobility shift (green arrows) indicates STK11 autophosphorylation. The pie charts associated with each lane display the relative % phosphorylated STK11 (green) versus unphosphorylated STK11 (red) determined by ImageJ analysis. Lane plots from ImageJ are included between the blots and pie charts. ( C ) STK11-dependent p53-mediated luciferase activity is plotted relative to the GFP empty vector control. Each variant was analyzed with respect to STK11 WT signal by unpaired student t-test ( n ≥ 5 for all samples, P ≤ 0.05). ( D ) We compared the luciferase-based functional assay and kinase assay results to 22 in silico predictive algorithms and the ClinVar database; ‘red’ boxes indicate a likely pathogenic prediction and “green” boxes indicate a likely benign prediction; for ClinVar, ‘gray’ boxes indicate conflicting reports or uncertain significance, while ‘white’ boxes indicate no information available. Only two of the variants resulted in uniform agreement across all predictive algorithms while also correlating with our functional results and the ClinVar database (G163R, D194Y). Four additional variants resulted in uniform agreement across all predictive algorithms while also correlating with our functional results, but were either absent from ClinVar, or reported as uncertain significance or conflicting impact (G56W, P179R, G242V and A397S).

Article Snippet: Antibodies directed against LKB1 (E-9; #sc-374334), p53 (D0-1; #sc-126), Actin (C-2; #sc-8432), STRADα (G-8; sc 515635), MO25 (#2716) and Tubulin (#2144) were obtained from Santa Cruz Biotechnology (Dallas, TX) and Cell Signaling Technology (Danvers, MA), respectively.

Techniques: Functional Assay, Variant Assay, Luciferase, Activation Assay, Labeling, Activity Assay, Transfection, Construct, Isolation, Immunoprecipitation, Purification, In Vitro, Kinase Assay, SDS Page, Western Blot, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Control, In Silico

Journal: Breast Cancer Research : BCR

Article Title: Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

doi: 10.1186/s13058-018-1044-5

Figure Lengend Snippet: Chemotherapy-treated patients with tumors harboring TP53 mutation fare equally well or better than patients with TP53 wild-type tumors. ( a ) Position and frequency of the 663 TP53 mutations present in the METABRIC dataset accessed through cBioportal. ( b ) Overall survival curves were created for patients in the METABRIC dataset with TP53 wild-type and mutant tumors from ( b ) all patients; ( c ) those who received chemotherapy (median survival 125 vs 129 months; ( d ) those who received chemotherapy plus radiation (median survival 144 vs 135 months); ( e ) those who received chemotherapy plus radiation but not hormone therapy; ( f ) those who received chemotherapy plus radiation plus hormone therapy. Survival curves were created for patients with TP53 wild-type ( g ) or mutant ( h ) tumors who received chemotherapy plus radiation and no hormone therapy, or chemotherapy plus radiation plus hormone therapy. Overall survival curves were created for patients with TP53 wild-type and mutant tumors from ( i ) PAM50 basal-like tumor cohort that received chemotherapy plus radiation but not hormone therapy; ( j ) the other PAM50 classifications combined [claudin low ( n = 39), HER2 ( n = 50), luminal A ( n = 1), luminal B ( n = 6), normal-like (n = 6)] that received chemotherapy plus radiation but not hormone therapy; ( k ) tumor cohort classified as “triple-negative” in the three gene classifier that received chemotherapy. Statistical differences in survival curves were calculated using both the Wilcoxon test (weighs early events more heavily) and log-rank (Mantel-Cox) tests (weighs events evenly over time). Shown below each survival curve is a table containing the sample size in each arm, the mean +/− standard error of the mean (SEM) and p value (unpaired, two-tailed Student’s t test) for tumor histological grade, tumor stage, tumor size, and Nottingham Prognostic Index

Article Snippet: Western blotting was performed as previously shown [ ] for p53 (D01, Cell Signaling, Danvers, MA, USA) and actin (BA3R, Thermo Fisher Scientific).

Techniques: Mutagenesis, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

doi: 10.1186/s13058-018-1044-5

Figure Lengend Snippet: TP53 mutation portends worse 5-year overall survival for patients who received hormone therapy, and those who received no chemotherapy. Overall survival curves were created for patients with TP53 wild-type and mutant tumors from cohorts who ( a ) received hormone therapy; ( b ) received hormone therapy but not chemotherapy; ( c ) did not receive chemotherapy. Overall survival curves were created for patients with TP53 wild-type and mutant tumors from cohorts who ( d ) were HER2+; ( e ) were classified as HER2 gain; ( f ) were classified as HER2 gain and received chemotherapy. Statistical differences in survival curves were calculated using both the Wilcoxon test (weighs early events more heavily) and log-rank (Mantel-Cox) tests (weighs events evenly over time). Shown below each survival curve is a table containing the sample size in each arm, the mean +/− SEM and p value (unpaired, two-tailed Student’s t test) for tumor histological grade, tumor stage, tumor size, and Nottingham Prognostic Index

Article Snippet: Western blotting was performed as previously shown [ ] for p53 (D01, Cell Signaling, Danvers, MA, USA) and actin (BA3R, Thermo Fisher Scientific).

