Journal: Frontiers in Pharmacology

Article Title: Early treatment with nootkatone prevents pressure overload-induced ventricular remodeling and heart failure

doi: 10.3389/fphar.2025.1702627

Figure Lengend Snippet: Early NKT administration downregulated the TGF-β1/Smad3 signaling pathway (n = 6–8). (A) mRNA expression of Col Ⅰ and Col Ⅲ in heart tissue. (B) Protein expression of Col Ⅰ, Col Ⅲ, HSP-90, TGF-β1, Smad3, and p-Smad3 in heart tissue. (C) Protein quantitative analysis of Col Ⅰ, Col Ⅲ, HSP-90, TGF-β1, Smad3, and p-Smad3. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, no significance.

Article Snippet: The membrane was incubated with primary antibodies against collagen type I (Col I, 1:500, ServiceBio, China), collagen type III (Col III, 1:500, ServiceBio, China), HSP-90 (1:2000, OriGene, China), phosphorylated Smad3 (p-Smad3, 1:4000, Wanleibio, China), Smad3 (1:4000, Proteintech, China), transforming growth factor (TGF-β1, 1:5000, Proteintech, China), and β-actin (1:2000, EPITMICS, China), followed by incubation with corresponding HRP-labeled secondary antibodies (ServiceBio, China).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Enhanced cytotoxic effect of radiation and temozolomide in malignant glioma cells: targeting PI3K-AKT-mTOR signaling, HSP90 and histone deacetylases

doi: 10.1186/1471-2407-14-17

Figure Lengend Snippet: Targeting PI3K-Akt-mTOR signaling. (A) U251 and T98G cells were pretreated with rapamycin (RPM) plus TMZ for 24 h and subjected to western blot analysis using the indicated antibodies. Pretreatment with rapamycin (100 nM) plus TMZ (25 μM) did not have a synergistic radiosensitizing effect compared to TMZ alone treatment on U251 and T98G cells. (B) U251 and T98G cells were pretreated with a dual inhibitor of class I PI3K and mTOR signaling, PI103 (0.4 μM), and TMZ (25 μM) for 24 h. PI103 effectively enhanced the radiosensitizing effect of TMZ in both U251 and T98G cells. (C) U251 and T98G cells were pretreated with the HSP90 inhibitor 17-DMAG (25nM) and TMZ (25 μM) for 24 h. 17-DMAG enhanced the radiosensitizing effect of TMZ in U251 and T98G cells. (D) U251 and T98G cells were pretreated with TMZ (25 μM) and LBH589 (20 nM) for 24 h. LBH589 effectively potentiated the radiosensitizing effect of TMZ. Points on survival curves represent mean surviving fractions from minimum three experiments performed in triplicate.

Article Snippet: Membranes were blocked in PBS containing 0.1% Tween 20 and 5% powdered milk and probed with primary antibody directed against p-EGFR (Tyr1068), p-Akt (Ser473), p-ERK (Tyr202/204), p-p70S6K (Thr421/Ser424), HSP70, HSP90, DNA-PKs (Thr2609), Rad51, caspase-3, LC3, MMP-2, E-cadherin, and EphA2 (Cell Signaling Technology, Inc.) at 1:1000 dilutions.

Techniques: Western Blot

Journal: BMC Cancer

Article Title: Enhanced cytotoxic effect of radiation and temozolomide in malignant glioma cells: targeting PI3K-AKT-mTOR signaling, HSP90 and histone deacetylases

doi: 10.1186/1471-2407-14-17

Figure Lengend Snippet: Impairment of DNA damage repair following irradiation. (A) U251 cells were pretreated with the indicated inhibitors plus TMZ before assessment of γH2AX foci formation. Mock-treated control cells were analyzed 6 h after irradiation with 6 Gy. Pretreatment of U251 cells with the dual inhibitor PI103, the HSP90 inhibitor 17-DMAG, or the HDAC inhibitor LBH589 plus TMZ caused marked prolongation of radiation-induced γH2AX foci formation 6 h after 6Gy irradiation. (B) Pretreatment of U251 cells with TMZ combined with PI103, 17-DMAG, or LBH589 attenuated p-DNA-PK expression.

