Journal: Veterinary pathology

Article Title: Immunohistochemical evaluation of vascular urinary bladder tumors from cows with enzootic hematuria.

doi: 10.1354/vp.46-2-211

Figure Lengend Snippet: Fig. 5. Hemangiosarcoma in bovine urinary bladder. Strong nuclear p53 reaction in the majority of neoplastic cells in a solid hemangiosarcoma. Streptavidin-peroxidase stain with Mayer’s hematoxylin counterstain. Bar 5 100 mm. Inset: Detail of the staining. Bar 5 30 mm. Fig. 6. Hemangiosarcoma in bovine urinary bladder. Urothelium covering endothelial tumor with strong nuclear p53 reaction in the majority of basal cells. Streptavidin-peroxidase stain with Mayer’s hematoxylin counterstain. Bar 5 100 mm. Inset: There is marked atypia of basal cells. Bar 5 30 mm. Fig. 7. Hemangioendothelioma in bovine urinary bladder. Urothelium covering endothelial tumor displays atypical uroplakin III reaction, with intense and diffuse staining of cells in the deeper layers (arrow). ImmPRESS- peroxidase with Mayer’s hematoxylin counterstain. Bar 5 50 mm. Fig. 8. Hemangiosarcoma in bovine urinary bladder. Heterogenous uroplakin III staining of urothelial cells in von Bru¨nn Nests within a hemangiosarcoma, with patchy intracytoplasmic positivity (asterisk); superficial cells display normal uroplakin III expression, more intense in the apical border of the cytoplasmic membrane (arrow). ImmPRESS-peroxidase with Mayer’s hematoxylin counterstain. Bar 5 30 mm. Inset: Detail of the staining. Bar 5 10 mm.

Article Snippet: CD31 Factor VIII– related antigen A0082/Dako, Glostrup, Denmark Proteinase K 10 minutes @ room temperature 60 minutes @ 1 : 300 Histostain-plus/Zymed Laboratories, San Francisco, CA CD31 PECAM-1 (M-20)/Santa Cruz Biotechnology, Inc, Santa Cruz, CA Steam 20 minutes @ 98uC in citrate 60 minutes @ 1 : 200 Mouse anti-goat IgG and 4plus immunoperoxidase fetection dystem/ Biocare Medical, Concord, CA Cyclin D1 18-0220, clone AM29/ Zymed Laboratories, San Francisco, CA Microwave 20 minutes in EDTA pH 8 Overnight @ 1 100 ImmPRESS/Vector Laboratories, Burlingame, CA p53 MCA1701, clone DO1/ Serotec Inc, UK Microwave 15 minutes in citrate Overnight @ 1 : 300 Histostain-plus/Zymed Laboratories, San Francisco, CA Uroplakin III PRO651108/clone AU1 Research Diagnostics, Inc, Concord, MA Proteinase K 10 minutes @ room temperature 60 minutes (ready to use) ImmPRESS/Vector Laboratories, Burlingame, CA at CARLETON UNIV on June 16, 2015vet.sagepub.comDownloaded from expression was detected in all endothelial tumors examined, including muscle-invasive hemangiosarcomas (Fig. 2).

Techniques: Staining, Expressing, Membrane

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: Stable silencing of Rac1 in renal epithelial cells ablates TGF-β1–mediated fibrotic reprogramming. Confluent control shRNA or Rac1 shRNA stably expressing cultures were untreated (−) or stimulated with TGF-β1 for 24 h and extracts processed by Western analysis for Rac1 (A, B), fibronectin (A, C), PAI-1 (A, D), CTGF (A, E), p21 (A, F), and GAPDH expression (A). Histograms (B–F) illustrate relative protein levels (mean ± sd) for 3 separate studies setting expression levels in untreated (−) control shRNA cells as 1 for each. Con, control. *P < 0.05, **P < 0.01.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Control, shRNA, Stable Transfection, Expressing, Western Blot

