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

Journal: Nature

Article Title: Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination

doi: 10.1038/s41586-018-0360-3

Figure Lengend Snippet: a) Percentage of MBP + oligodendrocytes generated from OPCs (OPC-5) at 72 h following treatment with methyl β-cyclodextrin (1 mM) for 30 min at 37 °C. n = 8 wells per condition, with > 1,000 cells analyzed per well. b) GC/MS-based quantitation of cholesterol (left) and desmosterol (right) levels in OPCs (OPC-5) treated with methyl β-cyclodextrin (Me-β-CD) at 1 mM or ketoconazole at 2.5 μM. n = 2 wells per condition. c, d) Percentage of MBP + oligodendrocytes generated from OPC-1 ( c ) and OPC-5 cells ( d ) at 72 h following treatment with the indicated purified sterol intermediates. n = 4 wells per condition, except n = 8 for DMSO and ketoconazole, with > 1,000 cells analyzed per well. Green text highlights metabolites that accumulate after treatments that enhance oligodendrocyte formation ( , ). e) Percentage of MBP+ oligodendrocytes generated from OPC­1 at 72 h following treatment with MAS-412 and MAS-414. n = 4 wells per condition, with > 1,000 cells analyzed per well. f) Representative images of OPC­5 cells treated 72 h with DMSO, MAS-412, or MAS-414 (3 μM). Nuclei are labeled with DAPI (blue), and oligodendrocytes are indicated by immunostaining for myelin basic protein (green). Scale bar, 100 μm. g) Percentage of MBP + oligodendrocytes generated from OPC-1 at 72 h following treatment with 2,2-dimethyl-zymosterol. n = 4 wells per condition except DMSO (n = 12), with > 1,000 cells analyzed per well. h) Representative images of OPC-5 cells treated 72 h with vehicle and 2,2-dimethyl-zymosterol (2.5 μM). Nuclei are labeled with DAPI (blue), and oligodendrocytes are indicated by immunostaining for myelin basic protein (green). Scale bar, 100 μm. i) Percentage of MBP+ oligodendrocytes generated from OPC-5 (left) and OPC-1 (right) at 72 h following treatment with FF-MAS or T-MAS. n = 4 wells per condition except DMSO and Ketoconazole (n = 8), with > 1,000 cells analyzed per well. j) Percentage of MBP + oligodendrocytes generated from OPC-5 and OPC-1 OPCs at 72 h following treatment with the indicated concentrations of cholesterol. n = 8 wells per condition, with > 1,000 cells analyzed per well. k, l) Percentage of MBP + oligodendrocytes generated from OPC-5 and OPC-1 at 72 h following treatment with the indicated concentrations of sterols that are structurally identical aside from the presence or absence of the 8,9 double bond (structures in panel o ). n ≥ 3 wells per condition (see dot plots as replicate values vary by condition), with > 1,000 cells analyzed per well. m) Percentage of MBP + oligodendrocytes generated from OPCs (OPC-5) at 72 h following treatment with the indicated small molecules or combinations of small molecules (ketoconazole, 2.5 μM; Ro 48–8071, 11 nM; liothyronine, 3 μM). n = 3 wells per condition, except DMSO n = 11, ketoconazole n = 13, liothyronine n = 8 & liothyronine + Ro 48–8071 n = 4, with > 1,000 cells analyzed per well. n) GC/MS-based quantitation of lanosterol levels in OPCs (OPC-5) treated 24 h with the indicated small molecules or combinations of small molecules at concentrations stated in m. n = 2 wells per condition. o) Structures of zymostenol, 8,9-dehydrocholesterol, 5α-cholestanol, and cholesterol. p) Total cell number as measured by counting of DAPI+ nuclei in the experiment presented in panel m . q, r) Percentage of MBP+ oligodendrocytes generated from OPCs (OPC­5 and OPC-1) at 72 h following treatment with the indicated small molecules or combinations of small molecules in two independent batches of OPCs (ketoconazole, 2.5 μM; MAS­412, 5 μM). In q , n = 16 for DMSO, 8 for Ketoconazole, and 4 for remaining bars. In r , n = 8 wells per condition. s) Luciferase reporter assays were used to assess if 2,2-Dimethylzymosterol (5 μM), Ketoconazole (2.5 μM), and TASIN-1 (250 nM) modulate human ERα, GR, LXRβ, NFkB, NRF2, PGR, PPARδ, PPARγ, RARα, RARγ, RXRα, RXRβ, TRα, TRβ and VDR transcriptional activity in agonist mode and ERRα, RORα and RORγ in inverse-agonist mode. n = 2 wells per condition and n = 3 wells per positive control conditions. t) Effects of sterols (2,2-dimethylzymosterol 5 μM, FF-MAS 10 μM) and small molecules (Ketoconazole 2.5 μM, TASIN-1 100 nM) on the NR2F1-mediated activation of a NGFI-A promoter driven luciferase reporter. n = 2 wells per condition. u) Effects of 2,2-dimethylzymosterol (5 μM) on NR2C2-mediated activation of a NGFI-A promoter driven luciferase reporter in comparison to cells transfected with reporter only, untreated, or treated with a previously reported positive control (all-trans retinoic acid, ATRA, 5 μM). n = 2 wells per condition. v) LSS, DHCR7, LDLR mRNA levels measured by RT-qPCR following 24 h treatment with DMSO, Mevastatin (2.5 μM), Ro 48–8071 (500 nM), Ketoconazole (2.5 μM), TASIN-1 (100 nM), or Amorolfine (100 nM). n = 2 wells. All bar graphs indicate mean +/− standard deviation, and panels a-n , and t-v are representative of two independent experiments.