Article Snippet: Membranes were blocked in PBS containing 0.1% Tween 20 and 5% powdered milk and probed with primary antibody directed against p-EGFR (Tyr1068), p-Akt (Ser473), p-ERK (Tyr202/204), p-p70S6K (Thr421/Ser424), HSP70, HSP90, DNA-PKs (Thr2609), Rad51, caspase-3, LC3, MMP-2, E-cadherin, and EphA2 (Cell Signaling Technology, Inc.) at 1:1000 dilutions.

Techniques: Irradiation, Control, Expressing

Journal: Frontiers in Oncology

Article Title: Rationally Repurposing Ruxolitinib ( Jakafi ® ) as a Solid Tumor Therapeutic

doi: 10.3389/fonc.2016.00142

Figure Lengend Snippet: (Ruxolitinib + afatinib) rapidly reduces CDC37 Serine 13 phosphorylation and after 6 h CDC37 total expression . (A,B) SUM149 cells were treated with vehicle control or with (ruxolitinib + afatinib) for 2 h or for 6 h after which cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed: (A) at 10× magnification to detect the total expression and phosphorylation levels of CDC37 and CDC37 Serine 13, respectively; (B) at 60× magnification the total expression of HSP90 and the total expression of CDC37, and the co-localization of the two proteins ( n = 3 ± SEM) * p < 0.05 greater decline than CDC37 protein expression. (C) SUM149 cells were transfected with empty vector control or with plasmids to express: HSP90, GRP78, HSP70, or HSP27, alone or in the indicated combinations. Twenty-four hours after transfection, cells were treated with vehicle control or with [ruxolitinib (1.0 μM) + afatinib (1.0 μM)] for 6 h after which cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed at 10× magnification to detect the total expression of MCL-1 and BCL-XL ( n = 3 ± SEM) * p < 0.05 lower decline than vehicle control. (D) SUM149 cells were transfected with empty vector control or with plasmids to express: HSP90, GRP78, HSP70, or HSP27, alone or in the indicated combinations. Twenty-four hours after transfection, cells were treated with vehicle control or with [ruxolitinib (1.0 μM) + afatinib (1.0 μM)] for 24 h. Twenty-four hours later, cell viability was assessed using a live/dead assay in a Hermes WiScan microscope at 10× magnification ( n = 3 ± SEM) * p < 0.05 greater survival than individual expression of chaperones.

Article Snippet: Antibodies used include: HSP90 (E289) (Cell Signaling); HSP90 (#2928) (Abcam); HSP90 (ab195575) Abcam; HSP90 3G3 (13495) (Abcam); GRP78 (50b12) (31772) (Cell Signaling); GRP78 (ab191023) Abcam; GRP78 (ab103336) Abcam; GRP78 (N-20) (sc-1050) Santa Cruz; HSP27 (G31) (2402P) Cell Signaling); HSP27 (EP1724Y) (ab62339) Abcam; HSP27 (H-77) (sc-9012) Santa Cruz; HSP27 (LS-C31836) Lifespan science Corp. Other antibodies were as used in prior studies by the laboratory.

Techniques: Expressing, Fluorescence, Transfection, Plasmid Preparation, Live Dead Assay, Microscopy

Journal: Molecular Cancer Therapeutics

Article Title: Cell Intrinsic Role of COX-2 in Pancreatic Cancer Development

doi: 10.1158/1535-7163.mct-12-0342

Figure Lengend Snippet: Figure 7. Decreased FKBP5 negative feedback leads to enhanced AKT activation in Pdx1-Creþ;K-rasG12D/þ;Ptenlox/þ; Cox-2lox/lox mice. A, FKBP5 mRNA expression in PDACs of Pdx1-Creþ; K-rasG12D/þ;Ptenlox/þ;Cox-2lox/lox

Article Snippet: The following primary antibodies were used: phospho-AKT(Ser473) (Cell Signaling; 1:50), Cytokeratin 19 (ab15463, Abcam; 1:100), COX-2 (SP21; Thermo Scientific, ready-to-use),GRP78(11587-1AP,ProteinTechGroup; 1:50), and FKBP5 (14155-1-AP, ProteinTech Group, 1:50).

