Ht22 cells

Mouse hippocampal HT-22 cells were purchased from EMD Millipore (La Jolla, CA, USA). Cells were cultured in DMEM (Gibco, Paisley, UK) containing 10% fetal bovine serum (Biowest, Nuaillé, France) and 1× Penicillin-Streptomycin (Gibco, Grand Island, NY, USA). Cells were maintained in a humidified 37 °C, 5% CO₂ incubator.

The human RPE cell line ARPE-19 [54 (link)] (ATCC, RRID: CVCL_0145) was cultured in 96-well plates (15,000 cells/well) in HyClone DMEM (GE Healthcare) containing 10% fetal calf serum (Linaris GmbH, Wertheim-Bettingen, Germany), 1% penicillin/streptomycin (Merck), 2.5% HEPES (Merck) and 1% non-essential amino acids (Merck). The uveal melanoma cell line OMM-1 (RRID: CVCL_6939) [55 (link)], provided by Dr. Sarah Coupland, was cultivated in 96-well plates (15,000 cells/well) in RPMI 1640 media (Merck), supplemented with 10% fetal calf serum (Linaris GmbH) and 1% penicillin/streptomycin (Merck). Immortalized hippocampal neuroblasts (HT-22 cells, RRID:CVCL_0321, Merck Millipore, Burlington, MA, USA) and SH-SY5Y cells (Depositor: JL Biedler, RRID:CVCL_0019, American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in 96-well plates in DMEM containing 10% fetal calf serum and 1% penicillin/streptomycin (4000 cells/well and 20,000 cells/well, respectively). Cell lines were treated 24 h after plating when the density reached at least 70% confluency. All cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere.

To study the expression at the plasma membrane, biotinylation assay of membrane proteins was used. In this assay, membrane proteins are labeled with biotin and subsequently immunoprecipitated with streptavidin beads to isolate the protein membrane fraction. Mouse hippocampal HT22 cells (Merck Millipore, Burlington, MA, USA), previously incubated with 1 mM glutamate (Sigma Aldrich, Darmstadt, Germany), were treated with EZlinkTM Sulfo-NHS-SS-Biotin (Thermo Scientific, Waltham, MA, USA) 0.5 mg/mL in cold 1X PBS for 15 min at 4 °C. Subsequently, the cells were washed twice with 200 mM Glycine in cold 1X PBS and twice with cold 1X PBS to inactivate and remove the excess of biotin, respectively. The cells were then lysed and centrifuged at 16′000 g at 4 °C for 15 min. Two milligrams of the supernatant were incubated with 50 μL Streptavidin Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 h at 4 °C, while forty μg of protein were kept for the input fraction. The beads were subsequently washed five times with 1X lysis buffer before elution with 50 μL of 2X NuPAGE sample buffer (Invitrogen, Carlsbad, CA, USA) plus 100 mM DTT at 37 °C for 30 min.

HT22 cells, an immortalized mouse hippocampal neuronal cell line, were purchased from Merck Millipore (Burlington, MA, USA). DMEM, containing 10% FBS and 50 units/mL penicillin/streptomycin, was used for cell culture, and the cells were grown at 37 °C in a humidified 5% CO₂ atmosphere.

HT22 cell is an immortalized mouse hippocampal neuronal cell line subcloned from parent HT4 cells that were originally immortalized from cultures of primary mouse hippocampal neurons [30 (link)]. HT22 cell is known to phenotypically resemble neuronal precursor cells, which has been used in cell model to study of hemin-induced injury in neuronal cells [7 (link), 29 (link)]. HT22 cells (Merck, USA) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in an atmosphere with 5% CO₂ at 37°C. After growing to 80% confluence, the HT22 cells were used for experiment. The HT22 cells were dealt with various concentrations of hemin (0, 10, 20, 40, 80, and 100 μM) over a period of 6 h to simulate the in vitro ICH model [7 (link)]. Subsequent studies were conducted using 20 μM of hemin. To evaluate the security of CHR, HT22 cells were dealt with various concentrations of CHR (0, 5, 10, 20, and 40 μM). To evaluate the neuroprotective effects of CHR, HT22 cells were dealt with CHR after treating them with 20 μM of hemin over a period of 6 h. Following this, they were incubated for 24 h. The negative control groups were dealt with an equal volume of solvent (Figure S1).

HT-22 cells were purchased from Merck KGaA (NJ, USA). Cells were maintained in normal conditions (DMEM containing 4.5 g/L glucose, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 5% CO₂ and 37 °C). When we performed quantification of glutathione (GSH), DMEM containing low glucose (1 g/L) and low FBS (1%) was used. In other experiments, DMEM was used at normal conditions.

For the in vitro experiments, we employed one subclone of the mouse hippocampal HT-22 cells, which were purchased from CHI SCIENTIFIC, Shanghai, China. HT-22 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, USA), MEM nonessential amino acids (Invitrogen), L-glutamine (Invitrogen), penicillin and streptomycin (Sigma), 0.01 mM β-mercaptoethanol (Invitrogen) and 1000 units/ml LIF (Millipore). Based on the database of GenBank, the reference sequence of Mus musculus Pias1 (NM_019663.3) was obtained. The gene insertion and transfection were performed by using a vector of PiggyBac (PB) in addition with 2 μg transposase using LTX (Invitrogen). The cell selection was continued for seven days by using 2 μg/ml puromycin.