Drr820a

The conditionally immortalized human podocytes (HPC) was a gift from Dr. Moin Saleem at Bristol University, UK. The cells were cultured in RPMI 1640 containing 10% fetal bovine serum and pennicilin/streptomycin (100 U/ml of each) (Gibco, USA) and 1% insulin-transferrin-selenium (ITS, Invitrogen). Other reagents used in the studies were as follows: podocyte transfection reagent (LipofecTAMINE 2000, Invitrogen); plasmid DNA extraction kit (Purelink HiPure Plasmid DNA Purification Kit, Invitrogen), antibodies against total p65, phospho-p65, total p38 and phospho-p38 (Cell Signaling Technologies), TLR9 and CD2AP (Abcam), podocin (Sigma), GAPDH (Kangchen), anti-rabbit IgG (Bioworld); puromycin aminonucleosides (Sigma); Alexa Fluor® 647 Annexin V and Propidium Iodide (Biolegend); Reverse transcription kit, DRR037A, and quantitative PCR kit, DRR820A (Takara); RIPA cell lysis buffer; BCA protein quantification kit (Bi-yun-tian, Shanghai); psiRNA-hTLR9 (Invivogen); RNA extraction kit (mirVana miRNA extraction kit, Ambion); E. coli DH5a competent cells (TransGen Biotech, Beijing).

Total RNAs of the shoots and roots were extracted from the samples of the three independent biological replicates for each of genotypes using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The first strand of cDNA was synthesized using 1.0 μg total RNA, 0.5 μg oligo d (T) 18 primer, and reverse transcriptase system (DRR037A; Takara, Dalian, China) at a total volume of 20 μL. The primers used for qPCR were designed according to the corresponding sequence on Primer 6, and the primers are listed in Table S1. Amplification through quantitative real-time polymerase chain reaction was performed with a Thermal Cycler Dice Real Time System III (Takarabio Inc., Otsu, Shiga, Japan) using an RT-PCR master mix (DRR820A; Takara) according to the user manual. The expression levels of the target genes were normalized using actin as an endogenous control. All reactions were run in triplicate. The quantification of gene expression levels relative to the control was determined by 2^−ΔΔCt method.