THP1 cells were stimulated with100 ng/ml GM-CSF and 100 ng/mL IL-4 for 5 days to differentiate into immature DCs. For differentiation into mature DCs, THP1 cells were stimulated with 200 ng/ml GM-CSF, 100 ng/mL IL-4, 20 ng/mL TNFα, and 200 ng/mL ionomycin for 5 days. After 5 days of differentiation, cells were electroporated with 20 nM siRNA targeting human IFT88 (Life Technologies), or siRNA Control (AM4611, Invitrogen) with the Neon Transfection System (Thermo Fisher Scientific). The protocol settings were: pulse voltage 1680 V, pulse width 20 ms, pulse number 1. The target sequences of siRNAs were as follows:
siIFT88 (human) #1;
Sense: 5'-CCAAGUGUCAAUAAGCAAAtt
Antisense: 5'-UUUGCUUAUUGAACACUUGGaa
siIFT88 (human) #2:
Sense: 5'-GGUAGCUAGUUGUUUCAGAtt;
Antisense: 5'-UCUGAAACAACUAGCUACCat.
Differentiated THP1 cells were labeled with FITC anti-human CD86 (374203, Biolegend), APC-Cy7 anti-human CCR7 (353211, Biolegend), APC anti-human CD11c (301620, Biolegend), Texas Red-PE anti-human HLA-DR (MHLDR17, Life Technologies), and Live/Dead Fixable Aqua Dead Cell Stain Kit (L34966, Invitrogen). Labeled cells were fixed with 4% PFA for 5 min, then cell markers were analyzed with a BD FACSAria II cell sorter (BD Biosciences). The gating strategy is shown inSupplementary Figure S9 .
siIFT88 (human) #1;
Sense: 5'-CCAAGUGUCAAUAAGCAAAtt
Antisense: 5'-UUUGCUUAUUGAACACUUGGaa
siIFT88 (human) #2:
Sense: 5'-GGUAGCUAGUUGUUUCAGAtt;
Antisense: 5'-UCUGAAACAACUAGCUACCat.
Differentiated THP1 cells were labeled with FITC anti-human CD86 (374203, Biolegend), APC-Cy7 anti-human CCR7 (353211, Biolegend), APC anti-human CD11c (301620, Biolegend), Texas Red-PE anti-human HLA-DR (MHLDR17, Life Technologies), and Live/Dead Fixable Aqua Dead Cell Stain Kit (L34966, Invitrogen). Labeled cells were fixed with 4% PFA for 5 min, then cell markers were analyzed with a BD FACSAria II cell sorter (BD Biosciences). The gating strategy is shown in