Techniques: Mutagenesis, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

doi: 10.1186/s13058-018-1044-5

Figure Lengend Snippet: TP53 wild-type, ER+ breast cancer cells made senescent by chemotherapy are sensitive to tamoxifen. ( a ) TP53 wild-type, ER+ cells as indicated were plated in triplicate at 80,000 cells per well in a 24-well plate and then treated with 250 nM doxorubicin for 24 h. Seven days later, 1 μM, 5 μM, or 10 μM tamoxifen (Tam) or ethanol vehicle (ETOH) was added as indicated in the figure, with ( gray bars ) or without ( black bars ) the pan-caspase inhibitor QVD. MTT assay was performed 24 h later. Proliferating cells were plated similarly but treated with tamoxifen the next day. ( b ) MCF-7 cells infected using a lentiviral CRISPR Cas9 system with non-targeting (NT) or TP53 guide RNAs were sorted and then plated and treated with 250 nM doxorubicin as in ( a ). Upper panel : light microscopy images were captured for untreated, proliferating cultures or treated cultures as indicated 8 days following treatment. Scale bar is 100 μm. Lower panels : western blot for p53 ( upper ) and actin ( lower ). ( c ). TP53 mutant, ER+ cell lines as indicated were plated, treated, and MTT assay performed as in ( a ). Statistical analyses of these data are shown in Additional file : Table S3. Data are representative of at least two independent experiments

Article Snippet: Western blotting was performed as previously shown [ ] for p53 (D01, Cell Signaling, Danvers, MA, USA) and actin (BA3R, Thermo Fisher Scientific).

Techniques: MTT Assay, Infection, CRISPR, Light Microscopy, Western Blot, Mutagenesis

Journal: NPJ Breast Cancer

Article Title: Retinoblastoma protein expression and its predictors in triple-negative breast cancer

doi: 10.1038/s41523-020-0160-4

Figure Lengend Snippet: Proportions of negative, low positive, and positive p53 by molecular features at presentation.

Article Snippet: Similarly, p53 staining was performed on TMA sections using a mouse monoclonal antibody to human p53 (clone D0–1, ImmunoTech, Hostivař, Czech Republic) and scored as negative (<1% nuclear staining), low positive (≥1% to <10% nuclear staining), or high positive (≥10% nuclear staining).

Techniques:

Journal: NPJ Breast Cancer

Article Title: Retinoblastoma protein expression and its predictors in triple-negative breast cancer

doi: 10.1038/s41523-020-0160-4

Figure Lengend Snippet: Molecular features at presentation by retinoblastoma protein status among the full cohort ( n = 180).

Article Snippet: Similarly, p53 staining was performed on TMA sections using a mouse monoclonal antibody to human p53 (clone D0–1, ImmunoTech, Hostivař, Czech Republic) and scored as negative (<1% nuclear staining), low positive (≥1% to <10% nuclear staining), or high positive (≥10% nuclear staining).

