Dxm1200f

ASCs were cultured in six-well plates in CCM until 70 % confluence. Medium was replaced with fresh osteogenic differentiation medium (ODM) consisting of CCM supplemented with 50 μM ascorbate 2-phosphate (Sigma, St. Louis, MO), 10 mM β-glycerol phosphate (Sigma), and 10 nM dexamethasone (Sigma). Where indicated, the FBS in ODM was substituted for charcoal dextran stripped fetal bovine serum (CDS-ODM; Atlanta Biologicals) and supplemented with log fold increases of 17β-estradiol (E₂) from 100 pM to 10 μM of estradiol. After 14 or 21 days, cells undergoing osteogenic differentiation in ODM or CDS-ODM were fixed in 10 % formalin for 1 h, washed with distilled water, and stained with 1 % Alizarin Red (pH 4.1) to visualize calcium deposition in the extracellular matrix as a marker of early osteogenesis. Images were acquired at 4 × magnification on Eclipse TE200 (Nikon; Melville, NY) with a digital camera (Nikon DXM1200F) using ACT-1 software (Nikon). For quantification, Alizarin Red was extracted from each well with 10 % cetylpyridinium chloride (CPC) and read at 584 nm (FLUOstar optima). To normalize to the amount of protein in each sample, protein extraction with RIPA buffer (Pierce, Thermo Scientific) and protein quantification with the BCA assay (Thermo Scientific) was performed according to manufacturer’s instructions.

Hearts were fixed with 4% paraformaldehyde and embedded in paraffin, and 6-μm thick sections were stained with hematoxylin and eosin or Masson’s trichrome [27 (link)]. Images were acquired using a Nikon ECLIPSE E600 microscope (Nikon Japann) with a 40x objective lens (Plan Apo40/0.95, Nikon, Japan) and a digital camera (Nikon DXm1200F, Nikon, Japan) and software (Nikon ACT-12.62, Nikon, Japan). The area of one image was 0.086 mm². The number of cardiomyocytes per high-powered field (× 400) was counted by two pathologists. To evaluate the fibrosis of the heart, four images were taken per group at 400x magnification with Masson’s Trichrome staining (MTS) slides. The images were analyzed with image analyzing software (analySIS, Soft Imaging Systems, Germany), and the MTS-positive area was calculated as following: MTS-positive area = (MTS-stained blue-colored area/total area) x 100 (%).

Terminal dUTP-mediated nick-end labeling (TUNEL) assays (Promega, Madison, WI, USA) were performed according to the manufacturer's protocol. Briefly, HepG2 cells were seeded in 8-chamber BD tissue culture slides (BD Bioscience Labware, Bedmord, MA) at 10% confluency. Celastrol or its derivatives were added to the culture medium at final concentrations of 5 μM. After 6 hours incubation, cells were washed twice with PBS, and then fixed in 4% paraformaldehyde for 25 minutes. Fixed cells were washed twice in PBS with 0.1% Triton X-100, and then incubated with TUNEL reaction mixture for 60 minutes at 37°C. After washing with 2xSSC, slides were immersed in PBS (with 5 μg/ml DAPI) for 5 minutes in the dark, and then washed with PBS. Fluorescence labeling was visualized and photographed (100x magnification) with a fluorescence microscope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan) and with a digital camera (Nikon DXM1200f, Nikon Corporation, Tokyo, Japan). For TUNEL staining of the patient-derived xenografts, 6-μm tissue sections were stained using the ApopTag Peroxidase in Situ Oligo Ligation Apoptosis Detection Kit (Chemicon International, Temecula, CA) according to the manufacturer's protocol.

We collected a stripe of tissue (0.5-cm wide in the longitudinal axis of the
flap). This sample was immersed in 4% neutral, phosphate buffered,
paraformaldehyde, for 48 h at room temperature.
Tissue was washed, dehydrated in graded concentrations of alcohol, and embedded
in paraffin. Four-micrometer-thick sections were mounted on glass slides and
stained with hematoxylin-eosin (HE). The sections were analyzed under the light
microscope Nikon Optiphot-2 (Nikon, Tokyo, Japan), coupled to a Nikon DXM 1200F®
(Nikon, Tokyo, Japan) video digital camera. Measurements were performed using
the ImageJ® (Media Cybernetics, Silver Spring, MD, USA). A blinded investigator
counted arterioles, inflammatory cells, and dermic appendages in ten fields per
specimen (x20).

A 6-μl drop of E. coli BL21 (DE3) co-transformed with the pUA66 promoter containing one of the different promoters and the pET29a-erxR plasmid (Supplementary Table 3), resuspended in LB growth medium, was spotted onto a Superfrost Plus slide (Fisher Scientific Ltd, UK) and visualized using a Nikon Eclipse 80i epifluorescence microscope (Nikon, France). Fluorescent images were acquired with a Nikon DXM1200F digital camera (Nikon, France), using Nikon ACT-1 software (Nikon, France). Fluorescence intensity was calculated by measuring the area, integrated intensity and mean gray value of the fluorescent bacteria and the background with ImageJ (National Institute of Health, USA). Corrected total cell fluorescence (CTCF) was calculated using the following formula: integrated density—(area of the cell × mean background readings). The average and statistical differences between the bacteria containing the plasmids with the different promoters and controls were calculated using the CTCF values of all the bacteria in four different fields of view. Images were processed to size, and brightness and contrast were adjusted after the measurements, using Adobe Photoshop cs5 (Adobe Systems Inc., California, USA).