Journal: Investigative ophthalmology & visual science

Article Title: METTL3-Mediated RNA m6A Modification Regulates the Angiogenic Behaviors of Retinal Endothelial Cells by Methylating MMP2 and TIE2.

doi: 10.1167/iovs.64.13.18

Figure Lengend Snippet: FIGURE 1. The profile of m6A modification in the retinas and retinal endothelial cells of OIR mice. (A) Dot blot analysis showcasing representative images of m6A modification levels in the retinas from both the OIR and control groups. (B) Quantitative assessment of m6A modification levels from the dot blot analysis (n = 9/group). (C) Western blot analysis detected the protein expression levels of Mettl3, Mettl14, Wtap, Fto, and Alkbh5 in the retinas of the OIR and control mice. (D) Quantification of respective protein expression levels (n = 9/group). (E) Dot blot representation and (F) subsequent quantification of RNA m6A levels in retinal endothelial cells from the OIR and control mice (n = 6/group). N, normal mouse; OIR, oxygen-induced retinopathy mouse; mRECs, mouse retinal endothelial cells.

Article Snippet: These membranes were incubated overnight at 4°C with specific primary antibodies: METTL3 (1:1000; Abcam), METTL14 (1:1000; Invitrogen), WTAP (1:1000; CST), FTO (1:1000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALKBH5 (1:1000; Novus Biologicals, Littleton, CO, USA), MMP2 (1:1000; CST), TIE2 (1:1000; CST), HIF-1α (1:1000; Abcam), and β-ACTIN (1:1000; Santa Cruz Biotechnology).

Techniques: Modification, Dot Blot, Control, Western Blot, Expressing

Journal: Investigative ophthalmology & visual science

Article Title: METTL3-Mediated RNA m6A Modification Regulates the Angiogenic Behaviors of Retinal Endothelial Cells by Methylating MMP2 and TIE2.

doi: 10.1167/iovs.64.13.18

Figure Lengend Snippet: FIGURE 2. RNA m6A modification levels increase in HRMECs under hypoxic conditions. (A) A representative dot blot image depict- ing RNA m6A modifications in HRMECs after four and 24 hours’ exposure to 1% O2, juxtaposed with normoxic conditions (21% O2). (B) Statistical analysis of m6A modification levels from dot blot results (n = 3/group). (C) RT-qPCR results displaying expression patterns of m6A methyltransferases (METTL3, METTL14, WTAP) and demethylases (FTO, ALKBH5) in HRMECs subjected to hypoxic and normoxic atmospheres (n = 3/group). (D) Western blot representation detailing protein levels of METTL3, METTL14, WTAP, FTO, ALKBH5, and HIF-1α in HRMECs under 21% O2 or 1% O2 exposure. (E) Relative protein expression quantification (n = 3/group). (F) Immunofluorescence reveals nuclear localization of METTL3 (green) in retinal endothelial cells, with enhanced fluorescence intensity under hypoxic conditions compared to normoxic states. DAPI (blue) marks cell nuclei. Scale bar: 100 μm.

Article Snippet: These membranes were incubated overnight at 4°C with specific primary antibodies: METTL3 (1:1000; Abcam), METTL14 (1:1000; Invitrogen), WTAP (1:1000; CST), FTO (1:1000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALKBH5 (1:1000; Novus Biologicals, Littleton, CO, USA), MMP2 (1:1000; CST), TIE2 (1:1000; CST), HIF-1α (1:1000; Abcam), and β-ACTIN (1:1000; Santa Cruz Biotechnology).

Techniques: Modification, Dot Blot, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Fluorescence

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A , B Quantification and representation of LKB1 and m6A modulators in KL relative to K lung cancer patients. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. C Spearman correlation analysis of ALKBH5 expression with the KRAS mutant lung cancer pathological characters. D Venn diagram showing the differentially expressed genes (DEGs) of LKB1 , m6A modulators, and readers between KL and K lung cancer. E Kaplan–Meier survival curve of patients with high and low ALKBH5 mRNA expression from the TCGA dataset. F Global m6A level regulation by LKB1 in lung cancer cell lines. G Western blot showing the LKB1 and ALKBH5 protein expression. H Co-immunofluorescence staining for LKB1 and ALKBH5 in A549 cells. Arrows show LKB1 positive and ALKBH5 negative cells. I LKB1 and m6A co-staining by LKB1 and/or ALKBH5 overexpression in A549 cells. Arrows show cells of LKB1 positive and m6A with strong (Second panel) or weak (Forth panel) intensity. Data were mean ± SD in F ( n = 5). * P < 0.05 (Student’s t -test). Si-CN, SiRNA-A; OE-CN, OE-c-Flag pcDNA3; OE, overexpression. KRAS Mut ; LKB1 Loss (KL); KRAS Mut ; LKB1 Wt (K).

