BMMSCs were seeded on twelve‐well culture dishes and grown to 80%–90% confluence followed by serum starvation for 2h. Cav1.2 (Santa Cruz Biotechnology, sc‐42689) or β‐catenin siRNA (Santa Cruz Biotechnology, sc‐29210) was transfected into BMMSCs at a final concentration of 50 nM, and overexpression plasmid of Cav1.2 (addgene, Plasmid #26572) was transfected into BMMSCs at 500 ng. For the control groups of siRNA or plasmid transfection experiments, scramble siRNA (RiboBio) or control overexpression vector pcDNA6/V5‐His (Invitrogen) was included. Lipo2000 (Invitrogen) was used as a transfection reagent according to the manufacturer's instructions. After transfection, the culture medium was substituted by normal culture medium and cells were harvested at 48 hr for RNA and 72 hr for protein extraction. For detection of the osteogenic differentiation capacity, transfection medium was removed in the next day and replaced by osteogenic induction medium.
Scramble sirna
Manufactured by RiboBio
Sourced in China
Scramble siRNA is a laboratory reagent used in RNA interference (RNAi) experiments. It serves as a negative control, with a sequence that does not target any known gene, to help researchers evaluate the effects of specific gene silencing in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Oxaliplatin, by RiboBio (2 mentions)
Oxaliplatin, by Merck Group (2 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
β catenin sirna, by Santa Cruz Biotechnology (1 mentions)
Pcdna6 v5 his, by Thermo Fisher Scientific (1 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
P ulk1, by Cell Signaling Technology (1 mentions)
Cytochrome c, by Proteintech (1 mentions)
Caspase 1, by Cell Signaling Technology (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
Nlrp3, by Cell Signaling Technology (1 mentions)
Trypsin, by Keygen Biotech (1 mentions)
Tnf α, by Proteintech (1 mentions)
26 protocols using scramble sirna
1
Modulating Osteogenic Differentiation of BMMSCs
2
Cellular Mechanisms of NLRP3 Inhibition
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The NLRP3 inhibitor MCC950, TNFα inhibitor Enbrel and aconitine were obtained from Shanghai Selleck Chemical Co., Ltd. (Shanghai, China) and Meilun Biotech. Co., Ltd. (Dalian, China). The primary antibodies against GAPDH, ULK1, TNFα, FADD, FasL, Cytochrome C, Caspase‐3 and ‐8, Bcl‐2, mTOR and LC3 were purchased from Proteintech Co., Ltd. (Wuhan, China). The primary antibodies against p62, p‐ULK1, IL‐1β, RIP1, RIP3, MLKL, Caspase‐1 and NLRP3 were purchased from Cell Signaling Tech. (Danvers, MA, USA). The foetal bovine serum and DMEM culture were obtained from GIBCO (NY, USA). DAPI and trypsin were purchased from Keygen Biotech (Nanjing, China). The H9c2 cell lines were transfected with siRNA by Lipofectamine 2000 (Invitrogen, USA) according to the manufacture's protocol. Scramble siRNA Ribobio Co., Ltd. (Guangzhou, China) and human BNIP3‐specific siRNA (Santa Cruz, Cat# sc‐37451) were transfected with a final concentration of 10 nmol/L to the transfection culture for 6 hours or cultured overnight. The other reagents were of analytical grade and used directly unless stated otherwise.
3
Characterization of lncRNA ZFAS1 and RALY in cancer cells
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ZFAS1 pcDNA3.1 vector (ZFAS1), RALY pcDNA3.1 vector (RALY) and empty vector (vector) were subcloned into the vector pcDNA3.1 (Invitrogen, Carlsbad, CA, United States). miR-193a-3p mimic, negative control oligonucleotides (mimic-NC), miR-193a-3p inhibitor, negative control oligonucleotide (NC inhibitor), small interfering RNA of ZFAS1 or RALY (si-ZFAS1, si-RALY) and scramble siRNA of ZFAS1 or RALY (siSCR) were purchased from RiboBio (Guangzhou, China). Cells were transfected using lipofectamine 2000 (Thermo Fisher, CA, United States) following to the manufacturer’s protocols. qRT-PCR was performed at 48–72 h later to determine the transfection efficiency. For further in vivo experiments, sh-ZFAS1 and sh-SCR were obtained from RiboBio (Guangzhou, China) and constructed into HB cell lines.
4
Silencing COL4A3 in Human NPC Cells
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The human 5-8F NPC cell line was donated by Sun Yat-Sen University Cancer Center and was cultured in RPMI-1640 medium (supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg-ml) (all from Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2 (Sanyo Eletcric Co., Ltd). Cells in the logarithmic growth phase were placed in a 6-well plate and incubated until 70-90% confluence.
Cells were transfected with specific short interfering (si)RNA targeting COL4A3 (designed and synthesized by Guangzhou Ribobio Co., Ltd.) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturers instructions. The following siRNA was used: 5′-GGGTAATCCTGGATTTCTA-3′. A scramble siRNA was also purchased from (Guangzhou Ribobio Co., Ltd) and used as a control siRNA (siNC). The cells were assigned to three experimental groups: Non-transfected group (control), siRNA negative control group (si-NC) and COL4A3 siRNA transfected group (si-COL4A3).
Cells were transfected with specific short interfering (si)RNA targeting COL4A3 (designed and synthesized by Guangzhou Ribobio Co., Ltd.) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturers instructions. The following siRNA was used: 5′-GGGTAATCCTGGATTTCTA-3′. A scramble siRNA was also purchased from (Guangzhou Ribobio Co., Ltd) and used as a control siRNA (siNC). The cells were assigned to three experimental groups: Non-transfected group (control), siRNA negative control group (si-NC) and COL4A3 siRNA transfected group (si-COL4A3).
