2.2. C. elegans

We employed standard nematode culture conditions [23]. All strains were maintained at 20 °C on Nematode Growth Media (NGM) agar supplemented with Escherichia coli (OP50 or transformed HT115). Strains employed in this work were as follows: N2 (wild-type), CL2166 (dvIs19[pAF15(gst-4::GFP::NLS)] III), SJ4100 (zcIs13[hsp-6::GFP]), VC228 nlg-1(ok259) X and cyp-14A4p::gfp::cyp-14A4 3′UTR [24].

Worms were treated with the compounds in two different ways, either in liquid culture or on solid medium (NGM agar plates). All compounds were dissolved in DMSO, which was used as control in all the experiments. In liquid the maximum DMSO concentration used was 1% while in solid media was 0.25%. Different stock solutions with different concentrations were prepared for the compounds, thus allowing to have the same final DMSO concentration in control and compounds treated animals in liquid or solid media.

In the first part of the screening C. elegans was cultured in liquid. The worms were synchronized by bleaching to obtain a large number of animals [25]. The screening was carried out on 96-well plates. Briefly, 10 mL of overnight grown OP50 bacteria were pelleted for 10 min at 4000 rpm; afterward, the supernatant was removed and the pellet was frozen in liquid nitrogen. Then, 10 mL of freshly prepared S-medium was prepared with the addition of following reagents: 8 μL Cholesterol (of 5 mg/mL stock solution in EtOH) and 50 μL 100× Penicillin/Streptomycin (P/S). The frozen bacterial-pellet was re-suspended with 4 mL of S-medium. The bacteria suspension was diluted in S-medium to obtain an OD600 = 2. We allowed the re-suspended bacteria to reach room temperature because lower temperatures could affect worms’ development. The final volume in every well of the 96-well plate was 250 µL, composed by: 140 µL of bacterial suspension, 10 µL compound under study plus H₂O, 60 µL of S-medium, 40 µL of solution containing L1 larvae (1 worm/5 µL). Each column of the plate was used for a different concentration of the compound (border rows, A and H, and columns, 1 and 12, were not used). The plate was covered with a lid.

Nematode growth media (NGM) agar plates with HT115 bacteria were prepared and spotted with bacteria. Before the addition of compounds, bacteria on the plates were killed by UV exposure for 25 min. Afterward, the compound at the desired final concentrations was spotted. Egg-lay was performed on the same plates and the phenotype and GFP induction were checked on the third and fourth day after egg-lay. After spotting, plates were dried overnight at room temperature and used the day after.

Plates were prepared accordingly to our previously established protocol [13]. Briefly, on the day of the screen (3 days after plating and 4 after bleaching) plates containing animals treated with the compounds under study were washed with S-basal (duplicate plates per condition). Nematodes were transferred into 15 mL collecting tubes and allowed to settle by gravity. Two to three washing were carried out to remove excess bacteria which could interfere with the imaging. Worms were finally resuspended in S- basal and aliquots of 150 μL of solution were transferred in each well of a 96-well plate. Prior to image acquisition, worms were anesthetized by adding 2 μL of 1 M NaN₃ to each well and the plate closed with a clear sealing film. Images were acquired with the ArrayScan VTI HCS Reader using a 2.5× objective and a 0.63× coupler using a 1-channel (GFP) assay. The Spot Detector BioApplication was used to quantify fluorescent intensity. Image acquisition was completed in about 25 min.

A stereo microscope (modular, Leica MZ10 F Wetzlar Germany) with maximum 40× magnification was used to investigate the worm’s phenotype, specifically any delay in development or change is size and pigmentation was monitored. When using GFP reporter strains the pictures were taken using a color digital camera (0.5×) connected to the microscope, then GFP intensity, as well as the Red Fluorescence of Nile Red, was quantified using the software Image-J (http://imagej.nih.gov/ij/, last accessed on 7 September 2021).

For area and GFP intensity measurements the Cellomics ArrayScan Reader platform was used [13,26]. In this study, the BioApplication SpotDetector was used to quantify the effect of natural compounds on animals’ phenotypes, specifically the size and induction of gst-4 or hsp-6::GFP transgene.

