Journal: The Journal of Biological Chemistry

Article Title: Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians

doi: 10.1016/j.jbc.2022.102455

Figure Lengend Snippet: Interaction of bryoporin with model lipid membranes. A , comparison of hemolytic activity of bryoporin, EqtII, and FraC at pH 7.4. The d ashed lines are fits of the logistic function and the midpoint was used to estimate EC 50 ( Table 1 ). B , hemolytic activity of bryoporin at different pH values. ( A and B ) show relative hemolysis ( i.e. , change in absorbance at 630 nm normalized to 1 after 20 min). Each measurement was repeated three times. Points represent mean value ± SD. C , SPR measurements of 500 nM bryoporin binding to lipid bilayers of various lipid compositions. The inset shows zoomed area, as marked. D , kinetics of calcein release from 20 μM LUVs of different lipid compositions induced by 100 nM bryoporin. The bold central line is an average of three measurements, shaded area shows SD. E , bryoporin binding to HeLa cells. 5 μg/ml of bryoporin was added to nontreated (control, left panel ) cells or cells pretreated with 20 mM MβCD ( central panel ) or 1.7 unit/ml SMase ( right panel ). Bryoporin was detected by fluorescently labeled antibody. Differential interference contrast images corresponding to each panel are shown below. The scale bar represents 20 μm. LUV, large unilamellar vesicle; SPR, surface plasmon resonance.

Article Snippet: Cells grown on glass coverslips were pretreated with 20 mM MβCD (Cyclolab), 1.7 unit/ml SMase from Staphylococcus aureus (Sigma), or DMEM/F12 medium without fetal calf serum as control at 37 °C for 30 min.

Techniques: Comparison, Activity Assay, Binding Assay, Control, Labeling, SPR Assay

Journal: Journal of Pharmaceutical Analysis

Article Title: Effects and translatomics characteristics of a small-molecule inhibitor of METTL3 against non-small cell lung cancer

doi: 10.1016/j.jpha.2023.04.009

Figure Lengend Snippet: In vivo effects and regulation of STM2457 on programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC). (A) The modeling and drug administration scheme of a pulmonary metastasis mouse model. (B) In the lung sections of a pulmonary metastasis mouse model, the effect of STM2457 on NSCLC progression and the regulation of STM2457 on the expression of both methyltransferase-like 3 (METTL3) and PD-L1 in vivo were evaluated using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining, respectively. HP: hydroxypropyl.

Article Snippet: In in vivo experiments, STM2457 was dissolved in a 20% ( m / V ) 2-hydroxypropyl-beta-cyclodextrin (Glpbio, Montclair, CA, USA) vehicle as the working solution with a concentration of 5 mg/mL.

Techniques: In Vivo, Expressing, Staining, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Cholesterol restricts lymphotoxin β receptor-triggered NF-κB signaling

doi: 10.1186/s12964-019-0460-1

Figure Lengend Snippet: Cholesterol replenishment rescues the effects of MβCD treatment on LTβR signaling and internalization. a Schematic description of cholesterol replenishment experiments. b Lysates of A549 cells treated as depicted in a were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control. Graphs depict densitometric analysis for the indicated proteins from Western blotting (protein levels normalized to vinculin). Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n = 5; ns - P > 0.05; * P ≤ 0.05; *** P ≤ 0.001 by ANOVA test. c Immunofluorescence staining of ligand-bound LTβR and EEA1 in A549 cells treated as depicted in A except Step 4, where cells were fixed and stained instead of cell lysis. Insets: magnified views of boxed regions in the main images. Scale bars, 20 μm. d Analysis of integral intensity and the number of LTβR- and EEA1-positive vesicles in cells treated as in C. Values are presented as fold change versus controls - vehicle-treated cells marked as a black line, set as 1. Data represent the means ± SEM, n = 3. ns - P > 0.05; * P ≤ 0.05; ** P ≤ 0.01 by one sample t-test

Article Snippet: Following stimulation with LTβR agonist was carried out in the absence of filipin for next 0.5 h and 1 h. Acute cholesterol depletion was performed using MβCD (C4555, Merck) at a concentration of 5 mM (short treatments – 0.5, 1 or 4 h) or 2.5 mM (long treatment – 6, or 8 h).

