iti 214  (MedChemExpress)

Journal: Science Advances

Article Title: Transcriptional determinants of lipid mobilization in human adipocytes

doi: 10.1126/sciadv.adi2689

Figure Lengend Snippet: ( A ) Representative images of cells stained with Hoechst (nuclei) and BODIPY (lipid droplets) at days 1 (top) and 14 (bottom) after adipogenic induction. Scale bars, 100 μm (merge) and 20 μm (inlay). Triglyceride (TG) quantification from the same time points is shown to the right. N.D., not detectable. ( B ) Isoprenaline (Iso.) and ANP responsiveness of adipocytes. Results are displayed as glycerol fold change versus unstimulated cells (basal). *** P < 0.001 from Tukey’s post hoc test after a one-way analysis of variance (ANOVA). ( C ) Representative immunofluorescence images of adipocytes transfected with nontargeting si Control or PLIN1 -targeting siRNA (si PLIN1 ). Cells were stained with Hoechst, BODIPY, and PLIN1 antibody. Scale bar, 20 μm. Glycerol release, PLIN1 intensity, and lipid droplets (LDs) per cell are presented to the right versus si Control . * P < 0.05 from a Welch’s two-tailed t test. Replicates are highlighted by dots and are based on at least three independent experiments. Bar charts are presented as means ± SEM.

Article Snippet: PDE1 inhibition was performed with 1 hour pre-incubation of adipocytes in media containing 1 μM ITI214 (MedChemExpress, HY-12501A), which was replaced with fresh assay media containing 1 μM ITI214 at the time of glycerol or cGMP induction.

Techniques: Staining, Immunofluorescence, Transfection, Control, Two Tailed Test

Journal: Science Advances

Article Title: Transcriptional determinants of lipid mobilization in human adipocytes

doi: 10.1126/sciadv.adi2689

Figure Lengend Snippet: ( A and B ) Representative Western blots of ZNF189, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cytosolic), and Lamin A/C (nuclear) in cytosolic (Cyt) and nuclear (Nuc) adipocyte fractions (A) and nuclear fractions from adipocytes electroporated with siRNAs targeting ZNF189 (si Z ) or nontargeting control (siC) (B). ( C to E ) Gene expression (C), adiponectin secretion (D), and glycerol levels (E) of siC and si Z adipocytes. Glycerol was measured in conditioned media following a 3-hour incubation under basal or stimulated conditions using either isoprenaline or ANP. ( F ) Overview of the ANP and isoprenaline signaling pathways. NPR, natriuretic peptide receptor; βAR, beta-adrenergic receptor; Gαs, Gs alpha subunit, AC = adenylate cyclase, PKG/PKA, protein kinase G and A; DG, diglyceride; MG, monoglyceride. ( G ) Representative Western blots for HSL phosphorylated at serine-660 (activating phospho-site; pHSL Ser660 ), total HSL (HSL), and GAPDH from basal, isoprenaline- and ANP-treated siC and si Z cells. ( H ) Principal components analysis of 307 lipid species measured by shotgun lipidomics of basal and ANP-stimulated siC and si Z adipocytes. Confidence ellipses display 95% confidence intervals. ( I ) Total triglycerides for the samples in (H) presented as fold change versus same siRNA basal companion. ( J and K ) Individual triglyceride species comparing siC versus si Z adipocytes. Volcano plots for ANP responsiveness of all triglycerides are shown in (J), and lipid abundance and ANP responsiveness are highlighted for the species with altered responsiveness following ZNF189 depletion in (K). In (J), adjusted P reflects FDR correction on multiple comparisons analyses performed using limma. Bar charts are presented as means ± SEM. In (C) to (E) and (I), replicates are highlighted by dots. For targeted analyses, data are based on at least three independent experiments. *** P < 0.001 for Welch’s t test [(C) and (I)] or Tukey’s multiple comparisons test (E).

Article Snippet: PDE1 inhibition was performed with 1 hour pre-incubation of adipocytes in media containing 1 μM ITI214 (MedChemExpress, HY-12501A), which was replaced with fresh assay media containing 1 μM ITI214 at the time of glycerol or cGMP induction.

