Imagej software

The viability of chondrocytes in GelMA and GelMA-GC hydrogels was determined on days 1 and 28 of free-swelling and day 14 of intermittent uniaxial loading (total 28 days of culture) using a live/dead assay described elsewhere.⁹⁷ (link) Briefly, after the push-out test, hydrogel constructs were sliced into two by a sterile scalpel, washed with PBS, and then incubated for 3 min at room temperature in PBS solution containing fluorescein diacetate (FDA; 1 μg/ml) and propidium iodide (PI; 1 μg/ml) (both from Sigma-Aldrich, USA). The samples were rewashed with PBS, and four Z-stack images per sample were acquired using a Zeiss Axio microscope (Carl Zeiss Axio Imager M2, GmbH, Germany) at each time point for each group (n = 4) and analyzed using Image J software (National Instruments, USA). Cell viability reported as a percentage of the total number of cells that were alive. Whole-slice images of hydrogels were also captured to represent an overall view of the impact of uniaxial loading on the top and bottom parts of hydrogel in cartilage-hydrogel constructs. To comprehensively examine the integration and cell viability of distinct cartilage-hydrogel areas, whole cartilage-hydrogel constructs were imaged on day 28 of culture with a Zeiss Axio microscope following a live/dead assay.

The viability of oBMSC on scaffolds and coverslips was analysed on days 7 and 21 using double staining with ethidium homodimer-1 and calcein-AM in PBS (4 and 2 µM, respectively) (Merck Life Science, Gillingham, UK). Briefly, the scaffolds and coverslips were moved to a new plate and washed, once, with PBS. Subsequently, 500 µL of working solution was added to each well, and the plates were incubated for 1 h (5% CO₂ and 37 °C). Afterward, the samples were gently rinsed twice with PBS and imaged with a confocal laser microscope (LSM 710, Zeiss, Oberkochen, Germany) at 488 and 543 nm. Cell viability was quantified using ImageJ software (National Instruments, Austin, TX, USA). Cells were manually counted using the multi-point tool. The percentage of viable cells were calculated according to the following equation: $$\left(\frac{\mathrm{L}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\right)\times 100.$$

A253 cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton-X-100 (Sigma-Aldrich) for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 30 minutes, all at room temperature. Then, cells were incubated with a mixture of 10 μg/mL mouse anti-galectin-3 antibody (#ab2785, Abcam, USA) in 2% BSA at 4°C overnight. The next day, cells were incubated with a mixture of 10 μg/mL Alexa Fluor-647 anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., USA) in 2% BSA at room temperature for 1 hour.
Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3 (#ab2785, Abcam) and rabbit anti-LAMP1 (#21997-1-AP, Proteintech) antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 1 hour.
Sections were subsequently counterstained with DAPI (#ab104139, Abcam). Images were acquired using a fluorescent microscope (Nikon, Japan) and analyzed using ImageJ software (public domain, source: National Institutes of Health, USA).

Dry seeds were mounted directly on specific ESEM microscope stubs fitted on a Peltier stage working at 2°C in a Quanta 200 FEG high resolution environmental scanning electron microscope (ESEM; FEI company) under a variable water partial pressure. Images were recorded using the FEI imaging system with an accelerating voltage of 5 kV, at a working distance of 4 mm. In order to control precisely and follow the kinetics of the mucilage secretion process, three successive increase/decreases of the partial pressure in the chamber were applied on the seed. Step by step, the vacuum was adjusted from 3 Torrs where the seeds are considered in extremely dry conditions (30% relative humidity) up to 6 Torrs corresponding to the full seed imbibition in water (100% relative humidity and condensation). Image analysis for slight brightness and contrast improvement was carried out using ImageJ software (W. Rasband, National Institutes of Health).

Total proteins were extracted with RIPA buffer (Thermo Fisher Scientific), supplemented with 1 mm phenylmethylsulfonyl fluoride (Sigma‐Aldrich), and 1× protease inhibitor cocktail (Sigma‐Aldrich). Proteins (50 μg) were resolved via SDS/PAGE and analyzed by western blot using the following antibodies: pRb (IF‐8), p53 (DO1), Myc (SC‐40), ERK2 (SC‐1647), p‐ERK (Tyr‐204) (SC‐7383), AKT1/2/3 (SC‐55523), and p‐AKT (Ser‐473) (SC‐7985‐R) obtained from Santa Cruz Biotechnology (Dallas, TX, USA). CCND1 (AB16663), GAPDH (AB8245), ODC1 (AB66067), P‐eIF4E (Ser‐209) (AB76256), 4EBP1 (AB2606), p‐4EBP1 (Thr‐70) (AB75831), and MNK1 (AB89223) were purchased from Abcam (Cambridge, UK). Antibody‐immobilized membranes were incubated with a corresponding horseradish peroxidase‐conjugated secondary antibody for 2 h. Finally, the immunoreactive proteins were detected using an enhanced chemiluminescent substrate (Millipore, Burlington, MA, USA) and imaged through a gel documentation system (ImageQuant LAS 500; GE Healthcare Life Science, Björkgatan, Sweden). Furthermore, the expression of proteins was quantified by measuring the density of bands using imagej software (National Institutes of Health, Bethesda, MD, USA).