Total RNA was isolated from HaCaT cells, cholesteatoma, and the corresponding skin with RNAiso plus (TaKaRa Biotechnology Co. Ltd., Dalian, China). The integrity, purity, and concentration of total RNA were analysed by spectrophotometry and gel electrophoresis. Then, the RNA was polyadenylated using E. coli Poly (A) Polymerase (New England Biolabs, MA) and reverse-transcribed into cDNA with a PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa). Finally, quantitative real-time PCR was carried out using a SYBR®Premix Ex Taq™II (TaKaRa) and 7500 real-time PCR system (Applied Biosystems, USA). The relative expression of gene levels was calculated using the 2–ΔΔCT method. U6 served as an internal control normalised the expression data of miR-125b. The sequences of the primers: miR-125b-5p: 5’-TCCCTGAGACCCTAACTTGTGA-3’ miR-reverse: n5’-GCTGTCAACGATACGCTACG-3’; miR-RT primer: 5’-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTTTTTTT-3’; U6 forward: 5’-CTCGCTTCGGCAGCACA-3’ and reverse: 5’-AACGCTTCACGAATTTGCGT-3’; HaCaT cells were transiently transfected with miR-125b mimics, miR-125b inhibitor, control miRNA, STAT3 siRNA, and control siRNA (all from RiboBio Co. Ltd., Guangzhou, China), using Lipofectamine® 3000 reagent (Invitrogen, Carlsbad, CA, USA).
Stat3 sirna
Manufactured by RiboBio
Sourced in China
STAT3 siRNA is a laboratory reagent used for gene silencing. It is designed to target and suppress the expression of the STAT3 gene, which is a transcription factor involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Mirna inhibitor, by RiboBio (2 mentions)
Mirna mimic, by RiboBio (2 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Control sirna, by RiboBio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Control mirna, by RiboBio (1 mentions)
Mir 125b mimic, by RiboBio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
E coli poly a polymerase, by New England Biolabs (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Mir 125b inhibitor, by RiboBio (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Empty vector control, by Genechem (1 mentions)
Empty vector control, by OriGene (1 mentions)
Scrambled sirna, by RiboBio (1 mentions)
Inhibitor nc, by RiboBio (1 mentions)
Si nc, by RiboBio (1 mentions)
Mimics nc, by RiboBio (1 mentions)
9 protocols using stat3 sirna
1
miR-125b Expression Analysis in Skin Conditions
2
STAT3 Silencing in Cell Culture
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STAT3 siRNA (5′-AGUCAGGUUGCUGGUCAAA-3′) and negative control siRNA (siN05815122147) were purchased from Ribobio, Guangzhou, China [28 (link)]. The cells were plated at 50% confluence, transfected with 50 nM siRNA overnight in Opti-MEM containing Lipofectamine 2000 (Invitrogen), and incubated for various amounts of time.
3
Transfection of miRNA and siRNA
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The miRNA mimic, miRNA inhibitor, STAT3 siRNA, and scrambled siRNA were synthesized by RiBoBio (China). The oligonucleotide sequences were listed in Table S1 (Additional file 1 : Table S1). The miRNA overexpression vector pCMV-MIR519A (MI0003182) and the empty vector control were obtained from OriGene (Rockville, MD, USA). The miR-519a sponge and empty vector control were purchased from GeneChem (China). Transfections were performed using Lipofectamine 2000 reagent (cat. no. 11668-019; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
4
Transfecting miRNA, siRNA in Neurons
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miRNA mimics, miRNA inhibitor, STAT3 siRNA, and their corresponding negative controls (mimics-NC, inhibitor-NC, and si-NC) were synthesized by Ribobio (Guangzhou, China). Transfections in neurons were conducted using either the CP reagent (Ribobio, Guangzhou, China) or Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol.
5
Gene Silencing and Overexpression Techniques
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The PTRH1 siRNA, STAT3 siRNA, and NC siRNA were designed and synthesized by Ribobio (Guangzhou, China). The PTRH1-shRNA and PTRH1-overexpression plasmid were designed and synthesized by ABlife. The siRNA and shRNA sequences are shown in Additional file 14 : Table S1. Lipofectamine™2000 (Invitrogen, CA, USA) was used for cell transfection followed the manufacturer’s protocol. Nonsilencing (NC)‐shRNA lentivirus (puromycin resistance) and PTRH1‐overexpression lentivirus (puromycin resistance) were purchased from GeneChem (Shanghai, China). Stable cell lines were selected with 0.5 μg/ml puromycin (PTRH1 oe cells) for 2 days.
6
Regulatory Mechanisms of LINC00265 and STAT3
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LINC00265 siRNA and negative control, miR-4500 mimics and inhibitor, STAT3 siRNA and negative control were purchased from Ribobio (Guangzhou, China). LINC00265 or STAT3 wild-type (WT) and mutant-type (MT) plasmids and corresponding blank plasmids were prepared in our laboratory. Molt-3 and SUP-B15 cells were seeded in 12-wells plates and cultured for 12 h before transfection. For transient transfection, LINC00265 siRNA, miR-4500 mimics, STAT3 siRNA, and recombinant plasmid were transfected using Lipofectamine 2000 (Invitrogen). Samples were collected after 24 h for the quantification of target gene expression.
7
miR-29a and STAT3 Regulation in EC Cells
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Human EC cell line HHUC was purchased from the Cell Resource Center of Shanghai Institute of Biosciences, Chinese Academy of Sciences. EC cells were stored in DMEM with 10% fetal bovine serum at 37 ℃ and in 5% CO 2 . miR-29a mimics or inhibitors, STAT3 plasmids (overexpressed STAT3 and STAT3-OE) or shRNA (knockdown of STAT3 and STAT3-KD) were transfected into 6-well plates. Liposome 2000 (Thermo Fisher Science) with untreated HHUC were used as the negative control (NC). miR-29a mimics, miR-29a inhibitors, STAT3 plasmids (pcDNA3.1-STAT3) and STAT3-siRNA were all purchased from Ribobio (RiboBio Co.Ltd., China).
8
siRNA-mediated STAT3 Knockdown
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A negative control small interfering RNA (siRNA) and siRNA-stat3 were obtained from RIBo. Transfection was performed according to the manufacturer’s protocol. The target sequence used in this study was as follows: 5′-GCACAATCTACGAAGAATCAA-3′.
9
Modulation of Hepatocyte Proliferation
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Treatments of this part were conducted as our previously described [61 (link)]. Briefly, BRL-3A cells were obtained from cell bank of the School of Basic Medicine of Peking Union Medical College (Beijing, China). The cells had been kept in a high glucose DMEM complete medium, supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1%streptomycin in a concentration of 5% CO2 incubator at 37 °C. miR-125a mimics (Ribobio, Guangzhou, China), miR-125a inhibitor (Ribobio, China), or their negative controls handled BRL-3A for 48 h, respectively. To further investigate the role of STAT3 in hepatocyte proliferation, cells were transfected with siRNA-STAT3 (siRNA1, 2, 3, Ribobio, China) or negative control for 48 h, respectively.
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