Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: The release of neutrophil extracellular traps (NETs) by neutrophils promotes migration and invasion of NSCLC. (A) The morphology of neutrophils isolated from healthy donors’ blood was observed by Giemsa staining (magnification, 1000×). (B) The neutrophil viability was assessed by trypan blue dye exclusion assays (magnification, 100×). (C) Representative images and quantification of NETs formation of neutrophils from healthy donors (HD) and NSCLC patients. MPO (red), cit-H3 (green), and DAPI (blue), respectively (magnification, 50×; scale bar, 200μm). (D) Representative images of NETs formation in NSCLC patients’ normal lung tissues and tumor tissues that were detected by con-focal microscopy. cit-H3 (red), Ly6g (green), and DAPI (blue), respectively (magnification, 200×; scale bar, 100µm and magnification, 400×; scale bar, 50µm). (E) Representative images of PMA-induced NETs formation of neutrophils from HD stained with MPO and cit-H3 were detected by immunofluorescence microscope; MPO (red), cit-H3 (green), and DAPI (blue), respectively (magnification, 50×; scale bar, 200μm). Transwell invasion (F) and wound healing assays (G) were performed to identify the effects of NETs on A549 and SK-MES-1 cells invasion (magnification, 100×) and migration (magnification, 50×). (H) Western blot analyzing the expressions levels of EMT markers protein (N-cadherin, E-cadherin, and Vimentin) in A549 and SK-MES-1 cells treated with NETs. ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Migration, Isolation, Staining, Microscopy, Immunofluorescence, Western Blot

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: MIR503HG is downregulated in NSCLC cells with NETs stimulation and is associated with poor survival of NSCLC. (A) Volcano plot illustrating the differentially expressed lncRNAs in A549 cells treated with or without NETs for 12 h (|log 2 fold change (FC)| > 2, P -value < 0.01). (B) Heat map showing the top 30 differentially down-regulated lncRNAs in A549 cells after treatment with NETs for 12 h. Red means up-regulated, blue means down-regulated, separately. (C) Relative expression of MIR503HG in A549 and SK-MES-1 cells with or without NETs stimulation. (D) MIR503HG is expressed at a lower level in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumor compared to corresponding adjacent normal lung tissues according to the TCGA database. (E) The expression of MIR503HG in 50 paired NSCLC tumors and normal tissues were quantified by qRT-PCR. (F) Kaplan-Meier analysis of metastasis risk of 50 NSCLC patients divided into two groups based on a middle cutoff of MIR503HG expression. (G) MIR503HG is mainly located in the nuclear of NSCLC cells. U6 snRNA (nuclear reserved) and GAPDH mRNAs (exposed to cytoplasm) were used as controls. Data are mean ± SD (n=3). ( ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: Overexpression of MIR503HG substantially reversed the metastasis-promoting effect of NETs on NSCLC in vitro and vivo. (A) Expression of MIR503HG was successfully up-regulated in A549 and SK-MES-1 cells. (B, C) Wound healing (magnification, 50×) and invasion assays (magnification, 100×) of NSCLC cells that stable transfection of MIR503HG vector versus control vector both treat with NETs 12 h. (D) Western blot analysis of the expression of EMT (N-cadherin, E-cadherin, Vimentin) in control and MIR503HG overexpressing A549 and SK-MES-1 cells after NETs treated 12 h. (E) Schematic diagram showing the experimental design of the effect of MIR503HG on NETs-induced metastasis. Representative images of the gross lung (F) and H&E staining (G) of metastatic lung nodules in mice specimens. (H) Quantification of the number, volume, and maximum size of metastatic lung nodules. Data are mean ± SD (n=5 nude mice in each group). (magnification, 100×; scale bar, 200 μm, magnification, 200×; scale bar, 50 μm). (I) Immunohistochemistry (IHC) detection of N-cadherin, E-cadherin, and Vimentin revealed EMT formation in the NETs-induced lung metastasis model (magnification, 100× and 400×). ( ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Over Expression, In Vitro, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot, Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: NETs promote migration and invasion of NSCLC by activating the NLRP3 inflammasome. (A) Up-regulated NSCLC-related pathways in response to NETs stimulated revealed by KEGG enrichment. (B) The mRNA expression of NLRP3, Caspase1, IL-1β and IL-18 in A549 cells was analyzed by qRT-PCR after NETs stimulation at different times. (C) Western blotting was used to analyze the expression of NLRP3 and Caspase1 in A549 and SK-MES-1 cells after NETs stimulation for different periods. (D) Immunofluorescence was used to observe the expression of ROS in A549 and SK-MES-1 cells treated with NETs for 12 h (magnification, 100×). The expression of NLRP3, Caspase1, IL-1β and IL-18 were detected by qRT-PCR (E) and Western blot (F) in A549 and SK-MES-1 cells after treating with NETs and NLRP3 inflammasome inhibitor MCC950, respectively. (G–I) Estimate the effect of NETs on the level of N-cadherin, E-cadherin, and Vimentin (Western blot) (G) in A549 and SK-MES-1 cells and the capacity of invasion (transwell invasion assays; magnification, 100×) (H) , migration (wound healing assays; magnification, 50×) (I) when inhibiting the expression of the NLRP3 inflammasome by MCC950. ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: NLRP3 inflammasome mediated the effect of MIR503HG to inhibit NETs-triggered metastasis of NSCLC. (A, B) The protein and mRNA expression of NLRP3 and Caspase1 in A549 and SK-MES-1 cells with MIR503HG overexpression were analyzed by Western blot (A) and qRT-PCR (B) after NETs were stimulated. (C) A negative relationship between MIR503HG and NLRP3 in NETs-induced NSCLC cells is presented by correlation analysis. (D) Overexpression of NLRP3 attenuated the effect of MIR503HG in inhibiting NETs-triggered EMT in NSCLC cells by Western blot. (E, F) Overexpression of NLRP3 effectively reverses the effect of MIR503HG in inhibiting NETs-triggered promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) . ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Expressing, Over Expression, Western Blot, Quantitative RT-PCR, Transwell Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: NLRP3 inflammasome induced by NETs promotes NSCLC progression is associated with the activation of NF-κB. (A) The protein expression level of p-p50, p50, p-p65, p65 in NSCLC cells treated with NETs was detected by Western blotting. (B) Nuclear translocation of NF-κB in A549 and SK-MES-1 cells treated with NETs or combined with DNase I were detected by con-focal microscopy (magnification, 3000×; scale bar, 5μm). Immunofluorescence assays (C) and Western blot (D) were used to detect the effect of NETs on NLRP3 inflammasome in A549 and SK-MES-1 cells after p50 knockdown (magnification, 200×; scale bar, 50 μm). (E) Downregulation of p50 attenuated the effect on promoting EMT of NETs in NSCLC cells by Western blot. (F, G) Downregulated p50 reverses NETs-induced promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) .

