Journal: Science advances

Article Title: Increased palmitoylation improves estrogen receptor alpha-dependent hippocampal synaptic deficits in a mouse model of synucleinopathy.

doi: 10.1126/sciadv.adj1454

Figure Lengend Snippet: Fig. 3. αS inclusion pathologies affect ERα distribution and palmitoylation. (A) Representative images (left) and quantification (right) of synaptophysin+ (top) and ERα + (bottom) puncta in the hippocampal SO of 3KL versus WT males/females and quantification of average %ERα area per mouse (five fields). (B) Higher-resolution images of ERα/αS+ in the hippocampal SO of 3KL versus WT males/females and size analysis of colocalizing particles. Arrowheads point to larger-sized ERα/αS foci in 3KL. (C) Representative images of ERα:Cav1 PLA in 3KL and WT and quantitation of PLA % area in the hippocampal SO (data points represent average % area per mouse over five subfields). (D) Confocal microscopy of cortical neurons labeled for ERα/hu-αS+/40,6-diamidino-2-phenylindole (DAPI) highlight ERα localization to hu-αS+ inclusions in 3KL (yellow arrowhead) versus nuclear ERα + in WT. (E) ERα localization to DLB (top) and PD (bottom) LBs in the transentorhinal cortex (top) and SN (surrounding neuro- melanin indicated), respectively. (F) Hypothesis: ERα is sequestered into abnormal perinuclear αS+ inclusions and depleted around synapses in 3KL mice (and human PD/ DLB). (G) WB image of palm-ERα (by ABE assay) in total brain lysates of 3KL versus WT and quantification (n = 5 to 6 per group). (A), (C), and (G) Dot plots show data from individual mice (and median); (B) boxplot based on analyzed fields (16 images per mouse; three mice per group). Statistics by two-way ANOVAs [(A), (C), and (G)] and nonparametric Kruskal-Wallis (B) with Bonferroni/Dunn corrections, respectively (see further table S3); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Sections were incubated overnight in diluted primary antibodies against ERα (TE111.5D11, Invitrogen, RRID: AB_2101962, 1:100 in Duolink Antibody Diluent) and Cav1 (no. D46G3; RRID:AB_2275453, Cell Signaling Technology; 1:100 in Duolink Antibody Diluent) at 4°C.

Techniques: Quantitation Assay, Confocal Microscopy, Labeling

Journal: Science advances

Article Title: Increased palmitoylation improves estrogen receptor alpha-dependent hippocampal synaptic deficits in a mouse model of synucleinopathy.

doi: 10.1126/sciadv.adj1454

Figure Lengend Snippet: Fig. 6. ERα expression level and palmitoylation contribute to increased hippocampal synaptic function in 15-month-old 3KL mice after ML348. (A) Average (M + F) traces of LTP (%fEPSP) in hippocampal sections of placebo- and ML348-treated 3KL males/females and after acute administration of the selective ERα antagonist MPP on sections of ML348-treated mice; (right) quantification of average %fEPSP slopes during the last 5 min of the measurement shown for male/female 3KL and combined (M + F). (B) Representative images and quantification of ERα:Cav1 PLA area in hippocampal SO subfields, shown for in 3KL male and female and combined (M + F). Scale bar, 20 μm. (C) Representative WB and ELISA-based quantification of palm-ERα in ABE-processed samples of male and female 3KL mice and combined (M + F; quantifi- cation in six samples from three mice per group). (D) Relative ESR1 mRNA level in placebo- and ML348-treated WT/3KL mice. (E) Heatmap summarizing hippocampal mRNA profiles for ERs and palmitoyl-acyl-transferases (also see fig. S6 for individual scatter blots). Dot plots show data from individual mice (and median). Statistics by two-way ANOVAs. Results of planned post hoc tests with Bonferroni correction are presented (see table S6); *P < 0.05 and ***P < 0.001.

Article Snippet: Sections were incubated overnight in diluted primary antibodies against ERα (TE111.5D11, Invitrogen, RRID: AB_2101962, 1:100 in Duolink Antibody Diluent) and Cav1 (no. D46G3; RRID:AB_2275453, Cell Signaling Technology; 1:100 in Duolink Antibody Diluent) at 4°C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: BMC Developmental Biology

Article Title: YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation

doi: 10.1186/s12861-019-0200-1

Figure Lengend Snippet: CDC25B distribution during oocyte maturation and interactions with YWHA proteins demonstrated by proximity ligation assays in oocytes and eggs. a Paired confocal images of equatorial sections through oocytes (left: immunofluorescence; right: brightfield) with rabbit anti-CDC25B. CDC25B is present thorught the oocyte and accumulates in the region of the nucleus across a two-hour time course during in vitro maturation of mouse oocytes. Control oocytes processed simultaneously in absence of the primary antibody showed minimal background fluorescence. These images are representative of two experiments that examined 6–12 cells at each time point. b Representative images of oocytes and eggs showing proximity ligation reaction spots that indicate interaction of YWHA protein isoforms with CDC25B at the molecular level. Each reaction spot represents a protein-protien interacton between a CDC25B and a YWHA protein (see for details). Except for the lower left images, the upper panel is an oocyte and the lower panel is an egg, each imaged with confocal fluorescence microscopy. The equatorial scan is a single scan and the compressed Z is obtained from combining consecutive cross-sectional images throughout each cell taken at 3 μm intervals. The corresponding brightfield image is included. The lower two left panels are representative of the experiment using PLA with different primary antibodies against CDC25B directed at different sites on the protien. Binding to the same protein is indicated by PLA spots (rabbit anti-CDC25B and goat anti-CDC25B; rabbit anti-pSer149 CDC25B and goat anti-CDC25B). No reaction spots are observed in the control (oocytes and eggs processed in absence of the primary antibodies). The images shown are representative of two experiments (oocytes and eggs from four animals) with similar results for a total of 7–10 oocytes and a total of 6–11 eggs for each condition and controls. Scale bars for all images represent 10 μm

Article Snippet: Following fixation and permeabilization, the cells were processed following the manufacturer’s instructions for the Duolink in situ PLA kit as previously described [ ] with primary antibodies for the YWHA isoforms (rabbit anti-14-3-3 isoform panel PAN017, AbD Serotec; diluted 1:200 in Duolink in situ antibody diluent) and with the primary antibody for CDC25B (goat anti-CDC25B, Santa Cruz, sc-6948; diluted 1:200 in Duolink in situ antibody diluent).

