*3.3. Assays with Fluorescent Reporters*

The insulin signaling pathway transmits signals in response to the environmental conditions that could change the expression of different genes related to stress and longevity, thus regulating important processes such as aging, metabolism, or dauer formation. The expression of some such genes, namely those encoding heat shock proteins (*hsp*-16.2 and *hsp*-70) and antioxidant enzymes (*sod*-3 and *gst-4*), was assessed in order to gain further insight into the mechanisms of action underlying the effects of Q on the modulation of lifespan and stress in *C. elegans*. With this aim, transgenic strains that express GFP under the control of *gst-4, sod-3, hsp-16.2*, and *hsp-70* promoters were employed. GFP expression levels were analyzed in animals grown in the presence or absence of quercetin under non-stress conditions for *Pgst-4::gfp* and *Psod-3::gfp* reporters, whereas for *Phsp-70::gfp* and *Phsp-16.2::gfp* reporters, worms were previously submitted to a heat shock (35 ◦C, 1 h) and further allowed to recover for 3 h (*hsp-70*) or 2 h *(hsp-16.2*) at 20 ◦C.

As it can be seen in Figure 6, the treatment with Q did not produce an increase in the expression of any of the studied genes (*gst-4, sod-3, hsp-16.2*, and *hsp-70*) in young worms of reproductive age. Indeed, there was even a decrease in the expression of *hsp-70* in the worms treated with Q (*p* = 0.018). The expression of *hsp-16.2* and *sod-3* was also studied using reporter strains in older worms (day 9), in order to establish if the mechanism of action underlying the effects of Q was dependent on the age of the worm. Similar results were obtained regarding the expression of *sod-3* (Figure 7) as compared to younger worms (Figure 6), finding no differences in the expression by the treatment with Q. However, the expression of *hsp-16.2* was increased by the treatment with Q at day 9 (*p* = 0.008) (Figure 7), which was not observed at day 2 (Figure 6).

Some of these genes have already been studied by other authors using fluorescent reporters to explore worm response to Q, specifically *gst-4* and *sod-3*. Opposed results were reported regarding the effects of Q on *sod-3*. Whereas, Kampkötter et al. [9] observed a decrease in the expression of *sod-3* in worms exposed to 100 μM Q, Grünz et al. [13] found that growing the worms in the presence of 100 μM of Q produced a significant increase in *sod-3* expression. As for the present study, no significant changes were detected in the expression of *sod-3* after treatment with 200 μM of Q in any of the two days studied. On the other hand, Kampkötter et al. [14] observed that the expression of *gst-4* was not modified by Q 100 μM under normal growth conditions, although it decreased the expression of *gst-4* when worms were subjected to oxidative stress with juglone 20 μM [14]. Those results would be in agreement with the observations made herein, where no increase in the expression of *gst-4* was found under normal growth conditions (200 μM Q).

The results for *Phsp-16::gfp* obtained at different ages of the worms could help to understand the effects of increased lifespan and improvement of resistance to stress induced by Q in *C. elegans*, since many heat shock proteins are regulated positively at the beginning of adult life to further decrease throughout life [33]. The obtained result might indicate that this decrease could be reversed by the treatment with Q. It is pertinent to indicate that in previous studies on longevity, greater survival started to be observed from approximately day 8 onwards in worms treated with quercetin in relation to non-treated worms [8], suggesting changes in *C. elegans* metabolism favored by prolonged exposure to the flavonol, among which, the upregulation of some heat shock proteins could be involved. Actually, the increased lifespan and thermotolerance observed in certain mutants, such as the long-lived *age-1*, have been explained by an increase in the regulation and accumulation of HSP-16 [34]. The involvement of *hsp-16.2* was also supported by the increase in the expression of *hsp-16.2* found in the RT-qPCR studies (Figure 5) and the loss of the improvement in resistance to thermal stress in the *hsp-16.2* mutant (Figure 4). On the other hand, the results showed a decrease in the reporter of *hsp-70* in worms treated with Q (Figure 6). Differences in the expression of genes encoding distinct heat shock proteins, were also found by Pietsch et al. [15], observing that Q produced an increase in the expression of *hsp-3, hsp-12.6, hsp-16.1*, and *hsp-16.41*, but a decrease of *hsp-70* and *hsp-17*.

