Reduced brain UCP2 expression mediated by microRNA-503 contributes to increased stroke susceptibility in the high-salt fed stroke-prone spontaneously hypertensive rat

UCP2 maps nearby the lod score peak of STR1-stroke QTL in the SHRSP rat strain. We explored the potential contribution of UCP2 to the high-salt diet (JD)-dependent increased stroke susceptibility of SHRSP. Male SHRSP, SHRSR, two reciprocal SHRSR/SHRSP-STR1/QTL stroke congenic lines received JD for 4 weeks to detect brain UCP2 gene/protein modulation as compared with regular diet (RD). Brains were also analyzed for NF-κB protein expression, oxidative stress level and UCP2-targeted microRNAs expression level. Next, based on knowledge that fenofibrate and Brassica Oleracea (BO) stimulate UCP2 expression through PPARα activation, we monitored stroke occurrence in SHRSP receiving JD plus fenofibrate versus vehicle, JD plus BO juice versus BO juice plus PPARα inhibitor. Brain UCP2 expression was markedly reduced by JD in SHRSP and in the (SHRsr.SHRsp-(D1Rat134-Mt1pa)) congenic line, whereas NF-κB expression and oxidative stress level increased. The opposite phenomenon was observed in the SHRSR and in the (SHRsp.SHRsr-(D1Rat134-Mt1pa)) reciprocal congenic line. Interestingly, the UCP2-targeted rno-microRNA-503 was significantly upregulated in SHRSP and decreased in SHRSR upon JD, with consistent changes in the two reciprocal congenic lines. Both fenofibrate and BO significantly decreased brain microRNA-503 level, upregulated UCP2 expression and protected SHRSP from stroke occurrence. In vitro overexpression of microRNA-503 in endothelial cells suppressed UCP2 expression and led to a significant increase of cell mortality with decreased cell viability. Brain UCP2 downregulation is a determinant of increased stroke predisposition in high-salt-fed SHRSP. In this context, UCP2 can be modulated by both pharmacological and nutraceutical agents. The microRNA-503 significantly contributes to mediate brain UCP2 downregulation in JD-fed SHRSP.

The SHRSP represents a suitable animal model for the investigation of the etiopathogenetic basis of hypertensive target organ damage. 1 Feeding SHRSP with JD accelerates both renal and cerebrovascular damage occurrence 2,3 with renal damage preceding stroke. 2,4,5 The gene encoding UCP2 maps nearby the lod score peak of STR1/stroke QTL identified on rat chromosome 1 in the SHRSP. 3 UCP2 is a inner mitochondrial membrane protein that exerts an antioxidant effect in various tissues by regulating fatty acid oxidation, mitochondrial biogenesis, substrate utilization and ROS elimination, 6 and is regulated by PPARα. 7 The latter, a member of nuclear receptor family of ligand-activated transcription factors, is known to regulate lipid and energy metabolism through the uncoupling proteins; 7 it also exerts anti-inflammatory and antioxidant effects in many cell types, including cardiovascular cells. 8 UCP2 downregulation associates with increased oxidative stress, atherosclerosis, vascular damage and shorter lifespan in mice. [9][10][11][12] UCP2 overexpression significantly prevented ROS production in endothelial cells and preserved endothelial function by reducing ROS levels. 13,14 Consistently with its ability to decrease endogenous mitochondrial ROS production and to maintain normal mitochondrial membrane potential and ATP levels, a neuroprotective effect of UCP2 has been previously described both in vitro and in vivo. [15][16][17][18][19][20][21] We previously reported an age-related spontaneous decrease of UCP2 gene and protein expression only in the brain of SHRSP, preceding spontaneous stroke occurrence at 1 year of age. 22 Of interest, we have shown that, in association with increased renal injury, JD significantly downregulates UCP2 gene and protein expression in the kidneys of SHRSP, but not of SHRSR. 23 Consistent findings were obtained in the kidneys of SHRSR/SHRSP-derived stroke congenic lines, depending on the genetic configuration of the transferred UCP2. 24 In vitro, UCP2 silencing in renal mesangial cells led to increased inflammation, oxidative stress and cell mortality. 23 Exposure of primary renal proximal tubular epithelial cells isolated from SHRSP to high-NaCl medium led to UCP2 downregulation and reduced viability, which was rescued by recombinant β-Actin UCP2. 24 Moreover, the PPARα-mediated upregulation of UCP2 gene and protein expression by BO sprouts juice, administered along with JD, completely prevented renal damage occurrence in SHRSP. 25 As expected, the selective inhibition of PPARα reduced the beneficial effects of BO on the renal injury of this strain. 25 Notably, fenofibrate, a compound that exerts renal and neuroprotection in various experimental settings through its impact on several antioxidant enzymes, [26][27][28] and that is also known to stimulate PPARα and UCP2 expression, 29 promoted protection from target organ damage in SHRSP. 30 Based on the above-mentioned observations, the aims of the present study were: (1) to assess for the first time the modulation of UCP2 in the brain of high-salt-fed SHRSP versus SHRSR, as well as in two SHRSR/SHRSP-STR1/QTL stroke congenic lines; (2) to explore the impact of PPARα and UCP2 expression modulation by BO and fenofibrate on the stroke susceptibility of high-salt-fed SHRSP; and (3) to explain part of the mechanisms underlying brain UCP2 downregulation upon JD in the stroke-prone strain.