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Activation Assay, Expressing, Western Blot, Translocation Assay, Microscopy, Immunofluorescence, Knockdown, Transwell Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: MIR503HG inhibits NETs-triggered NSCLC cells metastasis capacity and NLRP3 inflammasome activation dependently on NF-κB. (A) Western blotting was used to detect the changes of p-p50, p50, p-p65 and p65 in MIR503HG overexpression A549 and SK-MES-1 cells after NETs treatment. (B) qRT-PCR and (C) Western blotting analyses of up-regulating p50 in A549 and SK-MES-1 cells. (D) Analysis of the NLRP3 and Caspase1 protein levels in MIR503HG-overexpressed NSCLC cells transfected with p50-pcDNA3.1 and pcDNA3.1 vector by western blot with NETs stimulated. Transwell invasion (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) were performed to identify the effects of NETs on MIR503HG-overexpressed NSCLC cells invasion and migration transfected with p50-pcDNA3.1 and pcDNA3.1 vector. ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Activation Assay, Western Blot, Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Migration

Journal: International Journal of Molecular Medicine

Article Title: Ultrasound-targeted microbubble destruction technology delivering β-klotho to the heart enhances FGF21 sensitivity and attenuates heart remodeling post-myocardial infarction

doi: 10.3892/ijmm.2024.5378

Figure Lengend Snippet: KLB overexpression enhances the protective effects of FGF21 treatment on hypoxia-induced cardiomyocyte injury in vitro . (A) Viability of H9C2 cells exposed to hypoxia (HX), FGF21 and KLB overexpression (n=5). (B) Representative images of DHE staining (scale bar, 100 μ m), and DHE intensity was quantified to reflect ROS levels. (C) Representative TUNEL staining images of KLB-overexpressing H9C2 cells exposed to hypoxia and treated with FGF21 (n=4; scale bar, 100 μ m). (D) LDH leak from KLB-overexpressing H9C2 cells exposed to hypoxia and treated with FGF21 (n=8). (E) JC-1 staining of H9C2 cells (scale bar, 50 μ m). Mitochondrial membrane potential was estimated by the ratio of JC-1 aggregates (red, healthy mitochondria) and JC-1 monomers (green, depolarized mitochondria, n=5). ** P<0.01 and *** P<0.001 vs. the control (Con) group; ## P<0.01 and ### P<0.001 vs. hypoxia (HX). KLB, β-klotho; FGF21, fibroblast growth factor 21; AMI, acute myocardial infarction; LDH, lactate dehydrogenase.

