Journal: Nature Communications

Article Title: Loss of testosterone impairs anti-tumor neutrophil function

doi: 10.1038/s41467-020-15397-4

Figure Lengend Snippet: Representative images of lungs ( a ) and numbers of tumor colonies ( b ) in the lungs of male and female C57Bl/6 mice following a B16 tail-vein injection ( n = 13 per group). Representative images of lungs ( c ) and numbers of tumor colonies ( d ) in the lungs of B16-injected males, sham females, and females following ovariectomy ( n = 5 per group). Representative images of lungs ( e ) and numbers of tumor colonies ( f ) in the lungs of B16-injected sham males, castrated males, and female mice ( n = 6 per group). g Survival curve following tail-vein injection with B16 melanoma cells ( n = 9 male+sham and male+castration; n = 10 female). Representative images of lungs ( h ) and tumor colonies numbers ( i ) in YUMM1.7 injected sham males, castrated males, and female mice ( n = 10 per group). j Survival curves following tail-vein injections of YUMM1.7 melanoma cells ( n = 10 male+sham and male+castration; n = 9 female). Data in b , d , f , and i are mean ± s.e.m. by two-tailed unpaired t test ( a ) and 1-way ANOVA ith Bonferoni post-tests analysis ( d , f , and i ). Survival curve analysis ( g , j ) was performed using the two-sided Log-rank (Mantel-Cox) test. Data in a – b and e – f are representative of at least 3 independent experiments; data in c – d and h – j are representative of 2 independent experiments. The scale bar represents 0.5 cm for images of lung.

Article Snippet: B16, YUMM1.7, and LnCaP cells (ATCC, clone FGC, CRL-1740) were pelleted and lysed in ice-cold Lysis buffer consisting of Tris (25 mM; Bio Rad; catalog 161-0719), Triton x-100 (1%; Sigma: catalog T8787), Glycerol (10%; Fisher Scientific; catalog BP229-1), EGTA (4 mM; Sigma; catalog E-4378), pH adjusted to 7.8 with dilute o-phosphoric acid.

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of testosterone impairs anti-tumor neutrophil function

doi: 10.1038/s41467-020-15397-4

Figure Lengend Snippet: a Average immune cell content in the lungs of B16 and YUMM1.7 tumor-bearing mice, based on flow cytometry for the following cell types: CD11b − CD11c + F4/80 + alveolar macrophages, CD11b + CD11c − F4/80 + interstitial macrophages, CD11b + CD11c + F4/80 + dendritic cells, NK cells, NKT cells, CD4 + T cells, CD8 + T cells ( n = 12 male groups; n = 14 females). b Analysis of percent-activated immune cells by flow cytometry: CD69 + NKT cells, IFNγ + NKT cells, CD4 + CD44 + T cells, CD4 + CD69 + T cells, CD8 + CD44 + T cells, CD8 + CD69 + T cells ( n = 14 male groups; n = 16 females). Representative lung images ( c ) and tumor colonies numbers ( d ) in the lungs of Rag1 −/− male and female mice following B16 tail-vein injection. e – f Flow cytometry analysis of lung-infiltrating CD11b + Ly6G + neutrophils and ( e : n = 16 per group; f: n = 15 male+sham, n = 16 male+castration, female) g – h CD69 + NK cells and IFNγ + NK cells ( g : n = 13 male+sham, n = 15 male+castration, n = 11 female; h : n = 15 male+sham, n = 10 male+castration, n = 16 female). Ex vivo B16 cytotoxicity assays of lung isolated ( i ) NK cells and ( j ) neutrophils from tumor-bearing mice at effector cell:target cell ratios of 3:1 and 6:1. Data in a – b , d – h , and i – j are mean ± s.e.m. by 1-way ANOVA with Bonferoni post-tests analysis ( a – b and e – h ) or two-tailed unpaired t test ( d ). Data in a – b and e – h are representative of at least 3 independent experiments; data in c – d are representative of 2 independent experiments ( n = 8 per group); data in i – j are representative of 2 independent experiments (each point is from 3 pooled mice for an n = 15 per group). The scale bar represents 0.5 cm for images of lung.