Article Snippet: NR2C2 (Origene, MR221079) or NR2F1 (gift from Dr. Christian Schaaf) and NGFI promoter reporter plasmid (generous gift of C. Schaaf) were transfected using Lipofectamine 2000 (Thermo-Fisher, 11668027) as per manufacturer’s protocol.

Techniques: Generated, Gas Chromatography-Mass Spectrometry, Quantitation Assay, Purification, Labeling, Immunostaining, Luciferase, Activity Assay, Positive Control, Activation Assay, Comparison, Transfection, Quantitative RT-PCR, Standard Deviation

Journal: International journal of cancer

Article Title: TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

doi: 10.1002/ijc.29049

Figure Lengend Snippet: Figure 1. IHC staining results investigating TR4 level in tumor tissues of PCa patients. Clinical specimens with different Gleason scores were obtained from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China. IHC staining was performed using TR4 antibody (1:300). Left panels show imaging and quantification is shown on right. Magnification, 1003 (upper) and 4003 (lower). [Color fig- ure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Anti-TR4 antibody (PP-H0107B-00) was purchased from R&D systems (Minneapolis, MN).

Techniques: Immunohistochemistry, Imaging

Journal: International journal of cancer

Article Title: TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

doi: 10.1002/ijc.29049

Figure Lengend Snippet: Figure 2. Knockdown of TR4 decreased PCa cells migration/invasion. (a–c) Results obtained with C4-2-siTR4/C4-2 scramble (C4-2scr) con- trol cells. (d–f) Data obtained with TRAMP-C1-siTR4/TRAMP-C1 scramble (TRAMP-C1scr) control cells. (a, d) Knockdown efficiency of TR4 in the two cell lines. Upper and lower panels show TR4 level at protein and mRNA levels, respectively. (b, e) Migration test results and (c, f) represent invasion assay results. The migrated or invaded cells were stained with toluidine blue (1%) and positively stained cell numbers in randomly picked six areas were averaged. Experiments were repeated at least three times and mean 6 SEM values were shown in quanti- fication. p values presented in figures. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Anti-TR4 antibody (PP-H0107B-00) was purchased from R&D systems (Minneapolis, MN).

