Fig 2: SLC35D3 promotes white adipose tissue browning to ameliorate obesity via NOTCH signaling.SLC35D3 regulates obesity by interacting with the extracellular domain (ECD) of NOTCH1, which promotes NOTCH1 accumulation in the endoplasmic reticulum (ER) and inhibits NOTCH1 transport to the cell membrane, ultimately inhibiting NOTCH1 signaling. IngWAT, inguinal white adipose tissue.

Fig 3: SLC35D3 increased the ER accumulation of NOTCH1 by interacting with the NOTCH1 extracellular domain.a, b CoIP assay results showed the interaction between SLC35D3 and NOTCH1 (a), as well as the interaction between SLC35D3 and ECD (b). Representative images from three independent experiments are shown. c Direct interaction between SLC35D3 and ECD was further confirmed by pull-down assay. Representative images from three independent experiments are shown. d, e SLC35D3 overexpression promoted the colocalization of NOTCH1 and ER. Representative confocal images of cells are shown (d), as well as the quantification results of four independent parallel experiments (e). The inset in the right panel shows a magnified view of the indicated area. Scale bar, 20 μm. CALNEXIN, an ER marker protein. f, g SAKO mice showed higher levels of Notch1 at the cell membrane in IngWAT than control mice. Representative confocal images are shown (f), as well as the quantification results of six independent parallel experiments (g). Scale bar, 50 μm. Na-K ATPase, a plasma membrane marker protein. h–j Membrane proteins and endoplasmic reticulum proteins of IngWAT were isolated from SAKO and SAKI mice (n = 3 per group). The western blot results are shown (h), as well as the quantitative analysis results (i, j). Total NOTCH1 expression was normalized to β-Actin expression, membrane NOTCH1 expression was normalized to Na-K ATPase, and ER NOTCH1 expression was normalized to CALNEXIN. k–m Primary adipocytes were isolated from the IngWAT of SAKO mice and SAKI mice (n = 3 per group). Cell surface biotinylation assay results are shown (k), as well as the quantitative analysis results (l, m). Data are expressed as the mean ± SEM. Statistical significance was assessed by unpaired two-sided Student’s t test (e, g, i, j, l, m). The exact P values are shown in the figure. SAKO Slc35d3 adipocyte-specific knockout, SAKI Slc35d3 adipocyte-specific knock-in, OE overexpression, IngWAT inguinal white adipose tissue, ER endoplasmic reticulum. Source data are provided as a Source Data file.

Fig 4: SLC35D3 inhibited the activation of NOTCH1 signaling.a KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis results of IngWAT from WT and SAKI mice (n = 3 per group). P values are determined using the clusterProfiler R package’s built-in hypergeometric test, and adjusted P values are calculated using Benjamini–Hochberg correction. b GSEA (gene set enrichment analysis) results showed that the Notch signaling pathway was suppressed in the IngWAT of SAKI mice (n = 3 per group). The P values in GSEA are calculated empirically with permutation test by permuting the gene labels at random according to the GSEA algorithm, and FDR q values (corrected P values) are calculated by Benjamini–Hochberg FDR correction. The NES and FDR q values are shown in the images. NES, normalized enrichment score; FDR, false positive rate. c, e qPCR analysis of Slc35d3 and Notch1-related genes in IngWAT from SAKI mice and WT mice (c), as well as in IngWAT from SAKO mice and Flox mice (e); n = 6 per group. d, f Western blot showing the expression of Notch1-related proteins in IngWAT from SAKI mice and their controls (d), as well as in IngWAT from SAKO mice and their controls (f); n = 3 per group. Right panel, summary of the quantification of three independent experiments. g qPCR detection of NOTCH1 expression levels in HEK293T cells overexpressing SLC35D3; n = 6 per group. h Western blot analysis of NOTCH1 protein in HEK293T cells overexpressing SLC35D3. Right panel, summary of the quantification of three independent experiments. i Western blot analysis of NOTCH1 protein in HEK293T cells treated with CHX (cycloheximide). Right panel, summary of the quantification of three independent experiments. Values are the fold induction of gene expression normalized to the housekeeping gene β-Actin (c–i). Data are expressed as the mean ± SEM. Statistical significance was assessed by unpaired two-sided Student’s t test (c–i). The exact P values are shown in the figure. SAKI Slc35d3 adipocyte-specific knock-in, IngWAT inguinal white adipose tissue. Source data are provided as a Source Data file.