Results
Impact of 4 weeks JD feeding on brain UCP2 expression and related inflammatory and oxidative stress parameters in the four rat lines. Four weeks of JD feeding induced a significant UCP2 gene and protein expression downregulation only in the SHRSP brain (Figures 1a-c), as previously reported in the kidneys. [23][24][25] The (SHRsp.SHRsr-(D1Rat134-Mt1pa)) congenic line, derived from the SHRSP parental strain and carrying the SHRSR/STR1 chromosomal fragment, did not downregulate UCP2 under JD, differently from the SHRSP strain of origin (Figures 1d and e). Vice versa, the (SHRsr.SHRsp-(D1Rat134-Mt1pa)) congenic line, derived from the SHRSR parental strain and carrying the SHRSP/STR1 chromosomal fragment, significantly downregulated UCP2 under JD, differently from the SHRSR strain of origin (Figures 1f and g). These results confirmed the key role of UCP2 configuration (SP or SR) for the response to high-salt diet. Figure 2 shows the NF-κB protein expression level, a marker of inflammation, and the carbonylated protein level, a marker of oxidative stress, in the brains of the parental lines (SHRSR: panels a and b; SHRSP: panels c and d). Both inflammatory and oxidative stress markers were significantly increased only in the brains of JD-fed SHRSP. Figure 3 shows the same parameters in the two STR1/QTL stroke congenic lines, the one derived from the SHRSP (panels a and b), and the one derived from the SHRSR (panels c and d). Both inflammatory and oxidative stress markers were decreased in the SHRSP-derived stroke congenic line, carrying the SHRSR/ STR1 chromosomal fragment (panels a and b), whereas these markers increased significantly in the SHRSR-derived stroke congenic line carrying the SHRSP/STR1 chromosomal fragment (panels c and d). These results confirmed that, whenever UCP2 expression was downregulated, such as in JD-fed SHRSP and JD-fed (SHRsr.SHRsp-(D1Rat134-Mt1pa)), the degree of inflammation and of oxidative stress increased. Vice versa, no increase of these processes was detected in the brains of both JD-fed SHRSR and JD-fed (SHRsp.SHRsr-(D1Rat134-Mt1pa)), both carrying higher levels of brain UCP2 expression (as compared with the other two lines).
Impact of fenofibrate administration on brain UCP2 expression and on stroke occurrence in JD-fed SHRSP. Figure 4 shows the impact of JD plus fenofibrate versus JD alone on brain UCP2 gene and protein expression, and on NF-κB and oxidized total protein levels at the end of 4 weeks of treatment. Fenofibrate could restore UCP2 level (panels a and b) and decrease levels of both NF-κB and oxidative stress (panels c-e). Figure 5 shows the results of the stroke survival study performed with a long-term fenofibrate administration (3 months) in JD-fed SHRSP. The impact on UCP2 gene and protein expression levels in brains of JD plus fenofibrate treated SHRSP, as compared with animals receiving JD only and JD plus vehicle, is shown in the panels a and b of the Figure 5. Also at the end of 3 months of treatment, the parallel administration of JD and fenofibrate restored UCP2 level (panels a and b), and decreased levels of both NF-κB and oxidative stress despite the long-term treatment with JD (panels c-e). Importantly, fenofibrate fully protected animals from stroke occurrence over 3 months of follow-up (panel f). In contrast, occurrence of stroke events reached 100% by the seventh week of JD in both JD and JD plus vehicle treated rats, consistently with previous evidence. 3,5 SBP and BW values upon fenofibrate administration are reported in the Supplementary Table S1.