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: Rac1 is required for TGF-β1 to activate several non-SMAD pathways critical for fibrogenesis. Confluent control shRNA and Rac1 shRNA stably expressing cell cultures (maintained in low serum medium) were stimulated with TGF-β1 for 1–2 h. Cell extracts were immunoblotted with antibodies to p-p53Ser15 (A, B), pATMSer1981 (A, C), pEGFRY845 (A, D), p-cSrcY418 (A, E), and GAPDH (A). Plots (B–E) summarize the relative expression (mean ± sd) of the indicated markers setting the expression in control shRNA stable cells without TGF-β1 stimulation (−) as 1 (n = 3). Con, control. *P < 0.05, **P < 0.01, as indicated.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Control, shRNA, Stable Transfection, Expressing

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: Upstream role of p22Phox and p47Phox NOX subunits in promoting TGF-β1 noncanonical signaling. Serum-starved confluent control shRNA and p22Phox shRNA (A) or p47Phox shRNA (E) stably transduced HK-2 cells were treated with TGF-β1. Cellular extracts were immunoblotted with antibodies to p-p53Ser15, pEGFRY845, p-cSrcY416, and GAPDH following confirmation of p22Phox and p47Phox depletion compared to their respective vector control cells (A, E). Histograms in B–D and F–H represent the relative expression (mean ± sd) of indicated phospho-proteins for 3 separate studies. Con, control. *P < 0.05, **P < 0.01.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Control, shRNA, Stable Transfection, Plasmid Preparation, Expressing

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: Rac1 contributes to TGF-β1–mediated ROS generation via NADP(H) oxidases. A) The composition of different NOX proteins. Rac and p47Phox are common subunits of NOX1 and NOX2, whereas p22Phox is present in NOX1/2/4. Confluent HK-2 cells were stimulated with TGF-β1 for the times indicated with or without the Rac inhibitor EHT 1864 then incubated with 5 μM DCFDA for 15 min prior to scrape harvest. B) Histogram depicts relative DCFDA measurements (mean ± sd) in triplicate studies for each experimental condition. An equal number of cells were used to assess baseline fluorescence in the unstimulated (−) state and in response to TGF-β1; all measurements were done at the same time. *P < 0.05, **P < 0.01. Serum-deprived control shRNA and Rac1 shRNA stable HK-2 transductants were stimulated with TGF-β1 (15 min) for DCFDA analysis. C) Data illustrate relative free radical levels (mean ± sd) for each experimental condition for triplicate cultures. *P < 0.05, **P < 0.01. D) Plot depicts relative DCFDA measurements (mean ± sd) of serum-deprived control shRNA and p22Phox shRNA stably expressing HK-2 cells, which remained untreated (−) or stimulated with TGF-β1 (15 min); data plotted are for triplicate replicates for each condition in each of 3 separate experiments. Con, control. **P < 0.01.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Incubation, Fluorescence, Control, shRNA, Stable Transfection, Expressing

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: Rac1 and NADP(H) oxidases are key effectors of renal epithelial growth inhibition in response to TGF-β1. A) A schematic representation of study design for Rac1 involvement in renal epithelial cell cycle arrest. Briefly, subconfluent control shRNA or Rac1 shRNA stably transduced HK-2 epithelial cells at a similar density were serum-deprived for 1 d then incubated with TGF-β for 1 d followed by serum addition (1%) for 3 d to stimulate cell growth. B) Relative epithelial cell counts (mean ± sd) are plotted in setting the cell number in untreated (−) control shRNA cultures as 1. *P < 0.05 as indicated. N.S., not significant. C–F) To investigate the potential involvement of p22Phox and p47Phox subunits of the NADP(H) oxidases in TGF-β1–induced epithelial growth inhibition, study designs are adopted similar to above and the schematics (C, E) illustrate the experimental approaches. Semiconfluent and serum-starved control shRNA and p22Phox or p47Phox shRNA stably expressing renal epithelial cells at a similar density were incubated with TGF-β for 1 d followed by 3 d of 1% serum stimulation. Relative cell counts (mean ± sd) provided the comparisons of cell growth between control shRNA and p22Phox shRNA stable transductants (D) or control shRNA and p47Phox shRNA (F) stably expressing HK-2 cells setting the cell number in untreated control shRNA as 1 in each case. Con, control; NS, not significant; n = 3. *P < 0.05 as indicated.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Inhibition, Control, shRNA, Stable Transfection, Incubation, Expressing

