Sn 1002

For knockdown of specific proteins, cells were transfected for 72 h with siRNA (final RNA concentration, 25 nM) using DharmaFECT 2 reagents (Dharmacon), according to the manufacturer’s instructions. SMART pool siRNA against STAT6 (L-006690-00) (Dharmacon Thermo Scientific) and non-targeting control siRNAs (D-001810-10) were purchased from Dharmacon (Lafayette, CO, USA); siRNA against DNMT1 (1786–1), DNMT3b (1789–1), HIF-1α (3091–1) and a corresponding negative control (SN-1002) were purchased from Bioneer (Korea). siRNA against DNMT3a was chemically synthesized by Bioneer (Korea). The siRNA sequences are as follows: 5′-CAG GAG AUG AUG UCC AAC CC-3′ (sense) and 5′-GGG UUG GAC AUC AUC UCC UG-3′ (antisense).
STAT6 was overexpressed by transiently transfecting U373MG cells with pCMV6-Myc-DDK-STAT6 (RC210065; Origene Technologies) or mock control vector (PS100001; Origene Technologies) using TurboFectin 8.0 (OriGene Technologies, Inc.) according to the manufacturer’s recommendations. All assays were performed at least 48 h after transfection. STAT6 deletion mutants were generated from human STAT6 cDNA (RC210065, Origene) by PCR amplification using the appropriate primers. STAT6 deletion mutants (Δ1–260 (d1), Δ261–430 (d2), Δ431–522(d3), and Δ523–622 (d4)) were cloned into the pCMV6-entry vector (PS100001, Origene). Primer sequences are listed in Additional file 2: Table S4.

Small interfering RNAs (siRNA) for inhibiting the SPRY1 transcript, were purchased from Bioneer (Daejeon, South Korea). The siRNA sequence (5′ to 3′) was: siSPRY1: F – GAGAGAGAUUCAGCCUACU; and R - AGUAGGCUGAAUCUCUCUC. A scrambled-siRNA (silencer negative control, SN-1002; Bioneer, Daejeon, South Korea), was used to determine the effects of siRNA delivery and has no sequence similarity, and does not target human gene sequences. To knockdown SPRY1, GSC11 were seeded in a 6-well plate at a density of 5 × 10⁵ cells/well, and after 1 day, it was transfected with 50nmol/L of SPRY1 siRNA or scrambled-siRNA with Lipofectamine® RNAiMAX Reagent (Invitrogen, USA) and incubated for 2 days.

Rat mesangial cells were seeded into 6-well plates at 5 × 10⁴ cells/well and then transfected with either siRNA control (SN-1002, Bioneer, South Korea) or siRNA GLO-1 using Lipofectamine RNAi MAX (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h according to the manufacturer’s instructions. The cells were treated with 0.75 mM MGO and 10 nM Ex-4 for 4 h and then harvested for further analysis.