Journal: Nature Communications

Article Title: Single-cell analyses identify monocyte gene expression profiles that influence HIV-1 reservoir size in acutely treated cohorts

doi: 10.1038/s41467-025-59833-9

Figure Lengend Snippet: a Effects of IL1B on NF-κB activity were assessed using A549 NF-κB reporter cells. Cultures were treated with IL1B, TNFα, or LPS and infected with VSV-pseudotyped NL4-3, CH058, or Mock control. After 24 hrs the Alkaline Phosphatase Blue Microwell assay was performed with OD650 values relative to the corresponding no treatment control (NT) reflecting NF-κB expression which is shown on the y-axis. The data presents an average of n = 3 independent experiments ±SEM. Blue: IL1B, teal: TNFα, yellow: LPS. b PBMC from n = 3 participants were treated with IL1B or TNFα and examined for IκBα phosphorylation as described in the methods section. Graphs present the protein expression from these n = 3 participants as mean ± SEM. Corresponding representative uncropped images are provided in the Source Data file. White: no treatment, light blue: IL1B 10 ng/ml, medium blue: IL1B 50 ng/ml, dark blue: TNFα. c Effects of IL1B in vitro when HIV was quantified after a single round of infection. Plots show the relative proportions of pMorpheus-V5 latently (blue) or productively (orange) infected PBMC in cultures treated with IL1B prior to, simultaneously, or after transduction with Env viral particles carrying the indicated Env protein. The background obtained from uninfected cells (Mock) was subtracted from the values obtained for cells infected with the pMorpheus HIV-1 construct pseudo-typed with the indicated envelope proteins. The data represent n = 6 (X4) or n = 3 (R5 and R5X4) individual healthy participants, with error bars representing the average ±SEM. Corresponding gating strategies and representative plots are shown in Supplementary Fig. . d Effects of IL1B on spreading HIV-1 infection in cell culture. Using HIV-1 YU-2, bar plots display the relative p24-positive cell fractions after pre-treatment with increasing concentrations of IL1B (from 0.01-10.0 ng/ml, 10-fold increments) across n = 4 different participants. Significance was calculated using the Mann-Whitney U two-sided test. Orange: no treatment, yellow: different concentrations of IL1B treatment. e, f Bar plots display the average infectious virus yields ( e ) and p24 antigen levels ( f ) of n = 5 different participants at 4 days post-infection relative to the no IL1B treatment controls normalized to 100% ±SEM. Corresponding replication curves are shown in Supplementary Fig. . For all bar graphs shown, bar height and error bars represent mean values +/- SEM. Significant differences in all panels except ( d ), were determined using two-sided unpaired t -test analys e s. Asterisks indicate statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Source data and exact P values are provided in the Source Data file.

Article Snippet: A549-Dual TM Cells (InvivoGen; cat# s549d-nfis) were seeded at a density of 20,000 cells per well on 96-well plates.

Techniques: Activity Assay, Infection, Control, Expressing, Phospho-proteomics, In Vitro, Transduction, Construct, Cell Culture, MANN-WHITNEY, Virus

