Selected pathways over-represented among differentially expressed genes between OIR and OIS women^a.

<p>Selected pathways over-represented among differentially expressed genes between OIR and OIS women<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178485#t002fn001" target="_blank">^a</a>.</p

Effects of <i>KLF15</i> and <i>SLC25A10</i> knockdownon lipogenesis <i>in vitro</i>.

<p><b>A.</b><i>KLF15</i> and <i>SLC25A10</i> were knocked down using 40nM of siRNA in SVF-derived human adipocytes differentiated <i>in vitro</i> and expression of the genes evaluated using real-time PCR. Results were analyzed using Students t-test and are presented as relative fold change ± SD vs. negative control. <b>B.</b> SVF-derived adipocytes differentiated <i>in vitro</i> were transfected with 40 nM of siRNA against <i>KLF15</i> and <i>SLC25A10</i> for 48 hours followed by evaluation of basal and insulin-stimulated lipogenesis. Relative insulin-stimulated lipogenesis was calculated against non-targeting siRNA NegC at insulin-stimulated state. Induction of lipogenesis by insulin for NegC was minimum 3-fold in all experiments. Results are based on three to five biological/independent experiments.*p<0.05, **p<0.01 and ***p<0.001.</p

Clinical characteristics.

<p>Clinical characteristics.</p

Genes associated with adipocyte IR also associated with systemic insulin resistance or T2D.

<p>Genes associated with adipocyte IR also associated with systemic insulin resistance or T2D.</p

Glucose uptake and insulin signaling genes differentially expressed between OIR and OIS women.

<p>Glucose uptake and insulin signaling genes differentially expressed between OIR and OIS women.</p

Transfection screening of microRNAs identifies TNF-α as a key mediator of miRNA effects on human adipocyte lipolysis.

<p>(A) Human differentiated adipocytes were transfected with each individual miRNA Mimics or Negative Control (40 nM) for 48 h as described in experimental procedures. After 48 h, conditioned media was collected and glycerol release and TNF-α secretion were measured. Glycerol levels were evaluated by a bioluminescence method in three to six biological/independent experiments. TNF-α secretion values were detected by ELISA in at least, two biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. Statistical differences were analyzed by Student t-test comparing Mimics Neg. Cntl <i>vs</i>. Mimics of each miRNA: *p<0.05; **p<0.01; ***p<0.001. (B) MiR-145 was over-expressed in human differentiated adipocytes in a time-course experiment. Conditioned medium was collected at selected time-points post-transfection (6 h –12 h –24 h –48 h) for determination of glycerol (black line) and TNF-α secretion (black-broken line) levels. Cells were harvested for RNA and TNF-α mRNA expression (grey line) levels were determined. Results are indicative of three biological/independent experiments. Transfection efficiency showed ∼2×10⁴ fold-change up-regulation of individual miR-145 as compared to control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086800#pone.0086800.s001" target="_blank">Figure S1</a>). Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. of each time-point. Statistical differences were analyzed by Student t-test comparing Mimics miR-145 <i>vs</i>. corresponding Neg. Cntl at each indicated time-point: *p<0.05; **p<0.01; ***p<0.001.</p

miR-145 does not affect phosphorylation and protein content of PLIN1.

<p>(A) Representative blot of total protein content of PLIN1 after miR-145 over-expression in human differentiated adipocytes for 48 h. PLIN1 protein content was corrected by tubulin as described in experimental procedures. (B) Relative quantification of PLIN1 <i>vs</i>. tubulin (n = 3).</p

miR-145 increases HSL phosphorylation at activating residues (but does not change total protein content of HSL) and down-regulates PDE3B.

<p>(A) Representative blots of protein expression levels of phosphorylated HSL (Ser-552 and Ser-650) and total HSL in human differentiated adipocytes transfected with miR-145 mimics for 48 h. (B) Relative quantification by densitometry of above depicted blots for p-HSL (Ser-552, dark grey; Ser-650, light grey) and (C) total HSL. Equal amounts of total protein were loaded and separated by SDS-PAGE as indicated in experimental procedures. Total HSL levels were corrected by tubulin expression and p-HSL levels were corrected by total HSL protein content. Values are shown as mean of pooling results from three biological/independent experiments. (D) Relative expression of PDE3B after miR-145 over-expression in human differentiated adipocytes for 48 h. Results are representative of four biological/independent experiments. Values are shown as mean ± SEM. Statistical differences (<i>vs</i>. Neg. Cntl) were analyzed by Student t-test: **p<0.01; ***p<0.001.</p

ADAM17 is a miR-145 target that regulates TNF-α processing in human adipocytes.

<p>(A) mRNA expression levels of ADAM17 after miR-145 over-expression at 6 h –12 h –24 h –48 h. Results presented are obtained from three biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. at each corresponding time-point. (B) MiR-145 was over-expressed in human differentiated adipocytes for 48 h and whole cell lysates were analyzed by Western blot. TNF-α protein levels are expressed as ratio of TNF-α membrane bound (26 KDa) <i>vs</i>. soluble form (17 KDa). Results are representative of two biological/independent experiments. Values are shown as mean ± SEM. Statistical differences (<i>vs</i>. Neg. Cntl) were analyzed by Student t-test: **p<0.01; ***p<0.001.</p

miR-145 alters TNF-α signaling and induces tightly correlated changes in lipolysis and TNF-α.

<p>(A) Human differentiated adipocytes were transfected with mimics miR-145 or Neg. Cntl (40 nM) for 15 h. After incubation time (15 h), nuclei were isolated and 2.5 µg of nuclear extract was used to perform p65 transactivation assay as described by manufacturer. Figure depicts representative wells of Neg. Cntl and miR-145-treated adipocytes and its relative quantification graph of three biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. Statistical differences were analyzed by Student t-test: *p<0.05. (B) TNF-α receptor 1 (TNFR1) was silenced with siRNA for 24 h prior to co-transfection with miRNA Mimics (Neg. Cntl/miR-145) for additional 48 h in human differentiated adipocytes. After incubation time, conditioned medium was collected to measure glycerol release and cells were harvested for (C) TNF-α mRNA measurements. Results are representative of three biological/independent experiments. Values are shown as mean ± SEM. Statistical differences (<i>vs</i>. Neg. Cntl) were analyzed by Student t-test: **p<0.01; ***p<0.001. (D) Correlation between glycerol <i>vs</i>. TNF-α secretion (measured in conditioned medium/supernatant) and <i>vs</i>. (E) TNF-α mRNA levels in miR-145-transfected adipocytes <i>in vitro</i> for 48 h. Values are expressed as relative fold change <i>vs</i>. Neg. Cntl. (in D and E) after correction by transfection efficiency and were compared by linear regression. Each dot represents a technical replicate from 7–8 biological/independent experiments.</p