Journal: Journal of Advanced Research

Article Title: Negative pressure mechanical signal increases the phosphorylation of eNOS Ser1177 by upregulating HSP90 expression to promote wound angiogenesis

doi: 10.1016/j.jare.2025.07.041

Figure Lengend Snippet: NPWT modulates the biological behavior of HDMECs by regulating HSP90. (A) Volcano plot of differentially expressed proteins in the different groups of HDMECs. (B) Pie chart of differential protein cellular localization. (C) Heatmap of differentially expressed proteins in HDMECs from various groups. (D) GO analysis of upregulated proteins in HDMECs treated with NPWT. (E) mRNA expression of HSP90 after the application of siRNA. (F) CCK8 assay was used to measure the proliferation rate of HDMECs across different groups. n = 8. (G, H) Migration and lumen formation of HDMECs in each group after the application of the HSP90 inhibitor 17-AAG or siHSP90. n = 3. Scale bar = 200 µm. (I) Changes in NO levels in HDMECs across different groups. n = 3. Scale bar = 50 µm. (J) Variations in ROS levels across different groups. n = 3. Scale bar = 50 µm. (K, L) Apoptosis and necrosis levels of HDMECs in different groups. n = 3. Scale bar = 100 µm.

Article Snippet: Primary human dermal microvascular endothelial cells (HDMECs) were isolated from cryopreserved human dermal tissue via established methods [ ] and cultured in ECM (Sciencell) at 37 °C with 5 % CO 2 for 24 h. The cells in the corresponding groups were treated with 0.5 mM 17-AAG (MCE) or 0.5 mM 666–15 (MCE).

Techniques: Expressing, CCK-8 Assay, Migration

Journal: Aging Cell

Article Title: Decreased Glucose Metabolism and Declined Chaperones Are Unique Features Required for the Survival of Senescent Fibroblasts and Pyruvate Dehydrogenase Is a Potent Senolytic Target

doi: 10.1111/acel.70434

Figure Lengend Snippet: The abundance of chaperones and their transcription are decreased in senescent fibroblasts. Inhibiting Hsp90 by 17‐AAG caused the selective killing of senescent fibroblasts. (A–D) The summarization of protein changes in abundance of TRiC chaperone, Hsp70 family, Hsp90 family, and other chaperones in senescent BJ cells identified by our proteomic profiling. (E) Western blotting confirmed the protein levels of TCP1, Hsp70, and Hsp90 were decreased in senescent BJ and IMR‐90 cells. β‐Actin served as internal control. (F) RT‐PCR confirmed the transcription levels of TCP1, Hsp70, and Hsp90 genes were also decreased in senescent BJ and IMR‐90 cells. The RT‐PCR product of β‐Actin was as internal control. (G) The morphological changes of growing and senescent IMR‐90 cells at the indicated time of 17‐AAG treatment under the light microscopy. The senescent IMR‐90 cells without 17‐AAG treatment were stained with SA‐β‐gal. Cells were imaged at magnification 200×. (H) Inhibiting Hsp90 by 17‐AAG led to the selective killing of senescent IMR‐90 cells in a dose dependent manner. ** p < 0.01 by one‐way ANOVA. (I) The dose–response curves of 17‐AAG on proliferating and senescent BJ cells. *** p < 0.001 by one‐way ANOVA.

Article Snippet: Doxorubicin, 2‐deoxy‐glucose (2‐DG), CPI‐613, bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide (BPTES), and 17‐AAG were purchased from MedChemExpress.

Techniques: Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Light Microscopy, Staining

Journal: Aging Cell

Article Title: Decreased Glucose Metabolism and Declined Chaperones Are Unique Features Required for the Survival of Senescent Fibroblasts and Pyruvate Dehydrogenase Is a Potent Senolytic Target

doi: 10.1111/acel.70434

Figure Lengend Snippet: Inhibiting PDH, GLS1 and Hsp90 by the combination of CPI‐613+BPTES+17‐AAG gave rise to enhanced senolysis on senescent fibroblasts as well as the therapy‐induced senescent tumor cells. (A, B) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating (A) and senescent (B) BJ cells. For the dose of each compound in use, see the results 2.7 section for more details. ** p < 0.01 by Student's t ‐test. (C, D) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating and Dox‐induced senescent lung adenocarcinoma A549 cells. *** p < 0.001 by Student's t ‐test. (E, F) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating and Dox‐induced senescent cervical carcinoma HeLa cells. *** p < 0.001 by Student's t ‐test. (G) The morphological changes of senescent BJ induced by IR, senescent A549 and HeLa cells induced by Dox at the indicated time of CPI‐613+BPTES+17‐AAG treatment under the light microscopy. The senescent cells without treatment were stained with SA‐β‐gal. Cells were imaged at magnification 200×. (H) The schematic summarization of our findings. The activities of TCA cycle and chaperones are reduced in DNA damage induced senescent cells. Co‐inhibiting Hsp90 and TCA cycle with 17‐AAG+CPI‐613+BPTES combination leads to enhanced selective elimination of senescent cells, hinting TCA cycle and glutaminolysis are novel and potent targets for senolysis.

Article Snippet: Doxorubicin, 2‐deoxy‐glucose (2‐DG), CPI‐613, bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide (BPTES), and 17‐AAG were purchased from MedChemExpress.

Techniques: Light Microscopy, Staining

Journal: Aging Cell

Article Title: Decreased Glucose Metabolism and Declined Chaperones Are Unique Features Required for the Survival of Senescent Fibroblasts and Pyruvate Dehydrogenase Is a Potent Senolytic Target

doi: 10.1111/acel.70434

Figure Lengend Snippet: CPI‐613+BPTES+17‐AAG combination treatment reduced the p21 positive senescent cells and alleviated the physical dysfunctions in aged mice. (A) The experimental design for generating aged mice induced by D‐galactose via intraperitoneal injection and the combination treatment in the aged mice. For the dose of each compound in use, see the methods section for more details. (B) Representative images of aging mice on day 5 after the last combination treatment, the dull and shaggy coat hair and wrinkle formation observed in the aged mice were ameliorated in the treated mice. (C, D) Representative images of p21 stained liver, kidney, and lung tissues from aged mice with or without combination treatment and the quantification of p21 positive cells in liver, kidney, and lung tissues. The red arrows indicate the p21 positive cells, the scale bar stands for 30 μm. Data were presented as means ± SD, p value was calculated by two‐tailed Student's t ‐test. (E–G) Spleens dissected from 7 mice with or without combination treatment were displayed. The surface area and weight of spleens from each mouse group were measured and plotted in F‐G. The surface area of each spleen was calculated using the following formula: S = 5 × (0.524 × L × W × T) 2/3 . (H–M) Physical function measurements in aged mice with or without treatment with CPI‐613+BPTES+17‐AAG combination. Changes in body weight (H), food intake (I), grip strength (J), time on the rotarod (K), running distance on the treadmill (L), and treadmill endurance (M) were plotted. Results are shown as box‐and‐whisker plots with the median shown as a line in the middle, whiskers indicate the smallest and largest values. n = 7, 4 female and 3 male. g, gram; KJ, kilojoule; m, meter; N, Newton; s, second. p values were calculated by the two‐sided Welch's t ‐test and displayed on each plot.

Article Snippet: Doxorubicin, 2‐deoxy‐glucose (2‐DG), CPI‐613, bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide (BPTES), and 17‐AAG were purchased from MedChemExpress.

Techniques: Injection, Staining, Two Tailed Test, Whisker Assay