Image analysis software

Full-length AsPHYA mutant (K411L, T418D and D422R) genes were cloned into pBI121, and each construct was introduced into phyA-deficient Arabidopsis (phyA-201). After obtaining the homozygous lines, western blot analysis was carried out with crude extracts from 5-day-old dark-grown seedlings to assess the expression of AsphyA proteins using AsphyA-specific monoclonal antibody (oat22). For loading controls in this analysis, we used 1:10,000 polyclonal antibody against Arabidopsis translationally controlled tumour protein (AtTCTP; At3g16640) (ref. 48 (link)). For the photoresponse analyses of seedlings, seeds were sown on half strength MS medium and cold treated for 5 days in the dark. Hypocotyl lengths and cotyledon areas were then measured with 5- or 4-day-grown transgenic seedlings under continuous far-red light (cFR) with various fluence rates or at 10 μmol m⁻² s⁻¹, respectively. In addition, for the analysis of a FR-VLFR, seeds were exposed to red light (20 μmol m⁻² s⁻¹) for 1 h and transferred to dark condition at 21 °C for 1 day. Then, the seedlings were grown with hourly 5 min pulse of far-red light (0.5 μmol m⁻² s⁻¹) or in the dark for 3 days. The hypocotyls and cotyledons were photographed with a digital camera (Nikon), and then measured with Scion Image analysis software.

The in vivo chicken embryo CAM angiogenesis model was used as previously described. Leghorn fertilized eggs were incubated for 7 days at 37°C in the incubator, at which point a window was opened on the egg's shell to expose the CAM. The window was covered with tape, and the eggs were returned to the incubator. Different amounts of DYB‐03 (5, 10, and 20 µM) in a solution containing RPMI 1640 medium were applied to an area of 1 cm² (restricted by a plastic ring) of the CAM on day 9 of embryo development. 48 h after treatment and subsequent incubation at 37°C, CAMs were fixed in situ, excised from the eggs, placed on slides, and left to air dry. Pictures were taken through a stereoscope equipped with a digital camera, and the total length of the vessels was measured using image analysis software (software; Scion Corporation, Frederick, MD). Assays for each test sample were conducted three times, with each experiment including 6 to 10 eggs per data point.

Protein carbonylation was evaluated both in C2C12 myoblasts and C2C12 differentiated myotubes. After extraction, 20 μg samples of protein were denatured in 6% sodium dodecyl sulfate (Merck, Milan, Italy) and derivatized with 10 mM 2,4-dinitrophenyl hydrazine (DNPH) (Merck, Milan, Italy) for 15 min at room temperature. Samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad, Milan, Italy). Membranes were blocked with 1% bovine serum albumin (BSA) (Merck, Milan, Italy) in PBS containing 0.1% Tween 20 (PBST) and then probed overnight with specific primary antibody versus DNPH (1:5000 in PBST/1% BSA). After being washed with PBST, the membranes were incubated for 1 h in PBST containing the appropriate horseradish peroxidase-conjugated secondary anti-rabbit (1:5000; Cell Signaling, Danvers, MA, USA) and again washed. Enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA) was used to visualize the peroxidase-coated bands. Densitometric analysis was performed using the Scion Image analysis software. Regarding each experiment, the density of all bands shown in a lane was reported as the mean. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalization was performed [42 (link)].

Generation of intracellular ROS was measured with the fluoroprobe DCF-DA. Briefly, MMCs were incubated for 60 min at 37 °C with 10 μM DCF-DA in absence or presence of PFSE in a dose-dependently. Fluorescence intensity was measured by fluorescence microscopy (NIKON; excitation 488 nm, emission 513 nm). Average intensity for each experimental group of cells was determined using Scion Image Analysis software, and values were expressed as the above control.