Impact of BO administration on brain UCP2 expression and on stroke occurrence in JD-fed SHRSP. Supplementary Figure S1 shows the impact of JD plus BO versus JD alone on brain UCP2 gene and protein expression, NF-κB and oxidized total protein levels at the end of 4 weeks of treatment. BO restored UCP2 level (panels a and b) and decreased levels of both NF-κB and oxidative stress (panels c-e). Figure 6 shows UCP2 gene and protein expression levels, at different experimental times during the stroke survival study, in brains of JD plus BO treated rats, as compared with animals receiving JD only and JD plus BO plus PPARα inhibitor (panels

Chemiluminescence intensity (Arbitrary units)
ba nd 2 b a n d 3 b a n d 4 b a n d 5 to ta l __ ba nd 1 b a n d 2 b a n d 3 b a n d 4 b a n d 5 to ta l __ b a n d 1  Figure S2), and led to a significant delay of stroke occurrence (Figure 6e). In fact, 40% of rats survived until the 11th week of treatment. The PPARα inhibitor significantly counteracted the stimulatory effect of the BO juice on UCP2 expression, therefore leading to 100% stroke occurrence by the eighth week of treatment (Figures 6a-e), consistently with previous findings. 25 The SBP and BW values upon these treatments are reported in the Supplementary  Table S1.
Analysis of UCP2-targeted microRNAs upon JD versus RD in brains of SHRSR and SHRSP. Out of the compared UCP2-targeted microRNAs in the brains of the SHRSR and SHRSP strains upon the two diets, we detected a remarkable differential expression, very consistent with the parallel differential UCP2 expression, for the rno-microRNA-503. In fact, this miR was remarkably upregulated (42 folds) in the brain of JD-fed SHRSP whereas it was significantly downregulated in the brain of JD-fed SHRSR as compared with RD ( Figure 7a). No other miR showed a significant modulation in relation to the observed UCP2 expression changes. Based on the results of the microRNAs screening, we further explored the modulation of the microRNA-503 expression in our experimental groups. We discovered that SHRSP receiving either fenofibrate or BO along with JD showed a significant reduction of brain miR-503 expression level (Figures 7b and c). The expected interference by PPARα inhibitor was observed in SHRSP receiving JD and BO ( Figure 7c). Furthermore, we observed a significant downregulation of brain miR-503 level in the JD-fed SHRSPderived congenic line containing the SHRSR/STR1 fragment (Figure 7d), whereas the SHRSR-derived congenic line, containing the SHRSP/STR1 segment, showed a significant upregulation of miR-503 upon JD (Figure 7e). Therefore, the data obtained in the two STR1/QTL stroke congenic lines reinforced the evidence obtained in the parental lines of origin.
Impact of microRNA-503 overexpression on viability of HUVECs. The in vitro overexpression of hsa-miR-503 in HUVECs showed a marked UCP2 suppression with a linear dose-response (Figures 8a and b). Importantly, at a miR-503 concentration able to turn off UCP2 expression by 90%, a significant increase of cell mortality and a significant decrease of cell viability were observed (Figure 8c). The impact on cell viability was comparable to that obtained upon direct UCP2 silencing in HUVECs (Figure 8d).

