Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Schematic of the construct for recombinant human αVβ5 ectodomain protein production from mammalian cells. The domain boundaries are as follows: αV, 32–991; β5, 24–717. (B) and (D) Biolayerinferometry (BLI) measurement of binding of irisin to αVβ5. Purified irisin-His at the indicated concentration was infused over a sensor chip with immobilized clasped αVβ5 in the presence of 1 mM MgCl2 and 1 mM CaCl2. (C) Silver-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of affinity-purified αVβ5 fractions 7–20 collected following ion exchange. (E) A protein-elution profile of the indicated protein complexes from Superdex 200 increase 30/100 GL (optical density (OD)280). (F) Coomassie-stained SDS-PAGE and western blot with the indicated antibodies of the deglycosylated recombinant human Hsp90α and peak fractions from (E). (G) Fluorescence anisotropy measurement of binding of αVβ5 and the αVβ5/Hsp90α complex to 50 nM A488-irisin-His in the presence of 1 mM MgCl2 and 1 mM CaCl2. (H) Fluorescence anisotropy competition assay for the αVβ5/Hsp90α complex binding by irisin: 50 nM A488-irisin-His and 125 nM αVβ5 were mixed with varying concentrations of unlabeled irisin or irisin-His, and anisotropy was recorded. Probe alone: 50 nM A488-irisin-His.

Article Snippet: Rabbit Polyclonal Anti-Hsp90α , Invitrogen , Cat. # PA3–013.

Techniques: Construct, Recombinant, Binding Assay, Purification, Concentration Assay, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Affinity Purification, Western Blot, Fluorescence, Competitive Binding Assay

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Schematic of acute exercise and IF isolation procedure and processing. (B) Anti-Hsp90αwestern blot showing Hsp90α protein levels in IF samples taken from the mouse without acute exercise (basal), or from the mice rested for the indicated amounts of time after acute exercise. 10 μg of total IF protein was loaded for each sample as shown by Ponceau staining. (C) Anti-Hsp90α western blot showing Hsp90α protein levels in gastrocnemius muscle samples taken from the mouse without acute exercise (basal), or from the mice rested for the indicated amounts of time after acute exercise. 10 μg of total muscle protein was loaded for each sample as shown by Ponceau staining. (D) Anti-Hsp90αwestern blot showing Hsp90α protein levels in plasma samples taken from the same group of mice from (C). 10 μg of total plasma protein was loaded for each sample as shown by Ponceau staining. (E) Anti-Hsp90α and anti-HspA14 (control) western blot showing Hsp90α and HspA14 protein levels in plasma samples taken from five mice pre-exercise and post-exercise (1 hr). 10 μg of total plasma protein was loaded for each sample as shown by Ponceau staining. (F) Quantitative mass spectrometry showing the fold of changes of the indicated chaperone proteins identified in the IF samples from the exercised mice (1 hr post acute exercise) compared to the sedentary group (significant if FDR q-value < 0.05).q-values of the significantly upregulated genes are indicated in the bar graph.

Article Snippet: Rabbit Polyclonal Anti-Hsp90α , Invitrogen , Cat. # PA3–013.

