Lipofectamine rnaimax

Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778150) transfection was performed following the manufacturer’s protocols. In brief, 500 µl of Opti-MEM Reduced Serum Media (Thermo Fisher Scientific, 31985070) containing either Dharmacon’s siRNA targeting H1.0 (40 nM, Horizon Discovery, M-060325-01), H1.1 (80 nM, Horizon Discovery, M-049956-00), H1.2 (80 nM, Horizon Discovery, M-045246-00), H1.3 (80 nM, Horizon Discovery, M-051171-00), H1.4 (80 nM, Horizon Discovery, M-042536-01), H1.5 (80 nM, Horizon Discovery, M-049995-00) and Thbs4 (40 nM, Horizon Discovery, M-044016-01) or the appropriate concentration of siRNA scramble control (Horizon Discovery, D-001206-14) were mixed with 500 µl of Opti-MEM Reduced Serum Media containing 20 µl of Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778075). For the human skin fibroblast experiment, transfection was performed using Dharmacon’s siRNA targeting human H1.0 (40 nM, Horizon Discovery, M-017209-01). After incubating the reagents for 10 min at 37 °C, the solution was added to the cells and slightly agitated to mix. After 24 h of incubation at 37 °C, the siRNA reagent solution was removed and replaced with appropriate media according to the downstream experiment. siRNA sequences are listed in Supplementary Table 2.

To silence MyD88 expression in primary isolated KCs, mouse MyD88-siRNA or siRNA control (Santa Cruz Biotechnology Inc., Paso Robles, CA, USA) was used for transfection using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, siRNAs and Lipofectamine RNAiMAX were mixed and incubated at 22–28 °C for 5 min in the Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA, USA). The mixture was then transferred to a six-well plate with KCs (4 × 10⁵/well) at the final concentration of 30 nmol/L (siRNA) and 7.5 μL RNAiMAX per well. After siRNA transfection for 48 h, the cells were incubated with or without LPS (10 ng/mL; Sigma, St. Louis, MO, USA) or eritoran (10 μg/mL) for 6 h and subsequently collected for Western blot analysis.

For transient short interfering RNA (siRNA) transfection, cells at 70% confluence were transfected using Lipofectamine^® RNAiMAX (Thermo-Fisher, Waltham, MA, USA) in complete medium according to the manufacturer’s instructions, with a final siRNA concentration of 100 nM. The siRNAs were incubated with Lipofectamine^® RNAiMAX reagent for approximately 20 min and diluted with Opti-MEM^® medium. The siRNA-lipid complex was gently added to the culture medium. A total of 6 h later, the culture medium was replaced with fresh medium. A total of 48 h after transfection cells were trypsinized and plated for the different experiments. siRNAs were purchased from GenePharma (http://www.genepharma.com/). siRNA sequences were as follows: EGFR siRNA-1: 5′-CUGUGCGAUUCAGCAACAA-3′; EGFR siRNA-2: 5′-CCACCUAUCAGAUGGAUGU-3′; EGFR siRNA-3: 5′-CCCUGUCGCAAAGUUUGUA-3′; EGFR siRNA-4: 5′-GUGCUACGCAAACACAAUA-3′; and control non-targeting siRNA-NC: 5′-UUCUCCGAACGUGUCACGU-3′.

Silencer siRNAs targeting N-cadherin (siRNA ID: s2771) and scramble Silencer^® siRNA control were purchased from Thermo Fisher Scientific (Rochester, NY). Melanoma cells and CAFs were seeded in 6 cm dishes at an initial cell density of 1 × 10⁵ cells, cultured for 24 hours until 60–70% confluence, and then transfected with siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Rochester, NY). According to the manufacturer’s protocol, N-cadherin siRNA (10 μM) was diluted in 250 μl of Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Rochester, NY) and mixed with 15 μl of Lipofectamine RNAiMAX in 250 μl of Opti-MEM I reduced serum medium. After incubation at room temperature for 10 minutes, the mixture was added to the cells and incubated for three days. Afterward, the medium containing siRNA and RNAiMAX was replaced with regular DMEM culture medium.