Article Snippet: B16, YUMM1.7, and LnCaP cells (ATCC, clone FGC, CRL-1740) were pelleted and lysed in ice-cold Lysis buffer consisting of Tris (25 mM; Bio Rad; catalog 161-0719), Triton x-100 (1%; Sigma: catalog T8787), Glycerol (10%; Fisher Scientific; catalog BP229-1), EGTA (4 mM; Sigma; catalog E-4378), pH adjusted to 7.8 with dilute o-phosphoric acid.

Techniques: Flow Cytometry, Injection, Ex Vivo, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of testosterone impairs anti-tumor neutrophil function

doi: 10.1038/s41467-020-15397-4

Figure Lengend Snippet: Representative lung images ( a ) and numbers of B16 colonies ( b ) in the lungs of male and female mice treated with either control IgG or anti-Ly6G mAb ( n = 8 male+IgG, male+αLy6G, female+IgG, n = 7 female+αLy6G. c Percent IFNγ + NK cells in lungs of male and female IgG control or anti-Ly6G treated mice ( n = 5 per group). d Percent of lung neutrophils from tumor-bearing mice that have a mature, segmented appearance morphologically ( n = 18 per group; 3 pooled samples each). Percentage of lung neutrophils producing DHR123 without stimulation ( e ) and after PMA stimulation ( f ) ( n = 8 male+sham, n = 10 male+castration; n = 9 female). g FITC MFI of lung neutrophils after engulfing FITC-labeled zymosan A S. cerevisiae beads ( n = 5 per group). h Distance of neutrophils within 100 µM of a B16 tumor nodule in the lung of sham male, castrated male, and female mice ( n = 30 neutrophils/3 mice per male group, n = 44 neutrophils/4 female mice).Representative lung images ( i ) and number of melanoma tumor colonies ( j ) in lungs of mice receiving a BMT from the opposite sex ( n = 9 male to F and female to M, n = 8 female to F, n = 10 male to M). k Percent of lung-infiltrating neutrophils following a sex BMT ( n = 8 male to F, n = 9 female to M, n = 8 female to F, n = 10 male to M). l Percent of lung neutrophils producing DHR123 with PMA stimulation following a sex BMT ( n = 4 male to F, n = 14 female to M, n = 5 female to F, n = 14 male to M). Data in b – h and j – l are mean ± s.e.m. by 1-way ANOVA with Bonferoni post-tests analysis ( b , c , e – h , and j – l ) or z -score by difference of two proportions ( d ). Data in a – c and h are representative of 2 independent experiments and data in d-g and j-l are representative of at least 3 independent experiments. The scale bar represents 0.5 cm for images of lung.

Article Snippet: B16, YUMM1.7, and LnCaP cells (ATCC, clone FGC, CRL-1740) were pelleted and lysed in ice-cold Lysis buffer consisting of Tris (25 mM; Bio Rad; catalog 161-0719), Triton x-100 (1%; Sigma: catalog T8787), Glycerol (10%; Fisher Scientific; catalog BP229-1), EGTA (4 mM; Sigma; catalog E-4378), pH adjusted to 7.8 with dilute o-phosphoric acid.