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Expressing, Mutagenesis, Western Blot, Immunofluorescence, Staining, Over Expression

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A Western blot analysis shown that LKB1 and ALKBH5 were successfully knocked down in H1792 cells and overexpressed in A549 cells. B , C The measurement of global m6A by ELISA assay in H1792 ( B ) or A549 ( C ) cells, respectively. The colony formation ability ( D ) and cell migration ( E ) were reduced at both of basal level and LKB1 silenced level by ALKBH5 knockdown in H1792 cells, respectively. The reversible effect was found at both of basal level and LKB1 overexpressed level by ALKBH5 transfected in A549 cells using colony formation ( F ) and transwell assay ( G ). Data were mean ± SD ( n = 5) and were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison test for B – G . * P < 0.05. Bar = 5 mm in D and F ; Bar = 50 µm in E and G .

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Knockdown, Transfection, Transwell Assay, Comparison

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A 5mC DNA methylation was detected by ELISA assay. Data as mean ± SD ( n = 5), * P < 0.05 (Student’s t -test). B – D Representation and quantification of ALKBH5 expression at protein ( B , C ) and mRNA levels ( D ) in response to 5-aza treatment in A549 and H1792 cells for 72 h. Error bars, SD ( n = 4 for WB; n = 5 for qRT-PCR), * P < 0.05 as compared their corresponding controls (2-way ANOVA with Bonferroni multiple comparison post hoc test). E CTCF peak located in the CpGs Island of human ALKBH5 gene promoter. Bottom panel: the Methprimer histogram of CpG islands (CpGI, red box) and CpG dinucleotides (red vertical lines) in the regulatory region of ALKBH5 . F MeDIP assay showing the decrease of 5mC enrichment on the ALKBH5 promoter containing CTCF motif fragment by treated with 5-aza and LKB1 overexpression. G , H Representative bisulfite sequencing of four clones ( G ) and quantification of DNA methylation ( H ) of ALKBH5 promoter containing CTCF motif. n = 4 in F and H , mean ± SD, * P < 0.05 vs. A549 DMSO by one-way ANOVA followed by Tukey’s test in F and H . Si-CN, Si-RNA-A. OE-CN, OE-c-Flag pcDNA3. OE overexpression.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Comparison, Methylated DNA Immunoprecipitation, Over Expression, Methylation Sequencing, Clone Assay

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A – C Representative images and quantification of CTCF and ALKBH5 protein expression by WB and qRT-PCR in A549 cells with LKB1 overexpression and/or CTCF knockdown for 48 h. Error bars, SD ( n = 4 for WB; n = 5 for qRT-PCR), * P < 0.05 vs. Si-CN or OE-CN (one-way ANOVA with Bonferroni multiple comparison post hoc test). D Luciferase reporter assay of A549 cells transfected with pGL3-basic constructs containing serial LKBH5 promoters or deletion of CTCF peak fragment. n = 8/group, mean ± SD. * P < 0.05 by one-way ANOVA followed by Tukey’s test. E Reduction of ALKBH5 Luc:-1168 bp wild-type (WT) construct activities by treatment of 5-aza or LKB1 overexpression in A549 cells, but not for ALKBH5 Luc:-1168 bp deletion (Del). Data as mean ± SD ( n = 5), * P < 0.05 (Student’s t -test). F ChIP-qPCR showing that the ALKBH5 -CTCF peak region was occupied by the suppressor of CTCF and activators of histone modulators in A549 cells. G – J ChIP-qPCR analyses of 5-aza treated or LKB1 overexpressed A549 cells. n = 5/group, mean ± SD. * P < 0.05 vs. IgG in F , vs. A549-DMSO in G – J by one-way ANOVA followed by Tukey’s test.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Comparison, Luciferase, Reporter Assay, Transfection, Construct, ChIP-qPCR

