Oligofectamine transfection reagent

Specific siRNA targeting Cks1 (si
Cks1) and control siRNA (siCrtl) were purchased from Dharmacon (SMARTpools) and the cells were transfected with Oligofectamine transfection reagent (Life-Technologies-Invitrogen) following the manufacturer's instructions. Cells were analyzed 48 h after transfection.

GBM12 cells were kindly supplied by Dr. Jann Sarkaria (Mayo Clinic). All other cell lines were obtained from the ATCC and authenticated by the supplier. All lines were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. siUSP9X (#6308 S, Cell Signaling Technology, Danvers MA), siSurvivin (#6351, Cell Signaling Technology; (#4390824, Silencer Select S1458, Ambion), siRNA CTR (#6568, Cell Signaling Technology; Silencer^TM Select Negative Control, #4390843, Ambion) were transfected into cells using Oligofectamine™ Transfection Reagent (Invitrogen, Waltham MA) following the supplier’s protocols. All plasmids were transfected by using Lipofectamine™ 3000 (Invitrogen) following the supplier’s protocols.

Cells were seeded on glass slides (BD Falcon Cultureslides BD Biosciences) coated with 1% BD Matrigel Basement Membrane Matrix (BD Biosciences) in growth medium without antibiotics. Next day the growth medium was exchanged for OPTI-MEM I Reduced Serum Media (Invitrogen) and siRNA transfections were performed using Oligofectamine Transfection Reagent (Invitrogen) according to the manufacturer’s protocol. Four hours after transfection serum was added to a final concentration of 20%. Cells were allowed to grow for 72 h prior to the evaluation by immunofluorescence staining or western blot.
The following siRNA’s were used:
siGENOME siRNAs against mouse AmotL2 (0.32 μM, M-062016–01, Dharmacon/Thermo Scientific).
siGENOME siRNAs against mouse Par3 (0.64 μM, M-040036–01, Dharmacon/Thermo Scientific).
non-targeting siRNA Pool #2 (0.32/ 0.64 μM, D 001206–14, Dharmacon/Thermo Scientific).
siGENOME siRNAs against human AMOTL2 (0.32 μM, M-013232–00, Dharmacon/Thermo Scientific).
ON-TARGETplus siRNAs against human PAR3 (0.32 μM, L-015602–00, Dharmacon/Thermo Scientific).

Cells were seeded in 96-well plates at 3 × 10³ per well for MIA PaCa-2 and AsPC-1 or at 1 × 10³ per well for PCI-35. Twenty-four hours after seeding, the cells were transfected with small interfering RNAs (siRNAs) for LINC00941 (si#1, sense: 5′-GCCUCCAUAUUCAUGAACUtt-3′, antisense: 5′-AGUUCAUGAAUAUGGAGGCtg-3′, si#2, sense: 5′-CCAUUCAGCCUUGAACAUUtt-3′, antisense: 5′-AAUGUUCAAGGCUGAAUGGtc-3′, Thermo Fisher Scientific, Waltham, MA, USA) or Silencer Select Negative Control (Thermo Fisher Scientific) at 200 nmol/L using Oligofectamine Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Then, 24 h after transfection, the expression of LINC00941 was assayed by qRT-PCR. A colorimetric cell proliferation assay employing 0.05% MTT (Sigma) was carried out as described previously [12 (link)]. For the proliferation assay with forced expression of LINC00941, full-length LINC00941 cDNA was cloned into the pcDNA6/myc-HisA vector (Invitrogen). The cloned vectors were verified by DNA sequencing. The cells were transfected with the cloned vector or the empty vector at 1 ng/μL using Lipofectamine3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Finally, 24 h after transfection, the expression of LINC00941 was assayed by qRT-PCR, and an MTT assay was carried out in the same way.

Mammalian Caco2 cells were routinely cultured in complete growth media consisting of minimal essential medium (MEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM l-glutamine, 200 µg/ml⁻¹ 0.1 mM nonessential amino acids, streptomycin, and 100 U·ml⁻¹ penicillin (37°C, 5% CO₂). Transient transfection of Caco2 cells by microporation was performed using a Neon transfection system according to the instructions of the manufacturer (Invitrogen). For RNA interference (RNAi) analysis, small interfering RNA (siRNA) from Qiagen and Dharmacon (Table 3) was transfected into Caco2 cells with Oligofectamine transfection reagent (Invitrogen) according to the instructions of the manufacturer. EFA6A and ARNO siRNA have been previously described (3 (link), 5 (link)). The transfection mixture was replaced after 24 h with complete growth medium, and cells were cultured for 72 h in total. RNA interference (RNAi) efficiency was determined by Express One-Step SYBR GreenER qRT-PCR according to the instructions of the manufacturer (Invitrogen) with actin used as a relative control in each case.

ATF2 ON-TARGET siRNA (ATF2 siRNA 1; MQ-009871-00) and control non-targeting siRNA (siControl Non-Targeting siRNA #1; D-001210-01) were purchased from Dharmacon (Lafayette, CO). ATF2 Silencer siRNA (ATF2 siRNA 2; AM16708A) was purchased from Ambion (Ambion, ThermoFisher Scientific, Burlington ON). For silencing of ATF2, both ATF2 siRNAs and control siRNA were used at concentrations of 5 or 50 nM. HUVECs were transfected at 80% confluence in Opti-MEM® I reduced serum medium using Oligofectamine Transfection Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol.

Double‐stranded siRNAs against human FOXO3a (NM_001455.3), Akt (NM_001014431.1), AMPK (NM_001355028.1), PUMA (NM_001127240.2) were synthesized by Bioneer (Daejeon, Republic of Korea). Negative control siRNA was obtained from Bioneer (Cat. No.: SN‐1003). The sequence for siRNA duplexes used in this study are as follows: AKT: forward, 5′‐ GAC AAC CGC CAU CCA GAC U‐3′; reverse, 5′‐ AGU CUG GAU GGC GGU UGU C‐3′; AMPK: forward, 5′‐ CUG AGU UGC AUA UAC UGU A‐3′; reverse, 5′‐ UAC AGU AUA UGC AAC UCA G‐3′; FOXO3a: forward, 5′‐ CUA UCA UAU GGC AUU CUU A‐3′; reverse, 5′‐ UAA GAA UGC CAU AUG AUA G‐3′; PUMA: forward, 5′‐ GUA GAU ACC GGA AUG AAU U‐3′; reverse, 5′‐ AAU UCA UUC CGG UAU CUA C‐3′. SCC15 cells were seeded at 1.5 × 10³ cells/well in 96‐well microplates or 1 × 10⁶ cells/well in 100 mm dishes. After overnight incubation, the cells were transfected with small interfering RNA (siRNA) of target genes or with control siRNA using Oligofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the supplier's protocol, followed by drug treatment. Gene silencing efficacy of siRNA was measured by Western blotting.