Control shrna

Control, miR-4516 mimic, miR-4516 inhibitor, shRNA specifically targeting PART1, and scrambled negative Control shRNA were chemically synthesized by GenePharma Co. Transfection assays were performed for transient expression as previously described.21 (link) miR-4516 mimic: 5ʹ-GGGAGAAGGGUCGGGGC-3ʹ; miR-4516 inhibitor: 5ʹ-CCCUCUUCCCAGCCCG-3ʹ; miRNA control: 5ʹ-UAAGGCUAUGAAGAGAUAC-3ʹ; shPART1-1: 5ʹ-GAAAACGCAGCTACACCTGG-3ʹ; shPART1-2: 5ʹ-GACTACATATGCATTAAGG-3ʹ; Control shRNA: 5ʹ-AAGGCUAUGAAGAGAUAC-3ʹ. Breast cancer cells were transfected with 100 nM miR-4516 mimics, miR-control or miR-4516 inhibitors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
pMIR-PART1-WT or pMIR-PART1-Mutant and miR-4516 mimics were co-transfected into MCF-7 cells together with pRL-CMV Renilla luciferase reporter. After 48h, data was measured using luciferase assay kit (Promega, Madison, WI, USA). Firefly luciferase activity was normalized against Renilla luciferase activity.21 (link)

Primary epidermal melanocytes were obtained from the American Type Culture Collection (cat no. ATCC^® PCS-200-013) and cultured in Medium 254 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with human melanocyte growth supplement (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. Primary epidermal melanocytes (1×10⁶ cells per well) were transfected with the inhibitor control (5′-CAGUACUUUUGUGUAGUACAA-3′; Guangzhou Ribobio Co., Ltd.), miR-421 inhibitor (5′-GCGCCCAAUUAAUGUCUGUUGAU-3′; Guangzhou Ribobio Co., Ltd.), 0.2 µM control-shRNA (cat no. sc-108060; Santa Cruz Biotechnology, Inc.), 0.2 µM RIPK1-shRNA (cat no. sc-44326-SH; Santa Cruz Biotechnology, Inc.), miR-421 inhibitor + control-shRNA, or miR-421 inhibitor + RIPK1-shRNA for 24 h using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The transfection efficiency was determined by reverse transcription-quantitative PCR (RT-qPCR) 24 h after cell transfection.

Expression vector of LNCOC1 was constructed using pcDNA3.1 vector as backbone. miR-124 mimic, control miRNA, LNCOC1 knockdown negative control and LNCOC1 shRNA were synthesized by Sangon Biotech (Shanghai, China). Empty vector pcDNA3.1, control miRNA or LNCOC1 knockdown control RNA were used as negative control (NC). Following the manufacturer’s instructions of Lipofectamine 2000 (Lipo2000, Invitrogen), after SK-MEL-3 and A375 cells were harvested and counted, 10 nM expression vector, knockdown vector or 40 nM miRNA using were transfected with Lipo2000. miR-124 mimic, control miRNA, LNCOC1 shRNA and control shRNA were designed by Invitrogen, and the sequences were as following:
control miRNA: 5’-UUGUACUACACAAAAGUACUG-3’
miR-124 mimic: 5’-UAAGGCACGCGGUGAAUGCC-3’
LNCOC1 shRNA: 5’-AGTGCTCCTAGTGTTACCAGAG-3’
control shRNA: 5’-ACTAGCTAGGCATCGATATCAG-3’

At logarithmic growth phase, NSCLC cells were inoculated in a 6-well plate. Cells were transfected with HMGB1-siRNA or its control siRNA, HMGB1-shRNA or its control shRNA (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and then cultured at 37°C and 5% CO
₂ for 48 h. HMGB1 was subcloned into the pCDNA3.1 vector (Invitrogen) and cells were transfected with pCDNA-HMGB1 vector or empty vector using Lipofectamine 2000 (Invitrogen), and then cultured at 37 °C and 5% CO
₂ for 48 h. The siRNA sequences are as follows: HMGB1 siRNA, 5′-GCAGCCCUAUGAAAGAAATT-3′; control siRNA, 5′-UCCAAGUAGAUUCGACGGCGAAGTG-3′; HMGB1-shRNA, 5′-GGGAGGAGCAUAAGAAGAATT-3′; control shRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′.