Techniques: Knockdown, Migration, Control, Invasion Assay, Staining

Journal: International journal of cancer

Article Title: TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

doi: 10.1002/ijc.29049

Figure Lengend Snippet: Figure 3. Overexpression of TR4 increased PCa cells migration/invasion. Similar experiments as in Figure 2 were performed, but CWR22Rv1 TR4-cDNA/CWR22Rv1 vector control cells (a–c) and PC3 TR4-cDNA/PC3 vector control cells (d–f) were used in experiments. (a, d) Overex- pression efficiency of TR4 in the two cell lines. Upper and lower panels show TR4 level at protein and mRNA, respectively. (b, e) Migration test results and (c, f) represent invasion assay results. The migration and invasion assays protocols were similar as in Figure 2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Anti-TR4 antibody (PP-H0107B-00) was purchased from R&D systems (Minneapolis, MN).

Techniques: Over Expression, Migration, Plasmid Preparation, Control, Invasion Assay

Journal: International journal of cancer

Article Title: TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

doi: 10.1002/ijc.29049

Figure Lengend Snippet: Figure 4. TR4 regulates CCL2 expression at the transcriptional level. (a) qPCR analysis of testing expression levels of the PCa metastasis associated genes. Total RNAs were obtained from C4-2-siTR4 and C4-2scr control cells and mRNA expressions of indicated candidate mole- cules were determined by calculating siTR4/scr. (b) CCL2 ELISA test results. CCL2 concentrations in culture supernatants of the C4-2-siTR4/ C4-2scr control cells and CWR22Rv1 TR4 cDNA/CWR22Rv1 vector control cells were analyzed. (c) Luciferase assay using pGL3-CCL2 pro- moter containing TR4 binding element sequence. (d) ChiP assay results. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Anti-TR4 antibody (PP-H0107B-00) was purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Binding Assay, Sequencing

Journal: International journal of cancer

Article Title: TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

doi: 10.1002/ijc.29049

Figure Lengend Snippet: Figure 5. Blocking of CCL2/CCR2 pathway using the CCR2 antagonist (CCR2 A) inhibited TR4 mediated PCa cells migration/invasion increase. (a) C4-2 cells and (b) CWR22Rv1 cells data. Migration (left panel) and invasion (right panel) assay results were shown. C4-2 TR4 cDNA/C4-2 scramble control and CWR22Rv1 TR4 cDNA/CWR22Rv1 vector control cells were treated with either CCR2 A (25 nM) or DMSO (vehicle control) for migration/invasion assays. Experiments were repeated at least three times and Mean 6 SEM values were shown in quantification. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Anti-TR4 antibody (PP-H0107B-00) was purchased from R&D systems (Minneapolis, MN).

Techniques: Blocking Assay, Migration, Control, Plasmid Preparation

Journal: International journal of cancer

Article Title: TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

doi: 10.1002/ijc.29049

Figure Lengend Snippet: Figure 6. In vivo mice studies using the CWR22Rv1 xenograft mouse model. These luc-CWR22Rv1 cells were orthotopically injected into ante- rior prostates of nude mice (three groups). Group 1 (n 5 15) mice were injected with CWR22Rv1 vector control cells, Group 2 (n 5 7) and Group 3 (n 5 9) mice were injected with CWR22Rv1 TR4 cDNA cells. When tumors developed into palpable size (2 weeks), vehicle (DMSO, Group 1 and 2) and CCR2 antagonist (CCR2 A) (50 mg/Kg, Group 3) were i.p. injected into mice every other day for 4 weeks. (a) Incidence of metastases in different groups of mice at four weeks after cell implantation. (b) Incidence of metastases, mice with either pelvic lymph node metastasis or distant metastasis was defined as “meta,” mice without any evidence of metastasis was classified as “nonmeta” (left panel) and metastasis in lymph nodes and distant organs (right panel) were shown at sacrifice of mice (6 weeks after cell implantation). (c) H&E staining and luciferase staining results of metastatic sites obtained from the Group 2 mice. (d) (Continued next page) IHC staining for detect- ing TR4, CCL2 and MMP9 expressions in tumor tissues obtained from three groups mice. Quantification at right.

Article Snippet: Anti-TR4 antibody (PP-H0107B-00) was purchased from R&D systems (Minneapolis, MN).

Techniques: In Vivo, Injection, Plasmid Preparation, Control, Staining, Luciferase, Immunohistochemistry