Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet:

Article Snippet: Materials purchased from Proteintech Group (Wuhan, China) included antibodies against GAPDH (proteintech, 10494-1-AP, 1:5000), Beta Actin (proteintech, 66009-1-Ig, 1:1000), goat anti-rabbit IgG (proteintech, 66912-1-lg, 1:10000), Pro Caspase-8 (Abcam, ab108333, 1:10000), Caspase-9 (proteintech, 10380-1-AP, 1:1000), Cleaved Caspase-9 (Abcam, ab2324, 1 ug/mL), caspase-3 (proteintech, 19677-1-AP, 1:2000), cleaved caspase-3 (Abcam, ab2302, 1:100), Bcl-2 (proteintech, 12789-1-AP, 1:1000), Bax (proteintech, 50599-2-lg,1:1000), HSP90A (proteintech, 60318-1-Ig, 1:5000), Cdc37 (proteintech, 10218-1-AP, 1:200), CDK1 (proteintech, 19532-1-Ig, 1:200), Cyclin A2 (proteintech, 18202-1-AP, 1:5000).

Techniques: Recombinant, Magnetic Beads, RNA Extraction, Quantitation Assay, Protein Concentration, Software, Lysis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Analysis of CD44 and caveolin-1 regulation of high-molecular-weight hyaluronan (HMW-HA) binding to human pulmonary endothelial cells (EC). A: EC were grown to confluency and serum-starved for 1 h, and Triton X-100-soluble, Triton X-100-insoluble, and OptiPrep fractions were prepared. The 20% OptiPrep fraction represents the caveolin-enriched microdomain (CEM) fraction. Fractions were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-caveolin-1 (a), anti-fibrillarin (b), anti-cyclooxygenase (COX) IV (c), anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b, d), anti-Golgi reassembly stacking protein 65 (GRASP65, e), or anti-VEGF receptor (anti-VEGFR, f). B: EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 100 nM HMW-HA (5 min) or the CEM-abolishing cholesterol-depletion agent methyl-β-cyclodextrin (MβCD, 5 mM) for 1 h prior to 100 nM HMW-HA treatment (5 min). Cellular material was solublized in 4°C 1% Triton X-100, and soluble and insoluble fractions were obtained. Triton X-100-insoluble fraction was overlaid with 60%, 40%, 30%, and 20% OptiPrep and centrifuged at 35,000 rpm in an SW60 rotor for 12 h at 4°C. Triton X-100-soluble material and OptiPrep fractions were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-caveolin-1 (a), anti-CD44s (IM-7, standard domain, b), or anti-VEGF receptor 2 (anti-VEGFR2, c) antibody. The 20% OptiPrep fraction is the CEM fraction. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. C: immunoblot analysis of small interfering RNA (siRNA)-treated or untreated human EC. Cellular lysates from untransfected (control, no siRNA), scramble siRNA (siRNA that does not target any known human mRNA), caveolin-1 siRNA, or CD44 siRNA transfection were analyzed using immunoblotting with anti-caveolin-1 (a), anti-CD44 (IM-7, b), or anti-actin (c) antibody. Experiments were performed in triplicate, each with similar results, and representative data are shown. D: quantitation of fluorescein-conjugated HMW-HA binding to scramble siRNA-, annexin A11 siRNA-, CD44 siRNA-, or caveolin-1 siRNA-treated EC. Fluorescein-conjugated HMW-HA (100 nM) was added for 15 min to EC in serum-free medium, cells were washed 3 times in serum-free medium, and fluorescence intensity was quantified. Cells were counted utilizing a hemocytometer.

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Molecular Weight, Binding Assay, SDS Page, Western Blot, Small Interfering RNA, Transfection, Quantitation Assay, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Analysis of CD44 and caveolin-1 regulation of HMW-HA-mediated human EC barrier enhancement. A: EC were plated on gold microelectrodes, serum-starved for 1 h, and treated with PBS, pH 7.4 (control), or 10, 50, or 100 nM HMW-HA. Arrow indicates HMW-HA addition. Transendothelial electrical resistance (TER) trace represents pooled means ± SE from 3 independent experiments. B: HMW-HA induces CD44 receptor clustering. Human pulmonary microvascular EC were plated on 8-well glass cover slides and allowed to adhere for 48 h. Cells were then serum-starved for 1 h and incubated with 100 nM HMW-HA for 15 min and fixed in 4% paraformaldehyde or incubated with rat anti-CD44 (IM-7) antibody at 10 μg/ml for 2 h. Cells were then washed briefly in PBS, pH 7.4, and secondary anti-rat IgG (1 μg/ml; Sigma-Aldrich) was added for CD44 antibody cross-linking (36). Cells were incubated for 1 h before fixation in 4% paraformaldehyde. Cells were then immunostained for CD44 with use of a directly labeled mouse anti-CD44-Alexa 488 conjugate (Cell Signaling Technology). C: anti-CD44 antibody (IM-7) does not induce appreciable CD44 shedding in human EC. Human EC were serum-starved for 1 h prior to the addition of no antibody, rat IgG (1 μg/ml), or rat anti-CD44 (IM-7) antibody (1 μg/ml) for 3 h. EC medium was collected, concentrated, and immunoblotted with anti-CD44 (IM-7) antibody. D: percent inhibition of maximal HMW-HA-induced TER response (as described in A) with addition of normal rat IgG, anti-CD44 antibody, normal rabbit IgG, anti-CD44v10 antibody (10 μg/ml), or 5 mM MβCD. E: percent inhibition of maximal HMW-HA-induced TER response (as described in D) in human EC with scramble, annexin A11, CD44, or caveolin-1 siRNA treatment.

