Tlrl nacga23 02

2′3′-cGAMP was purchased from Invivogen. 2′3′-cGAMP (5 µg/ml) (Invivogen, tlrl-nacga23-02) was added to the cells complexed with LyoVec (Invivogen, Lyec-12) in order to aid internalization. ISD (Interferon stimulatory DNA)/LyoVec (Invitrogen, tlrl-isdc, 1 µg) was also used. Membrane lipid strips were obtained from Echelon. The following inhibitors were used: Fura-2 AM, Ca2+ selective fluorescent indicator (Abcam, ab120873), BX795 (TBK-1 inhibitor, Invivogen, tlrl-bx7, 1 µM), RU.521 (cGAS inhibitor, Invivogen, inh-ru521, 500 nM). GSK2998533 (100 nM) & GSK2683449 (100 nM) were provided by GlaxoSmithKline (GSK). PIK93 was also used (Sigma Aldrich, 1 µM)

Human osteoblasts (hObs) were purchased from, and authenticated by, Cell Applications (#406–05, San Diego, CA, USA), and human OSA cell lines (SAOS-2 (SAOS), SAOS-2-LM6 (LM6), MG63, U2OS) were purchased from the MD Anderson Characterized Cell Line Core (Houston, TX). hObs were cultured in human osteoblast growth medium (#417–500, Cell Applications, San Diego, CA, USA), and OSA cell lines were cultured in complete media consisting of Dulbecco’s Modified Eagles Medium (DMEM, SH30022.01, Cytiva, Logan, UT, USA) supplemented with 10% Fetal Bovine Serum (FBS, 83007–198, VWR) and 1% penicillin/streptomycin (K952-100ML, VWR, Sanborn, NY, USA). Cells were incubated at 37°C, with 5% CO₂. Mammalian (non-canonical) CDN, cyclic[G(2’,5’)pA(3’,5’)p] (2’3’-cGAMP, #tlrl-nacga23-02, InvivoGen, San Diego, CA, USA) was reconstituted using 1.5 mL endotoxin-free water.
For baseline mRNA and protein expression, cells were processed when they achieved ~90% confluency in a 6-well plate. For cGAMP stimulation experiments, cells were plated in 6-well plates at counts of 7 x 10⁵ cells/well for SAOS, and 5 x 10⁵ cells/well for LM6, MG63, and U2OS. After resting overnight, cells were then treated with 10 μg/ml cGAMP or control media. RNA was extracted after 16 hrs, and cell culture supernatant was collected after 24 hrs.