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: TGF-β1–induced PAI-1 up-regulation mediates renal epithelial cell proliferative restriction. A) Subconfluent and serum-starved control shRNA and PAI-1 shRNA stably expressing HK-2 cells were treated with TGF-β for 1 d prior to serum stimulation for 3 d to promote cell growth. B) Data depict relative cell counts (mean ± sd) setting cell number in untreated (−) control shRNA cultures as 1. Con, control; NS, not significant; n = 3. **P < 0.01.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Control, shRNA, Stable Transfection, Expressing

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: Rac inhibition results in decreased Rac1b, NOX, YAP/TAZ, p53 and EGFR signaling. Obstructed kidney extracts from EHT 1864–treated or vehicle-treated mice were Western blotted with antibodies against Rac1b (A, B), p22Phox (A, C), YAP/TAZ (A, D), pEGFRY845 (A, E), p-p53Ser15 (A, F), and GAPDH. Histograms illustrate renal levels (mean ± sd) of each protein between the (UUO + vehicle) vs. (UUO + EHT 1864) experimental groups (B–F); n = 5 mice per group. *P < 0.05, **P < 0.01.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Inhibition, Western Blot

Journal: The FASEB Journal

Article Title: Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways

doi: 10.1096/fj.201802489RR

Figure Lengend Snippet: A model for Rac1 involvement in TGF-β1–driven renal fibrosis. Rac1 is rapidly activated in response to TGF-β1 stimulation and likely facilitates the assembly of NOX1 and NOX2 signaling complexes in the renal cellular plasma membrane. NADP(H)-mediated generation of free radicals in response to TGF-β1 induces ATM and p53 phosphorylation as well as EGFR and c-Src activation. The resulting accumulation of p-p53Ser15 and pSMAD3 transcriptional complexes on the promoter of TGF-β1 target genes mediates fibrotic reprogramming and PAI-1–dependent cell cycle arrest. Rac1b and NOX subunit expression is dramatically increased in UUO-driven renal injury. Chemical blockade of Rac attenuated progressive renal fibrosis and Rac1b, NOX, YAP/TAZ, p53, and EGFR activation in the kidney. Therefore, Rac1 is a new non-SMAD control element of the TGF-β1 pathway and a novel therapeutic target against CKD.

Article Snippet: To generate stable Rac1, p22 Phox , p47 Phox , p53, transcriptional coactivator with PDZ-binding motif (TAZ), and plasminogen activator inhibitor-1 (PAI-1) knockdowns, semiconfluent HK-2 cells were infected with Rac1 short hairpin RNA (shRNA), p22 Phox shRNA, p47 Phox shRNA, p53 shRNA, TAZ shRNA, or PAI-1 shRNA or the corresponding empty vector (control shRNA) lentiviral particles (all from Santa Cruz Biotechnology, Dallas, TX, USA) using 5 μg/ml Polybrene in 10% FBS/DMEM for 24 h. Cells were allowed to recover for 24 h and then selected in 10% FBS/DMEM/5 μg/ml puromycin; medium was changed every 3 d. Rac1, p22 Phox , p47 Phox , p53, TAZ, and PAI-1 depletion was confirmed by Western blot analysis.

