Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: The optimal time-point for neutrophil differentiation. Neutrophils were differentiated from HL-60 cells by 1.25% DMSO induction for up to 10 days. At Day 0, 2, 4, 6, 8 and 10, the cells were analyzed as follows. A Wright’s staining. B Cell count. C Cell death analysis. Day 6 was the longest time-point when the cells showed typical neutrophil characteristics without a significant increase in cell death. This time-point (green arrow) was then selected as “the optimal time-point for neutrophil differentiation” and used in all subsequent experiments in this study. D and E At this optimal time-point, immunofluorescence staining was performed to examine surface CD11b, a marker for neutrophil phenotype. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Staining, Cell Counting, Immunofluorescence, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: COM crystal internalization by neutrophils. Neutrophils were incubated with plain or FITC-labeled COM crystals for up to 10 h. At 2, 4, 6, 8 and 10 h post-incubation, crystal internalization was investigated. A and B Microscopic examination of the internalized plain COM crystals. The cells containing positive birefringent crystals were identified and counted as COM-internalized cells from 15 random fields/sample. C and D Flow cytometric analysis of the internalized FITC-labeled COM crystals. The crystal-internalized cells were detected and quantified relative to 10,000 cells analyzed. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Incubation, Labeling

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Validation of LFQ proteomics data by Western blotting. Neutrophils were incubated at 37 °C with plain COM crystals for 10 h. In parallel, the cells incubated without crystals served as the control. The increase of hnRNP H ( A and B ) and the decrease of transketolase ( C and D ) in COM-treated neutrophils were confirmed by Western blotting. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Biomarker Discovery, Western Blot, Incubation, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Functional categories of significantly altered proteins. All COM-induced altered neutrophil proteins identified from LFQ proteomics were analyzed to get functional insights. A Biological processes. B Molecular functions. C and D Cellular components

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Functional Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Increased NETs formation in the COM-treated neutrophils. Neutrophils were incubated at 37 °C with plain COM crystals for up to 10 h. At 2, 4, 6, 8 and 10 h post-incubation, the DNA component was stained with Hoechst dye. The expulsed NETs were then imaged under a fluorescence microscope

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Incubation, Staining, Fluorescence, Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Increased activation and degranulation of the COM-treated neutrophils. Neutrophils were incubated at 37 °C with plain COM crystals for 1 h. In parallel, the cells incubated without crystals served as the control. A granule membrane protein, CD63 (as a neutrophil activation marker), was then examined. A and B Flow cytometric analysis of CD63-positive cells relative to 10,000 cells analyzed. C and D Immunofluorescence staining of CD63. The fluorescence intensity of CD63 was measured from 100 cells/sample. E Secretosy α-defensin (as a neutrophil degranulation marker) was quantified by ELISA. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Activation Assay, Incubation, Control, Membrane, Marker, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Increased ROS production in the COM-treated neutrophils. Neutrophils were incubated at 37 °C with plain COM crystals for 10 h. In parallel, the cells incubated without crystals served as the control. A and B ROS-positive cells were quantified relative to 10,000 cells analyzed using a flow cytometric-based DCFH-DA assay. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Incubation, Control, DCFH-DA Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Secretome from the COM-treated neutrophils enhanced macrophage migration. Neutrophils were incubated at 37 °C with plain COM crystals for 10 h. In parallel, the cells incubated without the crystals served as the control. The clear supernatant was collected and used as the neutrophil secretome to treat macrophages. A Diagram of the Transwell-based macrophage migration assay. B Fluorescence imaging of nuclei (stained with Hoechst dye) of the migrated macrophages under a Transwell insert. C The number of the migrated macrophages was counted from 15 random fields/sample. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Migration, Incubation, Control, Fluorescence, Imaging, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

doi: 10.1186/s12964-025-02345-2

Figure Lengend Snippet: Increased clearance of the COM-treated neutrophils by macrophages. Neutrophils were incubated at 37 °C with plain COM crystals for 10 h. In parallel, the cells incubated without the crystals served as the control. The prepared (floating) neutrophils were mixed (1:1) with (adherent) macrophages and co-incubated at 37 °C for 2 h. Thereafter, the remaining non-phagocytosed neutrophils were isolated from the adherent macrophages. A Macrophages were then imaged under an inverted phase-contrast microscope. B Sizes of macrophages were measured, and those larger than the maximal size of the untreated macrophages were identified and quantified as enlarged macrophages (likely to engulf neutrophils via phagocytosis) from 15 random fields/sample. C The number of remaining non-phagocytosed neutrophils was counted. Quantitative data are shown as mean ± SEM measured from three independent experiments

Article Snippet: Human neutrophils were differentiated from HL-60 cells (ATCC), which were cultured in complete RPMI 1640 medium (Gibco) (supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 60 U/ml penicillin G (Sigma-Aldrich) and 60 mg/ml streptomycin (Sigma-Aldrich)) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Incubation, Control, Isolation, Microscopy