Techniques: Western Blot, Immunofluorescence, Staining, Lysis

Journal: Cell Communication and Signaling : CCS

Article Title: Cholesterol restricts lymphotoxin β receptor-triggered NF-κB signaling

doi: 10.1186/s12964-019-0460-1

Figure Lengend Snippet: Cholesterol depletion by MβCD enhances LTβR-triggered activity of the NF-κB pathway and reduces internalization of the ligand-bound receptor. a, b Lysates of A549 cells preincubated for 1 h with MβCD or vehicle and stimulated for 0.5, 1 or 4 h with LTβR agonist (Ago) were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control. Graphs show densitometric analysis for the indicated proteins from Western blotting (protein levels normalized to vinculin). Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n = 6 (a), n = 4 (b); ns - P > 0.05; * P ≤ 0.05; ** P ≤ 0.01 by one sample t-test (in grey) and Student’s t - test (in black). c Immunofluorescence staining of ligand-bound LTβR and EEA1 in A549 cells upon 0.5 h stimulation with LTβR agonist in cells preincubated with vehicle (Veh.) or MβCD. Insets: magnified views of boxed regions in the main images. Scale bars, 20 μm. d Analysis of integral intensity and the number of LTβR- and EEA1-positive vesicles in cells treated as in B. Values are presented as fold change versus control - vehicle-treated cells marked as a black line, set as 1. Data represent the means ± SEM, n = 3. ns - P > 0.05; * P ≤ 0.05 by one sample t-test

Article Snippet: Following stimulation with LTβR agonist was carried out in the absence of filipin for next 0.5 h and 1 h. Acute cholesterol depletion was performed using MβCD (C4555, Merck) at a concentration of 5 mM (short treatments – 0.5, 1 or 4 h) or 2.5 mM (long treatment – 6, or 8 h).

Techniques: Activity Assay, Western Blot, Immunofluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Cholesterol restricts lymphotoxin β receptor-triggered NF-κB signaling

doi: 10.1186/s12964-019-0460-1

Figure Lengend Snippet: Cholesterol depletion enhances binding between LTβR and modified forms of TRAF2 and NEMO. a Western blot analysis of co-immunoprecipitates of anti-LTβR (IP:LTβR) and control antibodies (IP:IgG) from extracts of A549 cells stimulated with LTα1β2 for 0.5 h upon 1 h preincubation in medium containing either MβCD or vehicle. Antibodies against LTβR, TRAF2 were used for blotting. Input represents 10% of the lysates used for IP. h. e. – high exposure. Graph depicts the analysis of TRAF2 abundance (the main and modified forms of the protein) in LTβR co-immunoprecipitates upon stimulation with LTα1β2. The ratio of co-immunoprecipitated TRAF2 to immunoprecipitated LTβR was quantified. Data were normalized to the TRAF2-LTβR ratio in cells not treated with MβCD, which was assigned a value of 1. Data represent the means ± SEM, n = 3. ns - P > 0.05; * P ≤ 0.05 by one sample t-test. b Lysates of A549 cells preincubated for 1 h with MβCD and then stimulated or not for 0.5 h with LTα1β2 in the presence or the absence of MβCD were analyzed by Western blotting with antibodies against NEMO. h. e. – high exposure. Vinculin was used as a loading control. c Lysates of A549 cells preincubated for 4 h with TAK-243 or vehicle, treated or not for following 1 h with MβCD and then stimulated for 0.5 h with LTα1β2 were analyzed by Western blotting with antibodies against NEMO. h. e. – high exposure. Vinculin was used as a loading control. d Western blot analysis of immunoprecipitation performed as in A. Antibodies against LTβR and NEMO were used for blotting. Input represents 5% of lysates used for IP. Graph shows the abundance of modified NEMO in LTβR immunoprecipitates. Asterisk marks an unspecific band recognized by anti-LTβR antibody. Quantification as in A. Data represent the means ± SEM, n = 3. ns - P > 0.05; * P ≤ 0.05 by one sample t-test. e Lysates of A549 cells transfected with two control (Ctrl) or two TRAF2-targeting siRNAs and stimulated with Ago for 0.5 h were analyzed by Western blotting with antibodies against the indicated proteins. h. e. – high exposure. Vinculin was used as a loading control