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Incubation, Labeling, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Role of HMW-HA-induced recruitment of annexin A2 and protein S100-A10 to human EC CEM. A: HMW-HA induces caveolin-1 redistribution to EC-EC junctions. Human EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 100 nM HMW-HA (15 min), fixed in 4% paraformaldehyde, and stained with anti-caveolin-1 antibody, anti-vascular endothelial (VE)-cadherin antibody, or 4′,6-diamidino-2-phenylindole (DAPI). Overlay is a merged image of caveolin-1, VE-cadherin, and DAPI fluorescence, with yellow color indicating colocalization of caveolin-1 and VE-cadherin. B and C: EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 100 nM HMW-HA (5 min) and CEM fractions (20% OptiPrep layer). B: CEM fractions were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-annexin A2 (a), anti-protein S100-A10 (b), anti-filamin A (c), anti-filamin B (d), or anti-caveolin-1 (e) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. C: CEM fractions were solublized in immunoprecipitation (Ippt) buffer and immunoprecipitated with anti-annexin A2 antibody. Resulting immunobeads were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine (a) or anti-annexin A2 (b) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. D: EC were treated with no siRNA (control), scramble siRNA, annexin A2 siRNA, or protein S100-A10 siRNA for 48 h. EC lysates were obtained and run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-annexin A2 (a), anti-protein S100-A10 (b), or anti-actin (c) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. E: percent inhibition of maximal HMW-HA-induced TER response in human EC with scramble, annexin A11, annexin A2, protein S100-A10, or annexin A2 + protein S100-A10 siRNA treatment. Silencing both annexin II and protein S100-A10 is required for maximal inhibition of HMW-HA-induced TER in EC.

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Staining, Fluorescence, SDS Page, Immunoprecipitation, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: CEM proteins upregulated with HMW-HA stimulation

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Binding Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Annexin A2 and protein S100-A10 regulation of HMW-HA-induced filamin A/B recruitment to CEM and human EC barrier enhancement. A: immunoblot analysis of HMW-HA-treated (100 nM, 5 min) or untreated human EC lysates from scramble siRNA, annexin A2 siRNA, protein S100-A10 siRNA, or annexin A2 + protein S100-A10 siRNA transfection using anti-filamin A (a), anti-filamin B (b), or anti-caveolin-1 (c) antibody. Experiments were performed in triplicate, each with similar results, and representative data are shown. Silencing annexin A2 and protein S100-A10 blocks HMW-HA-induced filamin A and filamin B recruitment to CEM. B: EC were treated with no siRNA (control), scramble siRNA, filamin A siRNA, or filamin B siRNA for 48 h. EC lysates were obtained and run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-filamin A (a), anti-filamin B (b), or anti-actin (c) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. C: percent inhibition of maximal HMW-HA-induced TER response. EC were plated on gold microelectrodes and treated with scramble siRNA (control), annexin A11 siRNA, filamin A siRNA, filamin B siRNA, or filamin A + filamin B siRNA for 48 h. After EC were serum-starved for 1 h, 100 nM HMW-HA was added. Values represent pooled TER data ± SE at 30 min after addition of agonist from 3 independent experiments.

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Western Blot, Transfection, SDS Page, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Analysis of HMW-HA-mediated human EC actin cytoskeletal rearrangement. A: immunofluorescent images of HMW-HA-induced EC cortical actin rearrangement. EC were serum-starved for 1 h and either left untreated (control) or treated with 100 nM HMW-HA for 15 min or pretreated with 5 mM MβCD for 1 h and then with 100 nM HMW-HA for 15 min. Cells were then fixed and stained with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (to visualize F-actin) and analyzed using fluorescent microscopy. Observations are representative of the entire cell monolayer and were reproduced in multiple independent experiments (n ≥ 3 for each condition). B: immunofluorescent images of HMW-HA-induced EC cortical actin rearrangement as described in A. Human EC were treated with scramble siRNA (control), CD44 siRNA, caveolin-1 siRNA, annexin A2 siRNA, or protein S100-A10 siRNA for 48 h. After EC were serum-starved for 1 h, 100 nM HMW-HA was added for 15 min.

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Staining, Microscopy

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Quantitation of HMW-HA-induced cortical actin formation in human EC

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Quantitation Assay, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Proposed model of HMW-HA-induced vascular integrity. HMW-HA binding to CD44s in caveolin-enriched microdomains (CEM) in human EC (1) induces annexin A2 tyrosine phosphorylation and annexin A2/protein S100-A10 translocation to CEM (2). Annexin A2 and protein S100-A10 are crucial for subsequent HMW-HA-induced recruitment of filamin A and filamin B to CEM (3), actin cytoskeletal reorganization (cortical actin formation) (4), and EC barrier enhancement (5).

Article Snippet: Rabbit anti-von Willebrand factor (vWF) VIII antibody was purchased from Chemicon (Temecula, CA); rat anti-CD44 (IM-7, common domain) antibody from BD Biosciences (San Diego, CA); rabbit anti-caveolin-1 antibody from Cell Signaling Technology (Danvers, MA); mouse anti-annexin II antibody, rabbit anti-protein S100-A10 antibody, mouse anti-filamin A antibody, and goat anti-filamin B antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-fibrillarin, anti-cyclooxygenase (COX) IV, anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b), and anti-Golgi reassembly stacking protein (GRASP65) antibodies from Abcam (Cambridge, MA); recombinant human IL-2, goat anti-mouse ACE ectodomain antibody, and goat anti-vascular endothelial (VE)-cadherin antibody from R & D Systems (Minneapolis, MN); mouse anti-β-actin antibody, LPS, and OptiPrep from Sigma; secondary horseradish peroxidase-labeled antibodies from Amersham Biosciences (Piscataway, NJ); and DOTAP and DOPE from Avanti Polar Lipids (Alabaster, AL).

