Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet: Serum levels of total and HDM-specific immunoglobulins show a time-of-day response and sex-based differences to chronic HDM exposure Total IgE, Total IgG, HDM-specific IgE, HDM-specific IgG, HDM-specific IgG1, HDM-specific IgG2b, HDM-specific IgM, and HDM-specific IgA in the serum of chronic PBS- and HDM-exposed females and males at ZT0 and ZT12 were determined by ELISA. Data were expressed as ng/ml and mg/ml for total IgE and total IgG, respectively. HDM-specific immunoglobulins (IgE, IgG, IgG1, IgG2b, IgM, and IgA) in the serum were determined by commercially available ELISA kits (Chondrex, Inc.). Unable to detect HDM-specific IgG2a and IgG3 levels in PBS- and HDM-exposed mice. Data for the HDM-specific immunoglobulins were expressed as absorbance at 450 nm or μg/ml. Data are shown as mean ± SEM, Two-way ANOVA followed by Tukey’s multiple comparison test (n = 5/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to respective control (PBS) at ZT0 or ZT12; # p < 0.05, compared to HDM at ZT0 vs. ZT12; # # p < 0.01, compared to HDM at ZT0 vs. ZT12. Summary statistics for interaction between Treatment x Sex x Time were analyzed using generalized linear modeling using R (see Table S2 ).

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet:

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel bispecific antibody targeting CD3 and prolactin receptor (PRLR) against PRLR-expression breast cancer

doi: 10.1186/s13046-020-01564-4

Figure Lengend Snippet: PRLR-DbsAb stimulates T cell infiltration and the PD-L1 expression unregulated in tumor tissue. sc Tumor cells plus sc effector cells (E/T 1:4) model: ( a ) HE staining of tumor tissue strapped from mice treated with 3 mg/kg PRLR-DbsAb. The above image showed normal tumor tissue, and the following image showed degenerate necrotic tumor tissue; ( b ) Immunohistochemical staining of CD8 in tumor tissue from the mice of different group; ( c ) Immunohistochemical staining of PD-L1 in tumor tissue from the mice of different group. Sc tumor cells plus ip effector cells (1:1) model: ( d ) HE staining; ( e ) Immunofluorescent staining of CD8. Three independent experiments were conducted and representative data is shown in this figure

Article Snippet: Breast cancer cell lines (MDA-MB-231, MCF-7, SKBR-3, and T47D) were incubated with control isotype IgG, PE-conjugated mouse anti-Human PRLR (Sino Biological) or APC-conjugated mouse anti-Human PD-L1 (Sino Biological) on ice for 30 min and then washed twice with FACS buffer (PBS with 2% FBS).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel bispecific antibody targeting CD3 and prolactin receptor (PRLR) against PRLR-expression breast cancer

doi: 10.1186/s13046-020-01564-4

Figure Lengend Snippet: PD-1 inhibition increases PRLR-DbsAb mediated cytotoxicity of PRLR expression target cell. FACS histograms of PDL1 expression on different breast cancer cell lines including MDA-MB-231 ( a ) and T47D ( e ). The gray histogram is the fluorescent signal of non-specific IgG control antibody. Freshly isolated PBMCs and the target cells of MDA-MB-231 ( b ) or T47D ( f ) were incubated with or without addition of PD-1 mAb in different concentrations of PRLR-DbsAb at the ratio of 10:1 (E: T) for 20 hours. MDA-MB-231 ( c ) and T47D ( g ) cells were treated with 100 ng/ml PRLR-DbsAb at different time points (3, 10 and 20h) with or without adding PD-1 mAb and LDH release was detected. The expression of PD-L1 ( d and h ) on the corresponding effector cells and target cells is measured by Flow cytometer, respectively. ( i ) FACS histogram of the PD-L1 expression of target T47D with or without addition of 100 ng/ml PRLR mAb. ( j ) PD-L1 expression on CD4+ and CD8+ T cells. PBMCs and target T47D cells were incubated with 100 ng/ml PRLR-DbsAb at the ratio of 10:1 (E: T) for 20 hours. Three independent experiments were performed and the data was represented as the Mean ±SEM. *, ** and *** respectively indicate a statistically significant difference of at least P <0.05, <0.01 and <0.001

