miR-608 mimics (miR-608), miR-608 inhibitors (miR-608 inh) or their corresponding controls (miR-NC or NC inh) were obtained from genepharma (shanghai, China). To insert the 3ʹ-UTR of Notch1 and Notch2 into the pmiR-Glo dual-Luciferase reporter plasmid (Promega, Madison, WI, USA), a Cloning Kit (Vazyme Biotech, Nanjing, China) was used. The binding-site (CCACCCC) was mutated (GGUGGGG) and used as a control. According to the above methods, Notch1 and Notch2 expression plasmids containing their coding sequences (CDSs) were constructed. For cell transfection, a Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) was used. A GFP plasmid was used to determine the transfection efficiency, which is about 80%. The primers for plasmid construct were presented in Table S3
Inh nc
Manufactured by GenePharma
Sourced in China
The Inh-NC is a laboratory equipment product manufactured by GenePharma. It serves as a device for nucleic acid extraction and purification. The core function of the Inh-NC is to isolate and concentrate nucleic acid samples from various biological sources.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Mir nc, by GenePharma (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Mimic nc, by GenePharma (2 mentions)
Mim nc, by GenePharma (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Mir 608, by GenePharma (1 mentions)
Pmirglo, by Promega (1 mentions)
P3xflag cmv 10 vector, by Merck Group (1 mentions)
Hfob1, by American Type Culture Collection (1 mentions)
Hec 1b, by American Type Culture Collection (1 mentions)
Hec 1b, by Thermo Fisher Scientific (1 mentions)
Si nc, by GenePharma (1 mentions)
Rl95 2, by American Type Culture Collection (1 mentions)
Ishikawa, by American Type Culture Collection (1 mentions)
Mir 34b mimic, by GenePharma (1 mentions)
Short hairpin rna, by GenePharma (1 mentions)
12 protocols using inh nc
1
Notch Signaling Regulation by miR-608
2
miR-184 Modulates PDLSC Osteogenesis
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Isolated PDLSCs were seeded and transfected with 25 nM miR-184 mimics or negative control mimics (NC mimic), miR-184 inhibitor or NC inh (GenePharma, Shanghai, China). Two days after transfection, cells were harvested for functional assays.
For determination of effect of miR-184 on osteogenic differentiation of PDLSCs, lentiviral plasmid was designed for over-expression of miR-184. The lentiviral plasmid (LV-miR-184: pGC-LV-miR-184-GFP), as well as the negative control (LV-NC: pGC-LV-NC mimic-GFP), were constructed by Genechem (Shanghai, China). The PDLSCs were seeded and then transfected with the lentiviral plasmids (multiplicity of infection of 20). For the over-expression of NFI-C, full length NFI-C was constructed into p3xFLAG-CMV10 vector (Sigma Aldrich). The PDLSCs were seeded and then transfected with vectors (30 nM) or cotransfected with vectors and the lentiviral plasmids. Cells then underwent osteogenic differentiation mentioned before for 15 days.
For determination of effect of miR-184 on osteogenic differentiation of PDLSCs, lentiviral plasmid was designed for over-expression of miR-184. The lentiviral plasmid (LV-miR-184: pGC-LV-miR-184-GFP), as well as the negative control (LV-NC: pGC-LV-NC mimic-GFP), were constructed by Genechem (Shanghai, China). The PDLSCs were seeded and then transfected with the lentiviral plasmids (multiplicity of infection of 20). For the over-expression of NFI-C, full length NFI-C was constructed into p3xFLAG-CMV10 vector (Sigma Aldrich). The PDLSCs were seeded and then transfected with vectors (30 nM) or cotransfected with vectors and the lentiviral plasmids. Cells then underwent osteogenic differentiation mentioned before for 15 days.
3
Regulation of Osteoblast Differentiation by circRNA
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Human OS cell lines and osteoblast cell line HFOB1.19, available from the American Type Culture Collection (ATCC, Rockville, MD, USA) and the China Center for Type Culture Collection (CCTCC, Wuhan, China), respectively, were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, as well as 100 μg/mL streptomycin (Hyclone, Logan, UT, USA) at 37° C in 5% CO2. Besides, short hairpin RNA (shRNA) targeting circ_0081001 (sh-circ_0081001), its corresponding negative control (sh-NC), circ_0081001 overexpression plasmid (circ_0081001), and empty vector (negative control, NC) were designed by GeneChem (Shanghai, China). MiRNA-494-3p mimics (miRNA-494-3p mim), miRNA-494-3p inhibitors (miRNA-494-3p inh), and the corresponding negative controls (mim-NC, inh-NC) were available by GenePharma (Shanghai, China). Moreover, Lipofectamine® 2000 reagent was adopted to transfect the cells. The transfection efficiency, after 36 h, was measured employing quantitative real-time polymerase chain reaction (qRT-PCR).
