EMBO/soda-nel huggingface dataset:
m6A methylation promotes white to beige fat transition by facilitating Hif1a translation (2021)
Figure 5

Figure 5. FTO Regulates the Expression of HIF1A in an m6A-Dependent Manner.

(A) Western blot analysis of the protein levels of FTO, HIF1A, UCP1 and PPARGC1A in differentiated beige adipocytes from Ftoflox/flox mice infected with retroviruses MSCVhygro expressing Vec, Cre or together with lentiviruses pCDHpuro expressing wild-type (WT)- or mutant (MUT)-FTO.

(B) HPLC-QgQ-MS/MS quantification of the m6A/A in mRNA of siCtrl or siFto 3T3-L1 cells. FTO knockdown efficiency was measured by Western blot.

(C) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A peaks were in the dashed red box.

(D) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells.

(E) Western blot analysis of FTO in input and HA-RIP groups.

(F) RIP-qPCR analysis of the association of Hif1a with HA in 3T3-L1 cells overexpressing HA-tagged Vec or FTO. Enrichment of Hif1a with HA was measured by qPCR and normalized to input.

(G) Synonymous mutation sites in Hif1a.

(H) Western blot analysis of the protein levels of HIF1A, UCP1, PPARGC1A, PPARG and GFP in 3T3-L1 cells infected with lentiviruses pCDHpuro expressing Vec, WT- or MUT-HIF1A.

Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.
m6A methylation promotes white to beige fat transition by facilitating Hif1a translation (2021)
Figure 6

Figure 6. m6A reader YTHDC2 Mediates mRNA Translation of Hif1a.

(A) Western blot analysis of the protein levels of YTHDF1, YTHDF2 and HIF1A in siCtrl, siYthdf1 and siYthdf2 3T3-L1 cells.

(B) Western blot analysis of the protein levels of HIF1A in siCtrl, siIgf2bp1, siIgf2bp2 and siIgf2bp3 3T3-L1 cells.

(C) Western blot analysis of the protein levels of YTHDC2 and HIF1A in siCtrl and siYthdc2 3T3-L1 cells.

(D) Western blot analysis of the protein levels of YTHDC2 and HIF1A in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid.

(E) Western blot analysis of YTHDC2 in input and FLAG-RIP groups.

(F) RIP-qPCR analysis of the association of Hif1a with FLAG in 3T3-L1 cells overexpressing FLAG-tagged Vec or YTHDC2. FLAG was measured by qPCR and normalized to input.

(G) Polysome profiling of 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid.

(H) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid.

(I) Polysome profiling of siCtrl and siYthdc2 3T3-L1 cells.

(J) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in siCtrl and siYthdc2 3T3-L1 cells.

Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.
Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation (2022)
Figure 1

Figure 1. The RALF-FER module is involved in PSR-mediated immunity suppression in Arabidopsis.

(A) flg22-induced MAPK activation in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22 for 15 mins and roots were harvested. The MAPK level was determined with anti-pMAPK, and β-actin was used as a loading control.

(B) flg22-induced ROS production in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22, RALF23 or both flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown.

(C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test).

(D) Heatmap of differentially expressed defense-responsive genes after LP or RALF23 treatment (n = 3, n refers to technical replicates). Seven-day-old Col-0 seedlings were treated with either LP for 5 days or 1 μM RALF23 for 2 hours.

(E) Density figure showing the comparison of defense-responsive genes between LP treatment and RALF23 treatment (n = 3, n refers to technical replicates). The x- and y-axes represent changes in transcription level, and the red area indicates high density. Spearman correlations were used for the correlation analysis.

(F) RALF23 inhibits flg22-induced MAMP-responsive FRK1 gene expression in a Pi content-dependent manner. Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested. The relative expression of FRK1 was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test).

(G) Growth of Pto DC3000 in Col-0 and RALF23-OE lines under Pi-starvation conditions. Seedlings grown in either HP or LP media were root soaking inoculated with bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual roots was quantified at 0 dpi and 3 dpi. The data shown indicate the means ± SDs (n = 27, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test).

