Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing

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