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: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: Knocking down HDAC4 inhibits glutamine metabolism in A549 cells. (A) Glutamine uptake levels in A549 cells. (B) Glutamate production levels in A549 cells. (C) α‐Ketoglutarate production levels in A549 cells. (D) Measurement of GLS activity in A549 cells. (E) DCFH‐DA staining was used to measure the level of ROS in cells. (F) A kit was used to measure the GSH/GSSG ratio. (G) Western blotting was used to detect the expression of Nrf2, HO‐1, and NQO1. ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with three independent biological replicates. Statistical analysis was conducted using Student’s t ‐test for panels (A)‒(G). GLS, glutaminase; DCFH‐DA, 2 ′ ,7 ′ dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, oxidized glutathione; Nrf2, nuclear factor erythroid 2 related factor 2; HO‐1, heme oxygenase 1; NQO1: NAD(P)H quinone dehydrogenase 1. The original, uncropped western blot images for Figure are provided in the Supporting Information.

Article Snippet: The levels of total glutathione (T‐GSH) and GSSG in A549 cells were measured using a GSH/GSSG ratio detection kit (E‐BC‐K097‐M, Elabscience, China) following the manufacturer’s instructions.

Techniques: Activity Assay, Staining, Western Blot, Expressing, Analysis

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: HDAC4 promotes SLC38A2 expression through HIF‐1α. (A) Western blot detection of SLC38A2 expression in A549 cells with HDAC4 knockdown. (B) Western blot detection of SLC38A2 expression in A549 cells with HDAC4 overexpression. (C) Western blot detection of HIF‐1α expression in A549 cells with HDAC4 knockdown. (D) Western blot detection of HIF‐1α expression in A549 cells with HDAC4 overexpression. (E) JASPAR analysis of the binding sites of HIF‐1α and the promoter of SLC38A2. (F) ChIP‐qPCR detection of the enrichment level of HIF‐1α at the SLC38A2 promoter in A549 cells. (G) A promoter‐luciferase reporter assay was used to verify the binding of HIF‐1α to the SLC38A2 promoter. (H) RT‐qPCR detection of HIF‐1α mRNA expression in A549 cells with HIF‐1α overexpression. (I) Western blot detection of HIF‐1α expression in A549 cells with HIF‐1α overexpression. (J) Western blot detection of SLC38A2 expression in A549 cells. (K) RT‐qPCR detection of HIF‐1α mRNA expression in A549 cells with HIF‐1α knockdown. (L) Western blot detection of HIF‐1α expression in A549 cells with HIF‐1α knockdown. (M) Western blot detection of SLC38A2 expression in A549 cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with three independent biological replicates. Statistical analysis was conducted using Student’s t ‐test for panels (A)–(D), (F)–(I) and (K)–(L) and one‐way ANOVA for panels (J) and (M). HIF‐1α, hypoxia‐inducible factor‐1 subunit alpha; ChIP‐qPCR, chromatin immunoprecipitation quantitative polymerase chain reaction; IgG, immunoglobulin G; OE‐HIF‐1α, overexpression HIF‐1α; sh‐HIF‐1α, short hairpin RNA targeting HIF‐1α. The original, uncropped Western blot images for Figure –D, I,J, L,M are provided in the Supporting Information.

Article Snippet: The levels of total glutathione (T‐GSH) and GSSG in A549 cells were measured using a GSH/GSSG ratio detection kit (E‐BC‐K097‐M, Elabscience, China) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Analysis, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA

Journal: Frontiers in Immunology

Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

doi: 10.3389/fimmu.2022.1089369

Figure Lengend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

Article Snippet: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

Techniques: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

Journal: Frontiers in Immunology

Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

doi: 10.3389/fimmu.2022.1089369

Figure Lengend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

Article Snippet: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

Techniques: Cell Culture, Staining

Journal: Frontiers in Immunology

Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

doi: 10.3389/fimmu.2022.1089369

Figure Lengend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

Article Snippet: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

Techniques: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

Journal: Frontiers in Immunology

Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

doi: 10.3389/fimmu.2022.1089369

Figure Lengend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

Article Snippet: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

Techniques: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: TGF-β1 and MUC16 collaborate to induce the alveolar epithelial to mesenchymal transition. The A549 cell line transfected with control siRNA(−) or siRNA-MUC16 was stimulated for 48 h ( A – E ) or 72 h ( F ) with 5 ng/mL TGF-β1 to measure MUC16 ( A ), collagen type I ( B ), α-SMA ( C ), Slug ( D ), and Snail ( E ) mRNA expression by real-time PCR and collagen type I protein levels by Western blotting, quantification was performed by densitometry ( F ). Data are expressed as 2 −ΔCt for mRNA levels and relative to β-actin for protein levels. The results are expressed as means ± SE. Student t-test of three independent experiments performed in triplicate. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−).