Techniques: Clinical Proteomics, Membrane, Phospho-proteomics, Activation Assay, Expressing, Control

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: 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 aggregation , , . 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 ) . 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 ) . 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 ( λ ex/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 ( K d ) were then determined using a one-site-specific binding equation (Eq. ). 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) . 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, Fluorescence, 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: a , b Overlay of 15 N- 1 H HSQC maps of 19 μM WT ( a ) and 24 μM R248W ( b ) p53 DBD in H O/D O (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. ). d The five clusters of the two p53 DBD variants (WT and mutant R248W) that show high (>0.025) or medium (>0.015) CSP values . 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: 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: a Confocal fluorescence microscopy images showing thioflavin S (ThS) staining of mutant p53 (R248W) aggregates in MIA PaCa-2 cells treated with vehicle (0.02% DMSO) or ADH-6 (5 µM) for 0.5 or 6 h. Imaging experiments were performed in quadruplicate and representative images are shown. b Quantification of ThS-positive MIA PaCa-2 cells after treatment with vehicle or ADH-6. The number of positively stained cells in 3–5 different fields of view are expressed as % of the total number of cells ( n = 4 biologically independent samples). Data presented are mean ± SD. Statistical analysis was performed using two-tailed unpaired t -test. P < 0.0001 for ADH-6 vs vehicle at 6 h. c Confocal fluorescence microscopy images of ThS and PAb 240 antibody staining of R248W aggregates in MIA PaCa-2 cells treated with vehicle or 5 µM ADH-6 for 0.5 or 6 h. Images shown are representative of four independent experiments. d – f Quantification of PAb 240-positive MIA PaCa-2 cells after treatment with the indicated concentrations of ADH-1, ReACp53, or ADH-6 for 0.5 or 6 h relative to controls (vehicle-treated cells). The number of positively stained cells in 3–5 different fields of view are expressed as % of the total number of cells (mean ± SD; n = 4). Statistical analysis was performed using repeated measures two-way ANOVA followed by Holm-Sidak’s post hoc test. P < 0.0001 for ReACp53 (2.5–10 µM) vs vehicle at 6 h ( e ); P < 0.0001 for ADH-6 (2.5–10 µM) vs vehicle at 6 h ( f ). g Colocalization of FITC-labeled ADH-6 (ADH-6 FITC ) with PAb 240-stained R248W aggregates following incubation with the oligopyridylamide (5 µM) for 0.5 or 6 h. Colocalization was quantified using directional Pearson correlation coefficient, r , which measures pixel-by-pixel covariance in the signal level of two images . Scale bar = 5 µm. h , i Cellular thermal shift assay (CETSA) analysis of intracellular target engagement. Melting curves for p53 mutants R248W ( h ) and R175H ( i ) in MIA PaCa-2 and SK-BR-3 cells, respectively, in the absence or presence of the oligopyridylamides (mean ± SD; n = 3). *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls.

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: Fluorescence, Microscopy, Staining, Mutagenesis, Imaging, Two Tailed Test, Labeling, Incubation, Thermal Shift Assay, Drug discovery