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A , B Representative western blotting and quantification of SOX2, SMAD7, MYC, and ALKBH5 protein. Data as mean ± SD ( n = 4). C Mutation of m6A site or ALKBH5 overexpression released the SOX2, SMAD7 , and MYC gene posttranscriptional repression by LKB1 in A549 cells ( n = 4). D m6A-RIP analysis demonstrated that SOX2, SMAD7 , and MYC were subjected to ALKBH5-mediated m6A modifications ( n = 5). E YTHDF2-RIP-qPCR shown that YTHDF2 could occupy m6A sites of SOX2, SMAD7 , and MYC , which was mediated by ALKBH5 ( n = 4). F qRT-PCR was performed to indicate that knockdown of YTHDF2 significantly upregulated SOX2, SMAD7 , and MYC gene mRNA expression at basal level, LKB1 overexpressed or ALKBH5 silenced A549 cells ( n = 6). Data as mean ± SD. * P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for B – F . Si-CN, Si-RNA-A. OE-CN, OE-c-Flag pcDNA3. OE overexpression.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Western Blot, Mutagenesis, Over Expression, Quantitative RT-PCR, Knockdown, Expressing, Comparison

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A Representative immunostaining images and quantification of 5mC DNA in lung cancer patients with KW and KM. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. B , C High levels of ALKBH5 DNA methylation and mRNA expression in cases of LKB1 loss compared with that of LKB1 positive expression in KM patients. D – F The correlation of ALKBH5 protein with the global m6A modification ( D ), m6A levels of SOX2, SMAD7 , and MYC genes ( E , F ) in KM and KW groups. G – I Negative correlations of the m6A enrichment with mRNA expressions of SOX2 , SMAD7 , and MYC in KM group. J Representative immunostaining images and quantification of SOX2 , SMAD7 , and MYC in KM group with LKB1 loss or not. K The positive correlations of ALKBH5 with SOX2, SMAD7 , and MYC protein expression in KM group. Data as mean ± SD. KRAS mutation (KM). KRAS wild-type (KW). * P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for A , B , and C . Student’s t- test for J .

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Immunostaining, DNA Methylation Assay, Expressing, Modification, Mutagenesis, Comparison

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Loss of LKB1-induced DNA hypermethylation, which prevents CTCF binding on the ALKBH5 gene promoter, maintains ALKBH5 expression, and further represses global RNA methylation. Oncogenic SMAD7, SOX2 , and MYC are crucial targets of m6A meditated by LKB1 deficiency, and are involved in KRAS mutation lung cancer progression.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Binding Assay, Expressing, Methylation, Mutagenesis

Journal: Nature communications

Article Title: ALKBH5 controls the meiosis-coupled mRNA clearance in oocytes by removing the N 6 -methyladenosine methylation.

doi: 10.1038/s41467-023-42302-6

Figure Lengend Snippet: Fig. 9 | ALKBH5 manipulates maternal RNA clearance in GV-MII transition by regulating m6A demethylation. Illustrations summarizing the functions of ALKBH5 in oocyte maturation and the molecular mechanism regarding how ALKBH5 regulates maternal RNA decay in oocytes.

Article Snippet: Small interference RNA (siRNA) of murine Alkbh5 and Igf2bp2 were purchased from Santa Cruz (sc-141022) and GenePharma respectively.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: The mRNA N 6 -Methyladenosine Response to Dehydration in Xenopus laevis

doi: 10.3390/ani14223288

Figure Lengend Snippet: Summary of primary antibody information.

Article Snippet: ALKBH5 , Abbexa , Abx125516.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: The mRNA N 6 -Methyladenosine Response to Dehydration in Xenopus laevis

doi: 10.3390/ani14223288

Figure Lengend Snippet: Relative protein expression levels of ALKBH5 and FTO in livers from control and dehydrated X. laevis , as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Data were analyzed using a Student’s t -test; asterisks indicate values that are statistically significantly different from controls ( p < 0.05).

Article Snippet: ALKBH5 , Abbexa , Abx125516.