Techniques: Binding Assay, Translocation Assay

Journal: Microbial Cell Factories

Article Title: Evaluation of GFP reporter utility for analysis of transcriptional slippage during gene expression

doi: 10.1186/s12934-018-0999-3

Figure Lengend Snippet: Evaluation of frameshifted (− 1) gfp fusion’s usefulness to serve as transcriptional slippage reporter. a Details of the nucleotide sequences of the proximal part of gfp fusions. The names of plasmid constructs’ and reporter gene variants, nucleotide sequences with possible sites of insertion slippage events (indicated by arrows), and primary and slippage-induced amino acid sequences are shown. Reading frame of genes (− 1 or 0) is reflected in their names as suffix 0 or − 1, respectively. Actual (black, below) and native (green, above) amino acid numbering of the GFPA 6 0 hybrid is provided. b Relative fluorescence level of indicated GFP hybrids. All measurements were performed in three to five duplicate repetitions. Error bars represent standard deviations. c Western blotting of the total cell extracts of hybrids shown in b and GFP immunodetection with ECL chemiluminescence system. d Extraordinary slippage properties of T7 RNAP in contrast to E. coli RNAP. ER2566 cells with appropriate pET24a-(lanes 1–5) or pBAD24-derived plasmids (lanes 7–10) carrying − 1 frameshifted gfp genes were induced with 1 mM IPTG or 0.1% l -arabinose at 37 °C, respectively. Aliquot of cell extracts were western blotted and immunodetected with anti-GFP primary antibodies. Below the molecular weight of the GFP protein products in kDa are provided

Article Snippet: For fluorescence experiments E. coli ER2566 T7 phage RNAP IPTG inducible strain (F − λ − fhu A2 [lon] omp T lacZ ::T7 gene 1 gal sulA 11 Δ( mcr C- mrr )114::IS 10 R( mcr -73::miniTn 10 -Tet S )2 R( zgb -210::Tn 10 -Tet S ) end A1 [dcm]) was used (New England Biolabs, Ipswich, USA).

Techniques: Plasmid Preparation, Construct, Fluorescence, Western Blot, Immunodetection, Derivative Assay, Molecular Weight

Journal: Microbial Cell Factories

Article Title: Evaluation of GFP reporter utility for analysis of transcriptional slippage during gene expression

doi: 10.1186/s12934-018-0999-3

Figure Lengend Snippet: Analysis of polyA- and polyT-based gfp mRNA levels and stability. a Top panel: total mRNA was extracted from E. coli ER2566 bearing pBADmingfpA 6 0, pBADmingfpT 6 0 and pETmingfpA 6 0 plasmids after 10 min (lanes 1–3) and 30 min expression (lanes 4–6), respectively, generated by E. coli (lanes 1, 2, 4 and 5) or T7 phage RNAP (lanes 3 and 6). The membrane was probed with biotin labelled gfp DNA. Lane 7, no- gfp control bacteria. Arrow—prominent degradation product. Bottom panel: ethidium bromide-stained 16S rRNA are shown as loading control. b gfp transcripts stability. Rifampicin was added to an exponential culture of E. coli ER2655 growing in LB medium after 10 min of 0.1% L-arabinose induction. The mRNA levels were determined by RT-qPCR, using stable 16S rRNA as the internal standard. The circles represent gfpA 6 0 and triangles represent gfpT 6 0 transcripts. All mRNA levels were normalized to 1 at time = 0 (the points overlap). The data were fitted to an exponential decay. Standard error bars from three determinations are shown

Article Snippet: For fluorescence experiments E. coli ER2566 T7 phage RNAP IPTG inducible strain (F − λ − fhu A2 [lon] omp T lacZ ::T7 gene 1 gal sulA 11 Δ( mcr C- mrr )114::IS 10 R( mcr -73::miniTn 10 -Tet S )2 R( zgb -210::Tn 10 -Tet S ) end A1 [dcm]) was used (New England Biolabs, Ipswich, USA).

Techniques: Expressing, Generated, Staining, Quantitative RT-PCR

Journal: Molecular Metabolism

Article Title: DNA damage to β cells in culture recapitulates features of senescent β cells that accumulate in type 1 diabetes

doi: 10.1016/j.molmet.2022.101524

Figure Lengend Snippet: RNA-seq analysis reveals activation of the p53 transcriptional program during senescence in human islets . A) Overview of human islet DNA damage-induced senescence model. Islets isolated from a 44-year-old female donor (Donor 1) or from a 50-year-old female donor (Donor 2) were rested overnight and then divided into a total of 6 wells and cultured in the presence of vehicle (DMSO) or 50 μM bleomycin for 48 h (n = 3 biological replicates per group). The islets were then transferred to fresh drug-free media and cultured an additional 4 days prior to harvesting for RNA extraction and paired-end RNA-seq. B) Volcano plots of differentially expressed genes (fold-change cut-off and FDR <0.05) found indicating 1320 genes downregulated <0.5-fold and 142 genes upregulated >2-fold in bleomycin treated islets compared to controls from Donor 1 and 1139 genes downregulated and 480 genes upregulated from Donor 2. 645 genes were downregulated in common and 44 genes were upregulated in common in bleomycin-treated islets from both donors. C) Significant KEGG pathway terms of common differentially expressed genes from Donor 1 and Donor 2. D) Plot of normalized expression levels (FPKM) of selected significantly downregulated proliferation and cell cycle genes from Donor 1 and Donor 2. E) Plot of normalized expression levels of selected significantly upregulated p53 target genes and early senescence gene LMNB1 from Donor 1 and Donor 2. F) Plot of normalized expression levels of selected islet hormone genes, islet cell identity and hormone processing genes from Donor 1 (black and red bars) and Donor 2 (grey and blue bars). Only genes with an asterisk were found to be significantly different in expression. G) Plot of normalized expression levels of BCL-2 family anti-apoptotic genes from Donor 1 and Donor 2. H) Plot of normalized expression of selected significantly downregulated/unchanged SASP genes (Down/No change), SASP genes that were significantly upregulated in both Donors (Up in both), and SASP genes significantly upregulated only in Donor 2 but not Donor 1 (Up in Donor 2). Unsupervised hierarchal clustering heatmap of SASP gene normalized expression values (FPKM). Legend shows Row Z-score values. V1, V2, V3 and B1, B2, B3 were Vehicle-treated or Bleomycin-treated biological replicates from Donor 1. V4, V5, V6 and B4, B5, B6 are Vehicle-treated or Bleomcyin-treated biological replicates from Donor 2. For all barchart plots, data shown are mean ± SD of the n = 3 biological replicates in the RNA-seq datasets per sample group for each donor.