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: a – c Effects of ADH-6 on viability of cancer cells bearing WT or mutant p53. MIA PaCa-2 (mutant R248W p53) ( a ), MCF-7 (WT p53) ( b ), and SK-BR-3 (mutant R175H p53) ( c ), cells treated with increasing oligopyridylamide concentrations for 24 or 48 h. ( d – f ) p53 null Saos-2 cells before ( d ) and after transfection with p53 mutants, R248W ( e ) or R175H ( f ), treated with increasing concentrations of ADH-6 for 24 or 48 h. Cell viability in a – f was assessed using the MTS assay, with the % viability determined form the ratio of the absorbance of the treated cells to the control cells ( n = 3 biologically independent samples). Data presented are mean ± SD. Statistical analysis in a – f was performed using two-tailed unpaired t -test. P < 0.0001 for ADH-6 vs ADH-1 at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( a , c ); P < 0.0001 for ADH-6 treatment of Saos-2/R248W compared with untransfected cells (data shown in d ) at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( e ); P < 0.0001 for ADH-6 treatment of Saos-2/R175H compared with untransfected cells (data shown in d ) at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( f ). g , h Flow cytometry analysis of annexin V/propidium iodide (PI) staining of MIA PaCa-2 cells that were treated with vehicle (control), or 5 µM ADH-1, ReACp53, or ADH-6, for 24 h. The bottom left quadrant (annexin V−/PI−) represents live cells; bottom right (annexin V+/PI−), early apoptotic cells; top right (annexin V+/PI+), late apoptotic cells; and top left (annexin V−/PI+), necrotic cells ( g ). A summary of the incidence of early/late apoptosis and necrosis in the different treatment groups determined from the flow cytometry analysis of annexin V/PI staining (mean ± SD; n = 4) ( h ). Statistical analysis in h was performed using one-way ANOVA followed by Dunnett’s post hoc test. P < 0.0001 for ReACp53 vs vehicle (live and early apoptosis); P < 0.0001 for ADH-6 vs vehicle (live, early apoptosis, and late apoptosis). i Cell cycle distribution of MIA PaCa-2 cells treated w i th vehicle (control), or 5 µM ADH-1, ReACp53 or ADH-6, for 6 h as determined by measurement of DNA content using flow cytometry (mean ± SD; n = 4). Two-tailed unpaired t -test: P = 0.0071 and 0.0037 for ReACp53 vs vehicle (G0/G1 and G2/M, respectively); P < 0.0001 and P = 0.0004 for ADH-6 vs vehicle (G0/G1 and G2/M, respectively). ** P < 0.01, *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls.

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, Transfection, MTS Assay, Control, Two Tailed Test, Concentration Assay, Incubation, Flow Cytometry, Staining

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: a , b In vivo pharmacokinetics of ADH-6. Concentration of ADH-6 in plasma ( a ) and in MIA PaCa-2 xenografts ( b ) of mice ( n = 5–6 per group), after an intraperitoneal injection of the oligopyridylamide (15 mg kg -1 ), was quantified using LC-MS/MS . Shown are the circulation half-life ( T 1/2 ) ( a ) as well as the maximum (or peak) concentration ( C max ) in tumors and the time to achieve C max ( T max ) ( b ). Data presented are mean ± SD. c , d Design of the tumor reduction studies. A representative mouse bearing both MIA PaCa-2 (mutant R248W p53) and MCF-7 (WT p53) xenografts ( c ) and treatment schedule for the dual xenograft model ( d ). Once the tumor volume reached ~25 mm , the mice were randomized into the different treatment groups ( n = 8 per group), which were injected intraperitoneally with vehicle (0.02% DMSO), ReACp53 (716.4 µM), or ADH-6 (716.4 µM). Injections were done every 2 days for a total of 12 doses, with the first day of treatment defined as day 0. e Body weight changes of the tumor-bearing mice in the different treatment groups monitored for the duration of the experiment (mean ± SD; n = 8). f , g Tumor volume growth curves for the MIA PaCa-2 ( f ) and MCF-7 ( g ) xenografts in the different treatment groups over the duration of the experiment (mean ± SD; n = 8). Tumor volume was calculated using Eq. . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 ( f ). h , i Tumor mass analysis for the different treatment groups. After 25 days of treatment, four mice per treatment group were sacrificed and the tumor tissues were isolated and imaged ( h ) and subsequently weighed to determine the tumor mass ( i ). Data presented are mean ± SD, and statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 (MIA PaCa-2 xenografts; i ). j Hematoxylin and eosin (H&E)-stained xenograft sections from the different treatment groups following 25 days of treatment. Images on the right are magnified views of the boxed regions in the images on the left. Scale bar = 20 μm (50 μm for the magnified views). k – m Immunohistochemistry (IHC) analysis of the residual xenografts. Images of sections of MIA PaCa-2 and MCF-7 xenografts stained using the anti-p53 PAb 240 and DO-7 antibodies, respectively, from the different treatment groups ( k ). Images on the right are magnified views of the boxed regions in the images on the left. Scale bar = 20 μm (50 μm for the magnified views). Quantification of PAb 240 ( l ) and DO-7 ( m ) positive cells in 3–5 different fields of view expressed as % of the total number of cells (mean ± SD; n = 4). One-way ANOVA followed by Tukey’s post hoc test: P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 ( l ). n Survival curves for the vehicle, ReACp53 and ADH-6 treatment groups over 30 days ( n = 4 per group). Statistical analysis was performed using log-rank (Mantel-Cox) test. P = 0.0062 for ADH-6 vs vehicle. ** P < 0.01, *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls and between the different treatment groups.