Article Snippet: To evaluate the apoptosis of H9C2 cells or that of cells in the heart sections, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (cat. no. A321, Elabscience) was used, as previously described ( ).

Techniques: Over Expression, In Vitro, Staining, TUNEL Assay, Membrane, Control

Journal: International Journal of Molecular Medicine

Article Title: Ultrasound-targeted microbubble destruction technology delivering β-klotho to the heart enhances FGF21 sensitivity and attenuates heart remodeling post-myocardial infarction

doi: 10.3892/ijmm.2024.5378

Figure Lengend Snippet: Cardiac delivery of KLB enhances the antioxidant effects of FGF21 in the heart following AMI. (A) Western analysis and quantification of Nrf2 and KEAP1 expression in H9C2 cells (n=4). (B) The protein expression of HO-1, NQO1, Gstp1 and GCLM was assessed using western blot analysis (n=4). (C) Representative images of echocardiography at 4 weeks following AMI surgery. (D and E) The LVEF and LVFS were calculated (n=5). (F and G) DHE staining of the heart section (scale bar, 50 μ m) and quantification (n=5). ** P<0.01 and *** P<0.001 vs. AMI; # P<0.05, ## P<0.01 and ### P<0.001 vs. the AMI + KLB@cMBs + FGF21 group; ns, not significant. AMI, acute myocardial infarction; KLB, β-klotho; FGF21, fibroblast growth factor 21; CMB, cationic microbubble; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; Nrf2, nuclear factor erythroid 2-related factor 2; KEAP1, kelch-like ECH-associated protein 1; HO-1, heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; Gstp1, glutathione S-transferase pi-1; GCLM, glutamate-cysteine ligase modifier subunit.

Article Snippet: To evaluate the apoptosis of H9C2 cells or that of cells in the heart sections, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (cat. no. A321, Elabscience) was used, as previously described ( ).

Techniques: Western Blot, Expressing, Staining

Journal: International Journal of Molecular Medicine

Article Title: Ultrasound-targeted microbubble destruction technology delivering β-klotho to the heart enhances FGF21 sensitivity and attenuates heart remodeling post-myocardial infarction

doi: 10.3892/ijmm.2024.5378

Figure Lengend Snippet: Mitochondrial quality in the heart is improved by the UTMD-mediated KLB delivery and FGF21 treatment in rats post-infarction. (A) JC-1 staining of heart section post-infarction (scale bar, 100 μ m). (B) The ratio of JC-1 aggregates (red) and monomers (green) was used to assess mitochondrial membrane potential (n=5-6). (C) Mitochondrial OCR profile in H9C2 cells using an XF24 Extracellular Flux Analyzer. Oligomycin (1 μ mol/l), FCCP (4 μ mol/l) and rotenone (0.5 μ mol/l) plus antimycin A (0.5 μ mol/l) were added sequentially. (D) FCCP-related respiration, (E) basal respiration, (F) maximal respiration and (G) ATP turnover were calculated (n=4). *** P<0.001 vs. AMI; ## P<0.01 and ### P<0.001 vs. the AMI + KLB@CMBs + FGF21 group. CMB, cationic microbubble; UTMD, ultrasound-targeted microbubble destruction; KLB, β-klotho; FGF21, fibroblast growth factor 21; OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; AMI, acute myocardial infarction; HX, hypoxia.

Article Snippet: To evaluate the apoptosis of H9C2 cells or that of cells in the heart sections, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (cat. no. A321, Elabscience) was used, as previously described ( ).

Techniques: Staining, Membrane

Journal: Toxics

Article Title: Selenium Nanoparticles Attenuate Cobalt Nanoparticle-Induced Skeletal Muscle Injury: A Study Based on Myoblasts and Zebrafish

doi: 10.3390/toxics12020130

Figure Lengend Snippet: Cellular toxicity induced by cobalt and selenium nanoparticles. ( A ) Cells were treated with 5 μg/mL FITC-labeled CoNPs and SeNPs for 2 h. Confocal microscopy revealed fluorescent signals into the cells. Yellow arrows indicating cells that are filled with green fluorescence, while the culture medium contains free FITC-labeled CoNPs and SeNPs. ( B – D ) Viability of C2C12 cells treated with 5–80 μg/mL CoNPs, SeNPs, and mixing of CoNPs and SeNPs were determined by CCK-8. Data are presented as mean ± standard deviation of three identical experiments conducted in triplicate. * Statistically significant difference compared with the controls ( p < 0.05 for each).

