Double stranded sirna

TSCs were transfected with 62.5 nM double-stranded siRNA (GenePharma, China) using TransIT-X2 (Mirus Bio, WI, USA) according to the manufacturer’s instructions. Samples were analyzed 72 h after transfection. For CRISPR-Cas9 genome editing, TSCs were transduced by lentivirus generated from the LentiCRISPRv2-mCherry plasmid. The mCherry positive Cas9-expressing TSCs were isolated by fluorescence activated cell sorting (FACS) using BD FACSAria II cell sorter (BD Bioscience, CA USA). Two Alt-R CRISPR-Cas9 crRNAs (IDT, IA, USA), which targeted the Jmjd2b genomic sequence, were synthesized and separately annealed with the Alt-R CRISPR-Cas9 tracrRNA (IDT) to form the duplexed oligo. The Cas9-expressing TSCs were transfected with 90 nM duplexed oligo using TransIT-X2 (Mirus Bio). Two Jmjd2b-knockdown TSC lines were selected and verified by T7 endonuclease I digestion and Sanger sequencing (Supplementary Fig. 9). The Jmjd2b-knockdown TSCs were used for other experiments within 5 passages. The siRNA and crRNA sequences were listed in Supplementary Table 4.

For the overexpression assay, 5 × 10⁵ iPAM cells were seeded into 24-well tissue culture plates in RPMI 1640 medium containing 10% FBS. At 60% to 70% confluence, cells were transfected with Flag-CAV1, Flag-ARF6, or Flag-PPP2R1A expression plasmids and Flag-vector, respectively, using jetPRIME transfection reagent (Polyplus) according to the manufacturer’s instructions for 24 h. For siRNA-mediated knockdown, 5 × 10⁵ iPAM cells were seeded into 24-well tissue culture plates in RPMI 1640 medium containing 10% FBS. After the cells had grown to 60% to 70% confluence, they were transfected with 50 nM CAV1-targeting, ARF6-targeting, or PPP2R1A-targeting siRNA or negative-control siRNA using jetPRIME transfection reagent as recommended by the manufacturer for 24 h. Double-stranded siRNAs targeting CAV1, ARF6, and PPP2R1A and a negative control were designed and synthesized by GenePharma Co. (Shanghai, China) (Table 2).

Chemically synthesized, double-stranded siRNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). Sequence for CD147 siRNA (si-147) is 5′-GUUCUUCGUGAGUUCCUCtt-3′ [11 (link)]. siRNA for FAK (sc-35353) was purchased from Santa Cruz (Dallas, Texas), siRNAs for DOCK1 to DOCK11 were designed and synthesized by GenePharma Company. Lipofectamine2000 reagent was employed according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Silencer negative control siRNA (sncRNA) was used as a negative control.

HeLa cells were cultured in DMEM (Hyclone) and 10% fetal bovine serum (FBS, Gibco) supplemented with 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO₂. For starvation, cells were washed with PBS three times and incubated with Earle’s balanced salt solution (EBSS, Gibco) for 2–h at 37°C. Transfection of plasmids was carried out with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocols. Transfection of small interfering RNAs (siRNAs) was carried out with Lipofectamine RNAi MAX (Invitrogen). To knockdown CK1δ, double-stranded siRNAs were purchased from GenePharma. The following sequences were used: human CK1δ siRNA 5′-CGACCUCACAGGCCGACAATT-3′ and control siRNA 5′-UUCUCCGAACGUGUCACGUTT-3′.

Double-stranded siRNAs targeting c-Myc were obtained from GenePharma (China). MCF-7 and ZR-75-1 cells were transiently transfected with two c-Myc siRNA (designated siRNA1, siRNA2) and a control (designated CTRi) using Lipofectamine 3000 (GenePharma) according to the manufacturer’s instructions. Colony formation analysis was used to evaluate the effect of siRNA and Dual-luciferase reporter assays were performed at 48 h after siRNA treatment. The sequences of the siRNAs are as follows: siRNA1: sense 5′-UCCUGAGACAGAUCAGCAATT-3′ and anti-sense 5′-UUGCUGAUCUGUCUCAGGATT-3′; siRNA2: sense 5′-GGCGAACACACAACGUCUUTT-3′ and anti-sense 5′-AAGACGUUGUGUGUUCGCCTT −3′.

Primary hepatocytes were transfected with double-stranded siRNA or negative control siRNA (non-siRNA) using RFect transfection reagent (Bio-Tran Biotechnologies, China). Target sequences were as follows: sense 5′-GAAAUGAGCUG GUAAAGAATT-3′, antisense 5′-UUCUUUACCAGCUCAUUUCTT-3′ for TLR4 [18 (link)]. Double-stranded siRNAs were synthesized by Shanghai GenePharma (China).

ARPE-19 cells were transfected with double-stranded siRNA or negative control siRNA (non-siRNA) using Lipo6000 Transfection Reagent (Beyotime). Target sequences were as follows: 5′-GCCCUAUCCCUUUACGUCA-3′ for p65, 5′-GAGGACAUAUCAGUGGUGUUCAGCA-3′ for RelB and 5′-AAAUGUGAAGGGCGAUCAGCA-3′ for c-Rel. Double-stranded siRNAs were synthesized by Shanghai GenePharma (Shanghai, China).