Hacat

The human keratinocyte cell line, HaCaT, (AddexBio, San Diego, CA) was cultured in calcium-free DMEM (Gibco, Life technologies, Carlsbad, CA), supplemented FBS, 10% v/v (GE Hyclone Laboratories, Logan, UT) and penicillin-streptomycin with L-glutamine (Gibco). Cells were seeded in 24-well plates for 24h before transfection. Attractene reagent (QIAGEN) and 50nM of miR-15b-5p mimic or mimic negative control #1 (GE Dharmacon, Lafayette, CO) was used for transfection. Cells were harvested after 48h for RNA extraction.
The 3’UTRs of the WEE1 and IKBKB human genes were amplified by PCR from human genomic DNA with primers containing restriction sites for SacI and XbaI (Table 1) and cloned into the pmirGLO Dual-Luciferase miRNA Target expression vector (Promega Corporation, Madison, WI). 100ng of vector containing the UTRs and 25nM of miR-15b-5p mimic or mimic negative control #1 was used for transfections. Luciferase reporter assay was done using the Dual-Glo® Luciferase Assay system (Promega Corporation) 48h after transfection using manufacturer’s protocol.

Human oral tongue cancer cell lines, HSC-3 (JCRB Cat# JCRB0623, RRID:CVCL_1288, passages 4–12) and OSC-20 (Cat# JCRB0197, RRID:CVCL_3087, passages 4–12) were obtained from the Japanese Collection of Research Bioresources Cell Bank. The spontaneously immortalized human keratinocyte cell line HaCaT (RRID:CVCL_0038, passages 4–12) was purchased from AddexBio Technologies (Cat# T0020001, San Diego, CA). Human dysplastic keratinocyte cell line DOK (RRID:CVCL, 1180, passages 4–12) was purchased from Sigma-Aldrich (Cat# 94122104). The cell lines harbor mutations in TP53. Further information on the mutational status of the HSC-3, OSC-20 and DOK cell lines can be obtained from COSMIC, the Catalogue Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk).^{[98 (link)]} The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA, HSC-3, DOK, HaCaT) or DMEM/F12 (OSC-20) supplemented with fetal bovine serum (FBS) and penicillin/streptomycin (50 U/mL) for 48 hours. Cell lines were cultured in 75 cm² cell culture flasks at 37°C with 5% CO₂, 21% O₂ (normoxia). When cells reached 70–80% confluency, the culture medium was replaced with media supplemented with 10% exosome-depleted FBS (Gibco) and cultured for a further 48 hours for preparation of exosomes. Cell culture supernatant was collected and used for isolation of exosomes.

HEK293FT (Thermo Fisher, Waltham, MA, USA), HEK293A (Thermo Fisher, Waltham, MA, USA), A549 (ATCC, Manassas, VA, USA), HT1080 (ATCC, Manassas, VA, USA), HaCaT (AddexBio, San Diego, CA, USA), and HaCaT-GJA1+ cells were maintained and passaged in DMEM, high glucose, with L-Glutamine (Genesee Scientific, San Diego, CA, USA) supplemented with 10 % fetal bovine serum (FBS), non-essential amino acids (Life Technologies, Carlsbad, CA, USA), and MycoZap Plus-CL (Lonza, Basel, Switzerland) unless otherwise specified. HiPSC-CMs were obtained from Axol Bioscience and maintained in Cadiomyocyte Maintenance Basal Medium (Axol, Cambridge, UK) supplemented with Cardiomyocyte Supplement (Axol, Cambridge, UK) according to manufacturer’s instructions. Cells were maintained in a humidified atmosphere of 5 % CO₂ at 37 °C.