Article Snippet: Breast cancer cell lines (MDA-MB-231, MCF-7, SKBR-3, and T47D) were incubated with control isotype IgG, PE-conjugated mouse anti-Human PRLR (Sino Biological) or APC-conjugated mouse anti-Human PD-L1 (Sino Biological) on ice for 30 min and then washed twice with FACS buffer (PBS with 2% FBS).

Techniques: Inhibition, Expressing, Isolation, Incubation, Flow Cytometry

Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: Characterization of 293F cell-derived EVs carrying SARS-CoV-2 spike protein (A) Schematic of EV engineering strategy using 293F cells including SARS-CoV-2 spike expression construct (see Figure S1 ) with spike protein depicted in blue. Schematic created with Biorender.com . (B) Histogram plot of particle size distribution comparing 293F EVs and Spike EVs as determined by NTA. The number of particles falling within each size bin was normalized to total particle counts and are presented as the proportion of total particles. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student’s t test where p < 0.05 would be considered significant. (C) Mean and mode particle size of 293F EVs and Spike EVs as determined by NTA. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student's t test where p < 0.05 would be considered significant. n.s. indicates not significant. (D) Multiplexed bead-based profiling assay of 37 surface antigens on 293F EVs and Spike EVs confirming the presence of expected EV surface markers. Data represent the median fluorescent intensity (MFI) of APC signal (reflecting CD63-APC, CD81-APC and CD9-APC counterstaining) following the subtraction of isotype controls. N = 1 independent experiment. (E) Representative western blots characterizing the presence of SARS-CoV-2 spike protein in Spike EVs and markers known to be present in EV preparations (CD63, CD9, Flotillin, TSG101, GAPDH) as well as the absence of cell contaminant markers (GM130, Calnexin). (F) Confirmation of SARS-CoV-2 spike protein presence in Spike EVs through competitive Spike-ACE2 binding assay. Spike EVs inhibit the binding of fluorescently labeled, recombinant SARS-CoV-2 spike protein competitively, in a dose-responsive manner, while 293F EVs demonstrate little inhibition. Data are mean ± SEM. N = 2 independent experiments and 2 technical replicates per sample per run. (G) Transmission electron micrographs of unstained Spike EVs confirming expected EV morphology. SARS-CoV-2 spike protein presence on 293F Spike EVs was also visually confirmed using immunogold labeling where 10 nm gold particles are seen associating with 293F Spike EVs.

Article Snippet: Mouse Anti-Flotillin-1, Clone 18 , BD Transduction Laboratories , Cat#610820; RRID: AB_398139.

Techniques: Derivative Assay, Expressing, Construct, Two Tailed Test, Western Blot, Binding Assay, Labeling, Recombinant, Inhibition, Transmission Assay

Journal: Frontiers in Endocrinology

Article Title: Regulatory Actions of LH and Follicle-Stimulating Hormone on Breast Cancer Cells and Mammary Tumors in Rats

doi: 10.3389/fendo.2018.00239

Figure Lengend Snippet: Modulatory effects of gonadotropins on breast cancer (BC) cell expression/activation of follicular-stimulating hormone receptor (FSHR), LHR, moesin, and focal adhesion kinase (FAK). Protein extracts from (A) T-47D (estrogen-receptor positive) and (E) MDAMB-468 (estrogen-receptor negative) BC cells treated for 48 h with increasing concentration of LH and follicle-stimulating hormone (FSH) (5, 5 + 50, and 50 mUI/ml) were assayed using western blot analysis for their overall content of LHR, FSHR, moesin, p-Moesin T558 , FAK, p-FAKY 397 , and actin. (B–D,F–H ) LHR, FSHR, moesin, p-Moesin T558 , FAK, p-FAKY 397 densitometry values were adjusted to actin intensity, respectively, then normalized to the control sample. * P < 0.05 vs. corresponding control. The experiments were performed in triplicates and representative images are shown.