To analyze the effect of the compounds on worms’ fertility, the treatment was started from eggs. Gravid animals were allowed to lay eggs on plates containing the compounds for about 2 h, once ~40 eggs were reached the adults were removed and the eggs allowed to develop. As soon as the worms reached the L4 stage were individually transferred onto small (3 cm) NGM plates spotted with 50 μL of bacteria (HT115) at 20 °C. Animals were transferred to fresh plates every 24 h until day 7. Progeny in each plate was counted 48 h after hatching to facilitate the count of the larvae. The brood size of each animal was calculated as the sum of the total progeny in the whole fertile period.

The treatment with the compounds started from eggs similarly to fertility and lifespan assay. On the first day of adulthood 20–25 animals were transferred into a fresh plate with the same condition and exposed to 35 °C in an air incubator. The plates were sealed with parafilm to avoid desiccation. Survival was checked every hour until the death of all animals (about 8 h). Survival rate was estimated by using Kaplan-Meyer estimator (OASIS 2 [27], available at http://sbi.postech.ac.kr/oasis, last accessed on 22 Novermber 2021).

Juglone sensitivity assay was carried out, with slight modifications, as previously described in [28]. Specifically, a stock solution of 50 mm juglone (Calbiochem) was freshly dissolved in DMSO prior to addition to molten NGM. Plates were freshly made approximately 3 h before use and seeded with concentrated OP50 30 min before being used. To assess survival, age matched young adult animals growth on HT115 NGM plates, were transferred to 60 mm NGM plates containing either compounds or control (DMSO) and assayed over time. Animals that escaped the plates were excluded from the analysis (censored). Animals were scored as dead if they did not respond to repeated light prodding. Percentages alive for each condition were determined by averaging the fraction alive per plate at each time point and plotting graphically. Survival curves were realized by using Kaplan-Meyer estimator OASIS 2.

Survival curves and statistical analyses were carried out as previously described [29]. Briefly, survival analysis started from a synchronous eggs population and was carried out at 20 °C on solid NGM, at least 60 animals per condition were scored. Worms were treated with the compounds under study (or DMSO as control) from eggs throughout their lifetime.

Animals were scored as dead, alive or censored and transferred every day to fresh plates during the fertile period, and then every two or three days until death. Nematodes were considered dead when neither activity of their pharynx, nor response to touch was detected. Worms that died because of internal bagging, desiccation due to crawling on the wall of the plates, or gonad extrusion were considered censor. These animals have been included in the analyses until the time of censorship and are weighted by half in the statistical analysis. We used the Online Application for Survival analysis OASIS 2 for calculation of mean lifespan, standard deviation of the mean, and p values (Mantel–Cox regression analysis) from Kaplan–Meyer survival curves of pooled population of animals coming from at least two independent replicas. Data from survival analysis are summarized in Supplementary Table S2.

Locomotion was assessed by counting the number of body bends per minute for each worm on solid agar plates with no bacteria. One bend was counted every time the mid-body reached a maximum bend in the opposite direction from the bend last counted. Body bends were checked in at least 10 single worms in 2 or 3 independent biological trials [30].

A stock solution (0.1 mg/mL) of Nile red was prepared dissolving the powder (Invitrogen N1142) in 100% acetone. The working solution was prepared in S-basal and added on top of NGM 6-cm plates seeded with OP50 bacteria, at a final concentration of 50 nM. The plates were previously spotted with the investigated compounds (or DMSO as control) and let dry. Synchronous eggs were allowed to develop and grow until the worms became adults on the Nile Red plates. Plates were always stored in the dark. Adult worms were transferred on plates without Nile Red 30 min before imaging. Then, 10–15 animals randomly selected from an initial sample of 40 worms were mounted on microscope slides. Lysosomes content was quantified by fluorescence microscope. Fluorescence intensity was measured through ImageJ software by selecting the same area between the vulva and the pharynx in each worm, on grayscale using the red channel of the acquired images. The assay was performed in triplicate.