Lenti x expression system

To generate A549 cells, in which miR-32-5p can be stably overexpressed, a 500-bp amplified DNA fragment containing a primary hsa-miR-32-5p transcript was subcloned into a pLVX-IRES-Neo vector by restriction endonuclease XhoI and XbaI for expression through a Lenti-X expression system (Clontech Laboratories, Inc.). Subsequently, the miR-32-5p overexpression vector or empty vector were co-transfected with packaging plasmids into human embryonic kidney (HEK) 293T cells using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.). The empty vector was used as negative control (miR-neg). After 48 h, the packaged lentiviruses were harvested and used to infect A549 cells. The infected cells were cultured in medium for two days, and stable cells were selected by adding 300 μg/ml G418 (Thermo Fisher Scientific, Inc.).

Empty plasmids or various plasmids were transfected with the Hieff Trans™ Liposomal Transfection Reagent (Cat No. 40802ES08, Yeasen Biotech, Shanghai, China) according to the manufacturer’s instructions. HEK-293T STING stable expression cell lines were constructed using the Lenti-X™ Expression System (Cat No. 632164, Takara, Osaka, Japan). THP-1 Control/shSTING shut-down cell lines were constructed using the Lenti-X™ shRNA Expression Systems (Cat No. 632177, Takara, Osaka, Japan). TRAF3 scrambled siRNAs were synthesized by RiboBio (Guangzhou, China). siRNA was transfected using Lipofectamine RNAiMAX (Invitrogen, California, USA) according to the manufacturer’s protocols.

IRE1α shRNA plasmid was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PLKO-puro with mouse XBP1 shRNA plasmid was received as a gift from Dr. Ann-Hwee Lee, Ph.D. (Senior Staff Scientist at Regeneron Pharmaceuticals, Inc. Tarrytown, NY, USA). To establish stable cell lines expressing the IRE1–shRNA constructs, C2C12 cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific). The transfected cells were plated at low density and then selected using 3 μg/mL puromycin (Thermo Fisher Scientific); further, clonal colonies were isolated. IRE1 expression levels were analyzed by immunoblotting analysis. To establish shXBP1-transduced cells, lentiviral constructs were established for use with the Lenti-X Expression System (TaKaRa Bio Inc., Shiga, Japan). HEK293T cells were transfected with vectors using Lipofectamine 2000. After 48 h, the virus-containing supernatant was harvested. Transduction of C2C12 was performed with shIRE1 lentivirus (multiplicity of infection:10) in the presence of 5 µg/mL polybrene, and cells were selected with 3 μg/mL puromycin (Thermo Fisher Scientific) for five days. Single colonies were picked under light microscopy at the fifth passage.