Hl-1 cells were grown on cover slips pre-coated with 12.5 µg/ml bovine fibronectin in 0.02% gelatin solution. Transfection was performed using siPORT Amine (Applied Biosystems) according to manufacturer's protocol. 20 nM of miR-29 inhibitor cocktail (mirVana miRNA inhibitors for miR-29a, b and c) or 20 nM Allstars negative siRNA (Qiagen) was used for transfection. After 8 hours, cells were subjected to Rap treatment (10 nM) overnight. Coverslips were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 1% Triton-X, washed with PBS-T (1 mL Tween-20/L), and blocked with background sniper (Biocare Medical). Anti-MCL-1 antibody (Cell Signaling Technology) (1∶50 dilution) was added in fluorescent AB diluent (Biocare Medical) and incubated overnight at 4°C. After repeated washing with PBS-T, coverslips were incubated with Alexa Fluor 488 goat anti-rabbit (Invitrogen Inc.) (1∶200 dilution) for 1 hr at room temperature. After washing with PBS-T, coverslips were mounted on slides with Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and visualized using an Olympus IX51 microscope with a UC50 digital camera using cellSense software (Olympus, Center Valley, PA) at equal exposure times. Imaging was done at 60× magnification using oil immersion.
Allstars negative sirna
Manufactured by Qiagen
Sourced in Germany
AllStars Negative siRNA is a non-targeting control siRNA designed to be used as a negative control in RNA interference (RNAi) experiments. It does not target any known mammalian gene.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions)
Hiperfect transfection reagent, by Qiagen (2 mentions)
Succinimidyl ester with alexa fluor 532 a532, by Thermo Fisher Scientific (2 mentions)
Spio nps, by Thermo Fisher Scientific (2 mentions)
Maphosphamide, by Santa Cruz Biotechnology (2 mentions)
Clinical grade cisplatin, by Santa Cruz Biotechnology (2 mentions)
Ix51 microscope, by Olympus (1 mentions)
Anti mcl 1 antibody, by Cell Signaling Technology (1 mentions)
Siport amine, by Thermo Fisher Scientific (1 mentions)
Fluoroshield with 4 6 diamidino 2 phenylindole dapi, by Merck Group (1 mentions)
Background sniper, by Biocare Medical (1 mentions)
Cellsense software, by Olympus (1 mentions)
Miscript mirna mimic, by Qiagen (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Glomax 96, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
D glucose, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
11 protocols using allstars negative sirna
1
Immunofluorescence Assay for MCL-1 in Rap-treated Cells
2
Fluorescent Nanoparticles for Targeted Cancer Therapy
3
N2a Cell Transfection Protocol
4
Nanoparticle-Mediated Delivery of siRNA and Chemotherapeutics
5
Nanoparticle-Mediated Delivery of siRNA and Chemotherapeutics
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siRNA molecules and NPs were purchased as described in our previous study (10 (link)). The MXD3 siRNA sense strand sequence was 5'-AUGGACUAAAAGGACCCUUTT-3', and the antisense strand was 5'-AAGGUCCUUUAGUCCAUTT-3'. AllStars Negative siRNA (Qiagen, Hilden, Germany) was used as a negative control. Both MXD3 and control siRNAs were tagged with A488 on the 3' end of the antisense strand (Qiagen). SPIO NPs were purchased from OceanNanotech (San Diego, CA). The SPIO NPs had iron oxide cores measuring 15nm, and outer layers composed of amphiphilic polymer and polyethylenimine groups (PEI). Succinimidyl ester with Alexa Fluor 532 (A532) (Thermo Fisher Scientific) was used to label the SPIO NPs. Clinical-grade doxorubicin and vincristine were provided by the UC Davis Pharmacy. Maphosphamide and clinical-grade cisplatin were purchased from Santa Cruz Biotechnology (Dallas, TX) and the UC Davis Pharmacy, respectively.
6
Silencing Macrophage Gene Expression
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Concerning siRNA, we transfected macrophages with different siRNAs using the HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) in an optimum serum-free medium (Sigma-Aldrich, Taufkirchen, Germany). β-Catenin siRNA, FOSL2 siRNA, and AllStars negative siRNA as nonsilencing control (si_NS) were obtained from Qiagen (Qiagen, Hilden, Germany). According to the protocol provided by the manufacturer, we transfected the cells with siRNA for 6 hours in the serum-free medium. After 6 hours, we cultured the cells in a serum-containing macrophage medium for 24 hours.
Concerning shRNA, we transfected macrophages with different shRNAs using the jetPEI-Macrophage kit (Polyplus Transfection, Illkirch-Graffenstaden, France). We obtained β-catenin shRNA, EG5 positive control shRNA, and nonsilencing shRNA (sh_NS) from GE Dharmacon (Lafayette, CO, USA). According to the protocol provided by the manufacturer, we mixed shRNA (1.5 μg) and a transfection reagent (3 μl) for one well of a six-well plate and incubated for 30 min at room temperature (RT) to allow the formation of complexes. Subsequently, we added a mixture dropwise on the macrophages in a serum-containing medium, and we incubated the mixture for 24 hours at 37°C to allow transfection of cells.
Concerning shRNA, we transfected macrophages with different shRNAs using the jetPEI-Macrophage kit (Polyplus Transfection, Illkirch-Graffenstaden, France). We obtained β-catenin shRNA, EG5 positive control shRNA, and nonsilencing shRNA (sh_NS) from GE Dharmacon (Lafayette, CO, USA). According to the protocol provided by the manufacturer, we mixed shRNA (1.5 μg) and a transfection reagent (3 μl) for one well of a six-well plate and incubated for 30 min at room temperature (RT) to allow the formation of complexes. Subsequently, we added a mixture dropwise on the macrophages in a serum-containing medium, and we incubated the mixture for 24 hours at 37°C to allow transfection of cells.
