N9 cell lines derived from GBM patients, were established by Professor Fan of Beijing Normal University. TBD0220 cell lines derived from GBM patients, were established by Professor Kang of Tianjin Medical University (20 (link), 21 (link)). N9 and TBD0220 cells were cultured in DMEM/F12 supplemented with 1% Penicillin-Streptomycin and 10% FBS. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. All GBM cells, except in vivo cultures, were maintained for less than eight generations. Lentiviruses encoding the PTRF-EGFP fusion protein (GOSL87854) and luciferase (GCNL84615) were purchased from GENECHEM (Shanghai, China). Small interfering RNAs targeting PTRF were synthesized by GenePharma (Shanghai, China). Sequences of siRNAs were as follows: PTRF siRNA#1 5’-GCCGCAACUUUAAAGUCAUGAUCUA; PTRF siRNA#2 5’-AGGAGUCCCGCGCAGAGCGUAUCAA. Small interfering RNAs targeting NEAT1 were synthesized by GenePharma (Shanghai, China). Sequences of siRNAs were as follows: NEAT1 siRNA 5’- GAACUUUACUUCGUUAGAUTT. We treated cells with Actinomycin D (Selleck, S7418) at a final concentration of 1 μM.
Small interfering rna
Manufactured by GenePharma
Sourced in China
Small interfering RNAs (siRNAs) are double-stranded RNA molecules that play a crucial role in the RNA interference (RNAi) pathway, a natural mechanism for gene silencing. siRNAs are designed to target and degrade specific mRNA molecules, thereby reducing the expression of the corresponding genes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (19 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (10 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (6 mentions)
Luciferase reporter assay system, by Promega (4 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Mirna mimic, by GenePharma (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Plvx puro lentivirus, by Obio Technology (2 mentions)
Circrna1615 overexpression lentivirus, by Obio Technology (2 mentions)
Quikchange kit, by Thermo Fisher Scientific (2 mentions)
Lentivirus, by Genechem (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sirna, by GenePharma (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Jetprime transfection reagent, by Polyplus Transfection (2 mentions)
Lipofectamine 3000, by Polyplus Transfection (1 mentions)
Interferin reagent, by Polyplus Transfection (1 mentions)
Mutanbest kit, by Takara Bio (1 mentions)
70 protocols using small interfering rna
1
GBM Cell Lines and Molecular Tools
2
LINC00240 and DDX21 Plasmid Construction
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The human full-length LINC00240 (NR_026775.2) cDNA and truncated versions of LINC00240 cDNA were directly synthesized and cloned after the CMV promoter of pCDH-CMV-MCS-EF1-Puro. The full-length LINC00240 cDNA plasmid was named as LINC00240. After one HA-tag sequence was inserted after ATG of the CDS region of DDX21 (NM_004728.4), the cDNA was cloned into the pcDNA3.1 vector to generate the HA-tagged DDX21 plasmid (WT). Five truncated DDX21 plasmids (ΔDEADc, Δ1-398, Δ617-783, ΔGUCT, and ΔAdoMet) were mutants of the HA-tagged DDX21 plasmid with CDS region after deletion of the DEADc domain, 1-398aa, 329-1283aa, the GUCT domain, or the AdoMet domain, separately. Two shRNA hairpins targeting human LINC00240 (sh240-1 or sh240-2) or the control shRNA (Supplementary Table 2 ) were cloned into the pLKO.1 vector. The resultant plasmids were designated sh240-1, sh240-2, or shNC. All the plasmids were synthesized by Genewiz (Suzhou, China) and sequenced to confirm the orientation and integrity. Transient transfection of plasmid constructs or small interfering RNAs (GenePharma, Shanghai, China) (Supplementary Table 2 ) was performed with Lipofectamine 3000 or the INTERFERin reagent (Polyplus, 409–10).
3
Plasmid Constructs and siRNA for Parkin, OPTN, and TFEB Studies
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The FLAG, FLAG-Parkin, HA-Ub, GFP-OPTN, mCherry-C1 and GFP-TFEB plasmids were described before [22 (link)–27 (link)]. mCherry-Parkin was generously provided by Richard Youle (Addgene #23956); BFP-mito was generously provided by Gia Voeltz (Addgene #49151). FLAG-tagged and mCherry-tagged A46T, N254S and R275W Parkin mutants were generated by site-directed mutagenesis with a MutanBEST kit (Takara, Shiga, Japan). pcDNA3.1-mt-Keima was generated by inserting mKeima cDNA into the vector at the KpnI and EcoRI sites. All constructs were verified by sequencing. Small interfering RNAs targeting human OPTN and PINK1 were synthesized by Shanghai GenePharma.