Article Snippet: According to the manufacturer’s instructions, the levels of MDA (malondialdehyde), GSH (glutathione), and SOD (superoxide dismutase) in C2C12 cells were measured using a GSH ELISA Kit (Elabscience, Wuhan, China), MDA Content Detection Kit (Beyotime, Nantong, China), and SOD Activity Detection Kit (Beyotime, Nantong, China).

Techniques: Labeling, Confocal Microscopy, Fluorescence, CCK-8 Assay, Standard Deviation

Journal: Toxics

Article Title: Selenium Nanoparticles Attenuate Cobalt Nanoparticle-Induced Skeletal Muscle Injury: A Study Based on Myoblasts and Zebrafish

doi: 10.3390/toxics12020130

Figure Lengend Snippet: Protective effects of low-dose SeNPs on CoNP-induced oxidative stress in muscle cells. ( A ) C2C12 cells were exposed to control, 20 μg/mL CoNPs, 500 µM H2O2, 5 μg/mL SeNPs with or without H2O2, and mixing of Co and Se NPs. Oxidative stress condition induced by H2O2 treatment for 4 h. Intracellular ROS production was analyzed by H2DCFDA staining. ( B – E ) MDA, GSH, and SOD levels were determined in C2C12 cells from control, 20 μg/mL CoNPs, 500 µM H2O2, 5 μg/mL SeNPs with or without H2O2, and mixing of Co and Se NP-treated groups. Data are displayed as mean ± SD; * means p < 0.05 between two indicated groups (n = 10).

Article Snippet: According to the manufacturer’s instructions, the levels of MDA (malondialdehyde), GSH (glutathione), and SOD (superoxide dismutase) in C2C12 cells were measured using a GSH ELISA Kit (Elabscience, Wuhan, China), MDA Content Detection Kit (Beyotime, Nantong, China), and SOD Activity Detection Kit (Beyotime, Nantong, China).

Techniques: Control, Staining

Journal: Toxics

Article Title: Selenium Nanoparticles Attenuate Cobalt Nanoparticle-Induced Skeletal Muscle Injury: A Study Based on Myoblasts and Zebrafish

doi: 10.3390/toxics12020130

Figure Lengend Snippet: CoNP-induced apoptosis was inhibited by SeNPs in muscle cells. ( A , B ) The expression of caspase–3 and cleaved caspase–3 in C2C12 cells treated with 5 μg/mL SeNPs or 20 μg/mL CoNPs with or without SeNPs was analyzed by Western blotting. GAPDH was used as internal reference. Relative expression levels are shown in the graph. ( C , D ) The degree of apoptosis of cells subjected to different treatments was determined by annexin V-FITC/PI double-staining flow cytometry. The percentage of apoptotic cells was counted and compared between the different groups. Data are displayed as mean ± SD; * means p < 0.05 between two indicated groups (n = 3).

Article Snippet: According to the manufacturer’s instructions, the levels of MDA (malondialdehyde), GSH (glutathione), and SOD (superoxide dismutase) in C2C12 cells were measured using a GSH ELISA Kit (Elabscience, Wuhan, China), MDA Content Detection Kit (Beyotime, Nantong, China), and SOD Activity Detection Kit (Beyotime, Nantong, China).

Techniques: Expressing, Western Blot, Double Staining, Flow Cytometry

Journal: Toxics

Article Title: Selenium Nanoparticles Attenuate Cobalt Nanoparticle-Induced Skeletal Muscle Injury: A Study Based on Myoblasts and Zebrafish

doi: 10.3390/toxics12020130

Figure Lengend Snippet: Promotive effects of SeNPs on CoNP-induced inhibition of myogenic differentiation. ( A ) Immunofluorescence staining in SeNPs, CoNPs, and Co Se NP-treated C2C12 cells was analyzed at 4 days of differentiation to detect the effect of different nanoparticles on myogenic differentiation. Nuclei (DAPI, blue), F-actin (Green), and α-SMA (Red) were labeled; scale bar = 25 µm. ( B ) Quantification of α-SMA fluorescence intensity in ( A ) was performed. Data are displayed as mean ± SD. * means p < 0.05 between two indicated groups (n = 6). ( C – E ) mRNA expression of myogenic markers, MyoD, myogenin, and Myf5, in C2C12 cells treated with 5 μg/mL SeNPs, 20 μg/mL CoNPs with or without SeNPs was analyzed by qPCR. The control group was set to 1.0. Data are displayed as mean ± SD. * means p < 0.05 between two indicated groups (n = 6).

