Block it alexa fluor red fluorescent oligo

Gain- and loss-of-function studies were done in (day 0) D0 of differentiation. The miRCURY LNA™ microRNA mimic (Exiqon, Denmark) for miR-335-3p (MIMAT0004703), miR-335-5p (MIMAT0000765), and mimic control as well as miRIDIAN microRNA miR-335-3p and miR-335-5p hairpin inhibitors and miRIDIAN microRNA hairpin inhibitor control (Dharmacon) were used for gain- and loss-of-function studies in which 8 × 10⁵ cells were plated in each 3.5-cm tissue culture dish, 24 h before transfection. When cells reached 80% confluence, they were transfected by 50 nM siRNA or 5 nM mimic structures using Lipofectamine® 3000 reagent, based on the manufacturer’s instructions. The efficiency of siRNAs and microRNA mimics transfection was evaluated using BLOCK-iT Alexa Fluor Red fluorescent oligo (Invitrogen).

For siRNA transfection of MGE-neurons reverse lipofection with Lipofectamine RNAiMAX (Invitrogen) was used according to the manufacturer’s protocol. MGE-derived neurons growing on alternating stripes of EphB1-Fc and control protein were transfected with 10 nM mouse ephrin-B3 siRNA, containing a pool of 3 target-specific 20–25 nt siRNAs to knock down gene expression (Santa Cruz), in combination with 10 nM Alexa555-labeled RNA dublex (BLOCK-iT Alexa Fluor red fluorescent oligo; Invitrogen) to enable the visualization of the transfected interneurons. Transfection occurred for 5 h in antibiotics-free cell culture medium at 37°C and 5% CO₂ in a humid atmosphere. Then the medium was substituted by culture medium containing 10,000 U/ml penicillin and 10,000 μg/ml streptomycin. Transfected neurons were incubated for 2 DIV at 37°C and 5% CO₂.

iPSC-derived neurons were transfected with LipoStem (Thermofisher Scientific) on day 13 or 14 of differentiation according to the manufacturer’s protocol. Half of the culture medium was removed and stored before transfection. Transfection of cells was conducted using 15 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 14750100; Invitrogen (Carlsbad, CA, USA)) or 30 nM DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology). Then, 24 h after transfection, the medium was changed to previously collected conditioned medium, and cells were transferred to a live-imaging chamber (ALA Scientific Instruments, Farmingdale, NY, USA) to perform live-cell calcium imaging. To roughly estimate the transfection efficiency, cotransfection of cells was conducted using 15 nM control siRNA and 30 nM DNMT1 siRNA as above, and transfected cells were manually counted based on the presence of red fluorescence, yielding a transfection efficiency of 39.1 ± 2.3% 24 h after transfection.

To transfect the Stealth RNAi siRNA against DR3 into Bel-7402 and SMMC-7721, Lipofectamine™ RNAiMAX (Invitrogen Life Technologies) was used, and the negative control, the Stealth™ RNAi Negative Control Duplexes (Invitrogen Life Technologies) were used. BLOCK-It™ Alexa Fluor^® Red Fluorescent Oligo (Invitrogen Inc, USA) was used to facilitate assessment and optimize the delivery of double-stranded RNA oligonucleotides into Bel-7402 and SMMC-7721 cells, according to the manufacturer’s instructions. Reverse transfection was used to deliver Stealth RNAi siRNA, Red Fluorescent Oligo or negative control duplexes into two cell lines as follows: Lipofectamine™ RNAiMAX complexes were prepared according to the manufacturer’s instructions, cells were seeded at appropriate dilutions and incubated for 24 h to reach 30–50% confluence. The complexes were added to the cells (5×10⁵) and incubated for 48 and 72 h at 37°C in a CO₂ incubator until ready for gene knockdown assays.

Glioma cells were transfected with siRNA targeting Pyk2 (Qiagen Inc., Valencia, CA, USA, cat. #SI02649031, #100159039) and FAK (Qiagen Inc., cat. #S100301532, #103103156) using Lipofectamine RNAiMAX reagent (Invitrogen, cat. #13778-150) according to the manufacturer’s instructions. Cells (5.0 × 10⁴) were plated in 6-well plates and transfected with 20 nM Pyk2 siRNA or 20 nM FAK siRNA using 3 µL of Lipofectamine RNAiMAX in serum-free medium. Stealth RNAi negative control duplex (Invitrogen, cat. #12935) was used as control. The efficacy of transfection was controlled by Block-iT Alexa Fluor red Fluorescent Oligo (Invitrogen, cat. #14750) and by RT-PCR. Forty-eight hours after transfection, the cells were used for cell cycle, migration, and invadopodia assays.

Lipofectamine RNAiMAX (Invitrogen Life Technologies) was used to transfect the Stealth RNAi siRNA against Oct4 into CCE cells. The Stealth RNAi Negative Control Duplexes (Invitrogen Life Technologies) were used as a negative control. BLOCK-It Alexa Fluor Red Fluorescent Oligo (Invitrogen) was used to facilitate the assessment and optimize the delivery of double-stranded RNA oligonucleotides into the CCE cells. These siRNA sequences were submitted to a BLAST search to ensure that only the mouse Oct4 gene was targeted. The sequences of the three synthesized oligonucleotides were:

R1 sense 5’-CCAAUGCCGUGAAGUUGGAGAAGGU-3′ and anti-sense 5’-ACCUUCUCCAACUUCACGGCAUUGG-3′;

R2 sense, 5’-CCCGGAAGAGAAAGCGAACUAGCAU-3′ and anti-sense, 5’-AUGCUAGUUCGCUUUCUCUUCCGGG-3′;

R3 sense, 5’-CCAAUCAGCUUGGGCUAGAGAAGGA-3′ and anti-sense, 5’-UCCUUCUCUUAGCCCAAGCUGAUUGG-3′.

RNAi transfection was conducted according to the manufacturer’s instructions. Gene knockdown assays were performed after the complexes were added to the cells and incubated for 48 h at 37 °C in a CO₂ incubator.

The fluorescently labeled control siRNA (BLOCK-iT Alexa Fluor Red Fluorescent Oligo) was purchased from Invitrogen. The siRNAs targeting heat shock protein 90 (Hsp90) genes with Stealth modification were designed using BLOCK-iT RNAi Designer and purchased from Invitrogen. The Stealth modification is the chemical modification to siRNA, which enhances the potency and stability of siRNA and reduces the occurrence of off-target effect. The target sequence of each siRNA is listed in Table 1. Stealth RNAi GFP reporter control (Invitrogen), which targets enhanced green fluorescent protein, was used as the negative control siRNA (siRGFP). All the siRNAs were dissolved at the concentration of 20 µM in RNase-free distilled water and stored at −20°C until use.