Journal: Journal of Orthopaedic Translation

Article Title: The CD163/TWEAK/Fn14 axis: A potential therapeutic target for alleviating inflammatory bone loss

doi: 10.1016/j.jot.2024.09.002

Figure Lengend Snippet: TWEAK mediates the phenotype of osteoporosis in CD163 −/− mice . (A, B) Representative images and quantification of TWEAK from CD163 −/− male mice or their WT male littermates. Scale bar: 50 μm (top), 10 μm (bottom). (C, D) Representative immunoblotting and quantification of CD163 and TWEAK from CD163 −/− male mice or their WT male littermates. (E) Quantification of sTWEAK level in the peripheral circulation using ELISA. (F, G) Representative images and quantification of TWEAK from mice treated with or without LPS. Scale bar: 50 μm (top), 10 μm (bottom). (H, I) Representative images and quantification for proportion of TWEAK + cells in BMCs from CD163 −/− male mice or their WT male littermates. The numbers in the upper right corner represent the average proportion of TWEAK + cells. (J) Representative images of TWEAK + (red), CD68 + (green), and F4/80 + (green) cells from CD163 −/− mice. White arrow heads: the CD68-and TWEAK- double positive cells. Nuclei were stained with DAPI (blue). Scale: 50 μm (left), 10 μm (right). (K, L) Representative μCT images and quantification of femora from CD163 −/− mice treated with or without MTW-1. (M, N) Representative μCT images and quantification of femora from WT mice treated with 0.1%BSA (Vehicle), rTWEAK, or rTWEAK plus rCD163. Dots represent individual mice. The data are presented as mean ± s. e.m., ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The concentration of sTWEAK in serum samples were measured using a commercial mouse TWEAK ELISA kit (EL023987MO, CUSABIO, Wuhan, China) according to the manufacturer's directions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Characterization of thrombin generation after hepatic IRI and cytotoxicity of thrombin for hepatic SECs. (A) In the clinical study of patents who underwent hepatectomy (n = 14), plasma TAT levels were plotted at 2 time points: before and 1 day after hepatectomy. TAT levels were significantly elevated after operation. (B) In in vivo hepatic IRI models of mice, plasma TAT levels measured by ELISA was significantly increased after IRI compared with the naïve group (n = 6 in each group). (C‐a) In hepatic SECs cultured in the nonischemic condition, LDH cytotoxicity levels in the supernatant of cell cultures were elevated in a dose‐dependent manner with the addition of thrombin. The addition of 100 U/mL thrombin significantly increased LDH cytotoxicity compared with both groups of naïve and 10 U/mL thrombin (n = 4 in each group). (C‐b) In H‐R models of SECs in vitro, the addition of 100 U/mL thrombin also significantly increased LDH cytotoxicity compared with vehicle (n = 5 in each group). H‐R was established using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatic SECs in an H‐R model in vitro. (A) In H‐R models of pure cultured hepatic SECs, dabigatran treatment significantly decreased LDH cytotoxicity levels in the supernatant compared with vehicle (n = 5 in each group). (B‐a) Dabigatran treatment significantly increased TM expression in cell lysate measured by ELISA compared with vehicle (n = 5 in each group). (B‐b) By contrast, in TM expression in the supernatant of cell cultures measured by ELISA, there was no significant difference between the vehicle and dabigatran groups (n = 5 in each group). (C‐a) Based on Western blot analysis, dabigatran treatment did not significantly affect HMGB‐1 expression in cell lysate compared with the vehicle group (n = 5 in each group). Quantification of HMGB‐1 band intensities normalized to β‐actin. (C‐b) By contrast, HMGB‐1 levels in the supernatant of cell cultures measured by ELISA were markedly reduced by dabigatran treatment compared with the vehicle (n = 5 in each group). In H‐R models of this study, SECs were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatocytes in an H‐R model in vitro. In H‐R models of pure cultured hepatocytes, there was no significant difference between the H‐R + vehicle group and the H‐R + dabigatran group (A) in LDH cytotoxicity levels in the supernatant of cell cultures, in TM levels in cell lysate measured by ELISA (B‐a), in TM levels in the supernatant of cell cultures measured by ELISA (B‐b), in HMGB‐1 expression in cell lysate evaluated by Western blot analysis (C‐a), or in HMGB‐1 levels in the supernatant of cell cultures measured by ELISA (C‐b) (n = 5 in each group). In Western blot analysis, quantification of HMGB‐1 band intensities normalized to β‐actin. In H‐R models of this study, cells were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Effects of dabigatran for hepatocytes in a paracrine communication model between hepatic SECs and hepatocytes. (A) A scheme of our paracrine communication model between hepatic SECs and hepatocytes. SECs were pretreated with or without dabigatran followed by incubation in a serum‐starved medium in the H‐R condition; thereafter the supernatant of the SECs was harvested. Hepatocytes were pretreated with the supernatant of SECs followed by incubation in a serum‐starved medium in the H‐R condition. (B) LDH cytotoxicity levels in the supernatant of hepatocyte cultures were significantly lower in the group of H‐R + the supernatant of SECs pretreated with dabigatran than in the group of H‐R + the supernatant of SECs pretreated with vehicle (n = 5 in each group). TUNEL staining detected cellular death of hepatocytes after H‐R (C‐a) in the group of H‐R + the supernatant of SECs pretreated with vehicle and (C‐b) in the group of H‐R + the supernatant of SECs pretreated with dabigatran (original magnification ×200). (C‐c) The supernatant of SECs pretreated with dabigatran markedly reduced the number of TUNEL‐positive hepatocytes compared with the supernatant of SECs pretreated with the vehicle (n = 5 in each group).

