nr2c2  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) Volcano plot of m 6 A-IP-seq targets in shALKBH1 #1 As-Tr cells with NR2C2 as a top target labeled (black arrow). (B) HOMER motif analysis demonstrating the canonical m 6 A GGACU motif is enriched across experimental samples. (C) IGV visualization of increased m 6 A enrichment on representative site on the NR2C2 transcript. (D) qPCR analysis of the m 6 A levels on the NR2C2 transcript following m 6 A-IP in shNC and shALKBH1 #1 As-Tr cells. (E) qPCR analysis of the m 6 A levels on the NR2C2 transcript following m 6 A-IP of HaCaT control cells without or without 200 nM arsenic for 72 h and As-Tr cells. (F) CLIP-qPCR analysis of FLAG (ALKBH1) binding to sites on the NR2C2 transcript ( NM_001291694.2 , site 1: regions 1508243–1504892, and site 2: 150486–15048792, respectively) with an anti-FLAG or immunoglobulin G (IgG) antibody in shALKBH1 #1 As-Tr cells transfected with a construct expressing ALKBH1 (FLAG). (G) Immunoblot analysis of NR2C2 in shNC and shALKBH1 #1 As-Tr cells. (H) RT-qPCR of NR2C2 mRNA levels in shNC and shALKBH1 #1 As-Tr cells. (I) Immunoblot analysis of NR2C2 expression in HaCaT and A431 SCC cells. (J) Immunoblot analysis of NR2C2 expression in control (no As) and As-Tr cells. (K) Immunoblot analysis of NR2C2 expression in HaCaT cells treated with 200 nM arsenic for 72 h. (L) Immunoblot analysis of HA and NR2C2 expression in shALKBH1 #1 As-Tr cells expressing empty vector (EV), WT ALKBH1, catalytically inactive mutant ALKBH1 (D233A), and C118A and C207A mutant ALKBH1. (M) Immunoblot analysis of METTL3, METTL14, and NR2C2 expression in shALKBH1 #1 As-Tr cells transfected with control or METTL3/METTL14 siRNA. (N) Immunoblot analysis of YTHDF1 and NR2C2 expression in shALKBH1 #1 As-Tr cells transfected with control or YTHDF1 siRNA. (O) RNA immunoprecipitation (RIP) and qPCR analysis showing the binding of YTHDF1 to the NR2C2 transcript in As-Tr cells. (P) CCK8 assay of shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. (Q) Soft agar cloning assay of shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. (R) CCK8 assay of As-Tr cells with or without NR2C2 overexpression. (S) Soft agar cloning assay of As-Tr cells with or without NR2C2 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t test.

Article Snippet: NR2C2 ( NM_003298 ) Human Tagged ORF Clone , Origene , Cat# RC219184.

Techniques: Labeling, Control, Binding Assay, Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Mutagenesis, RNA Immunoprecipitation, CCK-8 Assay, Knock-Out, Cloning, Over Expression

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) SUnSET analysis of global translation levels across no As, Chr As, and As-Tr cells. (B) SUnSET analysis of global translation levels across HaCaT cells with ALKBH1 knockdown with or without arsenic treatment (200 nM, 72 h). (C) SUnSET analysis of global translation levels in As-Tr cells with ALKBH1 knockdown. (D) SUnSET analysis of global translation levels in shALKBH1 #1 As-Tr cells transfected with control or NR2C2 siRNA. (E) Immunoblot analysis of p-4EBP1 and 4EBP1 (total) in As-Tr cells with ALKBH1 knockdown. (F) Log2 fold change of SESN1 , SESN2 , and SESN3 mRNA expression in shALKBH1 #1 As-Tr cells as compared to shNC. Data taken from RNA-sequencing used as input for m 6 A-IP-sequencing. (G–I) IGV visualization of NR2C2 binding on SESN1 , SESN2 , and SESN3 transcripts taken from NR2C2 ChIP-seq data (ENCSR454GVT, ENCSR750LYM, ENCSR559ZKI) from ENCODE. (J) Immunoblot analysis of SESN1 and ALKBH1 expression in shNC and shALKBH1 #1 As-Tr cells. (K) Immunoblot analysis of p-4EBP1, 4EBP1 (total), SESN1, and NR2C2 expression in shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. **** p < 0.0001; Student’s t test.

Article Snippet: NR2C2 ( NM_003298 ) Human Tagged ORF Clone , Origene , Cat# RC219184.

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, RNA Sequencing, Sequencing, Binding Assay, ChIP-sequencing, Knock-Out