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: In Vivo, Drug discovery, Concentration Assay, Clinical Proteomics, Injection, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Isolation, Staining, Immunohistochemistry

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: miRNA expression profiling in H1299 cells expressing mutant p53 R273H using small RNA sequencing. ( A ) Immunoblot showing mutant p53 R273H level in H1299/ mutant p53 R273H stable cells. ( B ) Scatter plots showing a correlation of normalized read counts between biological replicates of individual samples. ( C ) Heat map showing normalized read counts of miRNAs differentially expressed ( p -value ≤ 0.05) between H1299/mutant p53 R273H and H1299/EV cells. Hierarchical clustering of samples is shown. Color bar indicates Z- scores of normalized read counts. Red color indicates high expression, green color indicates low expression. ( D ) Validation of the selected differentially expressed miRNAs in H1299/mutant p53 R273H cells using qRT-PCR. Bar graphs represent mean ± s.d.; n ≥ 2; two-tailed Student’s t -test: * p < 0.05. A relative comparison of qRT-PCR data with the deep sequencing results is shown.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Expressing, Mutagenesis, RNA Sequencing, Western Blot, Biomarker Discovery, Quantitative RT-PCR, Two Tailed Test, Comparison, Sequencing

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: Molecular and cellular functions enriched in H1299/mutant p53 R273H cells.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques:

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: Molecular networks enriched by miRNAs differentially expressed in H1299/mutant p53 R273H cells. Ingenuity Pathway Analysis (IPA) generated top three significantly enriched regulatory networks of mutant p53 R273H regulated miRNAs. The networks illustrate direct or indirect interactions between altered miRNAs and their target genes. Only the highlighted (green/red) miRNAs were present in our dataset. Green and red represent down-regulated and up-regulated miRNAs, respectively.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Mutagenesis, Generated

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: Molecular networks of anti-correlated miRNA-mRNA pairs in H1299/mutant p53 R273H cells derived from the integrated miRNA-mRNA expression analysis. Target genes of the up-regulated miRNAs (Red) were down-regulated (Green) [upper panel] and those of down-regulated miRNAs (Green) were up-regulated (Red) [lower panel] in the presence of mutant p53 R273H . The color intensity of the nodes indicates relative miRNA or mRNA expression levels in presence of mutant p53 R273H . The types of interaction between miRNAs and their respective target mRNAs are represented by arrows in dark red (experimentally validated), bright orange (highly predicted), and light blue (moderately predicted).

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Mutagenesis, Derivative Assay, Expressing

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: Validation of mutant p53 R273H -regulated miRNAs in the TCGA lung adenocarcinoma patient dataset. ( A ) Heat map showing normalized read counts of mutant p53 R273H -regulated miRNAs across TCGA lung adenocarcinoma patients bearing wild type and mutant p53. Based on TP53 mutation status, TCGA patients were categorized into two groups, wild type and mutant p53 patients. The relative expression levels of mutant p53-regulated miRNAs obtained in the present study were subsequently validated by comparing their normalized read counts between these two groups of patients. Patients with wild type p53 and mutant p53 are shown in black and red color letters respectively. miRNAs shown in red are significantly ( p < 0.05) altered between wild-type and mutant p53 patients with a pattern similar to that of our small RNA sequencing results. Heat map scale bar indicates Z-scores of normalized read count values. ( B ) Box-Whisker plots showing log2 transformed normalized read counts of two up-regulated and five down-regulated miRNAs in patients with mutant p53. p -values are indicated.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Biomarker Discovery, Mutagenesis, Expressing, RNA Sequencing, Whisker Assay, Transformation Assay