Article Snippet: To induce DDR and senescence, islets were cultured in media containing 50 μM bleomycin (Medchem express) or vehicle (0.2% DMSO) controls for 48 h, followed by removal and culture in drug-free media for 4 or 5 days, as previously [ ].

Techniques: RNA Sequencing Assay, Activation Assay, Isolation, Cell Culture, RNA Extraction, Expressing

Journal: Molecular Metabolism

Article Title: DNA damage to β cells in culture recapitulates features of senescent β cells that accumulate in type 1 diabetes

doi: 10.1016/j.molmet.2022.101524

Figure Lengend Snippet: DDR and SASP activation in senescent human islets and the EndoC-βH5 human β cell line . A) Overview of experiment on human islets. Islets from four different adult donors (ages 55, 36, 32 and 52) were treated as indicated with vehicle (DMSO) or 50 μM bleomycin for 48 h and cultured in drug-free media for an additional 4 or 5 days and harvested for either Western blot (2 donors) or Luminex assays (2 donors). B) Western blot analysis of persistent DDR on vehicle (DMSO) and bleomycin-treated islets from a 55-year-old male and a 36-year-old female at day 4 post-drug removal. Data are means ± SD of n = 3 biological replicates. C) Luminex assays for indicated SASP factors in the conditioned media from islets treated as in (A) from a 32-year-old male donor and 52-year-old male donor at day 5 post-drug removal. Data are normalized to islet RNA content and are means ± SD of n = 3 biological replicates. D) Overview of experiment on human female fetal-derived EndoC-βH5 cells. Cells were treated as indicated with 35 μM bleomycin or vehicle (DMSO) for 48 h, and then harvested for assays or cultured for an additional 2-5 days in drug-free media. Western blotting was carried out on cells after the 48 h after treatment, qRT-PCR was carried out 2 days after drug removal and Luminex assays were carried out 5 days after drug removal. E) Western blot analysis of DDR and early senescence markers in EndoC cells treated as indicated after 48 h in drug-containing media or vehicle media. Data are means ± SD of n = 3 biological replicates. F) qRT-PCR analysis of senescence genes CDKN1A, CDKN2A , prosurvival gene BCL2L1 (encoding BCL-XL) and SASP gene CXCL8 (encoding IL-8) in EndoC cells treated as in (A) at day 2 post-drug removal. Data are means ± SD of n = 3 biological replicates. G) Luminex assays of indicated SASP factors in the conditioned media from EndoC cells treated as in (A) at day 5 post-drug removal. Data are normalized to viable cell counts and are means ± SD of n = 4 biological replicates. For all panels, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, two-tailed T-tests.

Article Snippet: To induce DDR and senescence, islets were cultured in media containing 50 μM bleomycin (Medchem express) or vehicle (0.2% DMSO) controls for 48 h, followed by removal and culture in drug-free media for 4 or 5 days, as previously [ ].

Techniques: Activation Assay, Cell Culture, Western Blot, Luminex, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular Metabolism

Article Title: DNA damage to β cells in culture recapitulates features of senescent β cells that accumulate in type 1 diabetes

doi: 10.1016/j.molmet.2022.101524

Figure Lengend Snippet: Impact of DNA damage-induced senescence on GSIS and insulin content in mouse β cell lines, human EndoC-βH5 cells and human islets . A) GSIS assays and total insulin content of MIN6 or NIT1 cells treated with vehicle or etoposide (2 μM or 0.5 μM, respectively) at 72 h post-treatment. Data are means ± SD of n = 4 biological replicates. B) GSIS assays and total insulin content of EndoC-βH5 cells treated with vehicle or bleomycin (35 μM) for 72 h to induce senescence and cultured for 2 days in fresh drug free media before harvest at the day 2 post-drug removal time-point. Data are means ± SD of n = 5 biological replicates for vehicle samples or n = 6 biological replicates for bleomycin samples. C) GSIS assays and total insulin content of human islets from the three indicated donors treated with vehicle or 50 μM bleomycin for 48 h and then cultured in drug free media for 5 days and harvested at day 5 post-drug removal. Data are means ± SD of n = 5 or 6 biological replicates for each donor experiment. For GSIS panels, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, Two-way ANOVAs. For Insulin content panels, ∗p < 0.05, ∗∗p < 0.005, two-tailed T-tests.

Article Snippet: To induce DDR and senescence, islets were cultured in media containing 50 μM bleomycin (Medchem express) or vehicle (0.2% DMSO) controls for 48 h, followed by removal and culture in drug-free media for 4 or 5 days, as previously [ ].

