Fig 2: CCL27 rescues myeloma cells from bortezomib-induced cell death in a xeno-transplanted model(A) Chicken chorioallantoic membrane assay was used for in vivo testing and the setup of the 3D-onplant is summarized in the drawing. Tumor onplants containing myeloma cells and primary fibroblasts in a ratio of 11:1, bortezomib (1 nM) and/or chemokine (CCL27: 7.9 nM, CCL28: 8.1 nM) in a collagen matrix were positioned on the membrane and incubated for 5 days. Single myeloma xenografts were photographed using a stereo-fluorescence microscope (Olympus SZX10 at 12× magnification), excised, and GFP concentrations of single tumors were measured by Elisa. Values are shown as mean concentration in pg/ml ± SEM (n = 12). (B) Immunohistochemical analysis of cross-sections of NCI-H929, MM.1S and OPM-2 grafts in the chorioallantoic membrane model including control with and without bortezomib, CCL27 or CCL28 at the above mentioned concentrations; sections were stained with an anti-human Ki-67 antibody to detect growing myeloma cells. Pictures were taken using Zeiss Axiovert 200 M microscope at 200× maginification. The amount of Ki67 positive cells/tumor area are summarized in the graphs. **p < 0.01 and ***p < 0.001.

Fig 3: Behavioral alterations in H. oligactis animals undergoing aging. (A) Schematic view of the aging process induced in sexual H. oligactis cold‐sensitive (Ho_CS) animals that undergo gametogenesis upon transfer to cold (10°C). Animals from the closely related H. oligactis cold‐resistant strain (Ho_CR) also undergo gametogenesis within three weeks but do not exhibit any aging phenotype and remain fit over the following months (Tomczyk et al., 2017). (B) Contractility upon mechanical stimulation in Ho_CS animals maintained either at 18°C or at 10°C for 35 days. The contractility in response to tweezer stimulation of the peduncle region was recorded on live animals imaged with an Olympus SZX10 microscope equipped with a DP73 camera (movie). Contraction of the whole animal body takes place within 2 s in animals maintained at 18°C and within 10 s in Ho_CS animals maintained at 10°C for 35 days. (C) Light‐induced contractility of Ho_CS and Ho_CR animals maintained at 10°C (n = 15). The contractility was deduced from the time taken by the animal to elaborate a typical contraction response to a bright, directly focused light source. (D) Prey capture of Ho_CS and Ho_CR animals maintained at 10°C (n = 15) assessed from the number of Artemia fixed on tentacles of each animal normalized by tentacle number. In C and D, the legend codes are the same and the error bars correspond to standard error of the mean (SEM). Note that already two weeks after transfer to 10°C, Ho_CS but not Ho_CR animals show an extended contraction latency after light exposure and lose the ability to capture preys. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 4: In vivo testing of the treatments using zebrafish embryos. Adult wild fish were set in breeding tanks. Next day, the eggs were collected, washed, inspected and placed in embryo Danieau’s medium. (a) 48 h post-fertilization (48 hpf), at least, 10 embryos were placed in each well of a 48-well plate containing 400 µL embryo medium. The treatments, which included 25 µM curcumin, 50 ng/mL TRAIL or 25 µM curcumin + 50 ng/mL TRAIL, were added to the embryonic medium for 72 h. Post-treatment, the fish were anesthetized using 0.002% tricain and inspected under a microscope for any abnormalities or death. Representative light microscopy images were taken using an Olympus SZX10 microscope. (b) At least 25 larvae were injected with the ACHN cells labelled with vibrant Dil (red) in their yolk sac using a SYS-PV830 microinjector system. At 72 h post-injection, the larvae were anesthetized with 0.002% tricain then placed on a glass slide and imaged using a fluorescent Olympus SZX10 microscope. BF: bright field channel, RFP1: red channel, Scale bar is 2 mm, n = 3. (c) Statistical analysis using independent t-test to compare between the fluorescent intensity of embryos injected with untreated and pre-treated cells. N = 3 and *** indicates a significance level at p < 0.001.

Fig 5: Silencing gland-related NAC genes causes reduction of gland number. (A) Microscopic images of VIGS plant leaf by stereomicroscope (Olympus SZX10). The number indicates gland number per cm2. (B) QRT-PCR analysis of genes expression in VIGS plants (reference gene: actin).