4
Investigating ZFAS1 in Endometrial Cancer
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Immortalized stromal cell line (hEM15A) and endometrial carcinoma cell lines (HEC-1B, Ishikawa, KLE, and RL-952) were purchased from ATCC (Manassas, VA, USA). DMEM medium (HyClone, Logan, UT, USA) containing 10% of fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) was used to culture the cells at 37°C in a humidified incubator. The siRNAs targeting ZFAS1 (si-ZFAS1#1 or #2), si- vascular endothelial growth factor A (VEGFA), pcDNA-ZFAS1, miR-34b mimic, and inhibitor, as well as the negative controls (si-NC, pc-DNA, NC-mimic, and inh NC), were synthesized by GenePharma (Suzhou, China). HEC-1B was transfected with siRNAs, pc-DNAs, mimic, or inhibitor via Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
5
Transfection Methods for 3T3-L1 and AML12 Cells
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For transient transfection, 3T3-L1 adipocytes and AML12 cells were transfected with miR-1249-3p mimic, miR-NC, inhibitor, inh-NC, or siRNAs of SKOR1, SMAD6 (Genepharma, Shanghai, China) respectively, and their sequences are listed in Supplementary Table S3 . For SKOR1 knockdown, 3T3-L1 adipocytes and AML12 cells were transfected with short hairpin RNAs (Genepharma, Shanghai, China) targeting SKOR1 and negative control RNA duplex (Genepharma, Shanghai, China) using lipofectamine 3000 (Invitrogen, California, USA) according to the manufacturer’s instructions. For SKOR1 overexpression, Flag-SKOR1 (Genepharma, Shanghai, China) were transfected into 3T3-L1 adipocytes and AML12 cells using lipofectamine 3000 (Invitrogen, California, USA) according to the manufacturer’s instructions.
6
siRNA and miRNA Modulation in HT22 Cells
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The small interfering RNA (siRNA) targeting MALAT1 (si-MALAT1), negative control scrambled siRNA (si-NC), miR-195a-5p mimics, mimics control (mimics-NC), miR-195a-5p inhibitor (inh-miR-195a-5p), negative inhibitor (inh-NC), pcDNA3.1 targeting HMGA1 (pcDNA3.1-HMAGA1), pcDNA3.1 empty vector (pcDNA3.1-NC) were all purchased from GenePharma (Suzhou, China). The transfection of these sequences into HT22 cells was conducted by Lipofectamine 3000 reagent (Invitrogen) following the manufacturer’s instructions. After transfection, the original medium was replaced with fresh medium and cells were incubated for 48 hours for further analysis.
7
Cholangiocarcinoma Cell Line Manipulation
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Cholangiocarcinoma cell lines (CCLP‐1, RBE, QBC939 and HuCCT1) and normal control cell line HIBEC were preserved in the laboratory as previously described.28 Cells were cultured in 10% foetal bovine serum (FBS) loaded RPMI‐1640 medium (Invitrogen Life Technologies) and 100 μg/mL penicillin/streptomycin supplemented at 37°C with 5% CO2 in a humidified incubator. Small interfering RNAs targeting ZFAS1 (si‐ZFAS1‐1 and si‐ZFAS1‐2), USF1 (si‐USF1), negative controls (si‐ZFAS1‐NC, si‐USF1‐NC) were purchased from Shanghai GenePharma company, and so did miR‐296‐5p inhibitor and corresponding negative control (inh‐miR‐296‐5p, inh‐NC). Lipofectamine 2000 (Thermo Fisher Scientific, Inc) was used for transfection according to the manufacturer's instructions. The transfected cells were harvested 48 hours after transfection.