4
siRNA Silencing of COL10A1 Gene
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Small interfering RNAs were purchased from Shanghai Gene Pharma company. Cells were transfected with the appropriate small interfering RNA using the Lipofectamine 3000 Kit (Thermo Fisher Scientific, USA) following the instructions of the manufacturer. The medium was renewed after 8 hours of incubation. The siRNA sequences were shown below: siCOL10A1, 5′-GCAUGUGAAAGGGACUCAUTT-3′ sense and 5′-AUGAGUCCCUUUCACAUGCTT-3′ antisense; negative control (NC), 5′-UUCUCCGAACGUGUCACGUTT-3′ sense and 5′-ACGUGACACGUUCGGAGAATT-3′ antisense.
5
Cytotoxicity assay of CX-5461 and APR-246
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CX-5461 and APR-246 were purchased from Selleck Chemicals LCC. Both drugs were prepared in organic solvent, DMSO, and stored at −80 °C. Small interfering RNAs (siRNAs) were obtained from Shanghai GenePharma (Shanghai, China). Lipofectamine RNAiMAX was purchased from Life Technologies (Waltham, MA, USA). The CellTiter 96® AQueous One Solution Cell Proliferation Assay kit was purchased from Promega Corporation (Madison, WI, USA). The antibodies used in this study are listed in Table S1 .
6
Synthesis of Small Interfering RNAs
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Small interfering RNAs (siRNAs) were synthesized by GenePharma Company (Shanghai, China). The sequences of siRNA and primers were listed in Additional file 1 : Table S2.
7
Modulating miR-567 and ATG5 Expression
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miRNA mimics, inhibitors, and negative controls (NC or NC inhibitors) were purchased from GenePharma (Shanghai, China). To overexpress or knock down the expression level of miR-567 or ATG5, gene-specific overexpression plasmids (FulenGen, Guangzhou, China) or small interfering RNAs (GenePharma) were transfected into cells. Lipofectamine 3000 (Invitrogen, CAT. L3000008, Carlsbad, CA, USA) was used for transient transfection with a final concentration of 100 nM for in vitro assays. miRNA mimics, inhibitors, and NCs were synthesized and loaded into lentivirus, which were purchased from Shanghai Genechem Co., Ltd, for in vivo assays. The sequences of silencing sequences were shown in Supplementary Table S2 .
8
Silencing Piezo1 and SelK in Rat NP Cells
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Small interfering RNAs were constructed by GenePharma and used to inhibit the expression of Piezo1 and SelK (Table 3 ). Rat NP cells were cultured in six-well plates to 60–70% confluence and were transfected with 50 nM negative control (NC), Piezo1 or SelK siRNA using Lipofectamine 2000 (ThermoFisher) according to the manufacturer’s instructions. After 48 h, cellular lysates were obtained to analyze the expression of the genes of interest.
Sequences of SiSelKs
| SiSelK | Forward Primers, 5ʹ–3ʹ | Reverse Primers, 5ʹ–3ʹ |
|---|---|---|
| Si-1 | CUCGAAUGGUCAGGUGUUATT | UAACACCUGACCAUUCGAGTT |
| Si-2 | GAAUAGCAGAAUUUGUGGUTT | ACCACAAAUUCUGCUAUUCTT |
| Si-3 | GAUGUGCAGUUCUAUAAAUTT | AUUUAUAGAACUGCACAUCTT |
9
Silencing Piezo1 and SelK in Rat NP Cells
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Small interfering RNAs were constructed by GenePharma and used to inhibit the expression of Piezo1 and SelK (Table 3 ). Rat NP cells were cultured in six-well plates to 60–70% confluence and were transfected with 50 nM negative control (NC), Piezo1 or SelK siRNA using Lipofectamine 2000 (ThermoFisher) according to the manufacturer’s instructions. After 48 h, cellular lysates were obtained to analyze the expression of the genes of interest.
Sequences of SiSelKs
| SiSelK | Forward Primers, 5ʹ–3ʹ | Reverse Primers, 5ʹ–3ʹ |
|---|---|---|
| Si-1 | CUCGAAUGGUCAGGUGUUATT | UAACACCUGACCAUUCGAGTT |
| Si-2 | GAAUAGCAGAAUUUGUGGUTT | ACCACAAAUUCUGCUAUUCTT |
| Si-3 | GAUGUGCAGUUCUAUAAAUTT | AUUUAUAGAACUGCACAUCTT |
10
Overexpression and Knockdown Lentiviruses
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The control pLVX-Puro lentivirus, PIGT overexpression lentivirus and WTAP overexpression lentivirus were obtained from Generay Biotech (Shanghai) Co., Ltd, China. The control pLKO.1 shRNA lentivirus, PIGT shRNA lentivirus and GLUT1 shRNA lentivirus were obtained from Obio Technology Company (Shanghai, China). Small interfering RNAs (Shanghai GenePharma Co., Ltd, China) were transfected using lipo2000 (11,668,500; Invitrogen, Thermo Fisher Scientific). Empty vector and scramble siRNA were used as negative control. All the sequences of shRNA and siRNAs were listed in Additional file 1 : Table S2.
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