Thp 1

Human AML cell lines were purchased from ATCC (Kasumi-1, Mv-411, AML193, HL-60) or DSMZ (U937, OCI-AML3) all in 2017–2018. THP-1 and HEK-Blue-ISG-KO-STING were purchased from Invivogen in 2017–2018. Upon receipt, cells were expanded to make original cryopreserved stocks with each subsequent experiment using a new original stock. Human AML cell lines were cultured either in complete RPMI (10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, and 2 mmol/L l-glutamine) or as described in suppliers’ guidelines. THP-1 and HEK-Blue-ISG-KO-STING cells were cultured according to manufacturer's instructions (Invivogen). All cell lines were monitored for cell line authenticity at the MD Anderson Cancer Center in-house core using the Cytogenetics and short tandem repeat sequencing to assess for characteristics chromosomal/mutational status of respective cells lines. Cell lines were routinely tested for Mycoplasma at the MD Anderson Cancer Center in-house core using Lonza MycoAlert Kit. Cryopreserved primary bone marrow aspirates from patients with AML treated at MD Anderson Cancer Center were received as a gift from Drs. Gheath Al-Atrash and Jeffrey Molldrem.

U937 (ATCC, USA) and THP-1 (InvivoGen, USA) monocytic cell lines were grown in RPMI 1640 (Gibco, USA) supplemented with 10% FBS, 2 mM l-glutamine, and 100 U/mL of penicillin and streptomycin. In HG experiments, before HG stimulation, the cells were cultured in RPMI 1640 with 5.5 mM glucose for 48 h, and then were changed to 10, 20, or 30 mM glucose RPMI 1640 for indicated time points. 30 mM mannitol was used as an osmotic control. For the experiments using chemical inhibitors, Cs A, FK506, and U18666A were pre-treated for 24 h, while TG was pre-treated for 45 min. EGTA and BAPTA-AM were treated in the presence of HG stimulation. For the immunoblotting experiments measuring TFEB translocation by calcium inducers, GPN, H₂O₂, ionomycin, NAADP, and TG were stimulated for 20 min. The supernatants from U937 and THP-1 cells were collected for the detection of human IL-1β levels by ELISA (eBioscience, USA).

C57BL/6 mice of 6–8 weeks of age were euthanized by cervical dislocation. Animal experiments were performed in accordance with standard ethical guidelines and approved by the regional laboratory animal ethics committee in Malmö/Lund (Permit No. M36-13). Femoral and tibial bones were dissected. Skin and excess muscle tissue were removed. Femur and tibia were separated by a cut at the knee joint and bone marrow was flushed with 10 ml of phosphate-buffered saline (PBS; Life Technologies, Carlsbad, CA, USA) supplemented with 2% heat-inactivated fetal bovine serum (HI-FBS; Life Technologies, Carlsbad, CA, USA). Bone marrow was passed through 70 µm nylon cell strainer (Fisher Scientific, Lund, Sweden), centrifuged and resuspended and incubated in ammonium-sodium-chloride lysis buffer (ACK; Life Technologies, Carlsbad, CA, USA) for 5 min to lyse red blood cells. Cells were then washed with PBS with 2% HI-FBS, centrifuged and resuspended in RPMI 1640 culture medium supplemented with 10% HI-FBS and 1% penicillin-streptomycin.
For additional experiments, the human monocytic leukemia cell line THP-1 (InvivoGen, San Diego, CA, USA) was cultured in RPMI 1640 culture medium supplemented with 10% HI-FBS and 1% penicillin-streptomycin.