Techniques: Cell Culture, Two Tailed Test

Journal: PLoS ONE

Article Title: Elevation of Eosinophil-Derived Neurotoxin in Plasma of the Subjects with Aspirin-Exacerbated Respiratory Disease: A Possible Peripheral Blood Protein Biomarker

doi: 10.1371/journal.pone.0066644

Figure Lengend Snippet: Box plot of the eosinophil-derived neurotoxin expression levels in AERD and ATA groups as assessed by ELISA (Error bar: 25∼75 percentiles).

Article Snippet: From the identified candidate genes, we assessed the plasma levels of the eosinophil-derived neurotoxin using a quantitative Human Ribonuclease A2 ELISA Kit (Uscn Life Science Inc, Wuhan, CHINA).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: USP25 stabilizes STAT6 to promote IL-4-induced macrophage M2 polarization and fibrosis

doi: 10.7150/ijbs.99345

Figure Lengend Snippet: USP25 show high expression in M2 macrophages and fibrotic tissues. (A) Quantitative PCR analysis of USP family markers. (B) Western blot analysis of USP25 in homogenates from wild-type BMDM stimulated with IL-4 or vesicles. (C) Western blot analysis of USP25 in the lungs of mice following BLM induction. (D) Results for immunostaining of USP25 in BLM-induced lung sections. Scale bar, 20μm. (E) Western blot analysis of USP25 in the livers of mice following BDL induction. (F) Results for immunostaining of USP25 in BDL-induced liver sections. Scale bar, 100μm. (G) Results for co-immunostaining of USP25 and CD68 (macrophage marker) in the lung sections from patients with IPF and healthy subjects. Scale bar, 100μm. A total of nine IPF patients and nine control subjects were analyzed. Scatter plot indicates the USP25 + /CD68 + cell count (numbers/mm 2 ) in the lung sections from IPF patients and control subjects; each dot represents a patient. BLM, bleomycin; BDL, bile duct ligation; IPF, idiopathic pulmonary fibrosis. ****P < 0.0001

Article Snippet: Bleomycin hydrochloride (Cat: HY-17565A) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining, Marker, Control, Cell Counting, Ligation

Journal: International Journal of Biological Sciences

Article Title: USP25 stabilizes STAT6 to promote IL-4-induced macrophage M2 polarization and fibrosis

doi: 10.7150/ijbs.99345

Figure Lengend Snippet: USP25 deficiency protects mice against BLM-induced lung injury and fibrosis. (A) Histological analysis of the severity of lung fibrosis in mice after BLM induction. Representative results for H&E (top), Masson (center), and Sirius red (bottom) staining. Scale bar, 2000μm. (B) Bar graph showing the semiquantitative Ashcroft scores for the severity of fibrosis. (C) Bar graph showing the quantification of hydroxyproline content in the lungs of mice after BLM induction. (D) Western blot analysis of the fibrotic marker fibronectin. (E) RT-qPCR analysis of α-SMA and collagen Ⅰ . (F) Results for immunostaining of the fibrotic markers α-SMA, collagen Ⅰ and fibronectin in BLM-induced lung sections. Scale bar, 100μm. (G) Body weight changes during the course of BLM-induced fibrosis. BLM, bleomycin; Col I, collagen I; Fib, fibronectin. The data are represented as the means ± SD. *p < 0.05 and ****P < 0.0001.

Article Snippet: Bleomycin hydrochloride (Cat: HY-17565A) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Staining, Western Blot, Marker, Quantitative RT-PCR, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: USP25 stabilizes STAT6 to promote IL-4-induced macrophage M2 polarization and fibrosis

doi: 10.7150/ijbs.99345

Figure Lengend Snippet: USP25 deficiency attenuates M2 polarization in macrophages. (A) Western blot analysis of Arg 1 in the lung homogenates. (B) RT-qPCR results for analysis of Arg 1 expression in the lung. (C) Flow cytometry analysis of M2 macrophages in liver-derived macrophages. (D) RT-qPCR for analysis of Arg 1 , YM1 , Fizz1 , and VEGF expression in liver derived macrophages. (E) Flow cytometry analysis of CD206 expression in BMDMs following IL-4 stimulation. (F) Western blot analysis of Arg 1 in BMDMs after IL-4 induction. (G) RT-qPCR for analysis of Arg 1 and YM1 expression in the BMDMs after IL-4 induction. (H) Flow cytometry analysis of CD11c expression in BMDMs following LPS stimulation. (I) RT-qPCR analysis of IL-6 expression in the BMDMs after LPS induction. (J) The schematic representation for the macrophage adoptive transfer experiments. IL-4-stimulated WT M2 BMDMs were selectively transferred into both WT and USP25 -/- mice that had been pretreated with clodronate liposomes, via tail vein injection on the seventh day post BLM induction. (K) The severity of pulmonary fibrosis in WT and USP25 -/- mice after depletion of macrophages. Right: a bar graph figure showing the semiquantitative Ashcroft scores for the severity of pulmonary fibrosis. (L) Western blot analysis of fibronectin in the lungs of macrophage-depleted mice after BLM induction. (M) Results for adoptive transfer of WT macrophages into WT and USP25 -/- mice following BLM induction. Right: the semiquantitative Ashcroft scores relevant to the severity of fibrosis. (N) Western blot analysis of fibronectin expression. Arg 1, arginase 1; YM1, chitinase 3-like 3; Fizz1, found in inflammatory zone; VEGF, vascular endothelial growth factor; BMDMs, bone marrow-derived macrophages; BLM, bleomycin; Fib, fibronectin. The data are represented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001.

