ANTI-AGEING 133 n PGC1alpha n NRF1 n TFAM


350 300 250 200 150 100 50 0


Untreated *** ** * *** ** * ** ** *


min in phosphate buffer. After 2 h incubation with the primary antibody, the cells were washed, incubated with the anti- rabbit secondary antibody and then the amount of GDF11 produced was measured by a fluorescence reaction using the QuantaRed™ Enhanced Chemifluorescent HRP substrate (Thermo Fisher Scientific). The relative fluorescence units of each well were measured using the Multiwell Plate Reader Victor3 (Perkin Elmer, Woodbridge, Ontario, CA). To determine collagen production, 8 x 104


RrHEs 0.002% RrHEs 0.01% GDF11 100ng/ml


Figure 3: Gene expression analysis of the mitochondrial biogenesis markers, PGC1α, NRF1 and TFAM in fibroblasts. Cells were treated with the indicated compounds, and the expression level of the genes was analyzed by RT-PCR. The values obtained from the PCR band quantification were expressed as percentages to the untreated control, arbitrarily set as 100%. The error bars represent standard deviations, and the asterisks indicate statistically significant measures.


n Sirt3 n Sirt4


350 300 250 200 150 100 50 0


Untreated * ** *** ** *** ***


cells were seeded in 96-


well plates, and, after 16 h, treated for 24 h with the extracts. After washing in phosphate buffer, the cells were processed as above. The quantity of Procollagen I and III was measured by a colorimetric reaction, using 0.35 mg/mL solution of O- PhenylenDiamine (OPD) (Sigma-Aldrich) and 0.012% H2


O2 in 50 mM citrate buffer.


Gene expression analysis in HDF 8 x 104


cells were seeded in 6-well plates and RrHEs 0.002% RrHEs 0.01% TSA 10 ng/ml


Figure 4: Gene expression analysis of the mitochondrial Sirtuins, Sirt3 and Sirt4, in fibroblasts. Cells were treated with the indicated compounds, and the expression level of the genes was analysed by RT- PCR. The values obtained from the PCR band quantification were expressed as percentages to the untreated control, arbitrarily set as 100%. The error bars represent standard deviations, and the asterisks indicate statistically significant measures.


Compond Discoverer 2.1 Software (ThermoFisher) for the identification of the metabolites.


Cell cultures and reagents for cell bioassays Human dermal fibroblasts (HDF), deriving either from neonatal (nHDF) or 36 year old donors (36yoHDF) were maintained in DMEM supplemented with 10% of Fetal Bovine Serum (FBS) (Life Technologies Corporation, CA, USA), under 5% CO2


at


37 °C. nHDF derived from a Caucasian male donor (foreskin fibroblasts), while 36yoHDF from a Caucasian female donor. GDF11 recombinant protein was purchased from Cayman (Cayman Chemical, Ann Arbor, Michigan USA); Trichostatin A (TSA) and the inhibitor SB-431542 were from Sigma-Aldrich. Anti-GDF11 antibody was purchased from AbCam (Cambridge, UK)


April 2020


(#ab124721); anti-procollagen I and III antibodies (#sc166572 and #sc-166316) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, TX, USA). As secondary antibodies, we used a goat anti- rabbit IgG-HRP (#170-6515) from BioRad (BioRad, Hercules, CA, USA) and a goat anti-mouse IgG-HRP from BioRad (BioRad, Hercules, CA, USA) (#170-6516).


ELISA assays To measure GDF11 production, 12 x 104 cells were seeded in 96-well plates and, after 16 h, incubated with the rose extracts for 6 h. TSA, at 25 ng/mL, was used as positive control. After washing in phosphate buffer, cells were fixed for 10 min, washed with phosphate buffer (containing 0.5 mM CaCl2


, 1 mM MgCl2 ,


0.1% Triton X100) and treated with 0.5% non-fat dried milk (NFDM, Biorad) for 30


treated with the compounds to test for 24 h in DMEM plus 2% of FBS. RNA extraction was performed by the GenEluteTM Mammalian Total RNA Miniprep kit (Sigma-Aldrich) according to the manufacturer’s instructions. The cDNA was synthesised from 300 ng of RNA using RevertAid™ First Strand cDNA synthesis Kit (Thermo Fisher Scientific). RT-PCR was performed using gene specific primers and the QuantumRNA™ 18S internal standard (Thermo Fisher Scientific). The amplification reactions were performed according to the following scheme: 2 min at 94 °C, followed by 35 cycles of 94°C for 30 s, annealing temperature (specific for each gene) for 30 s, and 72 °C for 30 s, with a 10 min final extension at 72°C. The obtained PCR products were loaded on 1.5% agarose gel, and the amplification bands, visualised and quantified by the Geliance 200 Imaging system (Perkin Elmer, Woodbridge, Ontario, CA), were normalised to the amplification band corresponding to the 18S. The average values, obtained from three independent experiments, were converted into percentage values by considering the measure of the untreated control as 100%. The genes of GDF11, SIRT1, SIRT6, PGC1α, NRF1, TFAM, SIRT3 and SIRT4 were amplified using specific primers.


Smad2 reporter assay nHDF cells were seeded at a density of 3 x 103


in 96-well plates and after 16 h were transfected using X-tremeGeneTM


HP DNA


transfection Reagent (Roche Diagnostics S.p.A., Monza, Italy) with Smad2 reporter vector (Cignal SMAD reporter assay Kit, Qiagen Sciences, German Town, MD, USA) according to manufacturer’s instructions. After 24 h, the cells were treated with the rose extract, 100 ng/mL of recombinant GDF11, 1.9 μg/mL SB-431542 or their


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Gene expression (% to untreated cells=100)


Gene expression (% to untreated cells=100)


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