For flow cytometry analyses, cells were seeded in 12-well plates at a density of 2 × 105 cells/mL and incubated overnight.
Physicochemical parameters analyses, cells collection, DNA extraction, 16S rRNA gene Illumina sequencing and data processing were carried out as described previously (Xiao et al. 2015) and also see a detailed description in supplementary materials.
For western blot analyses, cells were collected from a 24-well plate 48 hours post-transfection.
For competitive analyses, cells were co-incubated with L-pyruvate at 4°C.
For immunocytochemistry analyses, cells were transfected with DNA constructs expressing GFP-tagged proteins and cultured in medium containing 0.5% fetal bovine serum for 48 hours to allow ciliogenesis.
For immunoprecipitation and western blot analyses, cells and supernatant were harvested 48 hours post-transfection, whereas for immunofluorescence assays, cells were analyzed 24 hours after transfection.
For flow cytometric analyses, cells were stained with Foxp3-PE/APC (eBioscience, San Diego, CA) and CD25 FITC BDD) according to the manufacturers instructions.
For quantitative flow cytometry analyses, cells treated with the ROS-sensitive fluorogenic probes were washed twice, trypsinized, resuspended in PBS, and immediately analyzed on a FACScan flow cytometer (Becton Dickinson).
For all flow-cytometric analyses, cells were stained with the appropriate combination of monoclonal antibodies in PBS containing 0.5% BSA and 2 mM EDTA for 20 minutes at 4°C.
For pathway analyses, cells were preincubated for 1 h with 20 µM PD98059 (Map Kinase Kinase/Erk Kinase 1 inhibitor) or 40 µM MG123 (proteasome inhibitor; Calbiochem, San Diego, USA).
In Schild plot analyses, cells were incubated with various concentrations of FP59 plus PA (constant at 12 nM) in the presence of different fixed concentrations of the LF protein being analyzed, which acted as a non-toxic competitor of FP59.