Techniques: Expressing, Control, Western Blot

Journal: Animals : an Open Access Journal from MDPI

Article Title: The mRNA N 6 -Methyladenosine Response to Dehydration in Xenopus laevis

doi: 10.3390/ani14223288

Figure Lengend Snippet: Relative protein expression levels of ALKBH5 and FTO in kidneys of control and dehydrated X. laevis , as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Data were analyzed a Student’s t -test; asterisks indicate that are statistically significantly different from controls ( p < 0.05).

Article Snippet: ALKBH5 , Abbexa , Abx125516.

Techniques: Expressing, Control, Western Blot

Journal: Cell death & disease

Article Title: METTL3 inhibition promotes radiosensitivity in hepatocellular carcinoma through regulation of SLC7A11 expression.

doi: 10.1038/s41419-024-07317-x

Figure Lengend Snippet: Fig. 4 METTL3-mediated m6A modification on SLC7A11 transcripts. A Metagene distribution of m6A-immunocipitated reads across the length of mRNA transcripti of MHCC-97H cell line; B Distribution of m6A peaks on functional regions of mRNA transcripts; C Histogram of the distribution of peaks on the 4 transcript function regions (5’UTR, 3’UTR, CDS and Intronic), with expected values in blue; D Statistics of the number of m6A peaks distributed on mRNA; E Identification of 25 representative motifs on the mRNA regions where m6A methylation occurred was performed using HOMER software; F Genome Browser screenshots of MeRIP-seq read density signals on SLC7A11 mRNA in MHCC-97H and Huh7 cells; G Gene-specific MeRIP-qPCR for shNC and shMETTL3 HepG2 and MHCC-97H cells, m6A positive regions were selected based on MeRIP-seq; H Relative mRNA levels of ALKBH5 and SLC7A11 after ALKBH5 overexpression in HepG2 cells were detected by qRT-PCR; I Protein levels of ALKBH5 and SLC7A11 after ALKBH5 overexpression in HepG2 cells were detected by western blot; J Overall m6A levels after overexpression of ALKBH5 in HepG2 cells were detected by m6A dot blot; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used: β-actin (66009-1-Ig, Proteintech), METTL3 (#86132S, CST), IGF2BP2 (11601- 1-AP, Proteintech), ALKBH5 (A22137, Abclonal), SLC7A11/xCT (#12691S, CST), GPX4 (#59735S, CST), FTH1 (#4393S, CST), CD71 (#13113S, CST), HO-1 (ab137744, Abcam), ACSL4 (22401-1-AP, Proteintech), Transferrin (#35293S, CST), FLAG (F1804, Sigma), IGF2BP3 (14642-1-AP, Proteintech), FTO (A3861, Abclonal), METTL14 (A8530, Abclonal).

Techniques: Functional Assay, Methylation, Software, Over Expression, Quantitative RT-PCR, Western Blot, Dot Blot

Journal: PLOS Pathogens

Article Title: The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome

doi: 10.1371/journal.ppat.1011917

Figure Lengend Snippet: ( A ) Zonation of the RNA demethylases ALKBH5, FTO and NDRG1 based on scRNA-seq data from mouse liver . ( B ) HepG2-NTCP cells were cultured under 18% or 1% oxygen for the indicated times and HIF-1α, HIF-2α, ALKBH5 or β-Actin protein expression measured. ALKBH5 expression was quantified by densitometry. ( C ) ALKBH5 and FTO transcript levels in HepG2-NTCP cells cultured under 18% or 1% oxygen for 72h. Data are expressed relative to a housekeeping gene B2M and presented as mean ± S.D. of n = 4 from 2 experiments. Statistical significance was determined using Mann-Whitney tests, with Bonferroni correction for multiple comparisons, **** p < 0.0001. ( D ) m 6 A-modified cellular RNAs. RNA was extracted from HepG2-NTCP cells cultured under 18% or 1% oxygen conditions for 72h and subjected to immuno-northern blotting using m 6 A antibody. Densitometric quantification of northern blots was performed and data expressed relative to 18% oxygen. Data is shown as the mean ± S.D. of 4 independent experiments with statistical significance determined using Mann-Whitney test. * p < 0.05. ( E ) Quantification of methylated HBV BCP-RNAs. HepG2-NTCP cells were transfected with HBV WT plasmid for 18h and cultured at 18% or 1% oxygen for 72h. Total cellular RNA was extracted and subjected to a Methylated RNA immunoprecipitation (MeRIP) assay and BCP-RNA and control CREBBP RNA measured by qPCR. Data are presented as the mean ± S.D. of n = 6 samples from 3 independent experiments with statistical significance determined using Mann-Whitney tests with Bonferroni correction for multiple comparisons. ** p < 0.01. ( F ) HepG2-NTCP cells were transfected with siRNAs (NC; Non-targeting, ALK; ALKBH5) for 6h, followed by delivery of HBV WT plasmid and cultured at 18% or 1% oxygen for 72h. RNA was extracted and BCP-RNA levels quantified by qRT-PCR. Data are expressed relative to a house keeping gene B2M and presented as the mean ± S.D. of n = 9 samples from 3 independent experiments. Statistical significance was determined using Mann-Whitney tests with Bonferroni correction for multiple comparisons. * p < 0.05, **** p < 0.0001.