Techniques: Control, Labeling

Journal: Nature Communications

Article Title: Loss of testosterone impairs anti-tumor neutrophil function

doi: 10.1038/s41467-020-15397-4

Figure Lengend Snippet: Representative lung tumor images ( a ) and number of lung tumor colonies in male mice and male mice following castration with and without testosterone (T) replacement at physiological doses ( b ) ( n = 7 male+sham, n = 8 male+castration, n = 9 female). c Percent of neutrophils in lung following castration with and without T replacement ( n = 10 male+sham, n = 12 male+castration, n = 13 female). Weight of YUMM1.7 subcutaneous tumors ( n = 10 male+sham, n = 9 male+castration, n = 10 male+castration+T, n = 9 female) ( d ) and number of spontaneous metastastic lung colonies ( n = 9 male+sham, n = 7 male+castration, n = 9 male+castration+T, n = 8 female) ( e ). Representative lung images ( f ) and number of lung tumor colonies ( g ) following a BMT with immune cells deficient in AR (AR Tfm mice). Percentage of neutrophils in the lungs of male mice receiving a BMT from either WT or AR Tfm mice ( h ) ( n = 10–15 per group). Percentage of DHR123-producing neutrophils following AR Tfm BMT without ( i ) and after PMA stimulation ( j ; n = 15 M C57→M Ly5.1, n = 10 M AR Tfm →M Ly5.1). Representative lung images ( k ) and number of B16 lung colonies ( l ) following implantation of either a placebo or slow-release flutamide pellet ( n = 9 male+placebo, n = 8 male+flutamide). Data in b – e , g – j , and l are mean ± s.e.m. by 1-way ANOVA with Bonferoni post-tests ( b – e ) analysis or two-tailed unpaired t test ( g – j , l ). Data in a – c are representative of at least 3 independent experiments and data in d – l are representative of 2 independent experiments. The scale bar represents 0.5 cm for images of lung.

Article Snippet: B16, YUMM1.7, and LnCaP cells (ATCC, clone FGC, CRL-1740) were pelleted and lysed in ice-cold Lysis buffer consisting of Tris (25 mM; Bio Rad; catalog 161-0719), Triton x-100 (1%; Sigma: catalog T8787), Glycerol (10%; Fisher Scientific; catalog BP229-1), EGTA (4 mM; Sigma; catalog E-4378), pH adjusted to 7.8 with dilute o-phosphoric acid.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: Loss of testosterone impairs anti-tumor neutrophil function

doi: 10.1038/s41467-020-15397-4

Figure Lengend Snippet: In castrated male mice, testosterone is diminished and can no longer activate the androgen receptor (AR). This causes a decrease in CXCR2, CD62L, and STAT3 expression, and an increase in CXCR4 and VLA4 expression. These changes lead to more banded, immature neutrophils and an overall reduced number of neutrophils in circulation. Once B16 cells are injected via tail vein and allowed to metastasize to the lung, castrated male mice have a poor recruitment of mature, segmented neutrophils which leads to an increase of ROS-producing neutrophils, fewer NK + IFNγ-producing cells, and results in increased tumor burden and decreased survival compared with sham-operated male mice.

Article Snippet: B16, YUMM1.7, and LnCaP cells (ATCC, clone FGC, CRL-1740) were pelleted and lysed in ice-cold Lysis buffer consisting of Tris (25 mM; Bio Rad; catalog 161-0719), Triton x-100 (1%; Sigma: catalog T8787), Glycerol (10%; Fisher Scientific; catalog BP229-1), EGTA (4 mM; Sigma; catalog E-4378), pH adjusted to 7.8 with dilute o-phosphoric acid.

Techniques: Expressing, Injection

Journal: Cancer Research

Article Title: Nuclear Alternate Estrogen Receptor GPR30 Mediates 17β-Estradiol–Induced Gene Expression and Migration in Breast Cancer–Associated Fibroblasts

doi: 10.1158/0008-5472.can-10-0408

Figure Lengend Snippet: Figure 1. Characterization of CAFs. A, deconvolution microscopy picture shows CAFs morphology in monolayer condition growth. Scale bar, 500 μm. B, CAFs were immunostained by anti-vimentin antibody. Scale bar, 75 μm. C, Western blot analyses of ERα, ERβ, GPR30, and EGFR protein expression levels in CAFs, SkBr3, and MCF-7 breast cancer cells and LNCaP prostate cancer cells. β-Actin antibody was used as a loading control. D, CAFs were transfected with the ER luciferase reporter plasmid ERE-luc alone and in combination with the wild-type ERα and treated with E2 for 18 h as indicated. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction on which the activity induced by E2 was calculated. Columns, mean of three independent experiments done in triplicate; bars, SD. °

Article Snippet: ERα (COOH-terminal domains, F-10), EGFR, pEGFRTyr1173, c-Fos, CTGF, cyclin D1, p-ERK 1/2, ERK 2, andβ-actin were purchased from Santa Cruz Biotechnology, and ERβ was from Serotec.