Discussion
Our study demonstrates that UCP2 gene and protein expression levels are significantly downregulated by Japanese style dietary feeding in brains of SHRSP but not in brains of its related control strain, the SHRSR. This phenomenon was associated with increased inflammation and oxidative stress. Accordingly, a SHRSR-derived stroke congenic line, carrying a fragment of the SHRSP-STR1/QTL (containing UCP2), showed brain UCP2 downregulation under JD feeding associated with increased inflammation and oxidative stress. Vice versa, brain UCP2 expression did not decrease, and both inflammation and oxidative stress were reduced upon JD in the reciprocal congenic line. Consistently, the administration of JD plus fenofibrate, known to stimulate UCP2 expression, 25 restored brain UCP2 levels, reduced oxidative stress and fully protected from stroke occurrence the high-salt fed SHRSP. The administration of BO sprouts extract, another stimulator of UCP2 expression, 25 was also able to delay significantly stroke occurrence in JD-fed SHRSP. The PPARα inhibitor antagonized the beneficial effect of BO, confirming previous evidence obtained in the kidneys. 25 The greater efficacy of fenofibrate versus BO on stroke protection may be explained by the additional molecular and pharmacological properties of the drug. Of note, NF-κB, which was characterized in the current study mainly for its important role in inflammation, is a ubiquitous transcription factor that, due to its wide range of gene targets, plays several other functions in mammalian cells, particularly in the nervous system. 31 Based on our results, UCP2 appears to play an important role in the high-salt diet-dependent increased susceptibility to cerebrovascular events, as well as it does for the increased susceptibility to kidney damage of SHRSP. 23-25 A common molecular mechanism, dependent on UCP2 suppression, may underlie the vascular damage observed in different organs of high-salt-fed SHRSP.
Uncoupling the proton flux through UCP2 is a critical pathway in the regulation of senescence. 8,32,33 The involvement of UCP2 in vascular diseases is known, being demonstrated in several pathological contexts, [9][10][11][12][13] and it represents the consequence of its key role in the clearance of ROS within the mitochondria. A neuroprotective effect of UCP2 has been previously associated with its role on oxidative stress. [16][17][18][19][20][21] An association of UCP2 with stroke has been described in other experimental settings. 34,35 A major strength of our data relies on the evidence obtained in the reciprocal SHRSR/SHRSP-STR1/QTL stroke congenic lines. The stroke phenotype of these lines clearly depends on the genomic configuration of the inserted chromosomal segment belonging to STR1, with evidence that the chromosomal fragment carrying UCP2 significantly interferes with stroke occurrence. 5 Herein, we report that the introgression of the stroke-prone STR1 chromosomal segment (carrying UCP2) within the stroke-resistant genomic background led to a suppression of UCP2 expression in contrast to the upregulation of the SHRSR strain. The opposite phenomenon was observed in the reciprocal congenic line, supporting the role of the stroke-prone UCP2 configuration to obtain downregulation in response to high-salt diet. We recently reported similar findings on UCP2 gene and protein expression with regard to renal damage in the same stroke congenic lines. 24 A fundamental demonstration of the role of UCP2 in stroke predisposition of SHRSP was provided by the significant protective impact of BO and fenofibrate administration, both stimulator of UCP2 expression, 25,26 toward stroke occurrence despite JD feeding. Of note, our data on fenofibrate as a UCP2 and stroke in SHRSP S Rubattu et al protective agent toward stroke confirm and extend previous findings obtained in the same animal model. 30 No evidence of UCP2 mutations between the two strains was obtained in our previous study. 23 In the attempt to unravel, at least in part, some of the mechanisms underlying brain UCP2 downregulation upon JD in the stroke-prone strain, we searched for mechanisms involved in the translational regulation of UCP2. As a result, we found that the UCP2 expression β-Actin

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UCP2 and stroke in SHRSP S Rubattu et al modulation upon JD in the brains of SHRSP and SHRSR was related to the microRNA-503. The latter turned out to be significantly upregulated in high-salt-fed SHRSP, whereas it was significantly decreased in the SHRSR upon the same diet. Consistent results were obtained in the two congenic lines, further supporting the role of this microRNA in UCP2 downregulation. Moreover, treatment with both fenofibrate and BO counteracted the increase of brain microRNA-503 level and the suppression of UCP2 expression in JD-fed SHRSP. Importantly, both treatments, by their ability to restore regular levels of both microRNA-503 and UCP2, significantly protected from stroke occurrence the JD-fed SHRSP. Finally, miR-503 overexpression in vitro abolished UCP2 expression and caused a high degree of cell mortality, consistently with what observed upon direct UCP2 silencing. Our results strongly suggest that miR-503 is a modulator of brain UCP2 expression in high-saltfed SHRSP and also in SHRSR. Of note, miR-503 exerts multiple actions. It is reported as an antiproliferative and antiangiogenetic factor, 36 and a cell cycle regulator; it is involved in cell adhesion, migration and angiogenesis processes. 37 Its upregulation in diabetes strongly associates with vascular damage. 36,38 On the other hand, a decrease of miRNA-503 upon losartan treatment is associated with an improvement of diabetic nephropathy in an animal model of spontaneous type 2 diabetes. 39 Herein, we report the first evidence that an increase of miR-503 associates with high-salt induced stroke occurrence, through its ability to modulate brain UCP2 expression, in an animal model of spontaneous hypertension and stroke and that, in turn, miR-503 can be decreased by both pharmacological and nutraceutical approaches to obtain protection from stroke. Further studies will address the interaction between PPARα and miR-503 in the UCP2 regulation. It will be also interesting to characterize the potential contribution of miRNA-503 in the prevention and/or amelioration of hypertensive target organ damage with the available therapeutic antihypertensive strategies. Our novel findings support the role of microRNAs in the endorgan damage promotion during hypertension. 40 We are also aware, as a limitation of the current study, that the role of other still unknown UCP2-targeted brain microRNAs remains to be determined in our experimental conditions.
