P s solution

Animal studies were approved by the Committee for Animal Care and Use of the Faculty of Pharmacy, Mahidol University (Protocol No. PYR004/2556 and PYR003/2560) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996). Isolation of cardiac fibroblasts was prepared as previously described (Nishida et al., 2007 (link)). Briefly, neonatal Sprague-Dawley rats were euthanized by decapitation and the hearts were removed and placed on ice. The atria of the hearts are quickly removed and the remaining hearts were cut into small pieces and digested by collagenase type A (Roche Diagnostics). The digested cells were plated on 10-cm dishes at 37°C for 2–3 h. Cardiac fibroblasts were attached to the bottom of the dishes during incubation time, whereas non-adherent myocytes were removed by changing the cultured media. Cardiac fibroblasts were cultured in DMEM containing 10% FBS, and 1% (v/v) P/S solution (Gibco). The purity of acquired cardiac fibroblast was then confirmed by both cellular morphology (thin and triangular cells) and immunostaining. Cardiac fibroblasts in the first and second passages were used for all experiments. The medium was changed to serum-free DMEM overnight before stimulation.

The gastric adenocarcinoma cell line MGC-803 and immortalized normal gastric epithelial cell line GES-1 were purchased from ATCC. Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and 1% P-S solution (Gibco, Grand Island, NY, USA). Cells were maintained at 37°C under 5% CO₂.

Experiments were performed using rat neuroblastoma cells (B-35, ATCC). Cells were cultured to 50% confluence in Advanced DMEM (Gibco) supplemented with 10% FBS (PanBiotech) and a 1% P/S solution (Gibco) at 37°C with 5% CO₂. The medium was exchanged every 3rd day. After the cells reached 50% confluence, we performed AAV2-shRNA transfection (with a previously selected construct of shRNA-HuR or shRNA scrambled control). The transfection medium was prepared by mixing the AAV stock (calculated as 10⁵ AAV particles per single cell) with Advanced DMEM supplemented with 2% FBS and 1% P/S. Cells were incubated with the transfection medium for 36 h at 37°C with 5% CO₂. Afterward, the transfection medium was replaced with fresh, fully supplemented culture medium (Advanced DMEM + 10% FBS + 1% P/S), and the cells were cultured for another 24 h. The transfection efficiency was evaluated by measuring the HuR protein content in fractionated Western blots.

After gene-edited HSPCs, the cells were differentiated towards erythroid lineage using a 14-day protocol. Erythroid differentiation media was made by IMDM (Gibco), 2% HyClone (GE Healthcare), 0.5% P/S solution (Gibco-Thermo Fisher Scientific), 3% AB serum (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), 3U/ml heparin (Sigma-Aldrich), and 200 μg/ml holo-transferrin (Sigma-Aldrich). From day 0 to day 6, the cells were cultured in this basal media supplement with 3 U/ml EPO (Amgen), 10 ng/ml SCF (EuroBiosciences GmbH), 1 ng/ml human interleukin 3 (hIL-3) (Eurobiosciences), and 1 × 10⁻⁶ M dexamethasone (Sigma-Aldrich). On day 6, cell culture media was exchanged by the basal media with 3 U/ml EPO (Amgen) and 10 ng/ml SCF (Eurobiosciences) until day 9. In the last 5 days of the protocol, the media was supplemented with 3 U/ml EPO (Amgen) and an increased concentration of holo-transferrin (200 μg/ml). The cultures were maintained under normoxic conditions.
Immunophenotype of in vitro differentiated erythroid cells was analyzed using anti-human CD71-FITC and anti-human CD235a-PE in a BD LSRFortessa^™ Cell Analyzer (BD Bioscience).
To quantify ATP production, CD235a⁺ erythroid cells were sorted and analyzed with CellTiter-Glo^® Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions and the luminescence was recorded using Genius Pro reader (Tecan).

Two human cholangiocarcinoma cell lines (RBE and HuH-28) were purchased from RIKEN Bioresource Center (Ibaraki, Japan) and maintained for the study in RPMI-1640 or minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) and P/S solution (Invitrogen) in a 5% CO₂ incubator at 37°C.

The human cholangiocarcinoma SSP-25 cells and HuH-28 cells were purchased from RIKEN Bioresource Center (Ibaraki, Japan) and preserved for the study in RPMI-1640 or minimum essential medium content with 10% FBS and P/S solution (Invitrogen, Carlsbad, CA) in 5% CO₂ incubator at 37°C. Gefitinib was purchased from Selleckchem, and lovastatin was purchased from Sigma.