Article Snippet: Bleomycin hydrochloride (Cat: HY-17565A) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry, Derivative Assay, Adoptive Transfer Assay, Liposomes, Injection

Journal: International Journal of Biological Sciences

Article Title: USP25 stabilizes STAT6 to promote IL-4-induced macrophage M2 polarization and fibrosis

doi: 10.7150/ijbs.99345

Figure Lengend Snippet: USP25 deficiency suppresses M2 macrophages TGF-β signaling. (A) RNA seq analysis results of WT and USP25 -/- mice IL-4 stimulated BMDMs. Differential gene clustering heat map. (B) RNA seq analysis results of alveolar macrophages from WT and USP25 -/- mice after BLM induction. KEGG pathway analysis bubble diagram. (C) ELISA results for IL-4, IL-13 and TGF-β1 levels in the BALF. (D) RT-qPCR results for Tgfbr1 and Tgfbr2 expression in the lungs after BLM induction. (E) Co-immunostaining of F4/80 and TGF-β1 in the lung sections. Scale bar, 50 μm. (F) ELISA results for TGF-β1 level in the mice serum after BDL induction. (G) RT-qPCR results for Tgfbr1 and Tgfbr2 expression in the livers after BDL induction. (H) ELISA results for TGF-β1 level in the cell culture medium. (I) RT-qPCR results for TGF-β1 expression in the BMDMs stimulated with IL-4. BALF, bronchoalveolar lavage fluid; BLM, bleomycin; TGF-β1, transforming growth factor β1. The data are represented as the means ± SD. **p < 0.01, ***p < 0.001 and ****P < 0.0001.

Article Snippet: Bleomycin hydrochloride (Cat: HY-17565A) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Immunostaining, Cell Culture

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Downregulation of TAP2 reduces the surface levels of peptide-HLA complexes in lung cancer cells. A Outline of the experimental strategy used to measure changes in the levels of selected HLA-peptide complexes in lung cancer cells with or without TAP1 and/or TAP2 downregulation using flow cytometry. B-G A549 lung cancer cells were transfected with scrambled/control siRNA or with TAP1 and/or TAP2 targeting siRNAs and left untreated (black histograms) or stimulated with IFNγ (blue histograms) or IFNγ + TNFα (red histograms). Panels B-D show the surface levels of HLA-A2-HER2 369-377 , and panels E–G show the levels of HLA-A2-MAGE3 271-279 . H–K A549 TAP2 knockout (KO) cells were transfected with an empty vector (KO + EV) or with a vector containing FLAG-TAP2 (KO + TAP2) and left untreated (black) or stimulated with cytokines IFNγ (blue) or IFNγ + TNFα (red). I, Graphs showing the levels of TAP2 protein analyzed by flow cytometry with or without TAP2 gene elimination, J-K, Surface levels of HLA-A2-HER2 369-377 or HLA-A2-MAGE3 271-279 in A549 cells with or without TAP2 gene elimination. For panels B-G and I-K, an isotype control antibody (IgG) was used as a background signal reference. Data presented as the mean ± s.d.; *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. For panels B-G, Scr transfected cells were used as a control for statistical comparison, and for I-K parental wild type (WT) cells were compared with TAP2 deleted cells (KO) and TAP2 deleted plus EV (KO + EV) or with TAP2 deleted with posterior TAP2 transfection (KO + TAP2). MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; Tx, treatment; ns, not significant

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Transfection, Control, Knock-Out, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: TAP2 downregulation protects cancer cells from tumor antigen-specific CD8 T-cell killing. A , B Schema and outline of the experimental strategy used to measure tumor antigen-specific killing of lung cancer cells by cognate CD8 + T-cells using flow cytometry, LDH release and MTT assay. C-L A549 lung cancer cells were transfected with scrambled siRNA or with TAP1 and/or TAP2 targeting siRNAs; and left untreated (black) or stimulated with IFNγ (blue) or IFNγ + TNFα (red). After treatment, target lung tumor cells were co-cultured with effector (CD8 + T-cells) cells in the ratios of 1:0, 1:2 and 1:5, respectively. Panel C shows the flow cytometry gating strategy to assess cell apoptosis in cancer cell/T-cell co-cultures using the markers CD3 (for CD8 + T-cells), EpCAM (for tumor cells) and Annexin V. Panels D-F show the percentage of EpCAM + and Annexin V + apoptotic cancer cells. Panels G-I show the percent of LDH release, and panels J-L represent the cellular viability using MTT assay. M-P A549 TAP2 knockout (KO) cells transfected with empty vector (KO + EV) or FLAG-TAP2 (KO + TAP2) and left untreated or stimulated with IFNγ or IFNγ + TNFα were co-cultured with tumor antigen specific CD8 + T-cells at different target cell (tumor): effector (CD8 + T-cell) cell ratios. An isotype control antibody (IgG) was used as a background signal reference. Data are presented as the mean ± s.d.; * , p < 0.05; ** , p < 0.01; *** , p < 0.001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. For panels D-L, Scr transfected cells were used as a control for statistical comparison, and for N-P wild type (WT) compared with KO and KO + EV or with KO + TAP2 cells. si, siRNA; scr, scrambled; E, effector CD8 T cells; T, target tumor cells; Tx, treatment; ns, not significant; WT, wild type. See also supplementary Fig. S5-S6

