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

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Strategy and materials in this study. (A) Diagram of the procedure in the study. The primary tumor cells and tumor organoids were generated from patients' tumor tissues and ROBO1-NK were constructed from patients' PBMCs. (B) IHC of ovarian tumor tissues. Negative control was used irrelevant IgG replacing primary antibody for incubating. Strong positive of ROBO1 expression on cell membrane was marked by red circles. (C) ROBO1 positively expressed in tumor tissues of the clinical ovarian cancer patient #01, #02, #03 and #04.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Generated, Construct, Negative Control, Expressing, Membrane

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Characters of ROBO1-NK cells. (A) Morphology comparison of PBMCs (upper panel), PBMC-NK cells (mid panel) and ROBO1-NK cells (bottom panel) under bright field. (B) Biomarkers determination of ROBO1-NK cells by flow cytometry. For the biomarkers CD34, CD45, CD3, CD56 and CD16, negative controls were used irrelevant IgG replacing primary antibody for incubating, presented as red peaks. For the ROBO1-CAR detection, negative control was used PBMC-NK incubating with anti-CAR antibody, presented as red peak. Samples in detection were presented as blue peaks.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Comparison, Flow Cytometry, Negative Control

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: ROBO1-NK lyse ovarian cancer cell line and primary cancer cells. (A) Positive expression of ROBO1 on SKOV-3 cells. (B) Rate of SKOV-3 cell lysis with different E-T ratio. E: number of effective cells; T: number of target cells. (C) Time curve of different NK cells, including Mock-CAR-NK (blue), PBMC-NK (green), ROBO1-NK (red) and control (medium, black), lysing SKOV-3 cells. (D) Lysis rate of primary ovarian tumor cells from patient #01, #02, #03 and #04 by PBMC-NK and ROBO1-NK after 24 h, detected with CCK-8 assay. (E) Photograph of PBMC-NK and ROBO1-NK lysing efficacy on primary ovarian tumor cells after 48 h co-culturing. The primary cells were presented as spindle-like form while NK cells were spherical form with clusters.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Expressing, Lysis, Control, CCK-8 Assay

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza modulated the neutrophil-associated TIME in the lungs. (A) Experimental setup. The mice were constructed as experimental metastasis models after a period of acclimatization. The mice were prepared as experimental metastasis models following an acclimatization period. Ginseng and Salvia miltiorrhiza extracts were administered daily to the relevant groups according to the schematic diagram, and lung tissues were collected on day 28. (B) Representative bioluminescence images of metastasis in the intravenously implanted LLC mice. (C) Representative H&E images of the tumor area in the lungs of the mice (scale bar: 50 μm). (D) Quantitative analysis of the lung tumor area ( n = 3). (E) Quantitative analysis of the number of lung metastatic nodules in tumor-bearing mice( n = 15). (F) Metastasis rates of LLC tumor-bearing mice ( n = 15). (G) GOBP analysis of intersecting genes in the control, model and G + S groups. (H) GSEA of genes related to neutrophil migration. (I) Representative images of immunohistochemical staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 50 μm). (J) Representative images of immunofluorescence staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 100 μm). All the data are presented as mean ± SEM. #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Construct, Control, Migration, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: CPT inhibited CD62E–mediated neutrophil adhesion and recruitment to metastases. (A) Representative images and quantification of HL–60 cell adhesion to HUVECs (scale bar: 200 μm). (B) Schematic diagram of the parallel plate flow chamber combined with the microscope. (C) Number of neutrophils firmly adhered to HUVECs. (D) Representative immunofluorescence images and quantification of CD62E in the lungs ( n = 3; scale bar: 50 μm). (E) Representative western blot images of CD62E levels in the lungs and quantitative data ( n = 3). GAPDH was used as a loading control. (F) The mRNA expression levels of TNF–α, ICAM1, and VCAM1 in lung tissues were determined by qRT–PCR ( n = 3). (G) The correlation between CD62E expression and neutrophil infiltration in LUAD and LUSC was analyzed via TIMER. (H) Representative images and quantification of Ly6G + neutrophils in lungs detected by flow cytometry ( n = 4). (I) Representative immunofluorescence images and quantification of Ly6G + neutrophils in the lungs (n = 3; scale bar: 50 μm). All the data are presented as mean ± SEM. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Microscopy, Immunofluorescence, Western Blot, Control, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Neutrophil RNA sequencing analysis and correlation analysis of NETs and lung cancer. (A) Schematic diagram of neutrophil isolation and RNA sequencing from mouse lung tissues. (B-C) Heatmap analysis of neutrophil-related genes and KEGG analysis of altered genes. (D) Results of partial GOBP analysis of intersecting genes in the control, model and G + S groups. (E) The expression of HIST3H3 in different cancers and adjacent normal tissues was analyzed via TIMER. (F–H) The correlation between copy number alterations in HIST3H3, ELANE, and MPO and neutrophil infiltration was analyzed via TIMER. (I) The correlations between cumulative survival and HIST3H3, ELANE, MPO and neutrophil infiltration in lung cancer patients were analyzed via TIMER. A two-sided Wilcoxon rank-sum test was used. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: RNA Sequencing, Isolation, Control, Expressing