Techniques: Isolation, Staining, Western Blot, Control, Mass Spectrometry

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Fluorescence confocal images showing A488-irisin binding in HEK293T cells. HEK293T cells were either transiently transfected with control vector or full-length αV and β5 plasmids. 2 nM Hsp90α was used for 1 hr pretreatment, and 2 nM A488-irisin-His was subsequently used for 5 min treatment. Scale bar: 20 μm. (B) Anti-phosphorylated FAK (Y397) and anti-FAK western blots showing the levels of integrin signaling upon irisin and/or Hsp90α treatments. HEK293T cells were transfected and treated in the same way as (A), except for the addition of the shown amounts of unlabeled irisin-His (0.1 nM or 1 nM) and Hsp90α (1 nM). Anti-αV and anti-β5 antibodies were used to probe the levels of the ectopically expressed αV and β5. (C) Immunofluorescence confocal images showing cell surface Hsp90α in SK-Mel2 cells. Live cells were treated with either control IgG or anti-Hsp90α at 4°C. Scale bar: 50 μm. (D) Quantification of the percentage of SK-Mel2 cells expressing cell surface Hsp90α in (C) (significant if p-value < 0.05 by unpaired t-test). (E) Fluorescence confocal images showing A647-irisin binding in SK-Mel2 cells. Live cells were pretreated with either control IgG or anti-Hsp90α at 4°C for 1 hr followed by 2 nM A647-irisin-His treatment at room temperature for 5 min. Scale bar: 50 μm. (F) Quantification of the percentage of A647-positive cells in (E) (significant if p-value < 0.05 by unpaired t-test). (G) Co-immunoprecipitation assay of endogenous cell surface αV and β5 using SK-Mel2 cells. Endogenous cell surface Hsp90α was captured by anti-Hsp90α in live cells at 4°C. (H) Crystal violet assay showing does-dependent inhibition of the cell viability of SK-Mel2 upon irisin treatment. Grey bar: control treatment with PBS. Concentrations of irisin-His used for the treatments were indicated (one-way ANOVA). (I) Crystal violet assay showing the inhibition of irisin-mediated effect in SK-Mel2 cells by anti-Hsp90α or control antibody. Grey bar: control treatment with PBS. 50 ng/mL of irisin-His was used (one-way ANOVA). (J) Western blot of mouse inguinal fat tissue lysates using the indicated antibodies to probe integrin signaling. Mice were given anti-Hsp90α antibody or control IgG (500 μg/kg) subcutaneously 24 hrs before a bolus injection of recombinant irisin (5 mg/kg) directly into the inguinal fat pads. The mice were sacrificed and inguinal fat tissues were harvested 20 min after irisin injection.

Article Snippet: Rabbit Polyclonal Anti-Hsp90α , Invitrogen , Cat. # PA3–013.

Techniques: Fluorescence, Binding Assay, Transfection, Control, Plasmid Preparation, Western Blot, Immunofluorescence, Expressing, IF-P, Co-Immunoprecipitation Assay, Crystal Violet Assay, Inhibition, Injection, Recombinant

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Flow charts of the steps used in three different methods for analyzing αVβ5-Apo and αVβ5/Hsp90α cryo-EM samples. (B) 2D classes (generated by method 1) of αVβ5 particles in each of the three conformational states and the numbers (quantified by all three methods) of particles in each state. (C) Quantification of the percentage of distinguished particles (“likely open” particles were not included) in each of the three conformational states. (D) Fluorescence anisotropy assay for A488-irisin binding by αVβ5, the αVβ5/Hsp90α complex in the presence of 1 mM MgCl2 and 1 mM CaCl2, or αVβ5 in the presence of 1 mM MnCl2. 50 nM A488-irisin-His was used in the assay. (E) Cartoon diagram showing a two-step process of the irisin action through αVβ5. Irisin alone has low affinity for the closed-state αVβ5. Hsp90α, Mn2+ ion, or other possible factors, “opens” αVβ5, allowing for high-affinity irisin binding and effective signaling transduction through its integrin receptor. (F) and (G) TALON pull-downs performed using 1 μM bead-bound clasped and tagged αVβ5. These were mixed with 2 μM untagged Hsp90α without bound nucleotide (Hsp90α-Apo) or Hsp90α charged with the indicated nucleotides (F), or Hsp90α nonhydrolyzing mutant (G95D) (G), and bound samples were analyzed by Coomassie staining and anti-Hsp90α western blot.

Article Snippet: Rabbit Polyclonal Anti-Hsp90α , Invitrogen , Cat. # PA3–013.

Techniques: Cryo-EM Sample Prep, Generated, Fluorescence, Binding Assay, Transduction, Mutagenesis, Staining, Western Blot

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: Rabbit Polyclonal Anti-Hsp90α , Invitrogen , Cat. # PA3–013.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Clone Assay, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation

Journal: Molecular and cellular endocrinology

Article Title: ZDHHC17 participates in the pathogenesis of polycystic ovary syndrome by affecting androgen conversion to estrogen in granulosa cells.

doi: 10.1016/j.mce.2023.112076

Figure Lengend Snippet: Fig. 1. ZDHHC17 controls the palmitoylation of HSP90α. (A) GRM02 cells were transfected with FLAG-tagged HSP90α, and then the cell lysates were immunoprecipitated by anti-Flag beads. The beads were washed three times with lysis buffer, separated by SDS‒PAGE and then stained with Coomassie blue. (B) Mass spectrometry analysis of proteins interacting with HSP90α in samples from (A). Several representative ZDHHC family member proteins that interact with HSP90α are shown in table. (C) Mass spectrometry analysis of a peptide derived from Flag-HSP90α immunoprecipitants to show the interaction between ZDHHC17 and HSP90α. (D) Endogenous ZDHHC17 interacts with endogenous HSP90α in GRM02 cells. (E) Endogenous HSP90α palmitoylation levels after transfection with HA-tagged empty vector or HA-tagged ZDHHC17 in GRM02 cells. (F) Representative immunoblot showing the knockdown efficiency of ZDHHC17 with siRNA targeting Zdhhc17. Scramble (Scr) siRNA was used as a negative control. (G) Endogenous HSP90α palmitoylation levels after transfection with scramble (Scr) or Zdhhc17-specific siRNAs in GRM02 cells. (H) GRM02 cells were transfected with WT Flag-HSP90α or Flag-HSP90α C598,599S mutant in the presence or absence of HA-tagged ZDHHC17 for 24 h. Cell lysates were harvested for ABE assay and immunoblot analysis.

