Scrambled sirna

Small interfering RNA (siRNA) duplexes targeting human SATB1, c-Jun, NFkB1, Sp1 sequence and scrambled siRNA were produced by Ribobio Co. Ltd. (Guangzhou, China) and their knock-down efficiency was validated by Real-time PCR and Western Blot. siRNA sequences were as follows:
SATB1, sense, 5′-GGAUAGUCUUUCUGAGCUA-3′, antisense, 5′-UAGCUCAGAAAG ACUAUCC-3′;
Sp1 si-1, sense, 5′-CCAACAGAUUAUCACAAAU-3′, antisense, 5′-AUUUGUGAUAAUCUGUUGG-3′;
Sp1 si-2, sense, 5′-GGCUGGUGGUGA UGGAAUA-3′, antisense, 5′-UAUUCCAUCACCACCAGCC-3′;
NFKB1, sense, 5′-GGGGCUAUAAUCCUGGACU-3′, antisense, 5′-AGUCCAGGAUUGUAGCCCC-3′;
c-Jun, sense, 5′-CUGCAAAGAUGGAAACGAC-3′, antisense, 5′-GUCGUUUCCAUCUUUGCAG-3′,
NC, sense, 5′-UUCUUCGAACGUGUCAC G-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′.

Human HCC cell lines Huh7 and Hep3B were maintained in our lab and cultured as previously described.6 The precursor miR‐301b‐3p in non‐viral vectors, lentivector‐mediated miR‐301b‐3p inhibitors and corresponding control clones were obtained from GeneCopoeia Inc. (Guangzhou, China). pcDNA3.1‐VGLL4 was constructed as previously described.24 VGLL4 siRNA (5′‐CAACGACCACGUCUCCAAAtt‐3′) and scrambled siRNA (5′‐ACAGACUUCGGAGUACCUG‐3′) were designed and purchased from RiboBio (Guangzhou, China). Lipofectamine^® 2000 Reagent (Invitrogen, Carlsbad, CA, USA) was used for vector transfection according to the manufacturer's protocols.

The coding sequences of STAT4 (Gene ID: 397261, accession number: NM_001197305.1) and KISS1 (Gene ID: 100145896, accession Number: NM_001134964.1) were cloned into pcDNA3.1(+) (ThermoFisher, Guangzhou, China) with the restrictive enzymes of KpnI and xhoI for STAT4; EcoRI and NotI for KISS1. The sequences of the primers for these coding sequences were shown in Table 2. STAT4-siRNA-1, STAT4-siRNA-2, STAT4-siRNA-3 and Scrambled-siRNA were synthesized and purified by RiboBio Co. Ltd. (Guangzhou, China).
According to our previous studies [40 , 41 (link)], the cell apoptosis was detected to by using an Annexin V-FITC Apoptosis Detection Kit (BioVision, Milpitas, CA, USA). Briefly, when GCs covered 30–50% of the triplicate in 6-well plates at 24 h prior to transfection, pcDNA3.1-STAT4, pcDNA3.1-KISS1, pcDNA3.1-Basic, STAT4-siRNA, and Scrambled-siRNA were transfected into the cells for 48 h, respectively. Then the transfected cells were harvested and treated by using Annexin V-FITC mix and were analyzed in a flow cytometer (Becton Dickinson Co., San Jose, CA, USA). All experiments were performed at least triplicate.

The ERβ (with target sequence GGTCCTGTGAAGGATGTAA) and scrambled siRNA were synthesized by RiboBio Co. (Guangzhou, China). EL4 and naïve CD4⁺ T cells were transfected with the siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Silence efficiency was assessed by quantitative PCR analysis after transfection for 24 h. The cells were then prepared for further analysis.

A vector encoding the human SRCIN1 open reading frame without the 3′-UTR (EX-Y4423-M68) was obtained from GeneCopoeia (Germantown, MD, USA), and an empty plasmid served as the negative control. The siRNA sequence (5′-AAGCTGTGTCTGTTGAGGCTG-3′) targeting human SRCIN1 was synthesized by RiboBio (Guangzhou, China), and a scrambled siRNA (RiboBio) was used as the negative control.