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza reduced the generation of NETs in lung tissues, and effective substances from ginseng were screened. (A) Representative immunohistochemical images of MPO expression in the lungs (scale bar: 100 μm). (B) Representative immunofluorescence images of H3cit and NE in the lungs (scale bar: 100 μm). (C–E) Quantitative analysis of MPO, H3cit and NE expression in the lungs ( n = 3). (F) Neutrophil MPO release was detected by ELISA. The chemical structure of Rg1 is shown on the right. (G) Representative images and quantification of extracellular DNA release after Sytox™ Green staining (scale bar: 50 μm). All the data are presented as mean ± SEM. ####p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reduced NET formation by inhibiting ROS production and ERK1/2 and MAPK phosphorylation. (A) Representative immunofluorescence images of the NET components H3cit and NE in neutrophils ( n = 3; scale bar: 50 μm). (B–C) Representative western blot images of H3cit expression in neutrophils and quantitative data. GAPDH was used as a loading control ( n = 3). (D-E) ROS production in dHL-60 cells (D) and neutrophils (E) was assessed by flow cytometry. The median fluorescence intensity of ROS was used for quantification. (F-G) Representative western blot images and quantification of p-ERK1/2, ERK1/2, p-MAPK and MAPK expression in dHL-60 cells (F) and neutrophils (G). GAPDH was used as a loading control. All the data are presented as mean ± SEM. # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reversed NET-induced invasion, EMT, and adhesion of LLC cells and increased vascular permeability. (A) LLC cell proliferation was detected via a CCK8 assay. (B) Schematic diagram of NET isolation and coculture with LLC cells in the transwell system. (C) Images of the transwell invasion assay (scale bar, 200 μm). The number of infiltrated cells and the OD values were used for quantification. (D-E) Representative immunofluorescence images and quantification of MMP9 in the lungs (n = 3, scale bar 50 μm). (F) Representative immunofluorescence images of E-cadherin and N-cadherin in LLC cells treated with or without Rg1 or NETs. (G-I) Representative immunofluorescence images and quantification of E-cadherin and N-cadherin in the lungs ( n = 3, scale bar 50 μm). (J) Schematic diagram of the adhesion assay of LLC cells by NETs. (K) Representative fluorescence images of the adhesion assay for DiI-labeled LLC cells trapped within PMA- or Rg1-treated neutrophils. (L) RNA-seq analysis of neutrophils. GSEA of genes related to the regulation of vascular permeability. (M) Schematic diagram of the endothelial permeability assay. (N) The effects of Rg1 or NETs on vascular permeability were tested via an endothelial permeability assay. (O-P) Representative immunofluorescence images and quantification of CD31 and FITC-dextran in the lungs ( n = 3; scale bar, 50 μm). All the data are presented as mean ± SEM. # p < 0.05, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Permeability, CCK-8 Assay, Isolation, Transwell Invasion Assay, Immunofluorescence, Cell Adhesion Assay, Fluorescence, Labeling, RNA Sequencing, Control