Article Snippet: Antibodies used were: FAK (dilution 1:1,000), p-FAK (Y397, 1:1,000), and moesin (clone 38, 1:1,000) (BD Transduction Laboratories, Lexington, KY, USA); Thr558-p-Moesin (sc-12895, 1:750), p-FAK-Tyr397 (sc-11765, 1:500), LHR (H-50, 1:500), FSHR (N-20, 1:500), and ACTIN (C-11, 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Concentration Assay, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Regulatory Actions of LH and Follicle-Stimulating Hormone on Breast Cancer Cells and Mammary Tumors in Rats

doi: 10.3389/fendo.2018.00239

Figure Lengend Snippet: LH/follicle-stimulating hormone (FSH) signal to moesin and focal adhesion kinase (FAK) via LHR/follicle-stimulating hormone receptor (FSHR). (A–C) T-47D breast cancer cells were transfected with shRNA vs. LHR and FSHR (shRNA LHR and shRNA FSHR) or with vehicle, and protein analysis for LHR, FSHR, actin, wild-type (FAK and moesin), or p-FAK and p-moesin was performed on cell lysates after treatment with LH and FSH in different concentrations (5, 5 + 50, and 50 mUI/ml) for 48 h. (B,D) The graphs display the quantitative analysis of the intensity of the bands, obtained as number of photons measured by the ChemiDoc digital imaging system and evaluated with the Quantity One Software (Bio-Rad).

Article Snippet: Antibodies used were: FAK (dilution 1:1,000), p-FAK (Y397, 1:1,000), and moesin (clone 38, 1:1,000) (BD Transduction Laboratories, Lexington, KY, USA); Thr558-p-Moesin (sc-12895, 1:750), p-FAK-Tyr397 (sc-11765, 1:500), LHR (H-50, 1:500), FSHR (N-20, 1:500), and ACTIN (C-11, 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, shRNA, Imaging, Software

Journal: Frontiers in Endocrinology

Article Title: Regulatory Actions of LH and Follicle-Stimulating Hormone on Breast Cancer Cells and Mammary Tumors in Rats

doi: 10.3389/fendo.2018.00239

Figure Lengend Snippet: Gonadotrophins’ signaling to moesin and focal adhesion kinase (FAK) increase T-47D cell migration and invasion. Breast cancer cells were transfected with 100 nM target shRNA for LHR and follicular-stimulating hormone receptor (FSHR), siRNA for FAK and 75 nM target phosphorothioate oligonucleotides antisense vs. moesin for 48 h and then treated with LH and/or follicle-stimulating hormone (FSH) (5 + 50 mUI/ml) for 48 h. (A,B) Cell migration distances were measured as mean migration distance (μm) ± SD. * P ≤ 0.05 vs. control. The experiments were performed in triplicates and representative images are shown. (C–H) Cell invasion was assayed using invasion chambers. Average of invading cells observed, photographed under the microscope at 100× magnification and counted in three different central fields of triplicate membranes. * P ≤ 0.05 vs. control. The experiments were performed in triplicates. Invasion indexes and representative images are shown.

Article Snippet: Antibodies used were: FAK (dilution 1:1,000), p-FAK (Y397, 1:1,000), and moesin (clone 38, 1:1,000) (BD Transduction Laboratories, Lexington, KY, USA); Thr558-p-Moesin (sc-12895, 1:750), p-FAK-Tyr397 (sc-11765, 1:500), LHR (H-50, 1:500), FSHR (N-20, 1:500), and ACTIN (C-11, 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Migration, Transfection, shRNA, Microscopy