Chalcone

All cell lines were grown in RPMI 1640 (PAN-Biotech P04-16500) supplemented with 10% heat-inactivated fetal calf serum (FCS; PAN-Biotech), penicillin/streptomycin (PAN-Biotech) (thereafter designated as RPMI-based medium) and cultivated at 37°C under 5% CO₂ in a humidified incubator. K562 cells (a kind gift from Daniela Männel, University of Regensburg, Germany; [62] (link)) were maintained in RPMI-based medium. The non-tumorigenic immortalized interleukin-3 (IL-3)-dependent mouse pro-B cell line Ba/F3 (a kind gift from Jacqueline Marvel, IFR 128 BioSciences Gerland-Lyon Sud, France; [63] (link)) was grown in RPMI-based medium supplemented with 2 ng/mL rmIL-3 (ImmunoTools). The Ba/F3-1*6 cell line stably expressing the constitutively active mouse STAT5A-1*6 mutant [64] (link) was generated according to German GenTSV (genetic engineering safety regulations; authorization AZ.55.1-8791.7.52) by electroporating Ba/F3 cells with a pcDNA3-based expression vector allowing expression of a mSTAT5A-1*6-FLAG fusion protein. Stably transfected cells were selected in IL-3-free medium in the presence of 500 µg/mL G418 (PAA). Individual IL-3-independent clones were isolated and characterized to verify mSTAT5A-1*6 transgene expression and constitutive activity, as originally described [64] (link). Clone Ba/F3-1*6 F7 was used for this study. Cells were maintained in RPMI-based medium supplemented with 500 µg/mL G418.
For cytokine stimulation of Ba/F3 cells, cells were washed twice in RPMI 1640 and rested in RPMI-based medium for 12 hours before addition of 5 ng/mL IL-3. Duration of IL-3 stimulation was contingent upon the downstream assay, as indicated in the figure legends. For chalcone and other inhibitor treatments, Ba/F3 cells were incubated with compounds or DMSO (vehicle) for 30 minutes prior to cytokine stimulation. Ba/F3-1*6 and K562 cells were treated for 60–90 minutes with the various compounds or DMSO (vehicle), as indicated. With the exception of cell viability assays (see below), all experiments were performed in the presence of equal amounts of DMSO.

Confocal microscopy was used to evaluate the damage to the cell wall caused by 2-chalcone, using fluorochrome Calcofluor White (Thermo Fisher Scientific) and T. rubrum ATCC 28189. Fungal suspensions were prepared at a concentration of 1 × 10⁶ cells/mL and added to 24-well plates containing sterile coverslips, along with sub-inhibitory doses of 2-chalcone (7.8 mg/L). After the incubation period (35°C, for 96 hours), the supernatant was removed and the coverslips were washed with PBS. The coverslips were covered with Calcofluor White solution (100 mg/L), and the plates were incubated again at 37°C, for 45 minutes, protected from light. Then, the coverslips were washed with PBS, removed from the wells, and mounted on 4 μL of Fluoromount-G (Sigma-Aldrich), which was previously deposited on microscopic slides. The slides were then observed using a confocal microscope (Carl Zeiss LSM 800 with Airyscan) with an image capture and processing program (Software ZEN BLUE 2.3 System) at the Faculty of Dentistry, UNESP-Araraquara, Brazil (Curcio et al., 2017 (link); Oliveira et al., 2020 (link)).

The brain was removed for histological examination and placed in fixative formalin for 24 h. Next, during the dehydration and clarification steps, the samples were embedded into molten paraffin. Using a microtome (Cambridge Medical Instruments, United Kingdom), 6-micron-thick sections were prepared. The samples were placed on a slide, and then half of the samples were stained with hematoxylin-eosin and the other half with thioflavin S.
The main goal of preparing sections of the hippocampus and staining them was to investigate possible changes in brain tissue and to quantitatively examine the amyloid fibrils formed in the absence and presence of silibinin and trans-chalcone in each section. Five tissue sections in each group were randomly examined microscopically. Since we observed that silibinin performed better than trans-chalcone in the initial tests, both DG and CA1 regions were checked for the former and DG for the latter.

Molecular docking was performed using the Autodock Vina software (USA). The molecular structure of silibinin and trans-chalcone was prepared from pubchem (https://pubchem.ncbi.nlm.nih.gov/) (Figure 1). Then, energy minimization was performed using Avogadro software (https://avogadro.cc) to provide a stable 3D structure. For insulin, two natural (3I40) and denatured (1SF1) structures were fetched from the RCSB protein database (https://www.rcsb.org). First, water molecules in the crystal structure were removed manually. Then, polar hydrogens and Gasteiger charges were added for both ligands and proteins using Autodock Tools software. All rotating bands of the active ligand were considered. Docking was performed blindly, and the search space was considered large enough for the entire protein, as all the protein surface was available to the ligand. The 2D protein-ligand interactions were demonstrated using LIGPLOT software (https://bio.tools). 3D images were also drawn using VMD software (https://www.ks.uiuc.edu).