*3.2. q-RT-PCR Analyses*

The influence of quercetin on some transcription factors and target genes of the IIS pathway was also explored by quantification of the expression of *daf-16, hsf-1, skn-1, daf-18*, and *hsf-16.2* by RT-qPCR in wild type worms, both submitted and not submitted to thermal stress (5 h, 35 ◦C) after growing 4 days in the presence of the flavonol. As it can be seen in Figure 5, the treatment with Q did not modify the expression of *daf-18* either after subjecting or not subjecting the worms to thermal stress. However, the results previously obtained with the mutants (Figure 4) indicated that *daf-18* could be involved in the protective effects of quercetin against stress. A possible explanation to this apparent contradiction could be that the result obtained in the *daf-18* mutant is rather reflecting the involvement of the protein kinase AGE-1, as the DAF-18/phosphatase and tensin homolog (PTEN)protein is responsible for dephosphorylating and inhibiting AGE-1/PI3K, counteracting its activity [21]. Another possibility is that DAF-18 was regulated by quercetin at the post-transcriptional or activity level.

**Figure 5.** Effect of Q on the expression of *daf-16*, *hsf-1*, *skn-1, daf-18*, and *hsp-16.2* genes in N2 *C. elegans* cultivated in the absence (controls) and presence of Q (200 μM) (**A**) under non-stressed conditions or (**B**) after subjecting them to thermal stress. The expression level was determined by RT-qPCR. *act-1* was used as housekeeping control. Nine independent experiments were performed. The results are presented as the mean values ± Standard Error of the Mean (SEM). Statistical significance was calculated using by one-way analysis of variance (ANOVA). The asterisk (\*) indicates significant differences at *p* < 0.05.

Similarly, the expression of *hsf-1* was not modified by the treatment with Q, neither in normal growing conditions nor after application of thermal stress (35 ◦C, 5 h) (Figure 5). This result would confirm that the heat transcription factor *hsf-1* is not involved in the effects of Q on stress resistance, supporting the above-described observations on *hsf-1* mutants (Figure 4). In a previous study on *hsf-1* loss of function mutants, Fitzenberger et al. [28] also found that this gene was not required to explain the ability of Q to prevent the glucose-induced reduction of survival in *C. elegans.*

The treatment with Q did not produce changes in the expression of *daf-16* (Figure 5). Together with the findings obtained in the assays with mutants, these results seem to confirm that the effect of Q on worm stress resistance is independent of *daf-16*. Other authors had already studied the involvement of *daf-16* in the effects of Q using knockout worms [10,11,13], finding that Q continued improving longevity and resistance to thermal and hydrogen peroxide-induced oxidative stress in *daf-16* mutants, which suggest that this gene was not essential for the effects of quercetin, observations that are consistent with the results obtained herein, either in worms subjected or not subjected to stress.

SKN-1 is the homologue of Nrf-2 transcription factor, which regulates oxidative stress response and lifespan, mobilizing the conserved phase 2 detoxification response [29]. No differences were found in the expression of *skn-1* between worms treated or not treated with Q and not subjected to stress (Figure 5A), which seems in agreement with the observations of Pietsch et al. [11], who reported that quercetin induced an increase in the lifespan of *skn-1* mutants under normal growth conditions, indicating that the effects of this flavonoid in the absence of stress are independent of that gene. However, the results obtained with the *skn-1* mutants (Figure 4) indicated that this gene is a mediator in the protective effects of Q against stress. Tullet et al. [29,30] suggested that SKN-1 is a transcriptional co-regulator of DAF-16 regarding resistance to oxidative stress and the expression of detoxification genes in response to a reduced IIS signal, but it extends worm half-life independently of DAF-16. This dual function of *skn-1* might be coherent with the results observed herein, where Q did not change the *skn-1* expression in absence of stress, although this gene seems to be necessary in the improvement of resistance to thermal stress mediated by Q.

The influence of Q on thermal shock proteins HSP-16.2, whose expression is influenced by the IIS, was also studied. As shown in Figure 5, the treatment with Q induced an increase in the expression of *hsp-16.2*, either without or with stress. However, the differences were more noticeable in the absence of stress, which could possibly be explained by the already strong induction of HSP-16.2 caused by the thermal stress, which would make the effect induced by Q treatment less relevant. Together with the results in the *hsp-16.2* mutants, where a decrease in the resistance to thermal stress was observed in quercetin-treated worms (Figure 4), it appears that thermal shock proteins can be involved in the protective effects of the flavonol against stress.

All in all, the obtained results showed that, at least in part, the IIS pathway would be involved in the improvement to thermotolerance induced by Q, entailing the genes *age-1, akt-1, akt-2, sgk-1, daf-2, daf-18, skn-1*, and *hsp-16.2*, but independent of *daf-16* and *hsf-1*. In addition, the involvement of these genes was not modified by the age of the worm. Previous studies by our group with epicatechin also showed that the enhanced stress resistance induced by this flavan-3-ol in *C. elegans* was also mediated by the IIS pathway, although it did not necessarily involve the same genes, as in that case the expression levels of the main transcription factors of the pathway (*daf-16, skn-1*, and *hsf-1*) were modified by the compound [31]. Actually, even compounds belonging to the same flavonoid class seem to act through different mechanisms. Thus, the lifespan extension produced by quercetin-3-*O*-diglucoside in *C. elegans* was explained by upregulation of the genes *daf-2, old-1, osr-1*, and *sek-1*, whereas no modification was produced in the expression of *daf-16, age-1*, and *sir-2.1* [12]. However, assays with a flavonol-rich extract obtained from *Baccharis trimera* concluded that the improvement in the stress resistance was independent of several stress-related signaling pathways (p38, JNK, and ERK) and transcription factors SKN-1 and DAF-16 [32].