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: TAOB-CMs increases lung cancer migration and EMT. TAOB-CMs enhanced the migratory ability of A549 and CL1–0 lung cancer cells. A and B, TAOB-CMs enhanced cell migratory ability, as determined by scratch wound healing assay (A) and Transwell system (B). C, TAOB-CMs enhanced cell invasion ability. D, TAOB-CMs caused EMT in cancer cells. E, the expression of BMP-2 in OB-CM, For A, the migration ability of lung cancer cells was assessed by wound healing assay. OB-CM (control group) and TAOB-CMs (20%) act as a chemoattractant of cancer migration. Quantification of cell migration was carried out by measuring the distance between the migratory fronts of cells in four random selected microscopic fields for each condition and time point. The degree of cell movement is expressed as the percentage of wound closure as compared with the zero time point. For B and C, the invasiveness and migration ability of A549 and CL1–0 cells were quantified by QCMTM 24-well cell migration and invasion assay. The cells were seeded in the upper inset, and the OB-CM (control group) and TAOB-CMs (20%) acted as the chemoattractant for cancer migration and invasion. For D, A549 and CL1–0 cells were treated with TAOB-CMs (20%) for 24 h, and then the expression of various proteins was assessed by immunoblot assay. For E, primary osteoblasts were treated RPMI 1640 (20%), A549-CM (20%), and CL1–0-CM for 24 h. The BMP-2 levels were assessed by BMP-2 ELISA kits. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Wound Healing Assay, Expressing, Invasion Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: BMP-2 is involved in TAOB-CM-mediated enhancement of migration and EMT in lung cancer. A and B, BMP-2 increased migratory ability, as determined by scratch wound healing assay (A) and Transwell system (B). C and D, BMP-2 increased the invasion ability (C) and EMT (D) of A549 and CL1–0 cells. E and F, noggin decreased TAOB-CM-mediated cell migration (E) and EMT (F). The migration ability of lung cancer cells was assessed by wound healing assay, in accord with the description under “Experimental Procedures.” BMP-2 (20 ng/ml for EMT assay) acts as the chemoattractant for cancer migration. For E and F, A549 and CL1–0 cells were pretreated with or without noggin for 1 h, and then OB-CM and TAOB-CMs were added for another 24 h. Cell migration was assessed by wound healing assay, and the expression of various proteins was determined by immunoblot assay. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Wound Healing Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: Sera from lung cancer patients increase lung cancer migration. A, the levels of BMP-2 in lung cancer patient sera. B, lung cancer sera enhance the migratory ability of lung cancer cells. C, depletion of BMP-2 decreased lung cancer patient serum-mediated cell migration. The levels of BMP-2 were assessed by ELISA. Horizontal bars represent means. The cells were treated with or without noggin for 1 h, and then culture medium containing healthy donor sera (15%) or lung cancer patient sera (15%) was added for another 24 h. Cell migration was assessed by wound healing assay. For C, BMP-2 depleted from lung cancer patient serum was performed using anti-BMP-2 and antibodies (4 μg/ml) and Sepharose A/G beads, following regular immunoprecipitation techniques. The migration ability of A549 and CL1–0 cells were quantified by QCMTM 24-well cell migration assay kit. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Immunoprecipitation, Cell Migration Assay

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: TAOB-CMs and BMP-2 increase the activation of MAPK and elevate the expression of Runx2 and Snail. A and B, TAOB-CMs (A) and BMP-2 (B) increase the phosphorylation of SMAD, ERK, and p38. C and D, TAOB-CMs (C) and BMP-2 (D) enhance the expression of Runx2 and Snail protein. Cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for the indicated times. The expressions of various proteins were determined by immunoblot assay. E, TAOB-CMs and BMP-2 enhance the expression of Runx2 and Snail mRNA. The cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for a specific time (3 h for snail and 12 h for E-cadherin). The expressions of mRNA were determined by quantitative PCR. F, noggin decreases TAOB-CM-mediated MAPK activation and Runx2 and Snail up-regulation. The cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for the indicated times. The expressions of mRNA and various proteins were determined by quantitative PCR and immunoblot assay. For F, A549 and CL1–0 cells were pretreated with or without noggin for 1 h and then treated with BMP-2 (20 ng/ml) for 6 h. The expression of various proteins was then assessed by immunoblot assay. The data shown are representative of three independent experiments. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: Runx2 is the upstream regulatory factor of Snail. A and B, inhibition of Runx2 decreases BMP-2-mediated Snail up-regulation and E-cadherin down-regulation (A), as well as cell migration (B). Cells were transfected with pLKO-AS2 or pLKO-AS2-RUNX2 shRNA. Stable clones were created by puromycin selection, and the efficacy of shRNA was assessed by RT-PCR. Cells were treated with BMP-2 (20 ng/ml) for the specified times (cell migration, 24 h; Runx2 and Snail, 6 h; E-cadherin, 24 h). Then the expression of various proteins was then assessed by immunoblot assay. C, overexpression of Snail reversed the inhibitory effect of Runx2 shRNA on BMP-2-mediated cell migration. Runx2-transfected A549 and CL1–0 cells were transected with pCMV or pSnail plasmid, and stable clones were established by G418 and puromycin. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05). The data shown are representative of three independent experiments.

