H9627

The transwell invasion assay was performed as previously described [39 (link)]. Briefly, cell culture inserts (6.5 mm, Falcon insert, 8-μm pore size; Becton Dickinson, Franklin Lakes, NJ, USA) were pre-coated with Matrigel (#356231, CORNING) diluted 1:20 in serum-free DMEM/F12 medium. Cells were accutased and resuspended in serum-free DMEM/F12 medium in the upper chamber (50,000 cells in 250 μL) and 800 μL of NSC medium containing growth factors was added to the lower chamber. After a 72-h incubation, cells remaining on the upper surface of the filter were removed, and cells that had invaded through the Matrigel to the lower surface were fixed with ethanol, stained with hematoxylin ((H9627, Sigma-Aldrich), and photographed. Data represent the mean of the number of cells counted in five random low-power fields (LPFs).
For transwell migration assays, NIH3T3 cells were cultured in DMEM/F12 media supplemented with 10% (v/v) FBS for 24 h, and the media was then used as conditioned media to attract cells through cell culture inserts precoated with gelatin (G1393, Sigma-Aldrich). After a 48-h incubation, cells that had migrated to the lower surface were fixed with ethanol, stained with hematoxylin (H9627, Sigma-Aldrich), and photographed. Data represent the mean of the number of cells counted in five random high-power fields (HPFs).

Formalin-fixed paraffin-embeded sections (4 µm) of whole emrbyos were stained with hematoxylin (Sigma, H9627) for 40 s and with eosin (Sigma-Aldrich, HT110116) for 30 s. The tissue sections were mounted with Permount mounting medium (Fisher Scientific, SP15-100). All procedures were conducted by Molecular Pathology Core, University of Florida. Images were generated with a Zeiss Axio Imager 2 microscope (Carl Zeiss, Inc., Thornwood, NY).

Formalin-fixed paraffin-embeded sections (4 μm) of the spleen, lymph node, and inguinal WAT were stained with hematoxylin (Sigma, H9627) for 40 s and with eosin (Sigma-Aldrich, HT110116) for 30 s. The tissue sections were mounted with Permount mounting medium (Fisher Scientific, SP15–100). All procedures were conducted by Molecular Pathology Core, University of Florida. Images were generated with a Zeiss Axio Imager 2 microscope (Carl Zeiss, Inc, Thornwood, NY). For adipose tissue analysis, adipocytes were counted for four nonoverlapping fields of view for three Dnmt3b^+/+ and Dnmt3b^CI/CI mice. Cells with single large lipid droplet were considered white adipocytes and cells with multiple small droplets—brown.