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, MTT Assay, Transfection, Cell Culture, Knock-Out, Plasmid Preparation, Control, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Downregulation of TAP2 alters intracellular immunomodulatory pathways via SOCS1 upregulation and TAP2 downregulation in human NSCLC is due to epigenetic changes. A A549 cells were transfected with scrambled siRNA or with TAP2 targeting siRNAs followed by targeted transcriptomic analysis. Graph shows differentially expressed genes in TAP2 silenced versus control cells ranked based on low (blue) to high (red) transcript expression. B , C SOCS1 protein expression by flow cytometry in A549 cells. B, Cells were transfected with scrambled siRNA or with TAP2 targeting siRNAs C, Cells were transfected with empty vector or with the full-length TAP2 including a FLAG octapeptide. D-G, A549 cells were transfected with TAP1/2 siRNAs as indicated in 3A and stimulated with IFNγ ( D , E ) or with IFNγ plus TNFα ( F , G ). Volcano plots representing differential expression of IFNγ pathway signature genes. H A549 cells were transfected with TAP1/2 siRNAs followed by stimulation with IFNγ plus TNFα. Phosphoprotein levels were measured and ranked based on low (blue) to high (red) expression in TAP2 silenced vs control cells. I-M Normal adjacent to tumor (NAT) and lung tumor tissues surgically resected from NSCLC patients with low TAP2 protein levels. I Schematic of the strategy for obtaining and analyzing single cell preparations. J Volcano plot showing the differential gene expression of paired lung cancer relative to NAT using RNA sequencing. K Fold change of the mRNA expression of TAP2 and SOCS1 in tumor samples relative to NAT, L Heatmap of ATAC-seq analysis representing chromatin accessibility in the TAP2 promoter region. M Predicted transcription factor (TF) binding sites with the highest affinity scores (log2 count) for the TAP2 promoter region. Data are presented as the mean ± s.d. ** , p < 0.01 determined by two-tailed unpaired Student’s t-test. FC, fold change; MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; TFs, transcription factors. See also supplementary Fig. S7

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Control, Expressing, Flow Cytometry, Plasmid Preparation, FLAG-tag, Quantitative Proteomics, Gene Expression, RNA Sequencing, Binding Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Myeloid cell-derived IL-4 reduces TAP2 expression via epigenetic remodeling in lung cancer. A TCGA NSCLC cohort analysis of TAP2 expression stratified by the median IL-4 mRNA levels. B A549 cells were treated with IL-4 for 0-24 h and TAP2 protein levels were measured using flow cytometry. C-I A549 cells were treated with IL-4, IL-4 + IFNγ or IL-4 with IFNγ + TNFα. C, TAP2 protein levels measured by flow cytometry, D, SOCS1 protein levels by flow cytometry, E, Surface levels of HLA-A2-HER2 369-377 complexes and F, HLA-A2-MAGE3 271-279 complexes measured by flow cytometry. G-I A549 cells (target) were treated with IL-4 ± IFNγ + TNFα and incubated with effector tumor antigen-specific CD8 + T-cells to measure, G, cancer cell killing by Annexin V positivity, H, LDH release and I, cellular viability using MTT assay. J-M Fluorescence images and signal measurement from multiplexed spatial analysis of protein and mRNA transcripts in NSCLCs from Cohorts #1 and #2. J-K, Representative captions of cytokeratin (CK, green), IL-4 mRNA (red), CD11b mRNA (white), TAP2 protein (yellow) and nuclei (blue). L, expression levels of IL-4 mRNA measured selectively in CK + tumor cells or in CD11b + myeloid cells. M, expression levels of TAP2 protein in CK + tumor cells stratified by the median IL-4 mRNA expressed in CD11b + myeloid cells. N-R A549 cells were treated with IL-4 ± IFNγ + TNFα and analyzed using ATAC-seq. N, ATAC-seq promoter peak enrichment values of the TAP2 gene. O, comparative analysis of the TAP2 ATAC-seq promoter peak enrichment after different cytokine treatments in A549 cells. P, heatmap of ATAC-seq analysis representing chromatin accessibility in the TAP2 promoter region after cytokine treatments. Q, promoter peak enrichment values of the SOCS1 gene. R, comparative analysis of the SOCS1 ATAC-seq promoter peak enrichment after cytokine treatments. Promoter regions were considered as the DNA sequences between the gene Transcription Start Site (TSS, + 3 kb) and Transcription End Site (TES, -3 kb). S-U Autologous single cell suspension cultures including cancer and immune cells from primary NSCLC tissues stimulated with IFNγ + TNFα followed by incubation with IL-4Rα (αIL-4R) or PD-1 blocking antibodies (αPD-1). S, levels of CD8 + /CD25 + T-cells measured by flow cytometry. T, percentage of EpCAM + /Annexin V + apoptotic cancer cells in autologous cell suspensions. U, percentage of EpCAM + /Annexin V + apoptotic cancer cells in autologous cell suspensions with selective elimination of CD3 + T-cells (TECS). Isotype (IgG) was used as a background signal control. Data are presented as the mean ± s.d. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. B-I and S-U, untreated cells were used as a control. E, effector CD8 + T-cells; FPKM, fragments per kilobase million; ns, not significant; MFI, mean fluorescent intensity; T, tumor; S, stroma. See also supplementary Figs. S8-S12

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Incubation, MTT Assay, Fluorescence, Suspension, Blocking Assay, Control, Two Tailed Test

