Sicontrol nontargeting sirna

HCC cells were transiently transfected with small interfering RNA (siRNA) using DharmaFECT (Dharmacon, Lafayette, CO). Twenty-one base pair siRNA duplexes targeting KLHL21 gene (siKLHL21-1: 5′-GTACAACTCAAGCGTGAAT-3′; siKLHL21-2: 5′-TGTCATTGCTGTCGGGTTA-3′) and a standard control (Dharmacon siCONTROL nontargeting siRNA) were synthesized by Dharmacon.

Subconfluent cells were transfected with a mixture of 3 μl/ml TransIT-KO transfection reagent (Mirus) and 50 nM siRNA diluted in serum free culture medium, according to the manufacturer’s protocol. siRNA targeting different sequences in the human netrin-4 gene were obtained from Qiagen (NTN 1–4). Two sets of 4 predesigned siRNA (SMARTpool, Dharmacon) were used to silence DCC and Unc5b gene expression. Two silencer validated neogenin siRNAs were purchased from Ambion (Autin, TX). Different siRNAs were used as controls: the siCONTROL non-targeting siRNA from Dharmacon and the Stealth RNAi negative control from Invitrogen.

Small interfering RNA was purchased from Sigma-Aldrich to deplete p65 proteins. siControl nontargeting siRNA was purchased from Dharmacon. PANC-1 GemR and BxPC-3 GemR cells were transfected with siControl and specific siRNA in 6-well plates at a final concentration of 100 nM using DharmaFECT1 transfection reagent (Dharmacon, USA) according to the manufacturer’s instructions and harvested 72 h later.

Transfection of the siRNA was performed using DharmaFECT 4 (Dharmacon). An siCONTROL nontargeting siRNA from Dharmacon was used as a negative control. The sequences for siRNA for SYCE2 and HP1α are listed in Table S3. The siRNA targeting the 3′ UTR of SYCE2 or HP1α was used for rescue experiments expressing exogenous FLAG-SYCE2 or functional analyses of HP1α mutants (V23M and KW41/42AA) under silencing of the endogenous HP1α protein.

Table S3 The targeted sequences of siRNA.

Cells were harvested at a density of 1 × 10⁶ cells per 100-mm-dish. Transfection was carried out using the Neon^TM transfection system (Invitrogen, Carlsbad, CA). Cells were transfected with 8 μg of plasmid DNA. siGENOME siRNA for human STS (target sequence sense: 5’-CAGUUCAUACAGCGGAACA-3’, antisense: 5’-UGUUCCGCUGUAUGAACU-3’) and siCONTROL nontargeting siRNA (Dharmacon, Lafayette, CO) were used for transfection. Cells were transfected with 50 nM siRNA using the Neon transfection system and cultured in 100-mm dishes in antibiotic-free RPMI with 10 % FBS for 24 h.

Three ON-TARGETplus siRNAs directed against human Nup35 were purchased from Dharmacon: siRNA 1, 5′-CUGCUGGUUCCUUCGGUUA-3′; siRNA 2, 5′-AGAUAAAAGUGGCGCUCCA-3′; siRNA 3, 5′-AGUUAUUUCUACC GACACA-3′. HeLa cells were plated at 1.5 × 10⁵ cells per well in 6-well plates and transfected the next day with a final concentration of 40 nM siRNA targeting Nup35 or non-targeting control siRNA (siCONTROL non-targeting siRNA, Dharmacon), using RNAiMAX (ThermoFisher Scientific) according to the manufacturer’s instructions. To generate stable Nup35 knockdown cells, a lentiviral vector and the following GIPZ Lentiviral (GE Healthcare, Dharmacon) shRNAs against hNup35 were used in this study:
GIPZ Lentiviral Human Nup35 shRNA:
V3LHS_364366, TGTCTGTCAGAAATAACCT
V3LHS_380787, TATGAGCTGGTACAACTGG
V3LHS_380788, TGGTTCAGATCCTAACGCG
Lentiviral vector stocks were produced by co-transfection of HEK293T cells with packaging plasmid ΔR8.2, MISSION shRNA or GIPZ Lentiviral shRNA, and pL-VSV-G at a ratio of 1:1:0.5, the culture medium was replaced at 12 h, and viral supernatants were collected and filtered at 48 h. HeLa cells were transduced with the filtered supernatant and selected in 2 ug/ml puromycin.