Article Snippet: According to the manufacturer’s instructions, the levels of MDA (malondialdehyde), GSH (glutathione), and SOD (superoxide dismutase) in C2C12 cells were measured using a GSH ELISA Kit (Elabscience, Wuhan, China), MDA Content Detection Kit (Beyotime, Nantong, China), and SOD Activity Detection Kit (Beyotime, Nantong, China).

Techniques: Inhibition, Immunofluorescence, Staining, Labeling, Fluorescence, Expressing, Control

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: TOE suppresses malignant phenotype of triple negative breast cancer cells. (A) The effect of TOE on cell proliferation in MDA-MB-231 and 4T1 cells. Cells were treated with varying concentrations of TOE for 24 hours, and cell proliferation was assessed using the CCK-8 assay. Data are expressed as the mean ± SEM (n = 3). Statistical significances were calculated via Student’s t-test. **p < 0.01 and ***p < 0.001 and ****p < 0.0001. (B) Transwell migration and invasion assay of MDA-MB-231 and 4T1 cells after treatment with TOE for 24 hours. Representative images of the migrated and invaded cells from randomly selected fields of Transwell inserts are shown on the left, while quantitative data for cell numbers are presented on the right. Cell numbers were calculated and expressed as the mean ± SEM of three independent experiments. Statistical significance was determined by t-test, with ** p < 0.01 and *** p < 0.001 and **** p < 0.0001 indicating significant differences between TOE-treated and DMSO-treated cells. Scale bar = 100 μm. (C) MA plot of DGEs in MDA-MB-231 treated with TOE. (D) Enrichment and scatter map of KEGG pathway of DGEs.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: CCK-8 Assay, Migration, Invasion Assay

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: Dig and AA inhibited tumor growth in breast cancer mouse models. (A) Inhibition of growth by Dig and AA in MDA-MB-231 and 4T1 cells for 24 h. MDA-MB-231 and 4T1 cells were treated with Dig and AA (at various concentration) for 24 hours, and cell proliferation was assessed using the CCK-8 assay. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical significance was determined using unpaired t -tests, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicating significant differences compared to the DMSO control. (B) Transwell migration and invasion assay of MDA-MB-231 and 4T1 cells with Dig and AA treatment for 24 h. Representative images from randomly selected fields of transwell inserts, and Scalebar = 100 μm. (C) Quantitative data from the Transwell migration and invasion assays. Cell numbers were calculated and are expressed as the mean ± SEM of three independent experiments. * p < 0.05,** p < 0.01, *** p < 0.001 and **** p < 0.0001, as determined by unpaired t -tests. (D) Diagrammatic representation of tumor volume measurement. The diagram illustrates the measurement method, including caliper-based measurements of length and width used to calculate tumor volume (Volume = 1/2 × length × width^2). (E) Tumor sizes at day 14. (F) The body weight changes of mice in the period of 14 days after different treatments. The body weight of mice was monitored every 2 days after Dig and AA treatment. Data are expressed as the mean ± SEM. No significant changes in body weight were observed, suggesting that the treatments did not cause overt toxicity in mice.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: Inhibition, Concentration Assay, CCK-8 Assay, Control, Migration, Invasion Assay

