Fig 1.
Dlc1 is Associated with PPARγ Defined Super-enhancers in both White and Brown Adipocytes.
(A) Venn diagram showing the overlap of the Top-100 super-enhancer associated genes in both white and brown adipocytes. Genome-wide binding profiles of PPARγ were used to define SEs. Representative SE associated genes with their ranks in the corresponding lineages were shown. (B) PPARγ binding peaks and their defined SEs at the Dlc1 gene locus in brown adipocytes, white adipocytes, brown adipose tissue and epididymal white adipose tissue. (C) ENCODE RNA-seq data showing the expression of Dlc1 gene in various mouse tissues.
Fig 2.
DLC1 is Required for White Adipogenic Differentiation.
(A) mRNA expression of Dlc1 gene during differentiation of 3T3-L1 white adipocytes. (B) qRT-PCR result showing the knockdown of Dlc1 gene expression in 3T3-L1 white adipocytes. Tbp was used as normalizing control for gene expression. (C) Oil-Red-O staining showing reduced lipid droplet formation in Dlc1 knockdown cells. (D) mRNA expression of adipogenic marker genes Adiponectin (AdipoQ) and Fabp4 in si-Dlc1 white adipocytes. (E) qRT-PCR result showing the knockdown of Dlc1 gene expression in primary iWAT SVF cells derived white adipocytes. (F) Oil-Red-O staining showing reduced lipid droplet formation in primary iWAT SVF cell derived white adipocytes upon Dlc1 knockdown. (G) mRNA expression of adipogenic marker genes Adiponectin and Fabp4 in si-Dlc1 white adipocytes derived from primary iWAT SVF cells. (H) Western blotting analysis of mature adipocyte marker perilipin in primary iWAT SVF cell derived white adipocytes upon Dlc1 knockdown. Data are presented as mean ± s.e.m. n = 3–4 biological replicates. Two-tailed Student’s t-test was used: ** P < 0.01.
Fig 3.
DLC1 Regulates Brown Adipogenesis and Brown Cell Function.
(A) mRNA expression of Dlc1 gene during differentiation of BAT-WT1 brown adipocytes and C3H10T1/2 derived brown adipocytes. (B) mRNA expression of Dlc1, general adipogenic, BAT-specific and mitochondrial genes in BAT-WT1 brown adipocytes upon Dlc1 knockdown. (C) Oil-Red-O staining showing reduced lipid droplet formation in Dlc1 knockdown BAT-WT1 brown adipocytes. (D) Oxygen consumption rates (OCR) in BAT-WT1 brown adipocytes with or without Dlc1 knockdown. Vertical green lines indicate the time points of Oligomycin, FCCP and Rotenone/Antimycin A (ROT/AA) injection. n = 3. (E) qRT-PCR measurements of the fold change of Ucp1 expression after isoproterenol treatment compared with basal levels without treatment. Dlc1 knockdown significantly impaired the upregulation of Ucp1 expression after isoproterenol treatment. (F) mRNA expression of Dlc1, general adipogenic and BAT-specific genes in Dlc1 knockdown brown adipocytes derived from C3H10T1/2 cells. (G) Oil-Red-O staining showing reduced lipid droplet formation in Dlc1 knockdown brown adipocytes derived from C3H10T1/2 cells. Data are presented as mean ± s.e.m. n = 3–6 biological replicates. Two-tailed Student’s t-test was used: * P < 0.05, ** P < 0.01.
Fig 4.
Adipogenic Role of DLC1 Involves the Rho Pathway.
(A) Western blots showing the absence of DLC1 protein in Dlc1-KO MEFs. Calnexin was included as a loading control. (B) Oil-Red-O staining of differentiated wild-type and Dlc1-KO MEFs. (C) Relative mRNA expression of adipogenic and BAT-specific genes in differentiated MEFs. (D) Western blots showing active RhoA levels in Dlc1-KO MEFs. Total RhoA was shown as a loading control. Band intensity was quantified using ImageJ and the ratio between Active / Total RhoA was presented as bar graph in the right panel. (E) Phalloidin staining of F-actin in MEFs (left panels) and quantification of F-actin stress fibers (right panel). n = 15. (F) Proposed model of DLC1-Rho pathway and the site of action for ROCK inhibitor Y-27632 and F-actin inhibitor Latrunculin-B. (G) Oil-Red-O staining to show the rescue of lipid droplet formation by the ROCK and F-actin inhibitors in Dlc1-KO MEFs. (H) qRT-PCR showing the restoration of adipogenic gene expression. Data are presented as mean ± s.e.m. n = 2–3 biological replicates. Two-tailed Student’s t-test was used: * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 5.
Dlc1 is a Direct Target of PPARγ.
(A) PPARγ ChIP-seq data revealed direct binding of this master regulator of adipogenesis at the promoter of Dlc1 gene in mouse BAT, eWAT, 3T3-L1 white adipocytes and C3H10T1/2 derived brown adipocytes. The positions (Relative to the transcription start site of Dlc1 gene) of PPARγ binding peaks (P1-P3) are indicated under the graph. (B) PPARγ ChIP confirmed its binding to Peak 1–3 at the promoter/ upstream region of Dlc1 gene in C3H10T1/2 cells (Brown adipocytes). Fabp4 gene promoter and a chromosome 15 (Chr.15) region were used here as positive/negative controls for PPARγ ChIP. (C-F) qRT-PCR results showing the knockdown of Pparg and the reduced expression of Dlc1 gene in (C) 3T3-L1 white adipocytes, (D) primary iWAT SVF cell derived white adipocytes, (E) BAT-WT1 brown adipocytes and (F) C3H10T1/2 derived brown adipocytes. Data are presented as mean ± s.e.m. n = 3–6 biological replicates. Two-tailed Student’s t-test was used: * P < 0.05, ** P < 0.01.