A5955

HEK293 cells were cultured in Dulbecco's modified Eagle's medium (D6429; Sigma–Aldrich, Inc) containing 10% fetal bovine serum (F2442; Sigma—ldrich, Inc), 1× antibiotic/antimycotic solution (A5955; Sigma–Aldrich, Inc), and 1× Plasmocin prophylactic (ant-mpp; InvivoGen). HEK293 Gαq/11 KO cells were cultured using the same conditions described previously and were a kind gift from Dr Asuka Inoue (41 (link)). UM cells (92.1, OMM1.3) were cultured in RPMI1640 (R8758; Sigma–Aldrich, Inc) containing 10% fetal bovine serum (F2442; Sigma–Aldrich, Inc), 1× antibiotic/antimycotic solution (A5955; Sigma–Aldrich, Inc), and 1× Plasmocin prophylactic (ant-mpp; InvivoGen). All cell lines were routinely tested free of mycoplasma contamination. VS-4718 (S7653), BYL719 (S2814), TGX221 (S1169), CAL101 (S2226), and BKM120 (S2247) were purchased from SelleckChem. FR900359 was prepared in the laboratory of Dr Evi Kostenis. CNO (4936) was purchased from Tocris, Inc EGF (E9644) was purchased from Sigma–Aldrich, Inc. All compounds were used at concentrations indicated in figure legends.

Cell culture was performed to verify the effects of ERa and ERb blockers on canine luteal cells derived from different timepoints in diestrus. Canine luteal cells were isolated from twelve healthy mongrel female dogs at early (day 20 p.o.), mid (day 40 p.o.), and late diestrus (day 60 p.o.; n = 4 animals/group). After washing with fresh phosphate buffered saline (PBS) containing 1% antibiotic-antimycotic solution (A5955, Sigma-Aldrich), CLs were minced. Fragments were transferred to 1 ml Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS; Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich.), 20 mM HEPES (Sigma-Aldrich), 1% antibiotic-antimycotic solution (A5955, Sigma-Aldrich), and 1 mg/ml collagenase type 1 (C0130; Sigma-Aldrich). Samples were incubated for 1 h with shaking (60 shakes/min) at 37°C. The suspension was centrifuged at 200 × g for 10 min, re-suspended in DMEM, and filtered through a cell strainer (70 μm; BD Falcon; BD Biosciences, Durham, NC, USA). The filtrate was centrifuged at 200×g for 10 min, re-suspended in DMEM (v/v) for 10 min, centrifuged at 200 × g for 10 min, and re-suspended in DMEM. Subsequently, cells were seeded in 24-well plates and incubated (5% CO2) at 37°C until 90% confluence.

To assess the optimal compression time in the POR scaffold, PDLFs were cultured for 4, 24, 48, and 72 h either on conventional 24-well cell culture plates (662-160, Greiner Bio-One GmbH, Frickenhausen, Germany) or on 24-well INO matrix plates (Porous POR PCL Morphology Single Type Plate, 811004-24, Biozym Scientific GmbH, Hessisch Oldendorf, Germany) in DMEM High Glucose (D5671, Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS (P30-3302, PAN-Biotech GmbH, Aidenbach, Germany); 1% antibiotic/antimycotic (A5955, Merck KGaA, Darmstadt, Germany); 1% L-glutamine (G7513, Merck KGaA, Darmstadt, Germany), and 100 µM ascorbic acid (A8960, Merck KGaA, Darmstadt, Germany). After 24 h of preincubation, the cells were either left untreated or compressed with ZnO₂ plates (2 g/cm²). Depending on the periods to be investigated, the plates were left on the cells for 4, 24, 48, or 72 h. After the corresponding incubation time, RNA was isolated and analysed with RT–qPCR. The supernatant was used for the LDH assay (Supplemental Materials).

Primary human PDLFs were isolated from the periodontal connective tissue of extracted human teeth of healthy donors that were free of decay and periodontal disease and were extracted for medical reasons. Tissue samples were cultivated in six-well cell culture plates (353046, Corning GmbH, Kaiserslautern, Germany) (37 °C, 5% CO₂, 100% H₂O) in complete media (DMEM High Glucose, D5671, Merck KGaA, Darmstadt, Germany) with 10% FBS (P30-3302, PAN-Biotech GmbH, Aidenbach, Germany); 1% antibiotic/antimycotic (A5955, Merck KGaA, Darmstadt, Germany); 1% L-glutamine (G7513, Merck KGaA, Darmstadt, Germany) and 100 µM ascorbic acid (A8960, Merck KGaA, Darmstadt, Germany) until proliferative outgrowth of adherently growing fibroblasts was observed. Cells were characterised by human PDLF-specific marker genes and a spindle-shaped morphology, as reported previously [49 (link),56 (link)]. For the experiments, human PDLFs from the third to fifth passage were pooled from six patients (male: 3; female: 3; age: 17–27 years).

To assess the optimal compression force in the POR scaffold, PDLFs were seeded in DMEM High Glucose (D5671, Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS (P30-3302, PAN-Biotech GmbH, Aidenbach, Germany); 1% antibiotic/antimycotic (A5955, Merck KGaA, Darmstadt, Germany); 1% L-glutamine (G7513, Merck KGaA, Darmstadt, Germany), and 100 µM ascorbic acid (A8960, Merck KGaA, Darmstadt, Germany) either on conventional 24-well cell culture plates (662-160, Greiner Bio-One GmbH, Frickenhausen, Germany) or on 24-well INOMatrix plates (Porous POR PCL Morphology Single Type Plate, 811004-24, Biozym Scientific GmbH, Hessisch Oldendorf, Germany) and loaded with ZnO₂ plates of different weights (2 g/cm², 4 g/cm², 6 g/cm²) (Figure 5c), for 48 h after 24 h of preincubation, under cell culture conditions. After the compression period, RNA was isolated and analysed with RT–qPCR. The supernatant was used for the LDH assay (Supplemental Materials).

Primary cultures of HGF were established through adaptation of previous works [20 ,21 (link)]. The biopsies were transported to the laboratory in phosphate-buffered saline (PBS; in mM: 137 NaCl, 2.7 KCl, 10 Na₂HPO₄, and 1.8 KH₂PO₄ [pH 7.4]), supplemented with 1% penicillin/streptomycin/amphotericin B (A-5955, Sigma Aldrich^®, Israel).
The explants were scraped to remove any epithelial tissue, cut into pieces, and submitted to digestion by incubation with 2% collagenase type I (Affymetrix^® 13820, Cleaveland, OH, USA) at 37 °C for 45 min. Subsequently, collagenase was carefully replaced by a solution of 0.25% trypsin (Gibco^® 25200056, Thermo Fisher Scientific, Paisley, Scotland), and digestion continued for 15 min. Afterwards, the fragments were distributed through a petri dish, and a few drops of DMEM (Dulbecco’s Modified Eagle’s Medium, D-5648, Sigma Aldrich^®, Saint Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich^®, F7524, Brazil), and 1% penicillin/streptomycin/amphotericin B were added. The cultures were maintained at 37 °C in a humidified incubator with 5% CO₂ and filled with a medium after 24 h. Cultures were monitored, and the medium was replaced every three days. Seventh to eighth passages HGF were used in this study.