Journal: bioRxiv

Article Title: Adenine DNA methylation, 3D genome organization and gene expression in the parasite Trichomonas vaginalis

doi: 10.1101/603894

Figure Lengend Snippet: A) Dot blot assay to detect 5mC shows lack of signal in the genomic DNA of highly-adherent strain B7268 and less-adherent strain G3. Left: gDNA dots stained with methylene blue for loading control. Right: 5mC signal. Chicken gDNA was used as positive control. B) Immunofluorescence assay using specific antibodies detects 5mC in the cytoplasm (RNA) but fails to detect 5mC in T. vaginalis nucleus of RNase-treated parasites. Nucleus was stained with DAPI. C) Immunofluorescence assay using anti-6mA specific antibody evidenced this modification in the cytoplasm (RNA) and nucleus (gDNA), or exclusively in the nucleus of RNase-treated parasites. Nucleus was stained with DAPI. D) Methylation sensitive RE assay shows the presence of methylated and un-methylated adenines in T. vaginalis gDNA. Lane 1: MW. Lane 2: undigested gDNA. Lane 3: MboI treated gDNA (cuts GATC sites). Lane 4: DpnI treated gDNA (cuts G6mATC sites). E) Anti-6mA dot blot assay of RNA-free gDNA shows similar abundance of 6mA in the genomic DNA of strain B7268 and strain G3. Left: gDNA dots stained with methylene blue for loading control. Right: 6mA signal. F) Quantification of methylated bases in the gDNA of adherent strain B7268 and less-adherent strain G3 using UHPLC followed by mass spectrometry shows high levels of 6mA and low levels of 5mC, but not differences among stains for both modifications. Each column represents the mean and SD of three independent experiments per group. Graphic is shown in logarithmic scale.

Article Snippet: Trichomonas vaginalis strains B7268 ( ) and G3 (ATCC PRA-98, Kent, UK) were cultured at 37°C in TYM medium supplemented with 10% horse serum, penicillin and streptomycin (Invitrogen) ( ).

Techniques: Dot Blot, Staining, Control, Positive Control, Immunofluorescence, Modification, Methylation, Mass Spectrometry

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3-WT and NLGN3-R451C increase neuritogenesis in mouse primary cortical neurons. (A) Representative images showing ectopic NLGN3-WT and NLGN3-R451C expression significantly increases neurite number and length in mouse primary cortical neurons. Scale bar = 50 μm. (B) Box plots of data demonstrating NLGN3-WT and NLGN3-R451C expression increases neurite number confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction or non-parametric Kruskal–Wallis H test with Dunn’s post-hoc test. (C) Box plots of data demonstrating NLGN3-WT and NLGN3-R451C expression increases neurite length confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction or non-parametric Kruskal–Wallis H test with Dunn’s post-hoc test.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Expressing

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3 and NLGN4X are expressed in developing human neurons and form nanoscopic growth cones clusters. (A) RT-qPCR data showing endogenous NLGN3 and NLGN4X mRNA expression levels increase as hNPCs differentiate into immature neurons, confirmed by t -test ( n = 3). (B) Data and representative western blots showing endogenous NLGN3 and NLGN4X protein expression levels increase as hNPCs differentiate into immature neurons, confirmed by t -test ( n = 3). (C) Representative super-resolution images of immature human neurons showing endogenous NLGN3 (upper) NLGN4X (lower) expression and localisation. NLGN3 and NLGN4X localize to the growth cone, particularly at the leading edge where they colocalise with F-actin (insets). Scale bar = 25 μm (whole cell), 5 μm (insets). (D) Representative super-resolution images of human neuronal growth cones ectopically expressing HA-tagged NLGN3 or NLGN4X. Clusters of HA-NLGN3/4X are visible at the growth cone leading edge based on high intensity puncta (open white arrows). Scale bar = 5 μm.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3/4X-WT but not ASD-associated variants increase neuritogenesis in developing human neurons. (A) Representative intensity images showing ectopic NLGN3-WT is highly localized to the leading edge of growth cones while the mutant variant is less localized but still present at the leading edge (open white arrows). Scale bar = 5 μm. (B) Representative intensity images showing ectopic NLGN4X-WT is highly localized to the leading edge of growth cones while NLGN4X-D396 is barely present in growth cones (open white arrows). Scale bar = 5 μm. (C + D) Representative images and data showing ectopic NLGN3 expression significantly increases neurite number and length in immature neurons, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction or non-parametric Kruskal–Wallis H test with Dunn’s post-hoc test ( n = 15). Scale bar = 25 μm. (E + F) Representative images and data showing ectopic NLGN4X expression significantly increases neurite number and length in immature neurons, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction or non-parametric Kruskal–Wallis H test with Dunn’s post-hoc test ( n = 15). Scale bar = 25 μm.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Mutagenesis, Variant Assay, Expressing