Techniques: Microscopy, Western Blot, Expressing, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection.

doi: 10.4049/jimmunol.1101231

Figure Lengend Snippet: FIGURE 8. UPEC-triggered IL-10 in a mixed coculture model of human bladder is monocyte derived. In cocultures of U937 monocytes and 5637 urothelial cells infected with UPEC for 5 h, then analyzed for CD45 by FACS (A) (percentage of CD45-positive cells in inset), isolated cell pop- ulations enriched for nonadherent monocytes (B) exhibited a 4.1-fold in- crease in IL-10 mRNA according to qRT-PCR (normalized to noninfected control cocultures). IL-10 mRNA in adherent cell populations, composed predominantly of urothelial cells, was unchanged after 5 h (C). GAPDH served as a reference gene.

Article Snippet: Cells were centrifuged and resuspended in wash buffer with mouse anti-human CD45 IgG conjugated to R-PE (NBP1-28512 [F10-89-4]; Novus Biologicals) at the concentration recommended by the manufacturer and incubated for 30 min in the dark at RT.

Techniques: Derivative Assay, Infection, Isolation, Quantitative RT-PCR, Control

Journal: Experimental eye research

Article Title: Differences between the neurogenic and proliferative abilities of Müller glia with stem cell characteristics and the ciliary epithelium from the adult human eye.

doi: 10.1016/j.exer.2011.09.015

Figure Lengend Snippet: Fig. 4. Comparison between the expression of glial and epithelial cell markers by immortalized Müller glia with stem cell characteristics and non-pigmented CE cells (A) RT-PCR analysis of Müller (MIO-M1 and MIO-M7) and non-pigmented CE cells (CE 6165 and CE 6213) for mRNA expression of retinal pigment epithelial (RPE) markers showed that both cell types expressed mRNA for the RPE cell markers ZO-1, RPE65, MITF-A and MERTK. However, non-pigmented CE cells showed higher expression of these markers than Müller stem cells. mRNA coding for CRALBP, a characteristic marker of Müller glia was expressed by Müller cells but not by CE cells. GAPDH primers were used as control. (B) Immunostaining of Müller and non-pigmented CE cells for markers of RPE. (i) Non-pigmented CE cells showed strong staining with a pan-cytokeratin antibody, whilst no expression of this marker was observed in Müller stem cells. (ii) A small proportion of Müller stem cells stained for RPE65 (less than 10%), whilst all CE cells stained strongly for this protein. (C) Staining for glial markers showed that Müller cells that expressed stem cell properties expressed the proteins CD44 and vimentin, whilst CE cells did not stain for these markers. (CE ¼ Ciliary Epithelium, EGF ¼ Epidermal Growth Factor. Scale bars represents 50 mm.)

Article Snippet: Primary antibodies were diluted in the blocking reagent and incubated with the cells overnight at 4 C. The primary antibodies used were: CD44 (F10-44-2; monoclonal; 1:1000; AbD Serotec, Oxford, U.K., http://www.abdserotec.com), CHX10 (SC-21692; goat; 1:200; Santa Cruz, U.S.A. http://www.scbt.com), Nestin (MAB5362; monoclonal; 1:1000; Chemicon (Millipore), Billerica, U.S.A. http://www.millipore.com/), RPE65 (ab13826; monoclonal; 1:200; Abcam, Cambridge U.K http://www.abcam. com), Pan-cytokeratin (MAB3406; 1:200; monoclonal; Chemicon (Millipore)), SHH (SC-9024; rabbit; 1:200; Santa Cruz), SOX2 (AB5603; rabbit; 1:1000; Chemicon (Millipore), Vimentin (SC130610; 1:500; rabbit; Santa Cruz).

Techniques: Comparison, Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Control, Immunostaining, Staining