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Incubation, TUNEL Assay, Staining

Journal: Hereditas

Article Title: USP8 protects rat-derived H9C2 cardiomyocytes from doxorubicin-triggered ferroptosis and cell death through deubiquitination-mediated stabilization of MDM4

doi: 10.1186/s41065-025-00527-z

Figure Lengend Snippet: Elevated USP8 expression protects H9C2 cardiomyocytes from Dox-mediated cytotoxicity and cell death. ( A ) H9C2 cardiomyocytes were subjected to transfection with oeUSP8 or oeNC before Dox exposure, and cell extracts were collected for immunoblot analysis of USP8 protein, with β-actin as a loading control. ( B-D ) H9C2 cardiomyocytes after 24 h transfection with or without oeUSP8 or oeNC were stimulated with or without 1 µM Dox for 24 h. ( B ) CCK-8 reagent was added to the cells, and absorbance was measured. Viability was determined relative to control cells. ( C ) Cells were harvested for evaluation of LDH release content using the assay kit. ( D ) Cells were collected for flow cytometry with Annexin V/PI staining, and cell death rate per group was quantified. ** P < 0.01, *** P < 0.001, ns: non-significant

Article Snippet: To assess cellular damage, LDH release was measured in H9C2 cardiomyocytes following either Dox treatment (1 μM, 24 h) or transfection prior to Dox exposure, using a commercial LDH Release Assay Kit as suggested by the vendor (Beyotime).

Techniques: Expressing, Transfection, Western Blot, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: Hereditas

Article Title: USP8 protects rat-derived H9C2 cardiomyocytes from doxorubicin-triggered ferroptosis and cell death through deubiquitination-mediated stabilization of MDM4

doi: 10.1186/s41065-025-00527-z

Figure Lengend Snippet: MDM4 inhibition reverses the cardioprotective effects of USP8 upregulation against Dox-triggered cell death and ferroptosis in H9C2 cells. ( A ) H9C2 cardiomyocytes were subjected to transfection with siMDM4 or siNC, and cell extracts were collected for immunoblot analysis of MDM4 protein. ( B-I ) H9C2 cardiomyocytes after 24 h transfection with or without oeUSP8 + siNC or oeUSP8 + siMDM4 were stimulated with or without 1 µM Dox for 24 h. ( B ) CCK-8 reagent was added the cells, and absorbance was measured. Viability was determined relative to control cells. ( C ) LDH release was measured in cell supernatants using the assay kit. ( D ) Cells were harvested for flow cytometry after Annexin V/PI staining, and cell death rate per group was quantified. ( E ) Intracellular Fe 2+ levels were determined using a commercial iron assay kit. ( F ) Cells were subjected to BODIPY-C11 probe staining and flow cytometry for the analysis of lipid ROS production. ( G and H ) Cells were processed for MDA and GSH expression quantification using the commercial kits. ( I ) Cell extracts were prepared for immunoblot analysis of GPX4 and SLC7A11. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: To assess cellular damage, LDH release was measured in H9C2 cardiomyocytes following either Dox treatment (1 μM, 24 h) or transfection prior to Dox exposure, using a commercial LDH Release Assay Kit as suggested by the vendor (Beyotime).

Techniques: Inhibition, Transfection, Western Blot, CCK-8 Assay, Control, Flow Cytometry, Staining, Iron Assay, Expressing