Article Snippet: The SBE Reporter kit (Cat#: 60654, BPSBioscience) was used for monitoring the activity of TGFβ/SMAD signalling pathway in A549 cells.

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

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: MUC16 modulates the effect of TGF-β1 on SMAD3 phosphorylation. ( A ) A549 and ( B ) MRC5 cell lines transfected with control siRNA(−) or siRNA-MUC16 were stimulated for 40 min with TGF-β1 10 ng/mL to measure p-Smad3 and for 48 h with TGF-β1 5 ng/mL to measure β-catenin by Western blot. A549 ( C ) and MRC5 ( D ) cells were stimulated with 10 ng/mL TGF-β1 for 40 min. Total protein was extracted and immunoprecipitated using MUC16 antibody and probed against p-Smad3 and MUC16 by Western blot (representative images are shown). ( E ) A549 cells were stimulated with 10 ng/mL TGF-β1 for 1 h. MUC16 and p-Smad3 co-localisation was analysed by confocal microscopy to generate a two-dimensional cytofluorogram that selected common localised points of both antibodies (white colour). Scale bars: 10 μm. ( F ) Smad Binding Element (SBE) measure following 10 ng/mL TGF-β1 stimulation during 18 h in A549 cells transfected with siRNA(−) or siRNA-MUC16. Data are expressed relative to Smad3 or β-actin protein level (A-D). The results are expressed as means ± SE of three independent experiments performed in triplicate. Student t-test was used. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−) + TGF-β1.

Article Snippet: The SBE Reporter kit (Cat#: 60654, BPSBioscience) was used for monitoring the activity of TGFβ/SMAD signalling pathway in A549 cells.

Techniques: Transfection, Western Blot, Immunoprecipitation, Confocal Microscopy, Binding Assay

Journal: Respiratory Research

Article Title: Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro

doi: 10.1186/s12931-024-02983-z

Figure Lengend Snippet: Determination of the minimal effective MOI (mMOI) of a ) A. actinomycetemcomitans and b ) A. aphrophilus based on NF-κB pathway activation and determination of the mMOI of c ) A. actinomycetemcomitans and d ) A. aphrophilus based on IL-8 production in 3-D lung epithelial cells. On the vertical axis % NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells is shown, on the horizontal axis the tested MOI is depicted. The mMOI was defined as the lowest MOI with anti-inflammatory activity (i.e. < 50% NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells as visualized by a dotted line, p < 0,05). e ) Western blot analysis of proteins (i.e. IκBα, p65 and phosphorylated (p)-IκBα) produced by 3-D A549 cells stimulated with LPS in the presence or absence of A. actinomycetemcomitans (targeted MOI 50:1) for 4 h (representative replicate is shown, image contains cropped blot – the full original blot is shown in Supplementary file_Western blot image). f ) Band intensity (normalised to β-actin) of Western blot at 4 h of 3-D A549 cells stimulated with LPS with/without A. actinomycetemcomitans . Results are expressed as a percentage of the positive control (i.e. LPS). NC: negative control (untreated 3-D A549 cells in serum-free GTSF-2 medium), A.ac. A. actinomycetemcomitans . Data represent mean ± SEM, * p < 0.05, n ≥ 3

Article Snippet: A previously developed organotypic 3-D lung cell culture model of the A549 alveolar epithelial cell line (ATCC CCL185) or the NF-κB–luciferase-transfected A549 recombinant stable cell line (BPS Bioscience, San Diego, CA, US) was used that exhibits in vivo-like phenotypic and functional properties of alveolar epithelial cells, including barrier function (localized expression of tight junctional markers), apical and basolateral polarity, and responds to infection in ways that are relevant to the infection process in vivo, including cytokine secretion [ , – ].

Techniques: Activation Assay, Activity Assay, Western Blot, Produced, Positive Control, Negative Control