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3/4X-WT effects growth cone structure by influencing actin filament organization. (A) Representative super-resolution images showing ectopic wildtype (WT) NLGN3 expression influences growth cone area and actin filament organization. Scale bar = 5 μm. (B) Data showing ectopic NLGN3-WT expression significantly increases growth cone area, filament number, filament length and filament bundle width while decreasing filament distance and anisotropy compared to control or ectopic NLGN3-R451C expression, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction or non-parametric Kruskal–Wallis H test with Dunn’s post-hoc test ( n = 15). (C) Representative super-resolution images showing ectopic NLGN4X-WT expression influences growth cone area and actin filament organization. Scale bar = 5 μm. (D) Data showing ectopic NLGN4X-WT expression significantly increases growth cone area, filament number, filament length and filament bundle width while decreasing filament distance but not anisotropy compared to control or ectopic NLGN4X-D396 expression, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction or non-parametric Kruskal–Wallis H test with Dunn’s post-hoc test ( n = 15). Filament distance was also found to be significantly increased between control and ectopic NLGN4X-D396 conditions suggesting a dominant negative effect.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Expressing, Dominant Negative Mutation

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3/4X-WT induces p21-activated kinase (PAK1) phosphorylation, likely via Shank3. (A) Representative blots and data showing ectopic NLGN3-WT expression in HEK293 cells increases phosphorylation of actin regulator proteins PAK1 and cofilin, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction ( n = 3). (B) Representative blots and data showing ectopic NLGN4X-WT expression in HEK293 cells increases phosphorylation of actin regulator proteins PAK1 and cofilin, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction ( n = 3). (C) Co-immunoprecipitation data showing NLGN3 and NLGN4X interact with Shank3 in HEK293 cells co-transfected with HA-NLGN3/4X-WT and Myc-Shank3-WT. (D + E) Representative blots and data showing ectopic co-expression of HA-NLGN3/4X-WT and Myc-Shank3-WT has a compounding effect on PAK1 phosphorylation compared to untransfected control and single transfection conditions, confirmed by parametric one-way ANOVA with Bonferroni post-hoc correction ( n = 3).

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Expressing, Immunoprecipitation, Transfection

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: Growth cone morphology changes induced by NLGN3/4X-WT are attenuated by PAK1 inhibition. (A) Representative images showing increases in growth cone area, filament number and decreases in filament distance induced by ectopic NLGN3/4X-WT expression are attenuated by PAK1 inhibition. Scale bar = 5 μm. (B) Data showing increases in growth cone area, filament number and decreases in filament distance induced by ectopic NLGN3-WT expression are attenuated by PAK1 inhibition, confirmed by two-way ANOVA with Bonferroni post-hoc correction ( n = 25). (C) Data showing increases in growth cone area, filament number and decreases in filament distance induced by ectopic NLGN4X-WT expression are attenuated by PAK1 inhibition, confirmed by two-way ANOVA with Bonferroni post-hoc correction ( n = 25).