Data information: All experiments were repeated three times with similar results.
Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation (2022)
Figure 2

Figure 2. PHR1 targets and activates RALF expression under Pi-starvation conditions.

(A) The PSR induces the cleavage of PRORALF23-GFP into mature RALF23-GFP. The 35S promoter-driven RALF23-GFP line was grown in either HP or LP media for 5 days and roots were harvested. Premature and mature RALF23 levels were determined with anti-GFP. β-actin was used as a loading control.

(B) The PSR activates RALF expression under LP conditions. Col-0 seedlings were grown in either HP or LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test).

(C) Expression pattern of RALFs in the shoots and roots under Pi-starvation conditions. The microarray data were obtained from Bustos et al, 2010.

(D) PHR1-binding site (P1BS, GNATATNC) prediction within 1000 bp of certain RALF promoters. The positions are relative to the start codon (+1).

(E) The PSR activates RALF expression in a PHR1-dependent manner. Col-0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test).

(F) PHR1 activates RALF promoters in plants. Depicted RALF promoter-LUC fusions (RALF24 as a negative control) were co-expressed together with 35S promoter-driven PHR1 or an empty vector control in Arabidopsis protoplasts. The relative LUC/REN reporter activity was quantified. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test).

(G) PHR1 directly binds to RALF promoters, as shown via ChIP-PCR. The Myc-labelled PHR1 overexpression line was immunoprecipitated with anti-Myc. ChIP analysis was performed on Col-0, CRY1-Myc and PHR1-Myc transformed plants, with Col-0 and CRY1-Myc transformed plants as negative controls. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test).

Data information: All experiments were repeated three times with similar results.
Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation (2022)
Figure 4

Figure 4. Analysis of bacterial colonization after LP stress and delivery of RALF23 by B. subtilis treatment.

(A-B) Relative Pseudomonas abundance in rhizosphere samples after (A), Pi-stress (LP) or (B) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).

(C-D) Relative Bacillus abundance in rhizosphere samples after (C), Pi-stress (LP) or (D) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test).

(E-F) Relative Geodermatophilus abundance in rhizosphere samples after (E), Pi-stress (LP) or (F) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test).

(G-H) Relative Methylobacillus abundance in rhizosphere samples after (G), Pi-stress (LP) or (H) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).
Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation (2022)
Figure 5

Figure 5. Bacterial colonization alleviates LP stress.

(A-B) Bacterial inoculation promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B. subtilis. (A), Images were taken 4 weeks after inoculation. (B), The seedlings were harvested, and the shoot fresh weight was measured 4 weeks after inoculation. The data shown indicate the means ± SDs (n = 30, n refers to the number of seedlings per group), Central bands: median, boxes: interquartile range, whiskers: max./min; n.s., not significant; *, p < 0.05, **, p < 0.01 (Student's t test).

(C) Pto DC3000 and B. subtilis upregulate phosphate absorption gene expression. Col-0, phr1 and fer-4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B. subtilis in Pi-sufficient or Pi-deficient condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); *, p < 0.05; **, p < 0.01 (Student's t test).

Data information: All experiments were repeated three times with similar results.
Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation (2022)
Figure 6

Figure 6. The fer-4 rhizosphere microbiome promotes growth in the next generation of plants under LP conditions.

(A) Venn diagram showing the total and overlapping numbers of genera enriched in the fer-4 mutant and under Pi starvation.

(B-C) The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (B), Images were taken 4 weeks after inoculation. (C), The seedlings were harvested, and the shoot fresh weight was measured 4 weeks after inoculation. The data shown indicate the means ± SDs (n = 7, n refers to the number of flowerpots (4 seedlings per flowerpot) per group). The different lowercase letters indicate statistical significance (one-way ANOVA).

(D) The fer-4 rhizosphere microbiome upregulated phosphate absorption gene expression. The roots of Col-0 were harvested after 4 weeks of inoculation with the Col-0 or fer-4 rhizosphere microbiome under LP condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test).