Article Snippet: Rabbit anti-HSP90α polyclonal antibody (13171-1-AP, 1꞉1000 dilution), rabbit anti-ZDHHC17 polyclonal antibody (15465-1-AP, 1꞉100 dilution for immunohistochemistry) and rabbit anti-ZDHHC15 polyclonal antibody (21627-1-AP, 1꞉400 dilution for immunoprecipitation) were purchased from Proteintech (Wuhan, China).

Techniques: Transfection, Immunoprecipitation, Lysis, Staining, Mass Spectrometry, Derivative Assay, Plasmid Preparation, Western Blot, Knockdown, Negative Control, Mutagenesis

Journal: Molecular and cellular endocrinology

Article Title: ZDHHC17 participates in the pathogenesis of polycystic ovary syndrome by affecting androgen conversion to estrogen in granulosa cells.

doi: 10.1016/j.mce.2023.112076

Figure Lengend Snippet: Fig. 5. ZDHHC17-mediated regulation of CYP19A1 expression is dependent on the HSP90α palmitoylation state. (A) HSP90α-knockout (KO) GRM02 cells were generated, and KO efficiency was evaluated by immunoblot analysis. (B) HSP90α-KO GRM02 cells were transfected with WT Flag-HSP90α or Flag-HSP90α C598,599S mutant in the presence or absence of HA-tagged ZDHHC17 for 48 h and then treated with DHT (100 nM, 12 h). Cell lysates were then collected for immunoblot analysis. In (B), data are presented as the mean ± SEM from three independent experiments, unpaired two-tailed Student’s t-test: ns indicates not significant, *P < 0.05.

Article Snippet: Rabbit anti-HSP90α polyclonal antibody (13171-1-AP, 1꞉1000 dilution), rabbit anti-ZDHHC17 polyclonal antibody (15465-1-AP, 1꞉100 dilution for immunohistochemistry) and rabbit anti-ZDHHC15 polyclonal antibody (21627-1-AP, 1꞉400 dilution for immunoprecipitation) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Knock-Out, Generated, Western Blot, Transfection, Mutagenesis, Two Tailed Test

Journal: Molecular and cellular endocrinology

Article Title: ZDHHC17 participates in the pathogenesis of polycystic ovary syndrome by affecting androgen conversion to estrogen in granulosa cells.

doi: 10.1016/j.mce.2023.112076

Figure Lengend Snippet: Fig. 6. A proposed working model illustrating how ZDHHC17-mediated regulation of HSP90α palmitoylation contributes to the conversion of androgen to estrogen. AR, androgen receptor; CYP19A1, cytochrome P450 family 19 subfamily a member 1; HSP90α, heat shock protein 90 alpha; ZDHHC17, zinc finger DHHC-type palmitoyltransferase 17.

Article Snippet: Rabbit anti-HSP90α polyclonal antibody (13171-1-AP, 1꞉1000 dilution), rabbit anti-ZDHHC17 polyclonal antibody (15465-1-AP, 1꞉100 dilution for immunohistochemistry) and rabbit anti-ZDHHC15 polyclonal antibody (21627-1-AP, 1꞉400 dilution for immunoprecipitation) were purchased from Proteintech (Wuhan, China).

