Fig 2: ALKBH1 demethylates DNA 6mA of HIF-1a.A, levels of the DNA 6mA modification were assessed via DNA dot blot in 3T3-L1 cells using a 6mA-specific antibody. Methylene blue detected DNA loading. ALKBH1 functions as a DNA demethylase in 3T3-L1 cells. si-Ctrl: control-siRNA 3T3-L1 cells, si-Alkbh1: Alkbh1 deficient 3T3-L1 cells, Lv-ALKBH1: ALKBH1-overexpressed 3T3-L1 cells, Vector: Empty vector in 3T3-L1 cells. B, enrichment of N6-mA peak on Hif-1a gene locus in Alkbh1 knockout embryonic stem cells. Data are from the published datasets GSE71866. C, ChIP-PCR analysis of immunoprecipitations of ALKBH1 associated Hif-1a fragments. D, representative images of Oil red O staining in 3T3-L1 cells and quantitation analysis (n = 3). Scale bar: 25 µm. E, Western blot analysis of HIF-1a, CEBPA and FABP4. Overexpression of HIF-1a partially rescued the decrease of adipogenic markers. F, qRT-PCR shows the mRNA level of Cebpa, Ppar?, Plin1, and Adipoq in si-Ctrl+Vector, si-Alkbh1+Vector, and si-Alkbh1+Lv-HIF1a group at 5 days of differentiation (n = 3). The p values were calculated by two-tailed Student' t test (C) and one-way ANOVA with Tukey's post hoc test (D and F). Scatter plots show individual data points ± SD.

Fig 3: Depletion of ALKBH1 inhibits adipogenic differentiation in vitro.A, Western blot analysis confirms successful knockdown of ALKBH1 in hMSCs (n = 3). B, CCK8 assay shows the viability of hMSCs is not affected after ALKBH1 depletion (n = 3). The absorbance was analyzed at 450 nm at 48, 72, and 96 h. C, representative images and quantitative analysis of oil red O staining. hMSCs were transfected with siRNA and subjected to adipogenic induction for 14 days. Lipid droplets were dissolved by isopropanol and quantified at 500 nm (n = 3). Scale bar: 25 µm. D, qRT-PCR analysis of the mRNA expression of adipogenic related genes CEBPA, PPARG, PLIN1, ADIPOQ after 2- and 5-day adipogenic differentiation (n = 3). E, Western blot shows successful knockdown of ALKBH1 in 3T3-L1 cells. F, CCK8 assay of viability of 3T3-L1 cells after knockdown of Alkbh1. G, representative images and quantitation of oil red O staining in 3T3-L1 cells after induction of 14 days (n = 3). Scale bar: 25 µm. H, qRT-PCR analysis of Cebpa, Ppar?, Plin1, Adipoq in Alkbh1-knockdown 3T3-L1 cells (n = 3). The p values were calculated by two-tailed Student's t test. Scatter plots show individual data points ± SD.

Fig 4: Overexpression of ALKBH1 promotes adipogenesis.A, Western blot confirms the successful overexpression of ALKBH1 in hMSCs. B, quantitative analysis of oil red O staining in hMSCs after 14 days of adipogenic differentiation (n = 3). Scale bar: 25 µm. C, qRT-PCR analysis of adipogenic related genes CEBPA, PPARG, PLIN1, ADIPOQ in ALKBH1-overexpressed hMSCs (n = 3). D, Western blot shows overexpression of ALKBH1 in 3T3-L1 cells. E, representative images and the OD values of oil red O staining in 3T3-L1 cells (n = 3). Scale bar: 25 µm. F, qRT-PCR analysis of Cebpa, Ppar?, Plin1, Adipoq in ALKBH1-overexpressed 3T3-L1 cells (n = 3). G, macroscopic identification and quantitative analysis of ectopic fats after 6 weeks of sternal subcutaneous injection. Nude mice were bilateral transplanted with Vector and Lv-ALKBH1 3T3-L1 cells (n = 6). Scale bar: 5 mm. H, hematoxylin and eosin staining (top) and GFP staining (bottom) of fat pads isolated from nude mice. Scale bar: 25 µm. The p values were calculated by two-tailed Student's t test. Scatter plots show individual data points ± SD.