7
Validation of miR-29b Targets in Mouse
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For validation of Tet1, 2, 3 and Dnmt3a targeting by miR-29b, segments of their 3′UTR including miR-29b seed sequences were amplified from mouse genomic DNA and cloned into pmirGLO Dual-Luciferase miRNA target expression vector (Promega). For miRNA seed mutagenesis of Tet3 3′UTR, mutations were predicted by ImiRP47 (link), a mutation generator program that enables selective disruption of miRNA target sites while ensuring predicted target sites for other miRNAs are not created. Tet3 3′UTR containing mutated seed sequences was synthesized by IDT and cloned into pmirGLO.
N2a cells were co-transfected with miR-29b-3p mimic (5′-UAGCACCA UUUGAAAUCAGUGUU-3′ from Qiagen), seed sequence mutated miR-29b-3p mimic (5′-UCAGCAAC UUUGAAAUCAGUGUU-3′ from Qiagen) or negative control (All Stars Negative siRNA, Qiagen) and 250 ng of pmirGLO with Lipofectamine® 2000 (Life Technologies) for 24 h. Cell extracts were prepared 24 h post-transfection, and luciferase activities of firefly and renilla were measured using a Dual-Luciferase Reporter Assay system (Promega) and a luminometer GloMax 96 (Promega). Firefly luciferase signals were normalized to Renilla luciferase signals, which serve as internal normalization control. Values were further normalized by that of an empty pmirGLO vector. The primer sequences used for cloning are shown in Supplementary Table S4 .
N2a cells were co-transfected with miR-29b-3p mimic (5′-U
8
Circadian Rhythm Disruption Assay
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Human U2OS cells stably expressing the pBmal-dLuc construct were reverse transfected with a total of 9.6 nM pooled siRNAs (n = 4/gene) using Lipofectamine/RNAiMAX (Invitrogen) transfection reagent and Opti-MEM media (Gibco), totaling 6 pmol/siRNA for each gene. All siRNAs were purchased from Qiagen. Allstars Negative siRNA (Qiagen) and AllStars Cell Death siRNA (Qiagen) were used as experimental controls. Transfected cells were seeded in triplicate at a density of 200,000 cells/mL in DMEM containing 10% FBS (Gibco), 0.1 mM nonessential amino acids (NEAA, Invitrogen), 1% L-Glutamine (Invitrogen) without antibiotics into 96-well plates and incubated for 24 h. siRNA/transfection medium was removed 24 h post-transfection and replaced with 250 µl of luminescence recording medium: phenol red-free DMEM (Sigma D-2902), sodium bicarbonate (Invitrogen), D-(+)-glucose (Sigma), 10 mM HEPES (Invitrogen), 1% pen/strep/L-Glutamine (Invitrogen), and 0.1 mM luciferin (Promega). The medium is supplemented with 0.1 µM dexamethasone (Sigma-Aldrich) to synchronize the cells to the same circadian phase. The Synergy 2 plate reader (BioTek) was used to measure luminescence every 1 h for 6 days. Period length was determined using the WaveClock algorithm implemented in R.
9
siRNA Knockdown of Mechanosensitive Targets
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HUAECs at 70% confluence were transfected with control siRNA (AllStars Negative siRNA, Qiagen, 1027280) and siRNAs against Piezo1, Gαq, Gα11, P2Y2, ITGA5, and ITGAV (all from Qiagen) by using Opti-MEM (Thermo Fisher) and Lipofectamine RNAiMAX (Invitrogen) for 6 h on two consecutive days as described (Wang et al., 2015 (link)). The targeted sequences of human siRNAs directed against RNAs encoding Piezo1, Gαq, Gα11, P2Y2, ITGA5, and ITGAV were Piezo1: 5′-CCAAGTACTGGATCTATGT-3′, 5′-GCAAGTTCGTGCGCGGATT-3′, and 5′-AGAAGAAGATCGTCAAGTA-3′; GNAQ: 5′-CAGGACACATCGTTCGATTTA-3′ and 5′-CAGGAATGCTATGATAGACGA-3′; GNA11: 5′-AGCGACAAGATCATCTACTCA-3′ and 5′-AACGTGACATCCATCATGTTT-3′; P2RY2: 5′-CCCTTCAGCACGGTGCTCT-3′, 5′-CTGCCTAGGGCCAAGCGCA-3′, and 5′-GCTCGTACGCTTTGCCCGA-3′; ITGA5: 5′-AATCCTTAATGGCTCAGACAT-3′ and 5′-CAGGGTCTACGTCTACCTGCA-3′; and ITGAV: 5′-GAGTGCAATCTTGTACGTAAA-3′ and 5′-CAGGCAATAGAGATTATGCCA-3′.
10
RNA Interference and Viral Infection in Cell Lines
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HeLa, HCT116 and A549 cell lines were seeded at a density of 4 × 104 cells/well in a 24-well plate and grown in 500 µl of complete cellular medium without antibiotics. After 24 h, cells were transfected with siRNAs (5–10 nM) in a serum-free medium using Lipofectamine RNAimax (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following siRNAs were used: LAMC1_1 (SI00035742), LAMC1_5 (SI02757475), LAMB1_4 (SI00035707) and LAMB1_9 (SI05109174), with AllStars Negative siRNA (SI03650318) used as negative control (all purchased from Qiagen). After 24 h, the medium was changed, and cells were grown for an additional 24 h to allow efficient gene silencing. The culture medium was then removed and replaced with 0.2 ml serum-free medium containing H-1PV at MOI 1 pfu/cell. Infection was performed for 4 h at 37°C to allow cell surface binding and internalisation of viral particles. H-1PV cell uptake was performed as described above.
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