In summary, we demonstrate that UCP2 expression downregulation by high-salt diet associates with increased stroke predisposition whereas UCP2 upregulation, by both nutraceutical and pharmacological agents, associates with a significant stroke protection in high-salt-fed SHRSP. Our data strengthen the role of UCP2 as a suitable therapeutic target for stroke. Notably, the microRNA-503 behaves as a key determinant of the dietary-dependent regulation of UCP2 expression in the brain of SHRSP. Thus, the microRNA-503 has a significant potential in unraveling the mechanisms underlying stroke pathogenesis and may reveal a promising therapeutic agent for this disease.

Materials and Methods
Effects of 4 weeks JD feeding on brain UCP2 gene and protein expression in SHRSP, SHRSR and in the SHRSR/SHRSP-STR1/ QTL stroke congenic lines. The following rat lines were used for this study: SHRSP, SHRSR, (SHRsp.SHRsr-(D1Rat134-Mt1pa)), (SHRsr.SHRsp-(D1Rat134-Mt1pa)). The latter two lines are congenic lines carrying the (D1Rat134-Mt1pa) chromosomal segment of STR1 (containing UCP2) in the SHRSR configuration within the SHRSP genomic background (SHRsp.SHRsr- (D1Rat134-Mt1pa)) and, viceversa, in the SHRSP configuration within the SHRSR genomic background (SHRsr.SHRsp- (D1Rat134-Mt1pa)). The preparation of the congenic lines has been previously reported. 5 By following our standardized experimental protocol, six-weekold male rats of both parental and congenic lines were fed with either RD or JD for 4 weeks (parental lines, n = 7 for each line at each treatment; congenic lines, n = 6 for each line at each treatment). At the end of 4 weeks of the dietary treatment, after SBP and BW measurement, animals were killed by cervical dislocation and brains were removed for molecular analyses. Tissue total RNA and proteins were extracted by following previously described procedures. [22][23][24][25] The UCP2 gene and protein expression levels, the NF-κB protein expression level and carbonylated total proteins level were assessed by previously described procedures. [22][23][24][25] Impact of fenofibrate administration on brain UCP2 gene and protein modulation and on stroke occurrence in JD-fed SHRSP. In order to fulfill the first aim, male SHRSP received, starting at 6 weeks of age, JD alone (n = 4), JD plus fenofibrate (150 mg/kg/die, n = 4) or vehicle (1% CMC, n = 4) administered via gavage for 4 weeks. In order to analyze the impact of fenofibrate on stroke survival, 6-week-old SHRSP received JD alone (n = 6), JD plus fenofibrate (n = 6) or JD plus CMC (n = 6). They were monitored for SBP, BW and stroke occurrence up to 3 months of the dietary plus fenofibrate treatment.
Brains of JD plus fenofibrate treated SHRSP, taken at the end of both 1 month and 3 months of treatment (n = 4 and 6, respectively), were analyzed for UCP2 gene and protein expression levels, NF-κB protein expression and oxidative stress levels. These molecular analyses were compared with those obtained in brains of 4-week JD-fed SHRSP from the above experimental setup and of 4-week JD plus CMC treated SHRSP.