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Restoration of TAP2 protein expression induces tumor cell surface antigenicity in lung cancer cells. A Schematic showing the strategy for high throughput screening of pharmacologic agents using TAP2 and PD-L1 immunostaining in A549 cells. B-D Representative plots showing the upregulation of TAP2 protein (red) or PD-L1 protein (blue) or both (green) in A549 cells treated with individual compounds from the Pharmakon 1600 library (B), Enzo 640 FDA library (C) and Enzo epigenetic library (D). The scores for each compound were calculated as TAP2 protein level fold change and normalized to the vehicle control (DMSO) treated group. E Summary of TAP2 protein inducer compounds selected based on representation of all 3 libraries. F-G Dose–response curves to determine EC 50 values for TAP2 protein upregulation in A549 cells using selected compounds shown in E. H Experimental outline for SAHA/Vorinostat treatment and analysis of A549 cells. I Levels of TAP2 protein measured by flow cytometry. J surface HLA-A2-HER2 369-377 complexes and K surface HLA-A2-MAGE3 271-279 levels measured by flow cytometry. L Levels of apoptotic cancer cell death (Annexin V staining) and cell viability (LDH release and MTT assay) of parental A549 cells co-incubated with tumor antigen-specific effector CD8 + T-cells using different effector to target cell ratios (1:0, 1:2 and 1:5) with or without treatment with 3.5 µM SAHA. An isotype control antibody (IgG) was used as a background signal reference. Data are presented as the mean ± s.d.; *p < 0.05; **p < 0.01; ****, p < 0.0001 determined by two-tailed unpaired Student’s t-test. FC, fold change; ns, non-significant; No-Tx, no treatment; MFI, mean fluorescent intensity; TKI, tyrosine kinase inhibitor. See also supplementary Fig. S13

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, High Throughput Screening Assay, Immunostaining, Control, Flow Cytometry, Staining, MTT Assay, Incubation, Two Tailed Test

Journal: Cell Reports

Article Title: Activation of the SARS-CoV-2 Receptor Ace2 through JAK/STAT-Dependent Enhancers during Pregnancy

doi: 10.1016/j.celrep.2020.108199

Figure Lengend Snippet: Ace2 Activation in Mammary Tissue during Lactation (A) Ace2 and Stat5 mRNA levels in mammary tissue from non-parous and L10 wild-type mice were measured by qRT-PCR and normalized to Gapdh levels. The Cish gene served as a control. Results are shown as the means ± SEM of independent biological replicates (n = 3). ANOVA was used to evaluate the statistical significance of differences between virgin and lactation mice. ns, not significant. (B and D) mRNA levels of genes in mouse mammary tissue at different stages of pregnancy and lactation were measured by RNA-seq. Day 6 of pregnancy (p6), day 13 of pregnancy (p13), day 18 of pregnancy (p18), L1, and L10. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to evaluate the statistical significance of differences in p6 and other developmental stages. *p < 0.05, **p < 0.001, ****p < 0.00001. (C) ACE2 protein level was analyzed by western blot in mammary tissues of virgin mice, as well as pregnant and lactating mice.

Article Snippet: mouse Ace2 probe (Mm01159006_m1) , Thermo Fisher scientific , Cat# 4351370.

Techniques: Activation Assay, Quantitative RT-PCR, Control, RNA Sequencing, Western Blot

Journal: Cell Reports

Article Title: Activation of the SARS-CoV-2 Receptor Ace2 through JAK/STAT-Dependent Enhancers during Pregnancy

doi: 10.1016/j.celrep.2020.108199

Figure Lengend Snippet: Occupation of a Candidate Ace2 Enhancer during Lactation (A and B) ChIP-seq data for STAT5, STAT3, GR, NFIB, MED1, and histone markers H3K27ac and H3K4me3 provided structural information about the locus including the Ace2 gene in L10 mammary tissue. Solid arrows indicate the orientation of genes. Black bars indicate GAS motifs (STAT binding sites). Orange shades indicate candidate regulatory elements.

Article Snippet: mouse Ace2 probe (Mm01159006_m1) , Thermo Fisher scientific , Cat# 4351370.

Techniques: ChIP-sequencing, Binding Assay

Journal: Cell Reports

Article Title: Activation of the SARS-CoV-2 Receptor Ace2 through JAK/STAT-Dependent Enhancers during Pregnancy

doi: 10.1016/j.celrep.2020.108199

Figure Lengend Snippet: Assembly of the Ace2 Enhancer during Pregnancy and Lactation (A and B) ChIP-seq data for transcription factors and histone markers provided enhancer structures of the Ace2 locus in mammary tissues at p6, L1, L10, and I24 after L10. Solid arrows indicate the orientation of genes. Orange and blue shades indicate regulatory elements. The Stat1 gene served as a ChIP-seq control.

Article Snippet: mouse Ace2 probe (Mm01159006_m1) , Thermo Fisher scientific , Cat# 4351370.

Techniques: ChIP-sequencing, Control

Journal: Cell Reports

Article Title: Activation of the SARS-CoV-2 Receptor Ace2 through JAK/STAT-Dependent Enhancers during Pregnancy

doi: 10.1016/j.celrep.2020.108199

Figure Lengend Snippet: STAT5 Is Required for Ace2 Expression and Enhancer Activation in Mammary Epithelium (A) Ace2 mRNA levels in mouse mammary tissue from wild-type and Stat5 mutant mice were measured by RNA-seq ( Yamaji et al., 2013 ). One-way ANOVA was used to evaluate the statistical significance of differences in WT and mutants. ***p < 0.0001. (B) STAT5 enhancers were profiled using ChIP-seq data from wild-type and Stat5 mutant tissue.

Article Snippet: mouse Ace2 probe (Mm01159006_m1) , Thermo Fisher scientific , Cat# 4351370.

Techniques: Expressing, Activation Assay, Mutagenesis, RNA Sequencing, ChIP-sequencing

Journal: Cell Reports

Article Title: Activation of the SARS-CoV-2 Receptor Ace2 through JAK/STAT-Dependent Enhancers during Pregnancy

doi: 10.1016/j.celrep.2020.108199

Figure Lengend Snippet:

Article Snippet: mouse Ace2 probe (Mm01159006_m1) , Thermo Fisher scientific , Cat# 4351370.

Techniques: Recombinant, Software