8
Circular RNA regulation of breast cancer
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Human embryonic kidney cells (HEK293T), human BC cell lines (MCF7, SKBR3, MDA-MB-231) and human mammary epithelial cell line MCF-10A were available from the American Type Culture Collection (Rockville, MD, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (Beyotime, Shanghai, China) with 10% fetal bovine serum (FBS; Beyotime, Shanghai, China), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China) at 37°C in 5% CO2. Circ_0048764 overexpression plasmid (pcDNA-circ_0048764), empty vector cDNA (pcDNA-NC), small interfering RNAs (siRNAs) targeting circ_0048764 (si-circ_0048764-1 and si-circ_0048764-2), siRNA negative control (scramble siRNA, si-NC), miR-1296-5p mimics and its control (mim-NC), miR-1296-5p inhibitors and its control (Inh-NC) were produced by GenePharma (Shanghai, China). The above vectors were subsequently transfected into SKBR3 and MCF7 cells by Lipofectamine®3000 (Invitrogen, Carlsbad, CA, USA). 24 h later, quantitative real-time polymerase chain reaction (qRT-PCR) was executed to detect the efficacy.
9
Glioma Cell Line Manipulation and Characterization
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Human glioma cell lines (U87, U251, SHG44, A172) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), as well as normal human astrocytes (NHAs). Cells were cultured with RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 U/mL penicillin/streptomycin (Invitrogen) in a humidified incubator with 5% CO2 at 37 °C.
To construct LINC00963-overexpressing cells, the full-length human LINC00963 sequence was amplified by PCR, and the product was subcloned into a pcDNA3.1 vector (Invitrogen) and named pcLINC00963. Specific siRNAs were designed to knockdown LINC00963 (si-RNA1, 5ʹ-GGTTCCTCATCTGCCAGTT-3ʹ; si-RNA2, 5ʹ-GGCGCAGTAACAATATAAT-3ʹ), and si-NC was used as negative control. A negative control (pcNC) was also constructed. MiR-506 mimic, miR-506 inhibitor and their corresponding negative controls (miR-NC and inh-NC) were purchased from GenePharma (Shanghai, China). BCAT1-expressing vectors (named pcBCAT1) were generated by GenePharma and used to restore the expression of BCAT1 in cells, and pcDNA was used as a control. The transfection procedure was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
To construct LINC00963-overexpressing cells, the full-length human LINC00963 sequence was amplified by PCR, and the product was subcloned into a pcDNA3.1 vector (Invitrogen) and named pcLINC00963. Specific siRNAs were designed to knockdown LINC00963 (si-RNA1, 5ʹ-GGTTCCTCATCTGCCAGTT-3ʹ; si-RNA2, 5ʹ-GGCGCAGTAACAATATAAT-3ʹ), and si-NC was used as negative control. A negative control (pcNC) was also constructed. MiR-506 mimic, miR-506 inhibitor and their corresponding negative controls (miR-NC and inh-NC) were purchased from GenePharma (Shanghai, China). BCAT1-expressing vectors (named pcBCAT1) were generated by GenePharma and used to restore the expression of BCAT1 in cells, and pcDNA was used as a control. The transfection procedure was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
10
Silencing circHIPK3 in ccRCC cells
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Three human ccRCC cell lines (A498, 786-O and 769-P) and a human renal proximal tubular epithelial HK2 cell line (HRPTEpiC) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at a constant temperature of 37°C in a humidified atmosphere containing 5% CO2.
In order to silence circHIPK3, shRNA target circHIPK3 or negative control (shNC), was inserted into a pLKO.1 vector (Biosettia). circHIPK3 overexpression plasmids containing wild-type or mutant miR-508-3p-binding sequences (circHIPK3 and mut-circHIPK3), as well as CXCL13 overexpression plasmids (oeCXCL13), were synthesized by Shanghai GenePharma Co., Ltd. miR-508-3p mimics (miR-508-3p) and negative control (miR-NC), as well as miR-508-3p inhibitors (inh-508-3p) and negative control (inh-NC), were also purchased from Shanghai GenePharma Co., Ltd. All plasmids or oligonucleotides were transfected into A498 and 786-O cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions.
In order to silence circHIPK3, shRNA target circHIPK3 or negative control (shNC), was inserted into a pLKO.1 vector (Biosettia). circHIPK3 overexpression plasmids containing wild-type or mutant miR-508-3p-binding sequences (circHIPK3 and mut-circHIPK3), as well as CXCL13 overexpression plasmids (oeCXCL13), were synthesized by Shanghai GenePharma Co., Ltd. miR-508-3p mimics (miR-508-3p) and negative control (miR-NC), as well as miR-508-3p inhibitors (inh-508-3p) and negative control (inh-NC), were also purchased from Shanghai GenePharma Co., Ltd. All plasmids or oligonucleotides were transfected into A498 and 786-O cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions.
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