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: Dig combined with PD-1 immunotherapy can promote immune stimulation of in situ 4T1 breast tumors. (A) HE staining of mouse tumor tissues (scale bar = 100 μm). (B) Representative flow cytometry plots showing tumor-associated macrophages (TAMs) (CD45.2+, CD11b+, F4/80+) were obtained after different treatments. (C) Representative flow cytometry plots showing tumor immune cells after different treatments, including CTLs (CD45+, CD3+, CD8+) and Th cells (CD45+, CD3+, CD4+). (D) Representative flow cytometry plots demonstrating tumor-infiltrating LAG-3+ exhausted T cells (CD3+, CD8+, LAG-3+) after different treatments. (E) Representative flow cytometry plots demonstrating tumor-infiltrating TIM-3+ exhausted T cells (CD3+, CD8+, TIM-3+) after different treatments. (F) Representative flow cytometry plots demonstrating tumor-infiltrating PD-1+ exhausted T cells (CD3+, CD8+, PD-1+) after different treatments. (G) The levels of TAMs were quantified through flow cytometry analysis (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.0033; Saline vs Dig: 0.0106; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.2039; Saline vs AA+PD-1: 0.9935; PD-1 vs Dig: 0.9961; PD-1 vs Dig+PD-1: 0.4395; PD-1 vs AA: 0.4330; PD-1 vs AA+PD-1: 0.0009; Dig vs Dig+PD-1: 0.2080; Dig vs AA: 0.7273; Dig vs AA+PD-1: 0.0028; Dig+PD-1 vs AA: 0.0109; Dig+PD-1 vs AA+PD-1: <0.0001; AA vs AA+PD-1: 0.0715. (H) The levels of CTLs were quantified by flow cytometry analysis (n = 5). The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.7941; Saline vs Dig: 0.2550; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.0161; Saline vs AA+PD-1: <0.0001; PD-1 vs Dig: 0.9237; PD-1 vs Dig+PD-1: 0.0013; PD-1 vs AA: 0.2253; PD-1 vs AA+PD-1: 0.0016; Dig vs Dig+PD-1: 0.0136; Dig vs AA: 0.7542; Dig vs AA+PD-1: 0.0164; Dig+PD-1 vs AA: 0.2253; Dig+PD-1 vs AA+PD-1: >0.9999; AA vs AA+PD-1: 0.2576. (I) Flow cytometry analysis quantified the levels of LAG-3+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. Flow cytometry analysis quantified the levels of LAG-3+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.0006; Saline vs Dig: 0.0030; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.5950; Saline vs AA+PD-1: >0.9999; PD-1 vs Dig: 0.9841; PD-1 vs Dig+PD-1: 0.0094; PD-1 vs AA: 0.0282; PD-1 vs AA+PD-1: 0.0005; Dig vs Dig+PD-1: 0.0019; Dig vs AA: 0.1152; Dig vs AA+PD-1: 0.0027; Dig+PD-1 vs AA: <0.0001; Dig+PD-1 vs AA+PD-1: <0.0001; AA vs AA+PD-1: 0.5676. (J) Flow cytometry analysis quantified the levels of TIM-3+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.0073; Saline vs Dig: 0.3784; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.9296; Saline vs AA+PD-1: 0.3206; PD-1 vs Dig: 0.4016; PD-1 vs Dig+PD-1: 0.0959; PD-1 vs AA: 0.0007; PD-1 vs AA+PD-1: <0.0001; Dig vs Dig+PD-1: 0.0011; Dig vs AA: 0.0698; Dig vs AA+PD-1: 0.0050; Dig+PD-1 vs AA: <0.0001; Dig+PD-1 vs AA+PD-1: <0.0001; AA vs AA+PD-1: 0.8546. (K) Flow cytometry analysis quantified the levels of PD-1+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: <0.0001; Saline vs Dig: 0.2205; Saline vs Dig+PD-1: 0.0276; Saline vs AA: 0.9984; Saline vs AA+PD-1: 0.8260; PD-1 vs Dig: 0.0043; PD-1 vs Dig+PD-1: 0.0469; PD-1 vs AA: <0.0001; PD-1 vs AA+PD-1: <0.0001; Dig vs Dig+PD-1: 0.9030; Dig vs AA: 0.1042; Dig vs AA+PD-1: 0.0181; Dig+PD-1 vs AA: 0.0108; Dig+PD-1 vs AA+PD-1: 0.0015; AA vs AA+PD-1: 0.9632. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. Saline.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: In Situ, Staining, Flow Cytometry, Comparison, Saline