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Inhibition, Migration, Transfection, shRNA, Clone Assay, Selection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Redox Biology

Article Title: Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer

doi: 10.1016/j.redox.2022.102395

Figure Lengend Snippet: (A) 2-ME significantly decreases A549 cell viability in a dose-dependent manner in the 2D culture model at concentrations less than or equal 100 μM. A549 cells were treated with serial dilutions of 2-ME at a concentration range from 100 μM to 0.43 μM for 24 h. Cell viability was determined by MTT assay. Presented values are the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells. (B) 2-ME reduces the cell viability of the A549 3D spheroidal models in a dose dependent manner with expenditures close to 200 μM. A549 cells were exposed to serial dilutions of 2-ME over a concentration range of 200 μM–0.78 μM for 48 h. Cell viability was determined by the WST-1 assay. Presented values are the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells. (C) IC50 values for the A549 cell line treated with 2-ME were >100 μM. These values were calculated by analyzing the relationship between concentrations and percentage (%) of inhibition using GraphPad Prism version 9.0 for Windows, GraphPad Software, CA, USA.

Article Snippet: Wild-type human lung epithelial carcinoma, A549 cell line as 3D cell culture spheroids were obtained by plating cells with an average density of 8000 cells/cm 2 in 96-well U-bottom and V-bottom Lipidure® cell culture plates (Amsbio, Abingdon, United Kingdom) coated with phosphorylcholine which is naturally found in cell membranes.

Techniques: Concentration Assay, MTT Assay, WST-1 Assay, Inhibition, Software

Journal: Redox Biology

Article Title: Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer

doi: 10.1016/j.redox.2022.102395

Figure Lengend Snippet: (A) A549 cells as well as cell medium alone were treated with 2-ME at 10 μM, 1 μM and 10 nM concentrations, respectively. 2-ME treated A549 cells generated higher total hydrogen peroxide levels as compared to untreated control A549 cells incubated with Nutrient Mixture F-12 Ham medium alone. The obtained result comparing ROS induction in the wells with no cells and in the wells with A549 cells, indicates an increased production of ROS by the cancer cells. Luminescence was determined with a Glo-Max® Luminometer from Promega (Mannheim, Germany). Statistical analysis was performed using the GraphPad TTEST function (GraphPad Prism 9 version 9.0.0.). For analysis, both the mean of average luminescence (RLU) and the standard deviation were calculated. (B) Time-dependent growth rate of 2-ME-treated A549 cells. A549 cells treated with 2-ME 10 μM concentration up to 24 h. The analysis of the obtained results evaluated by staining with propidium iodide (PI) was performed using the CytoSMART Analysis System (Lux 3, CytoSMART, Eindhoven, The Netherlands). Values are presented as the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells.

Article Snippet: Wild-type human lung epithelial carcinoma, A549 cell line as 3D cell culture spheroids were obtained by plating cells with an average density of 8000 cells/cm 2 in 96-well U-bottom and V-bottom Lipidure® cell culture plates (Amsbio, Abingdon, United Kingdom) coated with phosphorylcholine which is naturally found in cell membranes.

Techniques: Generated, Incubation, Standard Deviation, Concentration Assay, Staining

Journal: Redox Biology

Article Title: Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer

doi: 10.1016/j.redox.2022.102395

Figure Lengend Snippet: (A). A549 cells were pretreated with V5 peptide at a concentration of 100 μM for 4 h, and then treated with 1 μM 2-ME for 24 h. Values are the mean ± SD of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells. (B) The cells were treated for 24 h with either 100 μM PalmB or 10 μM 2-ME, and a combination of both. Both PalmB and 2-ME alone, significantly decreased A549 cell viability. A synergistic effect on the viability decrease was observed for treatment with both PalmB and 2-ME used in combination. The cell viability was determined by MTT assay. Values are presented as the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells C. Serum levels of critical estrogen metabolites analyzed in lung cancer patients (LC) as compared with healthy control (C) are presented as median, as well as quartile 1 and 3, with the minimum and maximum values, in the form of a typical box and whiskers plot. Statistically significant differences are marked with an asterisk * and refer to p-value < 0.05. It is worth noting that 2-ME serum level is significantly lower in lung cancer patients as compared with the control group. Based on table No 2.

Article Snippet: Wild-type human lung epithelial carcinoma, A549 cell line as 3D cell culture spheroids were obtained by plating cells with an average density of 8000 cells/cm 2 in 96-well U-bottom and V-bottom Lipidure® cell culture plates (Amsbio, Abingdon, United Kingdom) coated with phosphorylcholine which is naturally found in cell membranes.

Techniques: Concentration Assay, MTT Assay