Dimedone

Thiostrepton was purchased from EMB Biochemicals (Billerica, MA). Gentian violet was a kind gift from J. Arbiser (Emory University, Atlanta, GA). Dimedone, glutathione, N-acetyl-L-cysteine, and N-ethyl maleimide were purchased from Sigma (St. Louis, MO). Tris (2-carboxyethyl) phosphine (TCEP) was purchased from Thermo Scientific (Rockford, IL).

All chemical substances have been used as bought from Sigma-Aldrich and Merck Companies. Tetraethyl orthosilicate (tetraethoxysilane) (98% w/w), ethanol (99.5% w/w), (3-aminopropyl) triethoxysilane (95% w/w), HNO₃ (65% w/w), and silver nitrate (≥ 99.0%), potassium persulphate (99.99%), hydrazine hydrate (24–26% in H₂O (RT)), isatin (97%), acenaphthene quinone (97%), malononitrile (98%), ethyl cyanoacetate (98%), dimedone (95%), barbituric acid ethyl acetoacetate (99%), 4-hydroxycoumarin (98%), three-methyl-1H-pyrazole-five (4H)-one (98%), α-naphthol (99%), β-naphthol (99%), n-hexane (99%), ethyl acetic acid (99%), and desired derivations have been provided from the Sigma-Aldrich Company. The stem and roots of the Spear Thistle were purchased from a local shop in Tehran (in Iran). The leaves were purchased from a local shop in Tehran. The plant we used in this work is a plant that is found in abundance in local shops and is not wild and endangered. This study complies with relevant institutional, national, and international guidelines and legislation.

Whole cell protein lysates were prepared from 90% confluent cell cultures in 100 mm culture dishes (Sigma, St. Louis, MO). Briefly, cell pellets were resuspended in 100 mM 5,5-dimethylcyclohexane-1,3-dione (Dimedone, Sigma) and incubated at 37°C for 60 min under agitation. After washing with PBS, pellets were lysed in RIPA buffer (Cell Signaling) and then sonicated for 15 seconds using Sonic Dismembrator (ThermoFisher). Following sonication, supernatant was moved to a new tube and samples were incubated with 2 μg/mL primary antibody [mouse anti-Keap1, 1:100 (Abcam)] for 24 h at 4°C. Magnetic beads [Dynabeads M-280 anti-mouse IgG (Thermo Fisher Scientific)] was then added and samples were incubated for 3 hours at 4°C. After 3 washes, samples were analyzed by Western blotting as described above.
For co-immunoprecipitation of Cav-1/Keap1/Nrf2 complex, a direct immunoprecipitation method was used. Briefly, magnetic beads [Dynabeads M-280 anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific)] were incubated with primary antibody [anti-rabbit Nrf2 1:100 (Santa Cruz) or anti-mouse Keap1 1:100 (Abcam)]) for 6 h at 4°C. Beads were then washed to remove unbound primary antibody. Following treatment (400 μM H₂O₂, 1 h), cell lysates were added to the Dynabead/Antibody cocktail and incubated at 4°C overnight. Samples were washed twice with PBS and then analyzed by Western blot.

For the fabrication of the catalyst the following chemicals were applied: (3-chloropropyl) trimethoxysilane (CPTES), isatin (IS), triethylamine (TEA), guanidine (Gu), β-CD, p‐toluenesulfonyl chloride (p‐TsCl), K₂CO₃, toluene, EtOH and Bent. Bent was obtained from Madan Kavan Co. Iran. The other reagents were purchased from Sigma-Aldrich. For performing the ultrasonic-assisted chemical transformations, benzaldehyde, malononitrile, dimedone and urea (provided from Sigma-Aldrich) have been used.
Bent-Gu-CD synthesis was verified by Fourier transform infrared (FTIR), Thermo gravimetric analysis (TGA), X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive spectroscopy (EDS) and elemental mapping analysis. The FTIR spectrum were collected using PERKIN-ELMER Spectrum 65. For TGA, METTLER TOLEDO apparatus was employed. The tests were carried out under N₂ atmosphere at heating rate of 10 °C min⁻¹. XRD pattern of Bent-Gu-CD was gathered via Siemens, D5000 apparatus with Cu Kα as a radiation source. SEM /EDX analyses were carried out on MIRA 3 TESCAN-XMU. Brunauer Emmett Teller (BET) analysis was carried out via a Belsorp Mini II apparatus. Degassing was performed at 150 °C for 3 h.