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: NANOS1 Silencing Reduces TNF-α Expression and Cell Invasiveness. (A) Volcano plots of RNA-seq. Differential gene expression analysis was performed using RNA sequencing data from MDA-MB-231 cells with siNANOS1#1 silencing. The volcano plot shows the distribution of genes based on log-fold change versus the negative log-transformed p-value. Significant upregulated and downregulated genes are indicated with colored dots. Genes that meet the threshold of log-fold change (|logFC| > 2) and adjusted p-value < 0.05 are considered differentially expressed. (B) Heatmap of differential gene expression. (C) Chord diagram of GO enrichment results and related genes. (D) Top 15 KEGG pathways. The x-axis represents the gene ratio ( p < 0.05), and the y-axis represents the enriched terms. (E) The knockdown efficiency of siRNA and the expression of NANOS1 and TNF-α following Dig and AA treatment were quantified by qPCR. MDA-MB-231 and 4T1 cells were transfected with siRNA targeting NANOS1, followed by treatment with Dig and AA. Quantitative PCR was performed to assess the expression levels of NANOS1 and TNF-α. The data are expressed as the mean ± SEM, with statistical significance calculated using one-way ANOVA. (F) Dig and AA decreased the expression of NANOS1 protein (n = 3). Data are expressed as the mean ± SEM. Statistical significances were calculated via one-way ANOVA, * p < 0.05, ** p < 0.010, *** p < 0.001 and **** p < 0.0001 vs. DMSO. (G) Perforation migration and invasion assays of MDA-MB-231 and 4T1 cells after 24 h of siRNA treatment, scale bar = 100 mm. Cell numbers were calculated and are expressed as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, as determined by unpaired t-tests, were regarded as significant.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: Expressing, RNA Sequencing, Gene Expression, Transformation Assay, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Migration

Journal: Discover Oncology

Article Title: RBMS1 interference inhibits malignant progression of glioblastoma cells and promotes ferroptosis

doi: 10.1007/s12672-024-01430-1

Figure Lengend Snippet: RBMS1 silence inhibited the proliferation and promoted the apoptosis of T98G cells. A The transfection efficiency of sh-RBMS1 was examined with RT-qPCR and western blot. B The cell proliferation was detected using CCK-8 assay. C The colony forming ability was detected using colony formation assay. D The cell apoptosis was detected using flow cytometry. E The expression of proliferation- and apoptosis-related proteins were detected using western blot. ** P < 0.01 and *** P < 0.001 vs. sh-NC

Article Snippet: For the detection of lipid peroxidation in T98G cells, TBARS Assay Kits (cat. no. E-BC-K298-M; Elabscience) were performed according to the manufacturer’s instructions.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing

Journal: Discover Oncology

Article Title: RBMS1 interference inhibits malignant progression of glioblastoma cells and promotes ferroptosis

doi: 10.1007/s12672-024-01430-1

Figure Lengend Snippet: RBMS1 silence inhibited the migration, invasion and EMT process in T98G cells. A , B The cell migration and invasion were detected using wound healing and transwell assay. C The activities of MMP2 and MMP9 were detected using western blot. D The expressions of EMT-related proteins were detected using western blot. ** P < 0.01 and *** P < 0.001 vs. sh-NC

Article Snippet: For the detection of lipid peroxidation in T98G cells, TBARS Assay Kits (cat. no. E-BC-K298-M; Elabscience) were performed according to the manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Western Blot

Journal: Discover Oncology

Article Title: RBMS1 interference inhibits malignant progression of glioblastoma cells and promotes ferroptosis

doi: 10.1007/s12672-024-01430-1

Figure Lengend Snippet: RBMS1 silence promoted ferroptosis in T98G cells. A The lipid peroxidation was detected using TBARS Assay Kit. B The lipid ROS was detected using a BODIPY 581/591 C11 kit. C The total iron level was detected using corresponding assay. (D) The expressions of ferroptosis-related proteins were detected using western blot. *** P < 0.001 vs. sh-NC

Article Snippet: For the detection of lipid peroxidation in T98G cells, TBARS Assay Kits (cat. no. E-BC-K298-M; Elabscience) were performed according to the manufacturer’s instructions.

Techniques: TBARS Assay, Western Blot

Journal: Discover Oncology

Article Title: RBMS1 interference inhibits malignant progression of glioblastoma cells and promotes ferroptosis

doi: 10.1007/s12672-024-01430-1

Figure Lengend Snippet: RBMS1 silence inhibited the proliferation and promoted the apoptosis of T98G cells by promoting ferroptosis. A The cell proliferation was detected using CCK-8 assay. B The colony forming ability was detected using colony formation assay. C The cell apoptosis was detected using flow cytometry. D The expression of proliferation- and apoptosis-related proteins were detected using western blot. ** P < 0.01 and *** P < 0.001 vs. sh-NC, ## P < 0.01 and ### P < 0.001 vs. sh-RBMS1

Article Snippet: For the detection of lipid peroxidation in T98G cells, TBARS Assay Kits (cat. no. E-BC-K298-M; Elabscience) were performed according to the manufacturer’s instructions.

