ythdf1 shrna (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology
ythdf1 shrna
Ythdf1 Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ythdf1/YTHDF1+shRNA+Plasmid/pmc08495178-106-9-23
Average 92 stars, based on 1 article reviews
Ythdf1 Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ythdf1/YTHDF1+shRNA+Plasmid/pmc08495178-106-9-23
Average 92 stars, based on 1 article reviews
ythdf1 shrna - by Bioz Stars,
2026-11
92/100 stars
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1) Product Images from "N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells"
Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells
Journal: Redox Biology
doi: 10.1016/j.redox.2021.102151
Figure Legend Snippet: Primer Sequences used for qRT-PCR.
Techniques Used:
Figure Legend Snippet: m 6 A reader protein YTHDF1 promotes autophagy activation and BECN1 mRNA stability via recognizing the m 6 A binding site. HSC-T6 and HSC-LX2 cells were added sorafenib (10 μM), erastin (10 μM), and RSL3 (2.5 μM) and treated for 24 h. ( A ) Western blot was used to assay the protein levels of m 6 A readers (n = 3 in every group). ( B ) The mRNA levels of m 6 A readers were determined by real-time PCR (***, p < 0.001, n = 3 in every group). ( C ) YTHDF1 plasmid or control vector were transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h, and then were pretreated with Act-D (5 μg/ml) for indicated times. Real-time PCR was used to measure the remaining BECN1 mRNA levels (*, p < 0.05, n = 3 in every group). ( D ) YTHDF1 plasmid or control vector were transfected into HSC-LX2 and treated with erastin (10 μM) for 24 h. Western blot was used to determine the protein levels of BECN1 at different times under the CHX (100 μg/ml) treatment (n = 3 in every group). ( E ) The binding of YTHDF1 and BECN1 mRNA was measured by ribonucleoprotein immunoprecipitation (RNP IP) (**, p < 0.01, n = 3 in every group). ( F ) biotinylated transcripts of the BECN1 mRNA 3′-UTR, CDS, 5′-UTR were used for mRNA affinity isolation assay (n = 3 in every group). ( G ) HSC-LX2 cells were transfected with control vector or YTHDF1 plasmid and by erastin (10 μM) treatment for 24 h. The levels of m 6 A modification in BECN1 mRNA 3′-UTR, CDS and 5′-UTR were determined by MeRIP qPCR (**, p < 0.01, n = 3 in every group). ( H ) HSC-LX2 cells were stably transfected with empty vector (pGL3)or luciferase constructs carrying the genes encoding 5′-UTR-BECN1, CDS-BECN1, 3′-UTR-BECN1. Then cells were added erastin (10 μM) and treated for 24 h, and luciferase activities were measured (**, p < 0.01, n = 3 in every group). ( I ) BECN1 mRNA m 6 A binding site was showed. ( J ) Schematic representation of positions of m 6 A motifs and mutation in CDS within BECN1 mRNA were showed. ( K ) Binding of YTHDF1 with the BECN1-CDS-WT, BECN1-CDS-Mut1 (A437G) and BECN1-CDS-Mut2 (A1276G) in HSC-LX2 were analyzed by YTHDF1 RNP IP (**, p < 0.01, n = 3 in every group). ( L ) HSC-LX2 cells were transfected with control vector or YTHDF1 plasmid and BECN1-CDS-WT, BECN1-CDS-Mut1 and BECN1-CDS-Mut2 plasmid, and then were pretreated with Act-D (5 μg/ml) for indicated times. Real-time PCR was used to measure the remaining BECN1 mRNA (*, p < 0.05, n = 3 in every group). ( M ) After transfected with BECN1-CDS-WT, BECN1-CDS-Mut1 and BECN1-CDS-Mut2 plasmid, HSC-LX2 cells were treated with erastin (10 μM) for 48 h. The protein expression of BECN1 was measured by western blot (*, p < 0.05, ***, p < 0.001, n = 3 in every group).
Techniques Used: Activation Assay, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Transfection, Immunoprecipitation, Isolation, Modification, Stable Transfection, Luciferase, Construct, Mutagenesis, Expressing
Figure Legend Snippet: HSC-specific inhibition of m 6 A modification impairs erastin-induced HSC ferroptosis in murine liver fibrosis. Mice of 6 groups were treated with Vehicle, CCl 4, CCl 4 +VA-Lip-control-vector + Erastin, CCl 4 +VA-Lip-Mettl4-shRNA + Erastin, CCl 4 +VA-Lip-Fto-Plasmid + Erastin, CCl 4 +VA-Lip-Ythdf1-shRNA + Erastin. ( A ) Macroscopic examination was used to observe the pathological changes of the livers. Scale bars: 1 cm. Histopathological study was performed by H&E, Masson, and Sirius Red staining. Representative photographs were showed. Scale bars: 50 μm. (***, p < 0.001, n = 6 in every group). ( B ) Immunohistochemical staining of α-SMA was determined. Representative photographs were showed. Scale bars: 50 μm.(***, p < 0.001, n = 6 in every group). ( C ) Real-time PCR was measured to determine the mRNA expression of liver fibrosis markers (Acta2, Col1a1, Fn1, and Des) (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 6 in every group). ( D ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (n = 6 in every group, ***, p < 0.001, N.S., not significant). ( E, F ) Real-time PCR was used to determine the mRNA expression of autophagy markers (Becn1, Map1lc3b, Sqstm1, and Fth1) (*, p < 0.05, ***, p < 0.001, N.S., not significant, n = 6 in every group). ( G ) The mRNA expression of Ptgs2, iron accumulation and MDA production were determined (*, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant, n = 6 in every group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Techniques Used: Inhibition, Modification, Control, Plasmid Preparation, shRNA, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction, Expressing, Methylation
Figure Legend Snippet: m 6 A modification upregulation, autophagy activation, and ferroptosis induction occur in human HSCs receiving sorafenib monotherapy. ( A, B ) laser capture microdissection (LCM) was used to isolated the primary human HSCs from the collected liver tissue. ACTA2, FN1, COL1A1, METTL4, FTO, and YTHDF1 mRNA expression were determined by real-time PCR (No treatment, n = 10; Sorafenib treatment, n = 10, **, p < 0.01, ***, p < 0.001). ( C ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( D ) Real-time PCR was used to determine the mRNA expression of autophagy markers BECN1, MAP1LC3B, SQSTM1, and FTH1 (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( E ) The PTGS2 mRNA expression, iron accumulation, MDA production and GSH depletion were determined (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001).
Techniques Used: Modification, Activation Assay, Laser Capture Microdissection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Methylation
Figure Legend Snippet: m 6 A modification induces HSC ferroptosis by regulating autophagy signaling pathway. The upregulation of methylase METTL4 and the downregulation of demethylase FTO increased the levels of m 6 A modifications in BECN1 mRNA. m 6 A reader YTHDF1 promoted BECN1 mRNA stability via recognizing the m 6 A binding site, thus triggering autophagy activation, and eventually leading to HSC ferroptosis.
Techniques Used: Modification, Binding Assay, Activation Assay
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Plasmid Preparation:Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells Article Snippet: Anti-Mouse IgG (G-21040) and anti-Rabbit IgG (G-21234) were bought from Thermo Fisher Scientific. .. The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), shRNA:Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells Article Snippet: Anti-Mouse IgG (G-21040) and anti-Rabbit IgG (G-21234) were bought from Thermo Fisher Scientific. .. The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), Control:Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells Article Snippet: Anti-Mouse IgG (G-21040) and anti-Rabbit IgG (G-21234) were bought from Thermo Fisher Scientific. .. The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), |