Techniques: Ligation, Immunofluorescence, In Vitro, Control, Fluorescence, Microscopy, Binding Assay

Journal: BMC Developmental Biology

Article Title: YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation

doi: 10.1186/s12861-019-0200-1

Figure Lengend Snippet: FRET analysis of interaction of YWHAH with CDC25B and YWHAH with YWHAE. a ICR(CD1) oocytes were co-injected with mCherry-YWHAH and EGFP-CDC25mRNA. Expression of EGFP-CDC25B protein provides the donor fluorescence (green) in the FRET analyses. The mCherry-YWHAH serves as the acceptor fluorescent probe (red). Images show the donor emission with donor excitation (488 nm), the acceptor emission with donor excitation, and the acceptor emission with acceptor excitation (543 nm), and the corrected FRET image. Data from three regions of interest (shown by white boxes) were used to calculate the FRET efficiency, which, in this cell averaged 72%, and is visually represented in the heat map at the right. The figure is representative of 12 oocytes collected from three different females in three experiments. b ICR(CD1) oocytes were co-injected with mCherry-YWHAH and EGFP-YWHAE. EGFP-YWHAE protein provides the donor fluorescence (green) in the FRET analyses. The mCherry-YWHAH serves as the acceptor fluorescent probe (red). Images show the donor emission with donor excitation (488 nm), the acceptor emission with donor excitation, and the acceptor emission with acceptor excitation (543 nm), and the corrected FRET image. Data from three regions of interest (show by white boxes) was used to calculate the FRET efficiency, which in this cell averaged 27%, and is visually represented in the heat map at the right. The figure is representative of five oocytes examined in two experiments

Article Snippet: Following fixation and permeabilization, the cells were processed following the manufacturer’s instructions for the Duolink in situ PLA kit as previously described [ ] with primary antibodies for the YWHA isoforms (rabbit anti-14-3-3 isoform panel PAN017, AbD Serotec; diluted 1:200 in Duolink in situ antibody diluent) and with the primary antibody for CDC25B (goat anti-CDC25B, Santa Cruz, sc-6948; diluted 1:200 in Duolink in situ antibody diluent).

Techniques: Injection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) as a new tethering factor for ESR1 supporting its action in breast cancer

doi: 10.1101/2021.05.06.442900

Figure Lengend Snippet: ( A ) Interactions between ESR1 and HSP90 assessed by Proximity Ligation Assay (PLA; red spots) in HSF1-positive (HSF1+) and HSF1-negative (HSF1−) MCF7 cells created using DNA-free CRISPR/Cas9 system. Ctr, untreated cells; E2, 17β-estradiol treatment (10 nM). DNA was stained with DAPI. Scale bar, 20 μm. The mean number of spots per cell is shown in the boxplot below. The line dividing the box represents the median and the upper and lower side of the box shows the upper and lower quartiles, respectively. The whiskers show the highest and lowest values. ***p < 0.0001, **p < 0.001, *p < 0.05 (above the bar – compared to the corresponding control, between the bars – between cell variants). For detection of HSP90 and ESR1 by immunofluorescence see . ( B ) HSP90 and ESR1 levels assessed by western blot. Actin (ACTB) was used as a protein loading control. The graphs show the results of densitometric analyses (n=4). ***p < 0.0001, *p < 0.05.

Article Snippet: After washing, slides were incubated in Blocking Solution and immunolabeled (overnight, 4 °C) with primary antibodies diluted in the DuoLink® Antibody Diluent: rabbit anti-HSP90 (1:200; #ADI-SPA-836, Enzo Life Science) or rabbit anti-HSF1 (1:300; ADI-SPA-901, Enzo Life Sciences) and mouse anti-ERalpha (1:200; # C15100066, Diagenode, Liège, Belgium); negative controls were proceeded without one primary antibody or both.

Techniques: Proximity Ligation Assay, CRISPR, Staining, Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) as a new tethering factor for ESR1 supporting its action in breast cancer

doi: 10.1101/2021.05.06.442900

Figure Lengend Snippet: Both ESR1 and HSF1 are kept in an inactive state by the complexes of HSP90, p23, and immunophilins (I). Binding of E2 to ESR1 is connected with the release of the chaperone complex and activation of ESR1 leading to the phosphorylation of MEK1/2 followed by HSF1 activation. Oligomers of active transcription factors can bind to DNA and cooperate in the regulation of the transcription either directly or through chromatin re-organization.

Article Snippet: After washing, slides were incubated in Blocking Solution and immunolabeled (overnight, 4 °C) with primary antibodies diluted in the DuoLink® Antibody Diluent: rabbit anti-HSP90 (1:200; #ADI-SPA-836, Enzo Life Science) or rabbit anti-HSF1 (1:300; ADI-SPA-901, Enzo Life Sciences) and mouse anti-ERalpha (1:200; # C15100066, Diagenode, Liège, Belgium); negative controls were proceeded without one primary antibody or both.

Techniques: Binding Assay, Activation Assay