Techniques: Staining

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Stable expression of functional p53 in yeast cells. (A) Schematic representation of the domain organization of p53 protein. (B) Schematic representation of p53-mediated activation of various reporters in yeast and the corresponding phenotypes. (C) (i) Functionality of expressed p53 in yeast was assayed by measuring expression of three reporters placed under the control of p53RE. (ii) White/blue and white/red colony phenotypes assays were performed using the indicated strains after spotting them on X-Gal galactose or YPD plates. (iii) For the reporter HIS3 in strain SGY6004, the function of p53 was assayed by growing cells on SC−His plates harboring 3-AT to suppress the basal-level expression of HIS3. (D) Activity of p53 was monitored quantitatively by liquid culture assay using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate. Βeta-galactosidase units = 1,000 × OD420/(t × V × OD600), where t equals elapsed time (in minutes) of incubation, V equals 0.1 ml × concentration factor = 0.5, and OD600 equals the A600 of 1 ml of culture = 0.8. (E) Western blot analysis of p53 was performed using anti-p53 antibody (DO-1). (F) p53 expression in yeast cells. Expression of p53 from a galactose-inducible promoter in the yeast cell shows nuclear localization. The inset shows a magnified image of the p53 nuclear localization. Scale bar, ∼2 μm.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Expressing, Functional Assay, Activation Assay, Control, Activity Assay, Incubation, Concentration Assay, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Seeding effect of P8 fibrils (residues 250 to 257; PILTIITL) on functional p53. (A) Schematic representation of the loss of function of native p53 upon interaction with preformed P8 fibrils (Seed) (i), which leads to change in colony color (ii and iii) and a growth defect (iv), which is due to inactivation of transcription of three reporters, as indicated. (B) Growth curves of strain SGY6003 with (+) and without (−) seed were not found to be significantly different. (C) Internalization of FITC-labeled aggregated P8 fibrils and P8 monomer within the yeast cell. (i) FITC-labeled P8 fibril (green fluorescence) was used to transform yeast cells, similar to what was performed in panels A and E, and we obtained visual confirmation of the internalization of P8 fibrils and their localization in the cytoplasm. (Bottom left) Magnified image of the labeled seed (arrow) inside the cytoplasm. Scale bar, ∼2 μm. (ii) FITC-labeled P8 monomer (green fluorescence) was used to transform yeast cells, similar to what was performed in panels A and E, and we obtained visual confirmation of the internalization of P8 monomer and its localization in the cytoplasm (arrow). Scale bar, ∼2 μm. (D) Immunoprecipitation of p53 from cells with and without seed was performed using anti-p53 antibody. Dot blot analysis was performed with immunoprecipitated p53 using anti-p53, OC, and A11 antibodies separately. WCE, whole-cell extract; p53 IP, immunoprecipitated p53. (E) Immunofluorescence study showing colocalization of p53 and OC antibody in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds, which showed robust colocalized signal with OC antibody. Scale bar, ∼5 μm.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Functional Assay, Labeling, Fluorescence, Immunoprecipitation, Dot Blot, Immunofluorescence

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Transmission of aggregated p53 requires the presence of functional p53. Strain SGY6003(pGALp53LacZ) was allowed to pass through generations, as indicated, in inducing galactose (Gal) or noninducing dextrose (Dex) medium in the presence or absence of the seeds (P8) or p53 core domain. Finally, the cells were diluted and plated on the X-Gal galactose plate to visualize the colony color. Raf., raffinose.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Transmission Assay, Functional Assay

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Effect of p53 expression on yeast growth. (A) Growth curves of the yeast strain with and without GAL-p53 vector and with and without seeds on liquid minimal medium in the absence (left) or presence (right) of galactose. The A600 was measured every 2 h. The data points represent the means of the results of three independent experiments. The error bars indicate standard deviations from the mean. (B) Analysis of cell death induced in yeast. Addition of the seeds drastically reduced the percentage of cells stained with the death markers annexin V and PI. Scale bar, ∼5 μm.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Expressing, Plasmid Preparation, Staining

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Aggregation of native p53 requires preaggregated amyloidogenic peptides or the core domain. Loss of function of native p53 in the strain SGY6003(pGALp53LacZ) occurs in the presence of preaggregated amyloidogenic fibrils (PILTIITL) (A) or the core domain (B). However, monomeric amyloidogenic peptide, core domain, or aggregated scrambled peptide (ITLPITLI) failed to inactivate the native p53 function.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Loss of function of aggregated p53. (A) The level of LacZ mRNA was measured in cells with or without seeds using reverse transcription followed by real-time PCR analysis. Two independent reference genes, CDC19 and TAF10, were used for normalization and fold change in the mRNA levels. The data represent the averages of three independent real-time PCR analyses. The error bars indicate standard deviations from the mean. (B) (Top) ChIP of p53 from cells with or without seeds. Yeast cells were analyzed by PCR and visualized by agarose gel electrophoresis. Differences in amplification were observed in input, IP, and mock IP. TAF10 was used as a nonbinding control. (Bottom) Graphical representation of chromatin immunoprecipitation of p53 displaying percent enrichment/input by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent averages of the results of three independent real-time analyses. (C) Immunofluorescence study of chromatin spread of yeast cells with or without seeds. The cells without seeds, but not those with seeds, displayed p53 in a majority of the spread as chromatin bound. Scale bar, ∼3 μm. Statistical analysis of the total percent spread was calculated as represented graphically below.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Control, Chromatin Immunoprecipitation, Immunofluorescence

