Antibiotic antimycotic reagent

The PC12 cells used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI-1640 medium with 10% horse serum (Gibco, Carlsbad, CA, USA), 5% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 100 mg/mL antibiotic-antimycotic reagent (Gibco, Carlsbad, CA, USA) at 37 °C in a 5% CO2 incubator.

The fetal rhesus kidney (FRhK-4) cell is a HAV-permissive cell line derived from the rhesus monkey. Dulbecco’s modified Eagle’s medium (DMEM) containing 4% heat-inactivated fetal bovine serum (FBS, Gibco, Campinas, Brazil) and antibiotic-antimycotic reagent (Gibco, Campinas, Brazil) was used to maintain this cell line under a 5% CO₂ atmosphere at 37 °C.
The HAV strain HM-175/18f, clone B (VR-1402) was obtained from the American Type Culture Collection. Ten genomic equivalents (GE) copies of the HAV per cell were used to inoculate FRhK-4. Cells inoculated with the virus were maintained in DMEM supplemented with 4% heat-inactivated FBS and antibiotic–antimycotic reagent.
We purchased hemin, cobalt protoporphyrin IX (CoPP-9), andrographolide, zinc protoporphyrin IX (ZnPP-9), iron (III) chloride (FeCl₃), and carbon monoxide-releasing molecule (CORM-3) from Sigma-Aldrich (St. Louis, MO, USA). Biliverdin was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Primary cultures of human dental follicle cells (HDFCs) were established using the dental follicles obtained from four different patients presenting impacted canines and referred for surgical exposure.
After rinsing in MEM Alpha medium 1 × (α‐MEM; Gibco Life Technologies, Grand Island, NY, USA), tissues were minced using a sterile scalpel, cultured in α‐MEM supplemented with 10% (v/v) fetal bovine serum (Gibco Life Technologies), 2 mM Glutamax (Gibco Life Technologies), and Antibiotic‐Antimycotic reagent (Gibco Life Technologies) at 37°C in humidified air with 5% CO₂. After 48 h, non‐adherent cells were removed by changing the medium. Human dental follicle cells used at passages 3–5 were cultured in α‐MEM with or without osteogenic induction medium (50 mg ml⁻¹ of L‐ascorbic acid 2‐phosphate sesqui‐magnesium salt and 10 mM β‐glycerophosphate disodium salt hydrate) (Sigma‐Aldrich). After 14 d, HDFCs were cultured in α‐MEM containing 10⁻⁶ M forskolin (FSK; Sigma‐Aldrich) for 0, 24, and 48 h; the cells at the 0‐h time point were defined as controls. The FSK concentration was obtained from dose–response studies previously performed 19, 20.
The results are presented as the average of two different experiments performed in duplicate. The relative levels of RANKL and OPG transcription in FSK‐treated HDFCs were adjusted by standardization based on the levels of GUSB mRNA.

Human pancreatic cancer cell lines (PANC-1 and Capan-1) were purchased from the American Type Culture Collection (ATCC MD, Manassas, VA, USA). PANC-1 and Capan-1 were cultured in DMEM and RPMI 1640, respectively, and supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France) and 1% antibiotic–antimycotic reagent (Gibco, Waltham, MA, USA) at 37 °C and 5% CO₂. After one passage, gemcitabine was treated with PANC-1 and Capan-1 cells to establish a gemcitabine-resistant cell line. The surviving cells were subcultured and continuously treated with increasing gemcitabine concentrations (0.1 μM to 10 μM) for 3 months. After the gemcitabine-resistant cells stabilized, they were treated with the same concentration of gemcitabine for 3 months.

The mesothelioma cell lines used in this study (H2052, H2373, H2452, and H2052) and the control cell lines, Met5A (we note as Met5A AD) and 3T3, were obtained from either the ATCC (American Type Culture Collection) or in collaboration with Dr. Robert Kratzke (University of Minnesota). All mesothelioma cell lines lack functional p16INK4a. The cells were grown in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% newborn bovine serum (Sigma, St. Louis, MO, USA), and 1x concentration of antibiotic/antimycotic reagent (Gibco BRL, Grand Island, NY, USA) at 37 °C and 5% CO₂. The following antibodies were utilized (Actin, Bax, Bcl-xL, Bid, cdc2 (CDK1), CDK2, CDK4, CDK6, Cyclin A, Cyclin B1, Cyclin D1, Cyclin E1, FOXM1, Mcl-1, pRb (S780), Rb, Survivin, and XIAP (see Supplementary Materials for manufacturer and lot numbers). The CDK4/6 inhibitor, palbociclib, and auranofin were obtained from MedChemExpress (Monmouth Junction, NJ, USA).