S1810

The cells that used in this study is human hepatocellular carcinoma (HepG2) cell line (ATCC, HB-8065™) from Aretha Medika Utama Biomolecular and Biomedical Research Center, Bandung, Indonesia. It was grown in complete medium with composition: Modified Eagle Medium (MEM) (Biowest, L0416-500), fetal bovine serum (FBS) (Biowest, S1810) as much as 10% (v/v), antibiotic-antimycotic (Gibco, 15240062) as much as 1% (v/v), also nanomycopulitine (Biowest, LX16) addition) as much as 1% of (v/v). Acetaminophen (Sigma Aldrich, A7085) with concentration at 40 mM was used to induce the hepatotoxicity. When the cells were confluent, it was rinsed using PBS and detached using trypsin-EDTA (Gibco, 25200072) with incubation at 37 °C. In 6 well plates, the cells was seeded (5 × 10⁵ cells/well) and then incubated at the same temperature with 5% of CO₂ for 24 h. The cells was induced by RBLE and incubated again for 24 h. According to the treatment, it was divided into 5 groups: I) Normal Cells; II) DMSO1%; III) APAP 40 mM; IV) APAP 40 mM + RBLE 25 μg/mL; V) APAP 40 Mm + RBLE 100 μg/mL. Then it was centrifuged at 1600 rpm for 10 min. The supernatant was collected as sample for the Elisa assay (Luo et al., 2016 (link); Aouache et al., 2018 ; Lister et al., 2019b ).

ANC-1 (CRL-1469, ATCC, Manassas, VA, USA), MiaPaCa-2 (85062806, ECACC, Porton Down, UK), BxPC-3 (EP-CL-0041, Elabscience, Houston, TX, USA), MCF-7 (EP-CL-0149, Elabscience) and hNDF (C-12302, Promocell, Heidelberg, Germany) were cultured at 10,000 cells/cm² for no more than 15 passages in DMEM (DMEM-HXA, Capricorn, Ebsdorfergrund, Germany) or RPMI (RPMI-XA, Capricorn) (in the case of BxPC-3 cells) supplemented with 10% FBS (S1810, Biowest, Nuaillé, France), L-glutamine (X055, Biowest) and Penicillin/Streptomycin (L0022, Biowest). Cultures were maintained at 37 °C and 5% CO₂ in a humidified atmosphere.

Hair Follicle Dermal Papilla Cells (HFDPC) (C12071, Promocell, Heidelberg, Germany) were cultured at 10,000 cells/cm² from passage 2 to passage 6 in 75 cm² T-flasks in Follicle Dermal Papilla Cell Growth Medium (C26501, Promocell), which contains fetal calf serum, bovine pituitary extract, bFGF and insulin, supplemented with L-Glutamine (X0550, Biowest, Nuaillé, France) and 1% (w/v) Penicillin/Streptomycin (P/S) (L0022, Biowest). Human Foreskin Fibroblasts (HFF) and HeLa cells were cultured at 10,000 cells/cm² for not more than 10 passages in DMEM (DMEM-HXA, Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS (S1810; Biowest), L-Glutamine and P/S. Adipose-derived stem cells (ADSC) were cultured from passage 2 to passage 6 at 10,000 cells/cm² in ADSC Basal Medium (PT-3273, Lonza, Basel, Switzerland) with SingleQuots^TM Growth Supplement Kit (PT-4503, Lonza). Cultures were maintained at 37 °C and 5% CO₂ in a humidified atmosphere and passaged at 80% confluency.

Cells were seeded at 10,000 cells/cm² and cultured for 24h in EGM-2 MV medium for ECFCs and HAECs, and in DMEM/F12 supplemented with 10% SVF (S1810, Bio West, FRANCE) for fibroblasts. Cells were transduced with lentiviral vectors RL-EF1-OCT3/4, RL-EF1-SOX2, RL-EF1-KLF4, RL-EF1-v-MYC (Vectalys, Toulouse, FRANCE) with MOI 10 in EGM-2 containing 8 μg/ml polybrene (Sigma-Aldrich, St Louis, MO). Cells were cultured for 7 days in EGM-2 MV medium then transferred and seeded in EGM-2 MV medium for 24h at 40,000 cells/cm² into FIV wells (Fisher Scientific, Illkirch, France) containing irradiated Mouse Embryonic Fibroblasts. The following day, the medium was replaced by hESC medium. IPSC-derived cells appeared between day 11 and 20 and were transferred mechanically every week. Reprogramming efficiency was assessed with the Alkaline Phosphatase Staining Kit II (Stemgent, Cambridge, MA) in one FIV well 12–25 days after transduction depending on the cell type. Efficiency was calculated with 6 samples of ECFCs and 4 samples of HAECs. The number of positive Alkaline Phosphatase (ALP) colonies (red staining) was counted. The efficiency corresponded to the number of ALP positive colonies according to the total number of cells seeded.