Article Snippet: Following stimulation with LTβR agonist was carried out in the absence of filipin for next 0.5 h and 1 h. Acute cholesterol depletion was performed using MβCD (C4555, Merck) at a concentration of 5 mM (short treatments – 0.5, 1 or 4 h) or 2.5 mM (long treatment – 6, or 8 h).

Techniques: Binding Assay, Modification, Western Blot, Immunoprecipitation, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Cholesterol restricts lymphotoxin β receptor-triggered NF-κB signaling

doi: 10.1186/s12964-019-0460-1

Figure Lengend Snippet: Cholesterol depletion enhances LTβR-triggered expression of NF-κB target genes. a, b mRNA levels of the indicated NF-κB target genes in A549 cells preincubated for 1 h with vehicle or MβCD and then stimulated for 1 h (a) or 4 h (b) with LTβR agonist (Ago) (a, b) or lymphotoxin α1β2 (LTα1β2) (b). Values are presented as fold change versus control – unstimulated and untreated cells, set as 1. Data represent the means ± SEM, n = 4. ns - P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 by one sample t-test (in grey) or by Mann-Whitney or Student’s t-test (in black)

Article Snippet: Following stimulation with LTβR agonist was carried out in the absence of filipin for next 0.5 h and 1 h. Acute cholesterol depletion was performed using MβCD (C4555, Merck) at a concentration of 5 mM (short treatments – 0.5, 1 or 4 h) or 2.5 mM (long treatment – 6, or 8 h).

Techniques: Expressing, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: Cholesterol restricts lymphotoxin β receptor-triggered NF-κB signaling

doi: 10.1186/s12964-019-0460-1

Figure Lengend Snippet: Cholesterol depletion hyper-activates LTβR-dependent pro-inflammatory response a, b The concentrations of secreted CXCL8 were measured with ELISA in media collected from cells preincubated for 1 h with MβCD and then stimulated or not for 4 h (a) or 8 h (b) with Ago or LTα1β2 in the presence or absence of MβCD. Data represent the means ± SEM, n = 4. * P ≤ 0.05; ** P ≤ 0.01 by Mann-Whitney or Student’s t-test. c Lysates of A549 cells pretreated for 1 h with MβCD and then stimulated or not for 8 h with LTα1β2 or Ago in the presence or absence of MβCD were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control. Graph shows densitometric analysis for ICAM1 from Western blotting (protein levels normalized to vinculin). Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n = 4; ns - P > 0.05; * P ≤ 0.05; ** P ≤ 0.01 by one sample t-test (in grey) or by Mann-Whitney (in black). d, f Adhesion of Jurkat, NK cells, neutrophils and T lymphocytes to A549 cells (d) and HUVECs (f) treated as in a or e, respectively. Graphs represent quantification of immune cell adhesion to A549 and HUVECs relative to control (untreated) cells. Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n = 3 (d), n ≥ 3 (f); ns - P > 0.05; * P ≤ 0.05; ** P ≤ 0.01 by one sample t-test. e Lysates of HUVECs preincubated for 1 h with MβCD and then stimulated or not for 6 h with LTα1β2 in the presence or absence of MβCD were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control.

Article Snippet: Following stimulation with LTβR agonist was carried out in the absence of filipin for next 0.5 h and 1 h. Acute cholesterol depletion was performed using MβCD (C4555, Merck) at a concentration of 5 mM (short treatments – 0.5, 1 or 4 h) or 2.5 mM (long treatment – 6, or 8 h).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Western Blot