17 aag inhibitor  (MedChemExpress)

Journal: Cellular & Molecular Biology Letters

Article Title: Heat shock protein 90 stabilizes CD93 glycosylation to influence angiogenesis during diabetic wound healing

doi: 10.1186/s11658-025-00847-y

Figure Lengend Snippet: CD93 interacts with HSP90 to maintain its proangiogenic ability. A Peak map of MS detection in the CD93 IgG and CD93 IP groups. B Gene Ontology (GO) analysis of the MS results. BP, biological process; CC, cellular component; MF, molecular function. C Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the MS results. D Predicted schematic diagram of the interaction between HSP90 and CD93 via the AutoDock website. The black line indicates the boundary: the left represents CD93, and the right represents HSP90. E Local schematic representation of the interaction between HSP90 and CD93. Cyan represents CD93 (left), purple represents HSP90 (right), and yellow represents the hydrogen bonds between HSP90 and CD93. F Co-IP experiments were performed to identify the interaction between HSP90 and CD93. G Co-IP experiments were performed to identify the interaction between HSP27 and CD93. H CD93 protein expression after treatment with 50 nM 17-AAG with or without ad.CD93. G-CD93, glycosylated CD93; NG-CD93, nonglycosylated CD93. I Relative expression levels of glycosylated and nonglycosylated CD93 protein in ( H ) after normalization to β-actin. J Cell spheroid sprouting assay after treatment with 50 nM 17-AAG with or without ad.CD93; the lower column shows the local map of the top right quarter of the cell spheroids. Scale bar = 200 μm. K Length of the sprouts in ( J ). L Migration of ECs after treatment with 50 nM 17-AAG with or without ad.CD93. Scale bar = 200 μm. M The number of migrated ECs in ( L ). * P < 0.05; ** P < 0.01; *** P < 0.001, ns: not significant. Data represent three independent experiments and are shown as means (SDs)

Article Snippet: In addition, the HSP90 inhibitor 17-AAG (MCE) was delivered via intraperitoneal injection at a dosage of 100 mg/kg 2 weeks prior to skin wound construction, and recombinant CD93 (rCD93, R&D) was administered via local injections at the periphery of the wound for three consecutive days at a dosage of 80 ng.

Techniques: Co-Immunoprecipitation Assay, Expressing, Migration

Journal: Cellular & Molecular Biology Letters

Article Title: Heat shock protein 90 stabilizes CD93 glycosylation to influence angiogenesis during diabetic wound healing

doi: 10.1186/s11658-025-00847-y

Figure Lengend Snippet: HSP90 protects CD93 from ubiquitin‒proteasome-mediated degradation and facilitates FAK signaling pathway activation. ( A ) HSP90α mRNA expression after treatment with siHSP90α. ( B ) HSP90α protein expression after treatment with siHSP90α. ( C ) Relative HSP90α protein expression levels in ( B ) after normalization to that of β-actin. ( D ) Cell spheroid sprouting assay after treatment with siHSP90α-3 with or without ad.CD93. Scale bar = 200 μm. ( E ) Local magnification of the top right quadrant of the cell spheroids. ( F ) Statistical analysis of the length of the sprouts in ( D ). ( G ) CD93 protein expression after treatment with bortezomib with or without 17-AAG. ( H ) Relative protein expression of glycosylated and nonglycosylated CD93 in ( G ) after normalization to that of β-actin. G-CD93, glycosylated CD93; NG-CD93, non-glycosylated CD93. ( I , J ) Co-IP experiment testing the ubiquitination of CD93 after treatment with 17-AAG with or without bortezomib. ( K ) Schematic diagrams of the full-length CD93 plasmid and a truncated CD93 plasmid lacking the intracellular domain (CD93△), both of which are tagged with red fluorescent protein (RFP). CTLD, Sushi, epidermal growth factor-like, and mucin are extracellular domains of CD93. TM is a transmembrane domain. Cy is a cytosolic domain. ( L ) CoIP experiment to detect the interaction between HSP90 and CD93 or CD93△. ( M ) The expression of p-FAK and FAK after ad.CD93 treatment. ( N ) Relative expression of p-FAK and FAK in ( M ) after normalization to that of β-actin. ( O ) The expression of p-FAK and FAK after siCD93 treatment. ( P ) Relative expression of p-FAK and FAK in ( O ) after normalization to that of β-actin. ( Q ) The expression of p-FAK and FAK after treatment with 17-AAG with or without ad.CD93. * P < 0.05; ** P < 0.01; *** P < 0.001, ns = not significant. Data represent three independent experiments and are shown as means (SDs)

Article Snippet: In addition, the HSP90 inhibitor 17-AAG (MCE) was delivered via intraperitoneal injection at a dosage of 100 mg/kg 2 weeks prior to skin wound construction, and recombinant CD93 (rCD93, R&D) was administered via local injections at the periphery of the wound for three consecutive days at a dosage of 80 ng.