Article Snippet: Primary antibodies used in this study are ALKBH5 (Atlas Antibodies), β Actin (Sigma), HIF-1α (BD Bioscience), HIF-2α (NOVUS), HIF-1β (NOVUS), NDRG1 (CST), HBc (DAKO), anti-m 6 A polyclonal antibody (Synaptic Systems).

Techniques: Cell Culture, Expressing, MANN-WHITNEY, Modification, Northern Blot, Methylation, Transfection, Plasmid Preparation, RNA Immunoprecipitation, Control, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome

doi: 10.1371/journal.ppat.1011917

Figure Lengend Snippet: ( A ) HIF-α expression in ALKBH5 silenced cells. HepG2-NTCP cells were transfected with ALKBH5 specific siRNAs for 48h and cultured under 1% oxygen for 12 or 24h. Samples were probed for HIF-1α, HIF-2α, NDRG1, ALKBH5 or β-Actin proteins and expression quantified by densitometry. Data is expressed relative to 24h hypoxic siNC samples and represents the mean ± S.D. from 3 independent experiments. Statistical significance was determined using a two-way ANOVA. * p < 0.05, ** p < 0.01. ( B ) HIF-1α and HIF-2α mRNAs were measured by qRT-PCR and data presented relative to siNC hypoxic conditions. ( C ) Expression of HIF-regulated genes in ALKBH5 silenced cells. HepG2-NTCP cells were cultured as listed in and the indicated gene transcripts quantified by qRT-PCR with data expressed relative to siNC under hypoxic conditions. Data represent the mean ± S.D. of samples from 3 independent experiments. ( D ) HIF-1α protein expression in ALKBH5 silenced cells. HepG2-NTCP cells were cultured in 1% oxygen for 24h and treated with cycloheximide (CHX, 20 μg/mL) or MG132 (20 μM) for the indicated times. Samples were collected and probed for HIF-1α, HIF-2α, ALKBH5 and β-Actin by western blotting. Note: Different exposure times were used for the upper and lower panels.

Article Snippet: Primary antibodies used in this study are ALKBH5 (Atlas Antibodies), β Actin (Sigma), HIF-1α (BD Bioscience), HIF-2α (NOVUS), HIF-1β (NOVUS), NDRG1 (CST), HBc (DAKO), anti-m 6 A polyclonal antibody (Synaptic Systems).

Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: PLOS Pathogens

Article Title: The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome

doi: 10.1371/journal.ppat.1011917

Figure Lengend Snippet: 1. Hypoxia increases ALKBH5 protein expression and global reduction in m 6 A methylated host and HBV BCP RNAs. 2 . Hypoxia increases HDAC4 expression and stabilizes HIF-1α and HIF-2α expression that can activate HBV transcription via binding to the basal core promoter. 3. Reduced m 6 A methylation increases the half-life of HBV BCP-RNAs.

Article Snippet: Primary antibodies used in this study are ALKBH5 (Atlas Antibodies), β Actin (Sigma), HIF-1α (BD Bioscience), HIF-2α (NOVUS), HIF-1β (NOVUS), NDRG1 (CST), HBc (DAKO), anti-m 6 A polyclonal antibody (Synaptic Systems).

Techniques: Expressing, Methylation, Binding Assay