Techniques: Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Therapeutic effect of histone deacetylase 6 inhibitor for a mouse model of phenylketonuria

doi: 10.1038/s41598-025-29143-7

Figure Lengend Snippet: Western blot analysis of acetyl-HSP90, HSP90, and PAH protein expression in liver tissues from sham, treated, and wild-type groups following 12 weeks of TSA treatment. ( A ) Expression of acetyl-HSP90, HSP90, PAH, and GAPDH (used as a loading control). ( B ) Quantitative analysis of acetyl-HSP90 relative to HSP90 protein levels. ( C ) Quantitative analysis of PAH relative to GAPDH protein levels. The expression of PAE was quantified using the Image J software (Version 1.49) and expressed as arbitrary units (a.u.). Results are presented as mean ± SEM, with statistical significance indicated. ****P<0.0001.

Article Snippet: The antibodies used anti-acetylated HSP90 (600-401-981, Rockland Immunochemical, 1:500), anti-HSP90 (#4877, Cell Signaling, 1:1,000), and PAH (sc-271258, Santa Cruz, 1:2,000).

Techniques: Western Blot, Expressing, Control, Software

Journal: Scientific Reports

Article Title: Therapeutic effect of histone deacetylase 6 inhibitor for a mouse model of phenylketonuria

doi: 10.1038/s41598-025-29143-7

Figure Lengend Snippet: Mechanism of TSA in enhancing HSP90 acetylation and stabilizing misfolded PAH protein to improve phenylalanine metabolism.

Article Snippet: The antibodies used anti-acetylated HSP90 (600-401-981, Rockland Immunochemical, 1:500), anti-HSP90 (#4877, Cell Signaling, 1:1,000), and PAH (sc-271258, Santa Cruz, 1:2,000).

Techniques:

Journal: Frontiers in Immunology

Article Title: USP50 regulates NLRP3 inflammasome activation in duodenogastric reflux-induced gastric tumorigenesis

doi: 10.3389/fimmu.2024.1326137

Figure Lengend Snippet: USP50 stimulates HMGB1 release in DGR-induced gastric inflammation. (A, B) mRNA levels of HMGB1, HSP70, HSP90, S100A8, and S100A9 were detected via qRT-PCR in U937 cells (A) or RAW264.7 cells (B) treated with DMSO or TDCA (200μM, 24 h). (C-H) ELISA was used to measure the concentrations of HMGB1 (C) , HSP70 (D) , HSP90 (E) , S100A8 (F) , and S100A9 (G) in the supernatant of U937 cells and RAW264.7 cells, while ATP concentration (H) was surveyed by ATP assay kit. (I) The protein level of HMGB1 was detected in gastric tissues of Sham and GJ mice by western blotting. Image J was used for quantitative analysis, with GAPDH as loading control. (J) The level of HMGB1 between normal gastric tissues and gastric cancer tissues was analyzed via GEPIA database (gepia.cancer-pku.cn/). (K) The IHC staining of HMGB1 was performed with sections from BRG patients and GC patients. (L) IRS was calculated for (K) . (M, N) HMGB1 was assayed by ELISA in the supernatant of U937 cells with USP50 overexpression (M) or knock-down (N) . * P < 0.05, ** P < 0.01, and *** P <0.001. "ns" means "no significance" ( P >0.05).

Article Snippet: Human S100A8 (E-EL-H1289c), Human S100A9 (E-EL-H1290c), HSP90 ELISA Kit (E-EL-H1864c), Human HMGB-1 ELISA Kit (E-EL-H1554c), Mouse HMGB-1 ELISA Kit (E-EL-M0676c) were obtained from Elabscience Biotechnology Co., Ltd, China.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, ATP Assay, Western Blot, Control, Immunohistochemistry, Over Expression, Knockdown