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Inhibition, Expressing

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3/4X-WT clustering at growth cones is dependent on PAK1 signalling. (A) Representative images and data showing phosphorylated PAK1 intensity significantly increases in growth cone F-actin clusters of immature neurons ectopically expressing NLGN3/4X-WT, confirmed by t -test. Scale bar = 5 μm. (B) Representative images and data showing ectopically expressed HA-NLGN3-WT clusters are attenuated by PAK1 inhibition for cluster number, area, and intensity, confirmed by t -test ( n = 25). Scale bar = 5 μm. (C) Representative images and data showing ectopically expressed HA-NLGN4X-WT clusters are attenuated by PAK1 inhibition for cluster number, area, and intensity, confirmed by t -test ( n = 25). Scale bar = 5 μm.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Expressing, Inhibition

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: NLGN3/4X-WT dependent neuritogenesis is dependent on PAK1 phosphorylation. (A) Representative images and data showing ectopic NLGN3-WT mediated increases in neurite number and length are attenuated by PAK1 inhibition, confirmed by two-way ANOVA with Bonferroni post-hoc correction ( n = 25). Scale bar = 25 μm. (B) Representative images and data showing ectopic NLGN4X-WT mediated increases in neurite number and length are attenuated by PAK1 inhibition, confirmed by two-way ANOVA with Bonferroni post-hoc correction ( n = 25). Scale bar = 25 μm.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques: Inhibition

Journal: Human Molecular Genetics

Article Title: Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size

doi: 10.1093/hmg/ddab277

Figure Lengend Snippet: Model of NLGN3/4X clustering on growth cones and neurites dependent on NLGN-clustering/PAK1 feedback loop. Schematic diagram summarizing all primary findings of this research including the effects on neuritogenesis (upper—primary neurites = red, secondary neurites = blue, tertiary neurites = yellow), the effects on growth cone actin (lower left), and the molecular mechanism involved in both phenotypes (lower right). NLGN, neuroligin; GC, growth cone; WT, wild type; DMSO, dimethyl sulfoxide; FRAX, FRAX486; PAK1, p21-activated kinase.

Article Snippet: Immunoblotting: mouse anti-NLGN3 monoclonal (StressMarq, SMC-471D, 1:1000), rabbit anti-NLGN3 polyclonal (Synaptic Systems, 129 113, 1:1000), rabbit anti-HA polyclonal (ProteinTech, 51 064-2-AP, 1:1000), rabbit anti-NLGN4X monoclonal (Abcam, ab181251, 1:1000), mouse anti-Myc monoclonal (BioLegend, 626 801, 1:200), mouse anti-total cofilin monoclonal (ProteinTech, 66 057-1-lg, 1:3000), rabbit anti-phospho-cofilin (Ser3) polyclonal (Cell Signaling Technology, 3311, 1:1000), rabbit anti-total PAK1/2/3 (Cell Signaling Technology, 2604), rabbit anti-phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, 2606, 1:1000), rabbit anti-HRP (Life Technologies, G-21234, 1:10000), mouse anti-HRP (Life Technologies, A16078, 1:10000) ( Supplemental Material, Table S1 ) .

Techniques:

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: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

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: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

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: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

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: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

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: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

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

Journal: The Journal of Biological Chemistry

Article Title: The novel cardiac z-disc protein CEFIP regulates cardiomyocyte hypertrophy by modulating calcineurin signaling

doi: 10.1074/jbc.M117.786764

Figure Lengend Snippet: CEFIP is highly enriched in heart and skeletal muscle. A, confirmation of the specific cardiac- and skeletal muscle-enriched expression pattern of human and mouse CEFIP by Northern blotting of multiple human and mouse tissues. B, qPCR shows a 163-fold induction in heart and a 384-fold induction of CEFIP in mouse tissue normalized to spleen. C, Western blot analysis of CEFIP in mouse tissue extracts confirms the heart- and skeletal muscle-enriched expression pattern. The specificity of the antibody is confirmed by preincubation of the antiserum with the peptide that had been used as immunogen blocking specific reactivity. D, percentage identity of protein sequence between H. sapiens, M. musculus, Rattus norvegicus, Macaca mulatta, Canis lupus, and Bos taurus shows high conservation among these species.

Article Snippet: Northern blot analysis Multiple tissue Northern blots (BioChain) containing human or mouse poly(A) RNA were hybridized with [ 32 P]dCTP-labeled (Rediprime II Random Prime Labeling System, Amersham Biosciences) cDNA probes corresponding to the ORF of human or mouse CEFIP at 65 °C overnight.

Techniques: Expressing, Northern Blot, Western Blot, Blocking Assay, Sequencing