Silencer select predesigned

MEF and HEK cells were grown at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal calf serum (FCS). WT and Atg7KO MEF cells were gifts from M. Komatsu. Atg4BKO, Atg13KO, Atg14 and Atg16L1KO, and Ulk1/2DKO MEF cells were kindly provided by G. Marino, F. Reggiori, T. Saitoh, and S. Tooze, respectively, and have been characterized in ref. ^{19 (link)} and in Supplementary Fig. 2. To induce autophagy, cells were washed three times with Earle’s balanced salt solution (EBSS, Thermo Fisher) and then incubated in EBSS for 2 h in the presence or absence of 100 nM BafA1.
Transient transfections were performed using JetPrime transfection reagent (PolyPlus) according to the manufacturer’s instructions. RNA interferences were performed in MEF cells plated at 50–60% confluence. Cells were transfected with scrambled small interfering RNA or small interfering RNA (siRNA) against CHMP4B (5′-AAACAGUCCCUCUACCAAAtt-3′, 50 nM per dish, Silencer Select Pre-designed, Ambion). Cells were processed for immunofluorescence or for biochemical analyses 48 h after transfection (see below).

Logarithmically growing cells were seeded at a density of 3 × 10⁵ cells/6-cm dish and transfected with oligonucleotides by using Oligofectamine RNAi MAX (Invitrogen), according to the manufacturer’s instructions. TGFBI siRNAs were obtained from Ambion Life Technologies Corporation Silencer® select Pre-designed (Inventoried) siRNA Product (s14070, #ASO2L6OI), as the following sequences: TGFBI, 5′-GCAUGACCCUCACCUCUAUtt -3′. After 2 days, we passaged these cells to the other dishes and cultured more 2 days. Then we used these cells for other experiments.

At 24 h after seeding into six-well plates, Ca9-22 cells were transfected with negative control or Silencer Select Predesigned small-interfering (si)RNA targeting TLR2, TLR4, or ANGPTL2 (Life Technology, Tokyo, Japan) using Lipofectamine 3000 (Life Technology), according to the manufacturer’s instructions. After incubation for 48 h, cells were primed with rhIFN-γ for 12 h, washed three times with PBS, and treated with 30 ng/ml P. gingivalis LPS for 24 h.