**Figure 6.** Effect of Q on the expression of *gst-4, hsp-16.2, hsp-70*, and *sod-3* after cultivation of *C. elegans* in the absence and presence of Q (200 μM). (**A**) Representative fluorescence images of control and Q-treated worm strains. (**B**) Quantification of the relative fluorescence intensities of transgenic worms. Total green fluorescent protein (GFP) fluorescence of each whole worm was quantified using Image J software. Three independent experiments were performed. The results are presented as the mean values ± SEM. Approximately 35 randomly selected worms from each set of experiments were examined. Differences compared with the control (0 μM Q, 0.1% dimethyl sulfoxide (DMSO)) were considered statistically significant at \* (*p* < 0.05) by one-way ANOVA.

**Figure 7.** Effect of Q on the expression of *hsp-16.2* and *sod-3* in old worms (day 9 of adulthood) after cultivation of *C. elegans* in the absence and presence of Q (200 μM). Total GFP fluorescence of each whole worm was quantified using Image J software. Three independent experiments were performed. The results are presented as the mean values ± SEM. Approximately 35 randomly selected worms from each set of experiments were examined. Differences compared with the control (0 μM Q, 0.1% DMSO) were considered statistically significant at \*\* (*p* < 0.01).

The modulation of the subcellular localization of the DAF-16 forkhead transcription factor was examined using a transgenic strain expressing a fusion protein DAF-16::GFP. The treatment with Q did not affect the translocation of DAF-16 to the nucleus with respect to the control worms, neither under normal growth conditions nor after thermal stress (Figure 8). The localization of DAF-16 was also studied in older worms (day 9), obtaining similar results that at day 2 regarding its subcellular localization.

As discussed above, the treatment with Q did not produce an increase in the expression of *daf-16* in any of the assayed conditions, while it led to an increase in the resistance to thermal stress on *daf-16* mutant strains. All these results indicate that the effect of this flavonol in the improvement of worm resistance to stress is independent of *daf-16*. A similar conclusion has been obtained by some authors [10,11], although others observed greater translocation of DAF-16 from the cytosol to the nucleus following Q treatment [9,13,14]. In this respect, Saul et al. [10] suggested that the translocation of DAF-16 to the nucleus in response to quercetin could be more of a circumstantial effect than proof of an underlying longevity mechanism. In fact, although DAF-16 is a key factor in the control of stress response and longevity [17,35,36], it has also been pointed out that its translocation to the nucleus does not guarantee a longer lifespan, suggesting that DAF-16 would not necessarily act only in the regulation of longevity [37,38].

The finding that the effects of Q can be independent of *daf-16* but dependent on *daf-2* and other components of the IIS, such as *akt-1*, *age-1*, and *akt-2*/*sgk-1*, could appear surprising. Nevertheless, Hekimi et al. [37] and Lin et al. [39] pointed out that when the IIS pathway is altered in response to different environmental signals, besides the stress resistance genes, other unidentified signals are regulated by *daf-2*, which are not dependent on *daf-16*, but that are also essential for the extended longevity in *daf-2* mutants.

In the end, the obtained results indicated that two key transcription factors that could be involved in the expression of heat shock proteins, DAF-16 and HSF-1 [40], were not related with the effects exerted by Q on *C. elegans* (Figures 4 and 5). Actually, it is described that HSF-1 is not a key factor for all HSPs [41]. Mertenskötter et al. [42] showed that in the expression of chaperone genes, protein biosynthesis and protein degradation was positively influenced by the MAPK pathway and established the importance of this pathway in heat stress responses, possibly by a PMK-1-mediated activation of the transcription factor SKN-1 in *C. elegans.* Other authors have also found that components of related pathways, such as UNC-43, SEK-1, and OSR-1 are involved in the molecular mechanisms of the response to quercetin and other polyphenols [11,43,44]. Thus, the MAPK pathways could also be implicated in the effects of quercetin, which might contribute to explain the role of SKN-1 and the activation of certain HSPs observed in the present study.

**Figure 8.** (**A**) Effect of Q on DAF-16::GFP subcellular distribution and (**B**) representative pictures of the subcellular location of DAF-16::GFP, i.e., cytosolic, intermediate and nuclear. Transgenic worms expressing the fusion protein DAF-16::GFP were cultivated in the absence (controls) and presence of Q (200 μM) after subjecting or not subjecting the worms to thermal stress and evaluated at day 2 of adulthood.