Data information: All experiments were repeated three times with similar results.
E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2 (2022)
Figure 3

Figure 3. NEDD4L inhibits IL-17R signaling and MEKK2 expression.

(A-D) Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in HeLa cells transfected with Nedd4l siRNA (siNedd4l) or control RNA (siNC) (A), Nedd4l+/+ and Nedd4l-/- MEFs (B), primary lung epithelial cells (C), or Nedd4l+/+ and Nedd4l+/- astrocytes (D) , treated with IL-17 for indicated time.

(E) Immunoblot analysis of phosphorylated p65 subunit of NF-κB (P-P65), total P65, phosphorylated p38 (P-P38), total P38, MEKK2, NEDD4L, and actin in HeLa cells transfected with Myc-tagged NEDD4L，Myc- tagged NEDD4L-CA(C942A), or Myc- tagged Null plasmids, treated with IL-17 for indicated time.

(F)Immunoblot analysis of MEKK2, TRAF2, TRAF6, NEDD4L, and actin in Nedd4l+/+, Nedd4l+/-, and Nedd4l-/- MEFs.

(G)Immunoblot analysis of MEKK2 in lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with CHX (50 μg/ ml) for indicated time.

(H)The intensity values were measured by Image J software to calculate half-life of MEKK protein ( n = 3 biological replicates).

Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown. Significant differences were tested using a two-tailed Student's t-test. *P<0.05, **P<0.01. NS, no significance.
E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2 (2022)
Figure 4

Figure 4. NEDD4L ubiquitinates MEKK2 and inhibits the protein stability of MEKK2.

(A)Immunoblot analysis of NEDD4L, MEKK2, and MEKK3 immunoprecipitated with NEDD4L-specific antibody from lysates of HeLa cells pre-treated with 20μM MG-132 for 6 h and then treated with IL-17 for the indicated time.

(B)Immunoblot analysis of NEDD4L precipitated with GST-tagged Null or GST-tagged MEKK2 recombinant protein.

(C)Immunoblot of HA-tagged MEKK2 co-immunoprecipitated with anti-Myc antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted.

(D)Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l+/+, and Neddl4- /- treated with DMSO, 20μM MG-132, or 0.2 μM bafilomycin A1 (Baf A1) for 6 h. Densitometry of MEKK2 was relative to actin.

(E)Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro.

(F)Immunoblot analysis of K48-, K63-linked, and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2-specific antibody from lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with MG-132.

(G)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following precipitation of MEKK2 with Flag-tagged specific antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted.

(H)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6O, K11O, K27O, K29O, K33O, K48O, K63O mutant Ub.

(I)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6R, K11R, K27R, K29R, K33R, K48R, K63R mutant Ub.

(J)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, increased dosage of Myc-tagged NEDD4L, and HA-tagged wild-type, K27O, or K63O mutant Ub.

Data information: All experiments were performed three times, and representative data are shown.
E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2 (2022)
Figure 5

Figure 5. NEDD4L inhibits IL-17R signaling by targeting MEKK2.

(A, B) Immunoblot analysis of phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in HeLa cells (A) and MEFs (B) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, MEFs 100ng/ml) for indicated time.

(C, D) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (C) and MEFs (D) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, MEFs 100ng/ml) for indicated time (n = 3 technical replicates).

(E) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), NEDD4L, MEKK2 and actin in HeLa cells transfected with control RNA (siNC), Nedd4l siRNA (siNedd4l), Mekk2 siRNA(siMekk2) or Nedd4l siRNA plus Mekk2 siRNA (siNedd4l+ siMekk2), and stimulated with IL-17(50 ng/ml) for indicated time.

(F) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (p-p65), NEDD4L, MEKK2 and actin in Nedd4l+/+ and Nedd4l-/-MEFs transfected with Mekk2 siRNA (siMekk2) or control RNA (siNc), and stimulated with IL-17 (100 ng/ml) for indicated time.

(G, H) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (G) and MEFs (H) treated as described in (E) and (F) (n = 3 technical replicates).

Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.
E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2 (2022)
Figure 6

Figure 6. IL-17-induced MEKK2 Ser520 phosphorylation via TAK1 and promotes NEDD4L-mediated MEKK2 ubiquitination.

(A)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL-17 (50 ng/ml) for indicated time, and then incubated with or without calf intestinal alkaline phosphatasetime (CIAP) for 30 mins.

(B) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells transfected with HA-tagged MEKK2 S520A or control vector, and stimulated with IL-17 (50 ng/ml) for indicated time (n = 3 technical replicates).

(C) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), HA, and actin in HeLa cells treated as in (B). Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively.

(D)Immunoblot analysis of MEKK2 and TAK1 co-immunoprecipitated with anti-TAK1 antibody from lysates of HeLa cells treated with 50ng/ml IL-17 for indicated time.

(E)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1-specific (siTak1) or control RNA (siNC), and then stimulated with IL-17 (50 ng/ml) for indicated time.

(F)Immunoblot analysis of Myc-tagged NEDD4L co-immunoprecipitated with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing Myc-tagged NEDD4L and HA-tagged wild-type MEKK2 or mutant MEKK2 (S520A).

(G)Immunoblot analysis of total ubiquitination of HA-tagged MEKK2 and HA-tagged MEKK2(S520A) following precipitation of MEKK2 with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 or HA-tagged MEKK2(S520A) together with Myc-tagged NEDD4L and Flag-tagged Ub.

(H)Immunoblot analysis of HA-tagged MEKK2, HA-tagged MEKK2 (S520A), Myc-tagged NEDD4L, and actin in HEK293T cells transfected with the indicated plasmids with anti-HA, anti-Myc, or anti-actin antibody.

Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.
Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference. (2016)
Figure 1

Figure 1. NTN1 and DAPK1 are hypermethylated and downregulated in human breast cancers.

A, B DNA methylation level of NTN1 (A) and DAPK1 (B) 5’ regions (Illumina’s HumanMethylation450K Array (HM450) from The Cancer Genome Atlas breast cohort) in paired breast tissues (normal: green circles, tumor: red circles), n = 92, Wilcoxon matched-pairs signed rank test, P = 0.004 and P = 0.0008.

C, D NTN1 (D) and DAPK1 (G) gene expression in paired breast tissues (normal: green bars, tumor: red bars, RNAseq from TCGA breast cohort, n = 112 and 114, respectively.

E, F Correlation between NTN1 (E) and DAPK1 (F) gene expression and DNA methylation in the breast cancer cohort (TCGA, n = 807). Pearson correlation, P = 6.7.10-5, r = -0.14 for NTN1 (A) and P<10-16, r = -0.32 for DAPK1 (B), respectively.

G, H Tumor/Normal DNA methylation ratio of NTN1 (G) and DAPK1 (H) in human breast tumors (data extracted from TCGA cohort, paired samples) according to gene expression (downregulated FC ≤ 0.5, down, n = 33, or upregulated FC ≥ 1.3, up, n = 16). P = 3.10-2 and P = 3.10-4 two-sided Mann Whitney test, for NTN1 and DAPK1, respectively.
Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference. (2016)
Figure 2

Figure 2. DNA methylation and demethylation in mammary cell lines.

A Methyl-Cap-seq read density profiles of the 5’ end of DAPK1, UNC5B, and NTN1 in MDA-MB-231 (blue) and HMLER (green) cells. Red boxes represent the CpG islands (CGis); light gray boxes the regions analyzed by bisulfite-PCR-sequencing; dark gray boxes the regions analyzed by parallel sequencing of amplicons from bisulfite modified DNA; and black boxes the exons and UTR. Chromosome coordinates of each gene are given (black lines).

B, C Gene expression was measured by Q-RT-PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 µM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. **** P<0.0001, two-tailed unpaired Student’s t-test.

D, E Measurement of DNA hypomethylation of the DAPK1 (D) and NTN1 (E) promoters after decitabine treatment of MDA-MB-231 and HMLER cells. Over 1960 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.