Techniques: Activation Assay, Expressing, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: Heat shock protein 90 stabilizes CD93 glycosylation to influence angiogenesis during diabetic wound healing

doi: 10.1186/s11658-025-00847-y

Figure Lengend Snippet: The role of the HSP90–CD93 interaction in wound healing angiogenesis in vivo. A Wound healing in the control group, 17-AAG group, rCD93 group, and 17-AAG + rCD93 group at D0, D2, D4, and D6, respectively. Ruler = 5 mm. B Schematic diagram of overlapping wound healing at different times drawn via ImageJ. C Statistical analysis of the wound area in the four groups of mice at D0. D Statistical analysis of the wound area in the four groups of mice at D2. E Statistical analysis of the wound area in the four groups of mice at D4. F Statistical analysis of the wound area in the four groups at D6. G HE staining of wounds in the four groups at D0, D2, D4, and D6. The dotted lines delineate the boundary between the epidermal and dermal layers, and the length of the wound is marked by arrows. Scale bar = 1 mm. H Statistical analysis of the wound length at D7 in ( G ). I Masson staining of wounds in the four groups at D0, D2, D4, and D6. Blue represents collagen. Scale bar = 1 mm. J Statistical analysis of the collagen volume fraction in the four groups; the collagen volume fraction = collagen volume/total area. K Merged IF image of new vessels at D7. Red represents CD31 (a marker of new vessels), green represents CD93, and blue represents DAPI. Scale bar = 20 μm. See Supplementary Fig. S6A for fluorescence images of each individual channel. L Statistical analysis of the CD31-positive area at D7. M Merged IF image of new vessels and pericytes at D7; red represents CD31, green represents NG2 (a marker of pericytes), and blue represents DAPI. Scale bar = 20 μm. See Supplementary Fig. S6B for fluorescence images of each individual channel. ( N ) Statistical analysis of the NG2-positive area at D7. * P < 0.05; ** P < 0.01; *** P < 0.001, ns: not significant. Data are shown as means (SDs)

Article Snippet: In addition, the HSP90 inhibitor 17-AAG (MCE) was delivered via intraperitoneal injection at a dosage of 100 mg/kg 2 weeks prior to skin wound construction, and recombinant CD93 (rCD93, R&D) was administered via local injections at the periphery of the wound for three consecutive days at a dosage of 80 ng.

Techniques: In Vivo, Control, Staining, Marker, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis

doi: 10.1016/j.jbc.2025.110914

Figure Lengend Snippet: FKBP4 prevents CCT8 aggregation. A , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT1 and CCT8 proteins within cells was assessed using a filter trap assay. Cell lysates were filtered through a nitrocellulose membrane, and the retained proteins on the membrane were detected using specific antibodies against CCT1 and CCT8. B , quantitative analysis of CCT1 and CCT8 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. C , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT8, CCT1, CDK2, α-tubulin, and GAPDH within the cells. D , quantitative analysis of CCT8 and CCT1 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. E , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT2 and CCT3 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). F , quantitative analysis of CCT2 and CCT3 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. G , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT2, CCT3, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). H , quantitative analysis of CCT2 and CCT3 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. I , shFKBP4 HCT116 cells were harvested after 3 days of puromycin selection. The cell lysates were biochemically fractionated into Triton-soluble and -insoluble fractions as described in “ ”. The FKBP4 expression levels and Triton-soluble and -insoluble fractions of CCT8 were analyzed by western blotting. The GAPDH was used as a loading control. J , The ratio of the CCT8 Triton-insoluble form in ( I ). K , The primary structures of FKBP4 and FKBP5. L , after 48 h of transfection, vector, FLAG-tagged FKBP4 or FLAG-tagged FKBP5 expressing HEK293T cell lysates were precipitated by anti-FLAG antibodies, and the products were detected for the co-purification of the endogenous proteins. M , comparison of the interaction between Hsp90 and FLAG-tagged FKBP4 or FLAG-tagged FKBP5 in ( L ). N , following the knockdown of FKBP5 in HCT116 cells for 72 h, the aggregation of CCT8 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). O , quantitative analysis of the protein aggregation of CCT8, normalized to GAPDH, in shFKBP5 HCT116 cells. P , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP5, CCT8, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). Q , quantitative analysis of CCT8 protein expression, normalized to GAPDH, in shFKBP5 HCT116 cells. R , shFKBP5 HCT116 cells were harvested after puromycin selection for 3 days. The experimental procedure mirrored that of ( I ). S , the ratio of the CCT8 Triton-insoluble form in ( R ). T , HCT116 cells were treated with 17-AAG (20 μM) or ganetespib (2 μM) for 24 h, and CCT8 aggregation was assessed by filter trap assay. U , quantitative analysis of CCT8 protein aggregation with GAPDH serving as an internal control in ( T ). V , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of HSPA1A, CCT8, HSP90, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). W , quantitative analysis of CCT8 protein expression was normalized with GAPDH in ( V ). All data were analyzed with an unpaired two-tailed Student's t test. Each dataset is expressed as mean ± SD for n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: HCT116 cells were treated with the Hsp90 inhibitors 17-AAG (MedChem Express) and ganetespib (MedChem Express) at final concentrations of 20 and 2 μM, respectively.

Techniques: Knockdown, TRAP Assay, Membrane, Western Blot, Expressing, Selection, Control, Transfection, Plasmid Preparation, Copurification, Comparison, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis

doi: 10.1016/j.jbc.2025.110914

Figure Lengend Snippet: The model of the Hsp90-FKBP4 folding cycle. FKBP4 binds to Hsp90 through the C-terminal TRP motif, forming an Hsp90-FKBP4 protein complex to execute protein folding. FKBP4 can help fold CCT8, one of the TRiC subunits. CCT8, when well folded by the Hsp90-FKBP4 complex, can form a tubular chaperonin structure with other TRiC subunits. Functional chaperonin is known to be responsible for the folding of α-tubulin.

Article Snippet: HCT116 cells were treated with the Hsp90 inhibitors 17-AAG (MedChem Express) and ganetespib (MedChem Express) at final concentrations of 20 and 2 μM, respectively.

Techniques: Functional Assay