D3100

Absolute fitness was calculated as total fruit count (TFC) × silique length × survival. TFC was measured as the count of fruits (siliques) + flowers + buds per individual. Silique length was measured in ImageJ from digital images of harvested field plants taken using a Nikon D3100 as follows: each plant was placed flat on a white sheet of paper next to a ruler and pictures were taken using auto focus. After setting the scale in ImageJ using the ruler placed in each image, the segmented line tool was used to draw a line from the pedicle to the tip of the silique. For each plant, eight siliques were measured at random and these values were averaged to get a value for each plant. Survival was scored on a binary (0–1) scale. Plants that germinated, were transplanted into the field and survived to harvest were given a survival score of 1 and plants that germinated and were transplanted but did not survive to harvest were given a score of 0. Individuals that did not germinate or did not survive to transplantation were given an NA. Relative fitness was calculated for each GSL genotype within each environment relative to Col-0. To do this, average absolute fitness of a GSL genotype was divided by the average absolute fitness of Col-0 within a environment. Col-0 was chosen as the reference genotype given that it is the background genotype.

Galleria mellonella larvae were selected using as criteria similar size and weight ranging 0.10–0.15 (g). For the survival experiments, each group consisted of 20 larvae. The selected larvae were inoculated by injecting 10 μL of the different inoculum in the last left pro-leg with a Hamilton syringe (0.75 mm diameter needle) according to Fuchs et al. (2010 (link)). The control group was inoculated with PBS and the same number of larvae. The following control groups were used in the experiment. The first group included the larvae that received 10 μL of PBS to monitoring survival mortality related to trauma. A second group of larvae (SHAM) received no injection and no injury. All larvae were placed in sterile Petri dishes and kept in the dark at 37°C. Mortality was monitored once per day. The death of the larvae was assessed by the lack of movement, no response to stimulation and discoloration of the cuticle. Melanization was checked every 24 h with a NIKON D3100 camera and images were analyzed. Survival curves were plotted and statistical analyses were performed using the Log-rank (Mantel-Cox) test with Graph Pad Prism software. Statistical differences were set at p < 0.05.

Photographs of all experimental subjects were obtained at day 10 and day 24 of the experimental timeline. Zebrafish were anesthetized, placed in a dish of fish water, and photographed using a mounted camera (Nikon D3100 with a Nikon AF-S Micro Lens). Tumor area was measured at day 10 and day 24 using a traceable digital caliper (Fisher, 14-648-17). The pigmented tumor area was calculated by the longest measured length and width of the tumor. The drug response was quantified via the change from baseline tumor area using the RECIST (Response Efficacy Criteria in Solid Tumors) guidelines (Eisenhauer et al., 2009 (link)). The response rate for experimental cohorts was depicted via waterfall plots, and t-test statistics were applied for significance (Gillespie, 2012 (link)). Fluorescence imaging of FITC-dextran utilized a Nikon scope with a 0.5× objective lens (Nikon SMZ18-DSRi2)

Adult zebrafish used as recipients were immunosuppressed to avoid rejection of the donor material. They were anesthetized using a dual anesthetic protocol to minimize overexposure to tricaine [42 (link)]. Briefly, fish were treated with tricaine and then transferred to tricaine/isoflurane solution. Afterward, fish were treated with split sublethal dose (30 Gy) of X-irradiation (YXLON SMART 200E, 200 kV, 4.5 mA) 2 days before transplantation. Around 20 anesthetized fish per tumor were injected with 300,000 cells into the dorsal subcutaneous cavity using a 26S-gauged syringe (Hamilton, Reno, NV, USA). The syringe was washed in 70% ethanol and rinsed with PBS between uses. After transplantation, fish were placed into a recovery tank with fresh fish water and returned to the system. Finally, these transplanted fish were tracked weekly for a month, anesthetized, and photographed with a mounted camera (Nikon D3100 with a Nikon AF-S Micro Lens) to follow the growth of tumor cells in the injection site. The size of the pigmented tumor was then measured using Adobe Photoshop 2022 as the number of pigmented pixels. Tumor scoring was blinded, and experiments were independently repeated at least three times. For the DN-DUOX1 condition, five different tumors were transplanted, while for the GFP condition, three different tumors were used.

We attached markers to participants in 21 locations (ears, distal ends of metacarpal III, distal ends of phalanges, shoulder joints, elbow joints, hand joints, hip joints, knee joints, foot joints, vertex, upper margin of sternum, and navel). Furthermore, we asked the participants to assume an erect and immobile posture for photographs and to stand at attention in front of a wall with horizontal lines drawn at 10-cm intervals. We installed a digital camera (D3100, Nikon Co., Tokyo, Japan) at the height of 80 cm, 1 m away from the participants, and acquired still participants’ images from the coronal and sagittal planes. We performed a two-dimensional image analysis from the image of the sagittal plane [20 ,21 ]. We printed out an enlarged photo of each participant, and we drew a line on each segment from the marker. Further, we calculated the position of gravity of each segment obtained with reference to the mass ratio of each part and position of the center of gravity of the Japanese body [20 ]. Then, we calculated the body center of gravity (COG) from the mass ratio. We drew a midline from the COG and measured the distance between the line and ear position. The distance was calculated as the “degree of forward lean (cm)” of the head (Figure 1) [21 ].

Wound healing assays were conducted by means of the specific culture insert with respect to the supplier’s instructions (IBIDI, Germany). WT and TNFR2 KO-MSCs were seeded in the two separate wells of the insert at 15 × 10³ cells/ml in a final volume of 70 μl/well, to acquire a confluent layer within 24 h. To create a 500 μm homogeneous cell-free gap, culture inserts were carefully removed. The wounded area appearance was instantaneously captured by taking pictures (Nikon D3100, Japan) using objectives 4× and 10× of the microscope (Nikon ECLIPSE T5100) and continued every 2 h, until complete closure in WT-MSC group. Cells were incubated at 37°C in 5% CO₂. The decrease in wounded area was analyzed by measuring the scratch area size using the plugin MRI Wound Healing Tool for Image J (NIH, United States).