Techniques:

Journal: PLoS ONE

Article Title: Biological Responses of Three-Dimensional Cultured Fibroblasts by Sustained Compressive Loading Include Apoptosis and Survival Activity

doi: 10.1371/journal.pone.0104676

Figure Lengend Snippet: Fibroblasts were seeded to collagen sponge and incubated for 24(□) or 200 mmHg (▪) compression for 6 h. Total mRNA was extracted after WST-1 assay, and mRNA expression was assessed using real-time RT-PCR. The expression of the target genes in the 6 h–200 mmHg group relative to the value in the 6 h–0 mmHg group was calculated by the comparative Ct method using the 18S ribosomal RNA gene as an internal control. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Student’s t test between the 0 mmHg group and the 200 mmHg group, and statistical significance was taken as p <0.05. A value of p was expressed as: *; p <0.05, **; p <0.01, and ***; p <0.001. A: The transcription factors of various Hsps . B: various Hsps C: Bcl2 is an antiapoptotic gene, and Bax is a proapoptotic gene. D and E: Immunostaining for HSP90α. Representative sections of (D) the 6 h–0 mmHg group and (E) the 6 h–200 mmHg group. Higher expression and nucleus translocation of HSP90α was observed in the 200 mmHg group (E) when compared with the 0 mmHg group (D). Scale bars = 20 µm for all images.

Article Snippet: HSP90α, CD44, and COX2 immunostaining was performed on 3D cell culture as follows: the sections were incubated with anti-HSP90α rabbit polyclonal antibody (Lab Vision Corporation, Fremont, CA), anti-HCAM rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX), or anti-COX2 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA) at room temperature for 60 min (each antibody was diluted 1∶50) after quenching of endogenous peroxidase and antigen retrieval (3D cell culture samples were subjected to semi boiling for 10 min).

Techniques: Incubation, WST-1 Assay, Expressing, Quantitative RT-PCR, Immunostaining, Translocation Assay

Journal: PLoS ONE

Article Title: Biological Responses of Three-Dimensional Cultured Fibroblasts by Sustained Compressive Loading Include Apoptosis and Survival Activity

doi: 10.1371/journal.pone.0104676

Figure Lengend Snippet: Fibroblasts were seeded to collagen sponge and incubated for 24(A: HSP90α, B: HA, C: PGE 2 ) was measured by ELISA. A value of concentration was normalized by WST-1 value. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test between non-loaded group and each of loaded group, and statistical significance was taken as p <0.05. A value of p was expressed as: *; p <0.05, **; p <0.01, and ***; p <0.001.

Article Snippet: HSP90α, CD44, and COX2 immunostaining was performed on 3D cell culture as follows: the sections were incubated with anti-HSP90α rabbit polyclonal antibody (Lab Vision Corporation, Fremont, CA), anti-HCAM rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX), or anti-COX2 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA) at room temperature for 60 min (each antibody was diluted 1∶50) after quenching of endogenous peroxidase and antigen retrieval (3D cell culture samples were subjected to semi boiling for 10 min).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Inhibiting Heat Shock Protein 90 (HSP90) Limits the Formation of Liver Cysts Induced by Conditional Deletion of Pkd1 in Mice

doi: 10.1371/journal.pone.0114403

Figure Lengend Snippet: ( A, B ) Representative hematoxylin stained liver sections with immunohistochemical detection of HSP90α (brown) from three ( A ) wild type (wt) and ( B ) Pkd1 –/– mice. bd = bile duct, pv = portal vein, c = cyst. Scale bars = 100 µm; magnification, 20x.

Article Snippet: The membranes were then incubated for 1 hour at room temperature or overnight at 4°C in primary antibodies (1∶1000): anti-HSP70 (monoclonal, mouse, #ADI-SPA-820) or anti-HSP90α (polyclonal, rabbit, #ADI-SPS-771) (both from Enzo, Farmingdale, NY); or anti-pERK1/2 Thr202/Tyr204 (polyclonal, rabbit, #9101), anti-ERK1/2 (monoclonal, mouse, #4696), anti-pS6 Ser235/236 (monoclonal, rabbit, #4858), anti-S6 (monoclonal, mouse, #2317), anti-phospho AKT S473 (polyclonal, rabbit, #4060S) and anti-AKT (polyclonal, rabbit, #2920S) and anti-α/β-Tubulin (polyclonal, rabbit, #2148) (each from Cell Signaling, Danvers, MA).

Techniques: Staining, Immunohistochemical staining

Journal: PLoS ONE

Article Title: Inhibiting Heat Shock Protein 90 (HSP90) Limits the Formation of Liver Cysts Induced by Conditional Deletion of Pkd1 in Mice

doi: 10.1371/journal.pone.0114403

Figure Lengend Snippet: ( A ) Quantification of HSP90α and HSP70 expression levels among the Pkd1 –/– groups. n = 6–8 mice. * indicates comparisons to Pkd1 –/– , vehicle-treated mice: **, P ≤0.01; ***, P ≤0.001. ( B–E ) Negative correlations between HSP70, HSP90α expression in the drug treatment groups and ( B, D ) cyst volume/region of interest (ROI) ( P = 0.0046 for HSP70 and P = 0.0136 for HSP90) and ( C, E ) liver weight/body weight (lw/bw) ratios ( P = 0.0141 for HSP70 P = 0.0138 for HSP90). Dots represent individual mice; lines represent linear regression functions. n = 6–8 mice.