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Effect of P8 fibril on GFP-tagged functional p53. (A) Immunofluorescence assay to visualize p53-GFP in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds. However, in cells without any seeds, p53-GFP was colocalized with DAPI. Scale bar, ∼5 μm. (B) Graphical representation of ChIP of p53 displaying percent enrichment/input at the p53 binding element by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent the averages of the results of three independent real-time analyses. The error bars indicate standard deviations from the mean.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Functional Assay, Immunofluorescence, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Isolation of p53 amyloids from yeast cell lysate. (A) Amyloid-containing fractions were isolated from yeast cell lysates by sedimentation in SDS-containing lysis buffer. The p53 protein was detected in the total lysate, the SDS-soluble supernatant, and the SDS-insoluble pellet fraction by immunoblotting with anti-p53 antibody. A prestained molecular mass ladder was loaded in the middle lane. The blot was developed with the colorimetric substrate TMB (Genei, India). The blot was developed with anti-GPD to assess GPD levels in fractions with and without seeds as a loading control. (B) Lysates of yeast cells with/without seeds were analyzed by SDD-AGE and Western blotting. The p53 expression was detected by immunoblotting with anti-p53 antibody. Cell lysate from plus-seed cells showing higher-order SDS-resistant aggregates are marked in the gel.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Isolation, Sedimentation, Lysis, Western Blot, Control, Expressing

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Dominant character and non-Mendelian segregation by p53 amyloids. (A) Haploid pGALp53LacZ (+ Seed) and pADHp53ADE2 (− Seed) haploids were streaked on a YPD plate. The two strains were mated to obtain the desired combination. The resultant strain was streaked on YPD to check the colony assay, which was observed to be plus-seed cells, suggesting the presence of a dominant trait. (B) (Right) p53 amyloids in a diploid cell obtained by mating between the plus-seed and the minus-seed cells displayed non-Mendelian segregation, since all the meiotic spores of almost all the dissected tetrads displayed nonfunctional and aggregated p53, as shown by white colonies. (Left) Mating between the minus-seed cells (control) showing all the meiotic spores carrying functional p53, as shown by blue colonies.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Colony Assay, Control, Functional Assay

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Prion-like infectivity of p53 amyloids. Haploid recipient and donor cells of the indicated genotypes were mated, where donor cells were with (+Seed) or without (−Seed) seeds. Following heterokaryon formation, the cytoductants carrying recipient nuclei and a mixture of donor and recipient cytoplasm were selected based on the recipient nuclear genotype and growth on glycerol medium. The resultant cytoductants were transferred onto a YPD plate to assay ADE2 reporter (A) and onto a His–3-AT plate to assay HIS3 reporter (B).

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Infection

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Rescue of p53 amyloid due to overexpression of Hsp104. (A) Live-cell imaging to visualize p53-GFP signal in cells overexpressing Hsp104. Three types of cells were observed: type I, where cytoplasmic signal of p53-GFP was observed, suggesting localization of p53 aggregates in the cytoplasm; type II, where nuclear signal of p53-GFP was observed, suggesting functional nonaggregating p53 enters the nucleus; and type III, where both cytoplasmic and nuclear signals were observed, suggesting a fraction of p53 aggregates were rescued and became functional due to Hsp104 overexpression. Scale bar, 5 μm. (B) Field view showing all three types of cells. Scale bar, 3 μm. (C) Percentage distributions of the three types of cells with or without Hsp104. (D) Dose-dependent curing of p53 prion by Hsp104. (E) Growth defect observed due to inactivation of transcription of the HIS3 reporter for the cells harboring seeds. However, the defect was partially rescued when the same cells overexpressed Hsp104 (compare the rows indicated by the red and green arrowheads). (F) The growth rescue effect of Hsp104 in panel E was abolished upon treatment with GdHCl, and the growth of the treated cells became similar to the that of the plus-seed cells (compare the rows indicated by the red arrowheads).

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Over Expression, Live Cell Imaging, Functional Assay

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Proposed mechanism of p53 aggregation. Shown is a schematic representation of the yeast cell showing P8 fibril (seed)-mediated inactivation of p53 and its proposed aggregation pathway. When p53 amyloids (oligomer or mature fibrils) are formed, it may template the aggregation of the rest of the native p53 proteins in the cell and amplify the p53 amyloids by prion-like propagation. The aggregated p53 cannot enter the nucleus to activate the reporter, become stabilized in the cytoplasm, and infect the daughter cell through transfer of cytoplasm.

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Yeast strains used in this study a

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

doi: 10.1128/MCB.00118-17

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: The fixed nuclear spreads were blocked for 15 min with PBS supplemented with 0.1% bovine serum albumin (BSA) and 5% skim milk and incubated with primary antibodies and anti-p53 D0-1 antibody (Santa Cruz Biotechnology; 1:500 dilution) for 2 h. The slides were washed in PBS for 5 min, preadsorbed Dylight 488-conjugated goat anti-mouse secondary antibody was added at 1:200 dilution, and the slides were incubated for another 2 h. The slides were washed twice in PBS for 5 min each time and incubated with 1 μg ml −1 DAPI (4′,6-diamidino-2-phenylindole) for 15 min.

Techniques: Plasmid Preparation, Marker, Control, Binding Assay, Sequencing, Derivative Assay