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: Mutant p53 R273H -regulated miRNAs predict lymph node metastasis (LNM) in lung adenocarcinoma patients. ( A ) Relative expression of seven mutant p53-regulated miRNAs in lymph node positive (N + , n = 85) and lymph node negative (N 0 , n = 138) patients. Scatter plots showing normalized expression (RPM) of individual miRNAs in N + and N 0 group of patients. p -values are indicated. ( B ) Receiver operating characteristic (ROC) curve analyses for LNM prediction in lung adenocarcinoma patients using relative expression of miR-132, miR-147b, and miR-30d. Area under the receiver operating characteristic curve (AUC) is shown. p -values are indicated.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Mutagenesis, Expressing

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: Mutant p53 R273H -regulated miRNAs determine poor survival in lung adenocarcinoma patients. Kaplan–Meier analyses showing relative survival probabilities of lung adenocarcinoma patients with high (≥75th percentile) and low (≤25th percentile) expression of the individual mutant p53-regulated miRNAs. Log-rank p -value, hazard ratio, and median survival time are indicated. n indicates the number of patients in respective miRNA low and high groups.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Mutagenesis, Expressing

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: GOF Mutant p53 R273H -regulated miR-194 and miR-378a is down-regulated in NCI-60 cell lines with mesenchymal phenotype. Box-Whisker plots showing relative expression of miR-194 and miR-378a in mesenchymal and epithelial groups of NCI-60 cell lines. p -values are indicated.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Mutagenesis, Whisker Assay, Expressing

Journal: Genes

Article Title: Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53

doi: 10.3390/genes10110852

Figure Lengend Snippet: miR-X promotes oncogenic properties in lung cancer cells. ( A ) QRT-PCR data showing relative expression levels of miR-X in H1299/EV and H1299/mutant p53 R273H stable cell lines ( B ) QRT-PCR data showing ectopic expression of miR-X in H1299 cells transfected with pRNAU6.1-miR-X. H1299 cells were transfected with either empty pRNAU6.1 vector or with 250 ng of pRNAU6.1-miR-X. Forty eight hours post-transfection relative miR-X expression was evaluated using qRT-PCR. ( C ) Bar graphs showing relative proliferation of H1299 (left panel) and A549 (right panel) cells upon ectopic expression of miR-X as measured by WST-1 cell proliferation assay. Cells were transfected with either empty vector or miR-X expression plasmid and 16 h post-transfection WST-1 assay was performed for the day 1 and for the days indicated. Data represents average absorbance values at 450 nm of three independent experiments. ( D , E ) (Left panels) Histogram showing mean fluorescent intensities of empty vector and miR-X transfected H1299 (D) and A549 (E) cells stained with anti-Ki-67 antibody as measured by FACS analyses. (Right panels) Bar graphs showing relative percentages of Ki-67 positive H1299 (D) and A549 (E) cells transfected with control empty vector or with miR-X expression plasmid. ( F ) (Upper panel) Representative images of colonies formed by H1299 cells upon miR-X overexpression in clonogenic assay. (Lower panel) Quantification of the data that were obtained from the clonogenic assay. ( G ) (Left panel) Representative images of wound healing assay performed in H1299 cells transfected with either empty vector or miR-X expression plasmid. Images were captured at 0 h, 18 h, and 22 h after the scratch was introduced. (Right panel) Bar graph showing relative percentage of distance covered by the cells through 18 h post-scratch. Data represent mean ± s.d.; n = 3; two-tailed Student’s t -test: * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: To generate mutant p53 R273H expressing stable H1299 cell line, the cells were first transfected with pCMV-p53R273H expression plasmid (pCMV-Neo-Bam-p53 R273H, kindly provided by Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA) while using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Proliferation Assay, WST-1 Assay, Staining, Control, Over Expression, Clonogenic Assay, Wound Healing Assay, Two Tailed Test