5
In Vitro Ischemic Injury Model for Retinal Ganglion Cells
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We established an in vitro mimic of ischemic injury in RGCs according to our previously applied calcium ionophore/ATP depletion injury model (Zhang et al. 2021 (link); Ge et al. 2020 (link); Lee and Emala 2002 (link)). When RGCs were confluent over 90% of the plate, ischemia was simulated for 2 h by changing the medium to Hanks’ balanced salt solution (HBSS) with 10 mM antimycin A (a complex III inhibitor of mitochondrial electron transport; ab141904; Abcam, Cambridge, MA, US) and 2 mM calcium ionophore (A2318; Aladdin, Shanghai, China), which were dissolved in dimethylsulfoxide (DMSO). The complete growth medium was reapplied, and the cells were sustained for 0–4 h. For gene knockdown and overexpression experiments, 24 h before ischemic injury, when the density of the RGCs on the 24-well plate reached 80% to 90%, the cells were transfected lncRNA uc007nnj.1 siRNA/plasmid, miR-155-5p mimic or inhibitor, Tle4 siRNA, and scramble siRNA (Ribobio) using Lipofectamine 2000. After 6–8 h, the transfection solution was removed and replaced with a complete growth medium. Immunofluorescence staining of Tuj1 (1:500, GB11139; Servicebio, Wuhan, China) was used to identify the purity of RGCs.
6
siRNA Knockdown and Overexpression of H19
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The siRNAs (siRNA1 and siRNA2) for knockdown of H19 and the scramble siRNA for negative control were purchased from RiboBio (Guangzhou, People’s Republic of China). The constructed H19-overexpressing vectors (pcDNA3.1-H19) and pcDNA3.1 empty vectors were obtained from GenePharma (Shanghai, People’s Republic of China). For siRNA and vector transfection, cells were seeded into six-well plates at a density of 5 × 104 cells/well to reach about 50%–80% confluence for transfection. The transient transfection was performed by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. At 24 h after transfection, cells were processed for further experimentation.
7
Knockdown of Human Nrf2 Using siRNA
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The target small interfering RNA (siRNA) sequences directed against human Nrf2 were 5′-GAGAAAGAATTGCCTGTAA-3′ and 5′-TCCCGTTTGTAGATGACAA-3′. A scramble siRNA was purchased from RiboBio (Guangzhou, China) as control. Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. The final concentration of the siRNAs was 20 nmol/L.
8
SRPX2 Gene Silencing Protocol
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Three siRNAs targeting SRPX2 (named si-SRPX2_001, si-SRPX2_002, and si-SRPX2_003, respectively) and a nontargeting control siRNA (named scramble siRNA) were purchased from RiboBio (Guangdong, China). The specific SRPX2-targeted siRNA sequences were as follows: si-SRPX2_001: 5'-CAG ATG AAA GCT ACA ATG A-3', si-SRPX2_002: 5'-CAG ATG AAA GCT ACA ATG A-3', and si-SRPX2_3: 5'-GAG GAA ATC TTC ACA TTC A-3'. For transfection, Lipofectamine 3000 (Invitrogen, CA, USA) was used according to previously reported 18 (link).
9
Oxaliplatin-induced Neuropathic Pain Model
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Oxaliplatin was purchased from Sigma (USA) and dissolved in 5% glucose/H2O as a stock solution of 1 mg/ml as a previous study reported.14 (link) Rats were intraperitoneally administered Oxaliplatin at 4 mg/kg once per day for five consecutive days to induce mechanical allodynia. The control rats received an intraperitoneal injection of an equivalent volume of 5% glucose/H2O. Intrathecal injection of the neutralizing antibody against CXCL12 (8 µg, 10 μl; Torrey Pines Biolabs, Secaucus, NJ), neutralizing antibody against TNF-α (10 µg, 10 μl; R&D systems, Minneapolis, MN), a isotype IgG (8 µg, 10 μl; Sigma, Bellevue, WA), the IL-1 receptor antagonist (IL-1ra; 50 µg, 10 μl; R&D Systems, Minneapolis, MN), a STAT3 inhibitor S3I-201 (100 µg, 10 μl; Selleckchem, Houston, TX), scramble siRNA (1 nmol, 10 μl; Ribobio, China), or CXCL12 small interfering RNA (siRNA; 1 nmol, 10 μl; Ribobio, China) for consecutive 10 days was performed 30 min before Oxaliplatin administration. In addition, recombinant rat CXCL12 (2 µg, 10 μl; Signalway Antibody, College Park, USA) were intrathecally injected for consecutive 10 days.
10
Knockdown of Mouse Sart1 Gene
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Three siRNAs targeting mouse Sart1 and a nontargeting control siRNA (named si-Sart1_1, si-Sart1_2, si-Sart1_3 and scramble siRNA, respectively) were purchased from RiboBio (Guangdong, China). The specific Sart1-targeted siRNA sequences were as follows: si-Sart1_1: 5'-AGA CCA AAC GGA GAG TGA A-3', si-Sart1_2: 5'-CCC AGA AGA CAC CGT ATA T-3', and si-Sart1_3: 5'-GCG AAC ACC ATC ACC AAA T-3'. The expression plasmid (pCMV-Sart1 (GV657)) and NC plasmid were purchased from GeneChem (Shanghai, China). For transfection, Lipofectamine 3000 (Invitrogen, CA, USA) was used according to the manufacturer's instructions.
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