Ham f12 medium

A549 cells (American Type Culture Collection, ATCC) were grown in Ham F12 medium (GIBCO) and Vero cells (ATCC) in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO). Both media were supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, GIBCO) and 1% L-Glutamine (GIBCO). Cultures were performed without antibiotics and were tested negative for mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza) every 2 months.

Four human OS cell lines (MG-63, Saos-2, SW 1353, U-2 OS) and one normal osteoblast cell line, hFOB 1.19 were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). MG-63, Saos-2, and SW 1353 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, cat# 11965092, USA), U2 OS cells in McCoy’s 5A medium (Gibco by Life Technologies, cat# 16600082), and hFOB 1.19 cells in a mixture of DMEM and Ham F12 medium (1:1 ratio, Gibco). The cell culture environment was in a humid incubator.
The siRNAs (si-GNAS-AS1 and si-control) were delegated to Ribobio (Guangzhou, China) for design and synthesis. The miR-490-3p inhibitor (anti-miR) and corresponding negative control (anti-NC) were also synthesized by Ribobio (Guangzhou, China). Lipofectamine 2000 (Invitrogen, USA) was utilized to complete the transfection and qRT-PCR was utilized to evaluate the transfection efficiency.

The human epithelial cell line generated from alveolar lung adenocarcinoma A549, and was grown in HAM F12 medium (GIBCO BRL, USA), 10% inactivated fetal bovine serum (GIBCO BRL), glutamine (2 mM) and gentamicin (40 µg/ml), at 37 °C and 5% CO₂. All tests were conducted under conditions of sub-confluence (80–90%), with an initial number of 5 × 10⁵ cells/ml culture flask (25 cm²), unless otherwise indicated. This cell line expresses transglutaminase II^{33 (link)} and LPS or TNF-α induce an increase in its expression^{19 (link),20 (link)}. Therefore, for some experiments, cells were treated with LPS (24 h, 400 ng/ml) (Sigma, MA) or TNFα (24 h, 40 ng/ml).

Keratinocytes and fibroblasts were obtained from human biopsies at the Tissue Bank of the Community Center for Blood and Tissues of Asturias, according to Spanish law (RD 9/2017), and cells were grown as follows. Keratinocytes were cultivated with lethally irradiated 3 T3 cells in 75 cm² culture flasks. The culture medium was a 3:1 mixture of Dulbecco’s modified Eagle medium (DMEM, Gibco) and Ham-F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), insulin (5 mg/ml, Sigma Aldrich), hydrocortisone (0.4 mg/ml, Sigma Aldrich), triiodothyronine (1.3 ng/ml, Sigma Aldrich), cholera toxin (8 ng/ml, Sigma Aldrich) and adenine (24 mg/ml, Sigma Aldrich). Fibroblasts were cultivated in 75 cm² culture flasks with DMEM supplemented with 10% FBS, penicillin (5000 IU/ml, Sigma Aldrich) and G/streptomycin (5000 μg/ml, Sigma Aldrich). In both cases, cultures were incubated at 37 °C in a humidified 5% CO₂ atmosphere.
The P. acnes, S. aureus and S. epidermidis strains used were clinical isolates obtained from the Hospital Universitario Central de Asturias. Bacteria were grown in Brain Heart Infusion (Conda) at 37 °C in a shaking incubator, except P. acnes which was grown in anaerobic atmosphere.