Article Snippet: The membranes were then incubated for 1 hour at room temperature or overnight at 4°C in primary antibodies (1∶1000): anti-HSP70 (monoclonal, mouse, #ADI-SPA-820) or anti-HSP90α (polyclonal, rabbit, #ADI-SPS-771) (both from Enzo, Farmingdale, NY); or anti-pERK1/2 Thr202/Tyr204 (polyclonal, rabbit, #9101), anti-ERK1/2 (monoclonal, mouse, #4696), anti-pS6 Ser235/236 (monoclonal, rabbit, #4858), anti-S6 (monoclonal, mouse, #2317), anti-phospho AKT S473 (polyclonal, rabbit, #4060S) and anti-AKT (polyclonal, rabbit, #2920S) and anti-α/β-Tubulin (polyclonal, rabbit, #2148) (each from Cell Signaling, Danvers, MA).

Techniques: Expressing

Journal: Cell reports

Article Title: EPS8 Facilitates Uncoating of Influenza A Virus

doi: 10.1016/j.celrep.2019.10.064

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-HSP90α/β , Santa Cruz Biotechnology , Cat# sc-7947; RRID: AB_2121235.

Techniques: Virus, Recombinant, Western Blot, Luciferase, Plasmid Preparation, Software

Journal: Journal of Animal Science

Article Title: Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes ¹

doi: 10.1093/jas/sky213

Figure Lengend Snippet: Expression and subcellular localization of Hsp90α in maturing porcine oocytes. (A) RT-qPCR results showed Hsp90α mRNA expressed in maturing porcine oocytes and its level did not change significantly among different stages (0 h, 24 h, and 44 h of IVM corresponding to GV, GVBD, and MII stages). (B, C) Western blots showed Hsp90α protein was expressed and its level did not significantly change in porcine oocytes undergoing meiosis. (D) Subcellular localization of Hsp90α via immunofluorescent staining. Green, Hsp90α; blue, chromatin. GV, germinal vesicle; Pre-MI, pre metaphase I; MI, metaphase I; MII, metaphase II. Scale bar, 50 µm in the left three columns; 10 µm in the right column.

Article Snippet: Samples were then subsequently incubated with rabbit anti-Hsp90α polyclonal antibody (ABclonal, A0365, 1:50) and a fluorescein isothiocyanate ( FITC )–conjugated goat anti-rabbit secondary antibody (TransGen Biotech, HS111, 1:150).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Journal of Animal Science

Article Title: Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes ¹

doi: 10.1093/jas/sky213

Figure Lengend Snippet: Inhibition of Hsp90α by 17-AAG and antibody binding impaired porcine oocyte nuclear maturation. (A) Cumulus expansion was inhibited by adding 17-AAG into the maturation media (1 µM, 2 µM, and 4µM) to culture porcine COC for 24 h. Cumulus-free oocytes were visualized to be with first polar bodies (arrows) after adding 17-AAG (1 µM, 2 µM, and 4 µM) to mature porcine COC for 44 h. (B, C) GVBD rates (24 h) and PB1 rates (44 h) of oocytes derived from COC treated by 17-AAG at different concentration (1 µM, 2 µM, and 4 µM). (D) PB1 rate of GV oocytes denuded of cumulus (CDOs) treated by 2 µM 17-AAG in maturation medium for 44 h. (E, F) Western blots on Hsp90α protein level in oocytes derived from COC treated by 2 µM 17-AAG for 24 h and 44 h during IVM. (G, H) Microinjection of Hsp90α antibody into cytoplasm of porcine GV oocytes confirmed that immuno-inhibition of Hsp90α significantly decreased GVBD rate (24 h) and PB1 rate (44 h). (I, J) p-CDK1 (T161) levels in oocytes derived from COC treated by 2 µM 17-AAG for 24 h and 44 h during IVM. Scale bar, 200 µm. *Significant difference at P < 0.05 level, **P < 0.01, and ***P < 0.001. COC = cumulus–oocyte complexes.

