Fig 2: Mice homozygous for the floxed (“2lox”) allele show heart defects.(A) Genotyping of late stage embryos. Genomic DNA was isolated from 6 representative E18.5 embryos, and the Aof2 allele analyzed by PCR genotyping. Both the wild-type (253 bp) and 2lox (392 bp) alleles can be observed, with two animals homozygous for the 2lox allele. The size, in base pairs, of DNA size standard markers is indicated on the left of the image. (B) Embryos homozygous for the 2lox allele survive to birth, but die shortly thereafter, as shown by the lack of homozygous pups at day P21. The data represents the percent of each genotype present at the indicated time point; for E13.5, this is the total of 3 litters, while E18.5 and P21 represent data from 6 pooled litters each. (C) Representative photomicrograph (H&E staining) illustrating a ventricular septal defect in an E18.5 Aof2 2lox/1lox pup at low magnification (upper right hand panel; 1.0x) and higher magnification (lower right hand panel; 3.0x) compared to a wild type control animal (upper and lower left hand panels). (D) Sequence comparison of the wild-type and 2lox Aof2 alleles. Sequencing results in the regions with the two point mutations are presented for Aof2wt and Aof22lox alleles. The corresponding base pair sequence is written above the respective graphs. The A to G mutations at positions 1379 and 1483 (positions based on mRNA sequence) are highlighted in red, and indicated by the arrows.

Fig 3: Loss of H3K4me2 increases GS expression in adipose cells. (A) Fold change of individual amino acid (AA) omitted from PC of WT and TgGS mice (n = 3, *P < 0.05, **P < 0.01). (B) Schematic diagram of methionine cycle. (C) The protein levels of GS, LSD1, H3K4me2, and H3 in non-treated or 5FU-treated mouse adipose cells cultured with NM or CT26 cell supernatants were measured with Western blot. (D) WT mice and TgGS mice were intraperitoneally inoculated with MC38 cells and treated with 5FU or 5FU+GSK-LSD1 for 2 weeks, respectively. Then, the representative images of the mic-bearing tumors were showed. Arrow heads, tumors. (E) The tumors in (D) were isolated and weighed (n = 5, **P < 0.01). (F) Kaplan-Meier curves for the survival of 5FU-treated or 5FU+GSK-LSD1-treated WT mice or TGGS mice after MC38 tumor cell inoculation plotted against time (days after injection) (n = 10, **P < 0.01).

Fig 4: Domains of LASP1 differentially associate with Snail1 and its stabilizing proteins. (A) Top panel–Affinity purified full length LASP1 and its domains fused to GST were separated by 10% SDS-PAGE. Proteins were visualized by staining the gel with Imperial Blue Protein Stain; Bottom panel—A schematic depiction of the constructs of recombinant full length of LASP1 and its various domains that were bacterially expressed (B) Association of LASP1 and its domains with Snail1 upon stimulation of stably expressed CXCR4 by CXCL12. Notably, 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 200 µg of nuclear lysate derived from 293-CXCR4 cells that were transiently expressing Flag-Snail1-WT and stimulated with 20 nM CXCL12. The association of Flag-Snail1 with full length LASP1 and its domains were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3 (C) and (D) Association of LASP1 and its domains with proteins that contribute to the stabilization of the Snail1. Nuclear lysates were prepared from MDA-Bone-Un cells that were serum-starved and stimulated with 20 nM CXCL12. 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The association of endogenous A20, LSD1, AKT and GSK-3ß with full length LASP1 and its domains were analyzed by 10% SDS-PAGE followed by immunoblotting; n = 3.

Fig 5: H2A.Z binds to the upstream region of the SFRP1 promoter and reduces SFRP1 expression. A Graphical representation of the CpG sites in the SFRP1 promoter. B ChIP assay of KDM1A, H3K4me1 and H3K4me2 enrichment in the P3 region upstream of the SFRP1 promoter. IgG was used as the negative control. Relative fold enrichment = [%(ChIP/Input)]/[%(IgG/Input)]. C BSP analysis of the CpG site methylation rate. Each dot represents one CpG site. White represents the absence of methylation, and pale green to deep yellow represent increasing methylation rates. D Quantitative RT–PCR analysis of the SFRP1 mRNA transcript level relative to the GAPDH mRNA transcript level in the blank control, LV-shRNA-NC, LV-shR-H2A.Z and LV-NC-H2A.Z + 5-aza-dC groups. The data are expressed as the individual mean values or the mean ± SD of each group in three independent experiments. **p < 0.01, ***p < 0.001