Impact of BO juice administration on brain UCP2 gene and protein modulation and on stroke occurrence in JD-fed SHRSP. In order to fulfill the first aim, male SHRSP received, starting at 6 weeks of age, JD alone (n = 4) or JD plus BO sprouts extract (n = 4) for 4 weeks by following previously reported procedures. 25 In order to explore the impact of BO on stroke survival, male SHRSP received JD alone (n = 6), JD plus BO (n = 10), JD plus BO plus PPARα inhibitor (n = 6) as previously reported. 25 SBP, BW and stroke occurrence were monitored up to 3 months of the dietary plus BO juice treatment. Brains of SHRSP, taken either at the end of 4 weeks or at the time of stroke occurrence (between the 8th and 12th week of treatment upon BO; between the 7th and 8th week of treatment upon BO plus PPARα inhibitor), were analyzed for UCP2 gene and protein expression levels, NF-κB protein expression and oxidative stress levels. These molecular analyses were compared with those obtained in brains of 4-week JD-fed SHRSP from the above experimental setup.
All rats used for the experiments described in the paragraphs 1-3 were maintained at the animal facility of the Neuromed Institution in strict compliance with the guidelines set forth by the American Physiological Society. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Neuromed Institution. Climate was controlled, and temperature was set at 22°C. Diurnal 12-h cycles were kept automatically. Animals were housed two or three per cage with free access to RD (containing 22% protein, 2.7 mg/g Na + , 7.4 mg/g K + , 0.05 mg/g methionine) and tap water, unless stated otherwise. The JD contained 17.5% protein, 3.7 mg/g K + and 0.03 mg/g methionine (Lab. Piccioni, Milan, Italy), and 1% NaCl was added to the drinking water.
Analysis of UCP2-targeted microRNAs expression upon JD versus RD in brains of SHRSR and SHRSP. Based on the very limited knowledge of the rat UCP2 brain modulation by targeted microRNAs, we selected conserved predicted UCP2-targeted microRNAs with all miRSVR scores by performed in triplicate in brain extracts of both parental strains upon the two diets by specific gene expression Taqman assays (Life Technologies). Based on the evidence of a significant microRNA-503 upregulation in the brain of JD-fed SHRSP Figure 6 Impact of long-term administration of BO on brain UCP2 modulation, NF-κB protein expression, oxidative stress levels and on stroke occurrence in JD-fed SHRSP. as opposed to a significant downregulation in the brain of SHRSR (see Results section), the modulation of this miRNA was verified in JD-fed SHRSP upon fenofibrate, vehicle, BO, BO plus PPARα inhibitor administration, as well as in the brains of the two SHRSR/SHRSP-STR1/QTL stroke congenic lines (by analyzing the same rats used in the above described experimental groups).
In vitro hsa-microRNA-503 overexpression in HUVECs. In order to verify directly the impact of microRNA-503 on UCP2 expression levels, we performed a dose-response experiment in vitro. For this purpose, HUVECs (Lonza, Cambrex, Belgium) were seeded in 60-mm well plates (2 × 10 5 cells/well) and cultured in endothelial growth medium-2 (EGM-2; Lonza) to reach a 70-80% confluence. Then, serial concentrations of 12.5, 25, 50, 100, 200 and 400 nM of hsa-microRNA-503 mimic (Mission microRNA; Sigma-Aldrich (Milan, Italy)) were incubated in OPTIMEM reduced serum medium with a nucleic acid transferring agent (lipofectamine RNAiMAX reagent (Invitrogen, Milan, Italy)) in a final volume of 2 ml/well each for 20 min. Five hours later the complex containing medium was replaced with EGM-2 medium supplemented with 10% fetal bovine serum. Cells transfected with RNAiMax lipofectamine complex and mission miRNA negative control (Sigma-Aldrich) were used as control. Twenty-four hours after transfection cells were extracted for total RNA, by the RNazol procedure, 23 and used for the evaluation of both miR-503 and UCP2 expression levels by RT-PCR. A specific gene expression Taqman assay (Lifetech, Waltham, MA, USA) was used to assess miR-503 levels, as reported above. The RT-PCR of UCP2 was performed as reported above. Finally, we assessed the impact of miR-503 overexpression at 100 nM concentration (corresponding to 90% reduction of UCP2 expression) on cell apoptosis, necrosis and viability, as assessed by FACS. The results of FACS were compared with those obtained by performing UCP2 silencing with a specific siRNA in the same cell line (by following previously reported procedures 23 ).