Article Snippet: Samples were then subsequently incubated with rabbit anti-Hsp90α polyclonal antibody (ABclonal, A0365, 1:50) and a fluorescein isothiocyanate ( FITC )–conjugated goat anti-rabbit secondary antibody (TransGen Biotech, HS111, 1:150).

Techniques: Inhibition, Binding Assay, Derivative Assay, Concentration Assay, Western Blot, Microinjection

Journal: Journal of Animal Science

Article Title: Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes ¹

doi: 10.1093/jas/sky213

Figure Lengend Snippet: Suppression of Hsp90α disrupts cytoskeleton assembly of porcine oocytes. (A) Representative fluorescence images of porcine oocytes to show actin organization (green). Scale bar, 50 µm in the middle column; 10 µm in the right and left columns. (B) Quantitative analysis of relative actin fluorescence intensity from cytoplasm and membrane of oocytes. (C) Representative fluorescence images of porcine oocytes showing spindle (green) and chromosome morphology (red). MII oocytes from the control group displayed bipolar-shaped spindles and well-aligned chromosomes on the metaphase equator (a), whereas chromosome misalignment and spindle defects were frequently observed in 2 µM 17-AAG treatment group, which can be classified into four different types (b–e). (a) The normal bipolar-shaped spindle. (b) The monopolar spindle. (c) Spindle losing bipolar. (d) The weakened spindle. (e) The disturbed spindle. Scale bar, 10 µm. (D) Percentages of oocytes with normal spindle organization. (E, F) Western blots of p-ERK1/2 and β-tubulin in oocytes collected at 24 h and 44 h of IVM from the control and 2 µM 17-AAG–treated COC. *Significant difference at P < 0.05 level and **P < 0.01. Abbreviation: COC = cumulus–oocyte complexes.

Article Snippet: Samples were then subsequently incubated with rabbit anti-Hsp90α polyclonal antibody (ABclonal, A0365, 1:50) and a fluorescein isothiocyanate ( FITC )–conjugated goat anti-rabbit secondary antibody (TransGen Biotech, HS111, 1:150).

Techniques: Fluorescence, Membrane, Control, Western Blot

Journal: Journal of Animal Science

Article Title: Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes ¹

doi: 10.1093/jas/sky213

Figure Lengend Snippet: Effects of Hsp90α inhibition by 17-AAG on mitochondrial membrane potential and early apoptosis of porcine oocytes. (A) Representative fluorescence images taken from porcine oocytes stained with RH123. Scale bar, 100 µm. (B) The graphs to present the relative RH123 fluorescence intensity in oocytes. (C) Representative fluorescence images taken from porcine oocytes stained with Annexin V. Scale bar, 100 µm in the left and middle columns; 50 µm in the right column. (D) The graphs to show percentages of apoptosis positive oocytes. ***Significant difference at P < 0.001 level.

Article Snippet: Samples were then subsequently incubated with rabbit anti-Hsp90α polyclonal antibody (ABclonal, A0365, 1:50) and a fluorescein isothiocyanate ( FITC )–conjugated goat anti-rabbit secondary antibody (TransGen Biotech, HS111, 1:150).

Techniques: Inhibition, Membrane, Fluorescence, Staining

Journal: Journal of Animal Science

Article Title: Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes ¹

doi: 10.1093/jas/sky213

Figure Lengend Snippet: Inhibition of Hsp90α by 17-AAG and antibody binding induces fragmentation of parthenotes. (A) Morphology of cleaved parthenotes and blastocysts from mature oocytes after 17-AAG treatment for 44 h during IVM. (B) Cleavage, fragmentation, and blastocyst percentages of parthenotes from mature oocytes after 17-AAG treatment for 44 h during IVM. (C) Cell number per blastocyst derived from 17-AAG–treated mature oocytes. (D) Morphology of cleaved parthenotes and blastocysts from mature oocytes after anti-Hsp90α antibody microinjection. (E) Cleavage, fragmentation, and blastocyst percentages of parthenotes from mature oocytes after anti-Hsp90α antibody microinjection. (F) Cell number per blastocyst derived from anti-Hsp90α antibody microinjected mature oocytes. Scale bar, 100 µm. *Significant difference at P < 0.05 level, **P < 0.01, and ***P < 0.001.

Article Snippet: Samples were then subsequently incubated with rabbit anti-Hsp90α polyclonal antibody (ABclonal, A0365, 1:50) and a fluorescein isothiocyanate ( FITC )–conjugated goat anti-rabbit secondary antibody (TransGen Biotech, HS111, 1:150).

Techniques: Inhibition, Binding Assay, Derivative Assay, Microinjection