Journal: Journal of Nanobiotechnology

Article Title: Concurrent induction of pyroptosis and immunogenic cell death by capsaicin/graphene nanocomplex for enhanced breast cancer immunotherapy

doi: 10.1186/s12951-025-03439-2

Figure Lengend Snippet: Induction of cancer cell pyroptosis by natural product Cap in vitro and in vivo. A Chemical structures of Cap, Arv, and Olv, and their cell-killing effects on the breast cancer cell lines EMT6 and Py8119 and the osteosarcoma cell line K7M2 after 24 h incubation. B Representative optical microscope images to compare the EMT6 cell morphological changes induced by various concentrations of Cap, Arv, and Olv. “l” represents a low concentration of 50 μg/mL, and “h” represents a high concentration of 100 μg/mL. Dox is used as a positive control. Red arrows indicate the swollen and blebbing cells. The scale bar is 25 µm. C Representative confocal images acquired on an Operetta High Content Screening System (Perkin Elmer) to demonstrate the dynamics of capsaicin-induced morphological and fluorescence intensity changes of EMT6 cells from 0 to 1000 min incubation time. Annexin V-FITC and PI were added to the cells before imaging. D Fluorescent images to demonstrate the Cap-induced morphological and fluorescence intensity changes in Py8119 and K7M2 cells. E Western blotting to show the cleavage of GSDME-FL to GSDME-NT in EMT6 cells treated by 50 (l) and 100 (h) μg/mL of Cap and its analogs Arv and Olv for 7 h. Quantitative analysis of GSDME-NT expression intensities was calculated by ImageJ software. F H 2 DCFDA assay to evaluate the intracellular ROS by Cap, Arv, and Olv in EMT6 cells. G ELISA assays to determine the released LDH (top panel) and IL-6 (upper panel) levels in cells. H Immunoblotting to demonstrate the GSDME-NT cleavage and cleaved caspase-3 expression levels in EMT6-bearing mice after 3 days of 5 mg/kg Cap intratumoral injection. The ratios of GSDME-NT/Vinculin and cleaved caspase-3/Vinculin in different treatment groups were calculated by Image J. I Determination of IL-6 (top panel) and IL-1β (upper panel) in EMT6-bearing mouse serum using ELISA assays. Data represents mean ± SD, n ≥ 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The cell viability detection kit (CCK-8) was acquired from Mei Lun Biotechnology Co., Ltd, and H 2 DCFDA (HY-D0940) was obtained from MedChemExpress.

Techniques: In Vitro, In Vivo, Incubation, Microscopy, Concentration Assay, Positive Control, High Content Screening, Fluorescence, Imaging, Western Blot, Expressing, Software, Enzyme-linked Immunosorbent Assay, Injection

Journal: Journal of Nanobiotechnology

Article Title: Concurrent induction of pyroptosis and immunogenic cell death by capsaicin/graphene nanocomplex for enhanced breast cancer immunotherapy

doi: 10.1186/s12951-025-03439-2

Figure Lengend Snippet: ICD effects induced by GO in vitro and in vivo. A Various GO samples from a range of sizes (GO-1, GO-2, and GO-3) and surface groups of C=O content (GO-3, GO-4, and GO-5) used in this study. B Representative AFM images (left panel) and DLS data (right panel) to demonstrate the sheet-like GO-1, GO-2, and GO-3 with an average size of ~500 nm, ~1000 nm, and ~1500 nm, respectively, with similar heights of 1 nm. C XPS data to show different surface groups and their content in GO-3, GO-4, and GO-5 samples, in terms of C=O contents with ~15%, ~10%, and ~5%, respectively. D Cell killing of GO samples and the correlation analysis between cytotoxicity and GO size (left panel) and C=O content (right panel). E H 2 DCFDA assay to evaluate abiotic ROS generation of all GO samples. F Evaluation of the photothermal capacity of GO-3 to grow temperature (left panel) and kill EMT6 cells with NIR laser irradiation (808 nm, 1.7 w/cm 2 , 5 min) (right panel). G Representative fluorescent images (left panel) and flow cytometry analysis (middle panel) showing CRT exposure, along with ELISA data (right panel) quantifying HSP70 levels in EMT6 cells treated with GO-3 and NIR light. Tunicamycin (Tun) was used as a positive control. The scale bar is 25 μm. H Quantification (left panel) and representative IHC staining images (right panel) for CRT as well as HMGB1 to confirm ICD induction in EMT6-bearing mice intratumorally injected 20 mg/kg GO under NIR irradiation 3 days late. The quantitative analysis was performed by calculating the percentage of integrated density across all areas in IHC images using ImageJ. Data represents mean ± SD, n = 3 for cell study and n = 6 for animal study. *** p < 0.001, **** p < 0.0001. The scale bar is 100 µm

Article Snippet: The cell viability detection kit (CCK-8) was acquired from Mei Lun Biotechnology Co., Ltd, and H 2 DCFDA (HY-D0940) was obtained from MedChemExpress.

Techniques: In Vitro, In Vivo, Irradiation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Positive Control, Immunohistochemistry, Injection