Techniques: CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot

Journal: Discover Oncology

Article Title: RBMS1 interference inhibits malignant progression of glioblastoma cells and promotes ferroptosis

doi: 10.1007/s12672-024-01430-1

Figure Lengend Snippet: RBMS1 silence inhibited the migration, invasion and EMT process in T98G cells by promoting ferroptosis. A , B The cell migration and invasion were detected using wound healing and transwell assay. C The activities of MMP2 and MMP9 were detected using western blot. D The expressions of EMT-related proteins were detected using western blot. *** P < 0.001 vs. sh-NC, # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. sh-RBMS1

Article Snippet: For the detection of lipid peroxidation in T98G cells, TBARS Assay Kits (cat. no. E-BC-K298-M; Elabscience) were performed according to the manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Western Blot

Journal: Genes

Article Title: Transcriptional Activation of the TREM2 Gene by ZEB2 in a Zinc Finger-Dependent Manner

doi: 10.3390/genes16111329

Figure Lengend Snippet: The 5 kb upstream region and 5′-UTR of TREM2 recapitulate its cell type-specific expression. ( A ) Schematic representation of the reporter construct ( T2-5k-u-mCherry ). The CMV promoter in the mCherry-N3 vector was replaced with a fragment containing the 5 kb sequence upstream of the TREM2 transcription start site and its 5′-UTR. ( B ) T2-5k-u-mCherry was transfected into HEK293, THP-1, and HMC3 cells. Representative fluorescence images of mCherry expression are shown. Nuclei were counterstained with Hoechst33342. Scale bars, 50 μm. ( C ) Schematic diagram of the T2-5k-mCherry construct lacking the TREM2 5′-UTR. ( D ) Fluorescent images of THP-1 cells transfected with T2-5k-u-mCherry or T2-5k-mCherry . Scale bars, 50 μm. ( E ) Quantification of mCherry-positive cells relative to Hoechst-positive cells using the IN Cell Analyzer. Error bars represent SDs; n = 3; Welch’s t -test. ( F ) Fluorescence images of HMC3 cells transfected with T2-5k-u-mCherry or T2-5k-mCherry . Scale bars, 50 μm. ( G ) Quantification of mCherry-positive cells relative to Hoechst-positive cells analyzed as in ( E ). Error bars represent SDs; n = 3; Welch’s t -test.

Article Snippet: The HMC3 cell line (EP-CL-0620) was obtained from Elabscience (Houston, TX, USA).

Techniques: Expressing, Construct, Plasmid Preparation, Sequencing, Transfection, Fluorescence

Journal: Genes

Article Title: Transcriptional Activation of the TREM2 Gene by ZEB2 in a Zinc Finger-Dependent Manner

doi: 10.3390/genes16111329

Figure Lengend Snippet: ZEB2 overexpression or knockdown modulates TREM2 mRNA and protein expression. ( A ) RT-qPCR analysis of ZEB2 mRNA in doxycycline-treated inducible EGFP and EGFP-ZEB2 cells. Expression was normalized to ACTB . Error bars represent SDs; n = 3; Welch’s t -test. ( B ) TREM2 mRNA levels normalized to ACTB , quantified from the same samples as in ( A ). Error bars indicate SDs; n = 3; Welch’s t -test. ( C ) ZEB2 siRNAs were transfected into HMC3 cells. ZEB2 mRNA levels were measured by RT-qPCR and normalized to B2M . Error bars indicate SDs; n = 5; Tukey’s test. ( D ) TREM2 mRNA levels quantified as in ( C ). Error bars indicate SDs; n = 5; Tukey’s test. ( E ) Membrane-bound protein fractions from HMC3 cells were analyzed for TREM2 protein levels following ZEB2 knockdown. APP served as a loading control. ( F ) Quantification of ( E ). Error bars indicate SDs; n = 5; Welch’s t -test. ( G ) Relative YY1 mRNA expression in HMC3 cells transfected with YY1 siRNA. YY1 mRNA levels were normalized to B2M. Error bars indicate SDs; n = 3; Welch’s test. ( H ) TREM2 mRNA levels in HMC3 cells transfected with siZEB2 or siYY1. Error bars indicate SDs; n = 5; Tukey’s t-test. ( I ) Relative luciferase activity induced by EGFP-ZEB1. Error bars indicate SDs; n = 3; Welch’s test.

Article Snippet: The HMC3 cell line (EP-CL-0620) was obtained from Elabscience (Houston, TX, USA).

Techniques: Over Expression, Knockdown, Expressing, Quantitative RT-PCR, Transfection, Membrane, Control, Luciferase, Activity Assay