Journal: Signal Transduction and Targeted Therapy

Article Title: The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

doi: 10.1038/s41392-019-0100-7

Figure Lengend Snippet: SK-MEL-24; GFP/Fb spheroid and SK-MEL-24; bcat-GFP/Fb spheroid cultures were grown for 96 h. Representative bright-field images of SK-MEL-24; GFP/Fb spheroid a – d and SK-MEL-24; bcat-GFP/Fb spheroid cultures e – h were taken using a Carl Zeiss Axiovert 100 TV inverted microscope at ×5 magnification at 24, 48, 72, and 96 h. i Ki67 immunofluorescence staining (red) of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids embedded in paraffin. The nuclei were stained with DAPI (blue). Scale bar: 200 μm. j Quantification of Ki67-positive cells per square millimeter of SK-MEL-24; GFP/Fb spheroid sections and SK-MEL-24; bcat-GFP/Fb spheroid sections. A minimum of ten randomly selected fields was counted for each spheroid type. k Flow cytometry was used to analyze apoptosis and death of SK-MEL-24 melanoma cells in both SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids using annexin V and PI staining. l Percentages of SK-MEL-24 cell subpopulations based on PI and annexin V staining in SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids are shown. m – p α-SMA m–n and FSP-1 o – p immunostaining was performed on 5-μm-thick paraffin sections of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids. Pictures were taken using a Nikon microscope at ×20 magnification. Scale bar: 200 μm. q Collagen content in SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids was visualized by picrosirius red staining. Pictures were taken using an inverted light microscope at ×20 magnification. r Quantification of collagen content was performed by collagen extraction and colorimetric measurement. Collagen content was normalized to total protein content for each sample. s – v Fibronectin s – t and vimentin u – v immunostaining was performed on 5-μm-thick paraffin sections of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids. Pictures were taken using a Nikon microscope at ×20 magnification. Scale bar: 200 μm. w – z Photographs were taken at ×40 magnification. Scale bar: 100 μm.

Article Snippet: Picrosirius red staining was performed according to the manufacturer’s instructions (American MasterTech, Lodi, CA).

Techniques: Inverted Microscopy, Immunofluorescence, Staining, Flow Cytometry, Immunostaining, Microscopy, Light Microscopy, Extraction

Journal: PLoS ONE

Article Title: Tumor Necrosis Factor Receptor Associated Factor 6 Is Not Required for Atherogenesis in Mice and Does Not Associate with Atherosclerosis in Humans

doi: 10.1371/journal.pone.0011589

Figure Lengend Snippet: Lethally irradiated 6 week old TRAF6 +/+ /LDLR −/− mice received TRAF6-deficient (hatched bars, N = 21) or competent fetal liver cells (white bars, N = 21), TRAF6 +/− /LDLR −/− mice received TRAF6-deficient fetal liver cells (black bars, N = 22) only. Subsequently, all groups consumed high cholesterol diet (HCD) for 18 weeks. Sections of the aortic roots were analyzed for macrophage- (A), lipid- (B), smooth muscle cell- (C), collagen (D), and T cell-content (E). Mac-3-, oil-red-O-, α-actin-, picrosirius red, and CD4-positive staining in per cent of total wall area is displayed as mean±SEM.

Article Snippet: Air dried and formalin-fixed frozen sections were incubated for 3 h in 0.1% solution of picrosirius red (Polysciences) in saturated aqueous picric acid (Sigma-Aldrich).

Techniques: Irradiation, Staining

Journal: Reproductive Sciences

Article Title: Cervix Stromal Cells and the Progesterone Receptor A Isoform Mediate Effects of Progesterone for Prepartum Remodeling

doi: 10.1177/1933719118820462

Figure Lengend Snippet: Top: Photomicrographs of progesterone receptor (PR) stained cells and counterstained cell nuclei (CN) in cervix sections from nonpregnant (NP), pregnant (days 15 and 18 postbreeding), and day of birth postpartum (PP) mice. Scale bar is 25 µm. Middle: Density of PR cells normalized to CN per area to account for variability in cell nuclei density due to heterogeneity of tissue morphology within and among sections in individuals, as well as within and among groups. Data are the mean ± SE (n = 5-21/group). Bottom: As inversely related to cross-linked collagen in the extracellular matrix,2 optical density of birefringence of picrosirius red-stained cervix sections cells was normalized to CN per area. Data are mean ± SE (n = 4-10/group; *P < .05 vs NP mice or **vs NP, day 15 and day 16 postbreeding groups by 1-way ANOVA). See Methods for details about staining and analyses. SE indicates standard error; ANOVA, analysis of variance.

Article Snippet: Additional sections were stained with a picrosirius red stain kit (PK-4000; Polysciences, Warrington, Pennsylvania) to assess extracellular collagen organization and counterstained with hematoxylin (SH26; Fisher Scientific, Asheville, NC) as described previously.

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders

doi: 10.1172/JCI138634

Figure Lengend Snippet: Five-week-old BL10.mdx mice with mechanically induced fibrosis in the TA muscle were treated with ActRIIB:ALK4-Fc (10 mg/kg) or vehicle (PBS) twice weekly for 4 weeks. (A) TA muscle sections stained with Picrosirius red. Scale bars: 50 μm. (B) Percentage area in TA sections occupied by fibrotic tissue. Data are means ± SEM (n = 4–7). Group differences were assessed by 1-way ANOVA with Tukey’s adjustment. ***P < 0.001. For respiratory studies, 7-month-old D2.mdx mice were injected s.c. with ActRIIB:ALK4-Fc (10 mg/kg) or vehicle (PBS) twice weekly for 4 weeks. (C) Percentage change in tidal volume after exposure to hypoxic stress (10% O2) for 30 minutes. Data are means ± SEM (n = 3–5 per group). Group differences were assessed by 1-way ANOVA with Tukey’s adjustment. **P < 0.01 vs. WT. (D) Specific force generated by diaphragm muscle ex vivo. Data are means ± SEM (n = 6–10 per group). Group differences were assessed by 1-way ANOVA with Tukey’s adjustment. ***P < 0.001.

Article Snippet: Sections of paraffin-embedded TA muscle were stained with Picrosirius red (0.1% in saturated picric acid; Rowley Biochemical Inc.) to determine muscle fibrosis or were incubated with a rabbit anti-dystrophin antibody (1:2000; Abcam), followed by a polymer secondary antibody to identify dystrophin.

Techniques: Staining, Injection, Generated, Ex Vivo