Techniques: Western Blot, Control, Gene Expression, Incubation, Protein-Protein interactions

Journal: Science Advances

Article Title: Transcriptional determinants of lipid mobilization in human adipocytes

doi: 10.1126/sciadv.adi2689

Figure Lengend Snippet: ( A ) Volcano plot for transcriptomic analysis of ZNF189 -depleted adipocytes (si Z ) versus control cells (siC). Adj. P reflects FDR correction on multiple comparisons analyses performed by limma. ( B ) Overrepresentation analysis for results from (A). ( C ) Peak density plots of average peak values shown atop heatmaps of all ZNF189 peaks in immunoglobulin G (IgG) (left) and ZNF189 (right) chromatin immunoprecipitation followed by sequencing (ChIPseq). Scores are peak signal relative to IgG. ( D ) Top five enriched known motifs by homer analysis of ChIPseq peaks identified in (C). *** P < 0.001. ( E ) Binding distribution of ChIPseq peaks from (C). Enrichment per genomic region was assessed with hypergeometric testing and regions enriched with P < 0.05 are shown in green. ( F ) Activating or repressing function prediction by BETA. P value reflects gene activation (up-regulated) or repression (down-regulated) prediction for ZNF189 by a Kolmogorov-Smirnov test. ( G ) Direct gene target prediction by BETA. Values are −log(rank product), which reflect a P value for the combined score for binding potential. ( H ) ChIP signal at the top three BETA-predicted target genes. TTS, transcription termination site; 3′UTR, 3′ untranslated region; ncRNA, noncoding RNA; GPCR, G protein–coupled receptor.

Article Snippet: PDE1 inhibition was performed with 1 hour pre-incubation of adipocytes in media containing 1 μM ITI214 (MedChemExpress, HY-12501A), which was replaced with fresh assay media containing 1 μM ITI214 at the time of glycerol or cGMP induction.

Techniques: Control, Chromatin Immunoprecipitation, Sequencing, Binding Assay, Activation Assay

Journal: Science Advances

Article Title: Transcriptional determinants of lipid mobilization in human adipocytes

doi: 10.1126/sciadv.adi2689

Figure Lengend Snippet: ( A ) Left: ZNF189 ChIP signal shown above tissue-wide cap analysis of gene expression signals [expressed as relative log expression (RLE)] at the two PDE1B promoters (encoding PDE1B1 and PDE1B2 ). Right: Gene expression of PDE1B1 and PDE1B2 across tissue/cell samples from the FANTOM5 database. Adipose samples are highlighted in green. ( B and C ) PDE1B mRNA (B) and protein (C) levels for si ZNF189 (si Z ) versus control cells (siC). ( D ) Sequence alignments for PDE1B1 and PDE1B2 shown above protein domains from InterPro. Purple characters represent deviations from the canonical protein isoform. Conserved protein domains are indicated with colored boxes. ( E to H ) PDE1B mRNA (E) and protein (F) as well as cGMP (G) and glycerol (H) in cells engineered to overexpress PDE1B2 under a doxycycline (doxy)-inducible promoter. In (G) and (H), cells were incubated with ANP. ( I and J ) Effects of the PDE1 inhibitor ITI214 on ANP-stimulated cGMP levels in untransfected (I) or siC versus si Z adipocytes (J). Bar charts are expressed relative to the indicated controls and are presented as means ± SEM. In these panels, replicates are highlighted by dots and are based on at least three independent experiments. Statistical analyses were performed using Welch’s two-tailed t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: PDE1 inhibition was performed with 1 hour pre-incubation of adipocytes in media containing 1 μM ITI214 (MedChemExpress, HY-12501A), which was replaced with fresh assay media containing 1 μM ITI214 at the time of glycerol or cGMP induction.

Techniques: Gene Expression, Expressing, Control, Sequencing, Incubation, Two Tailed Test

Journal: Science Advances

Article Title: Transcriptional determinants of lipid mobilization in human adipocytes

doi: 10.1126/sciadv.adi2689

Figure Lengend Snippet: ( A ) Left: ZNF189 ChIP signal shown above tissue-wide cap analysis of gene expression signals [expressed as relative log expression (RLE)] at the two PDE1B promoters (encoding PDE1B1 and PDE1B2 ). Right: Gene expression of PDE1B1 and PDE1B2 across tissue/cell samples from the FANTOM5 database. Adipose samples are highlighted in green. ( B and C ) PDE1B mRNA (B) and protein (C) levels for si ZNF189 (si Z ) versus control cells (siC). ( D ) Sequence alignments for PDE1B1 and PDE1B2 shown above protein domains from InterPro. Purple characters represent deviations from the canonical protein isoform. Conserved protein domains are indicated with colored boxes. ( E to H ) PDE1B mRNA (E) and protein (F) as well as cGMP (G) and glycerol (H) in cells engineered to overexpress PDE1B2 under a doxycycline (doxy)-inducible promoter. In (G) and (H), cells were incubated with ANP. ( I and J ) Effects of the PDE1 inhibitor ITI214 on ANP-stimulated cGMP levels in untransfected (I) or siC versus si Z adipocytes (J). Bar charts are expressed relative to the indicated controls and are presented as means ± SEM. In these panels, replicates are highlighted by dots and are based on at least three independent experiments. Statistical analyses were performed using Welch’s two-tailed t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: PDE1 inhibition was performed with 1 hour pre-incubation of adipocytes in media containing 1 μM ITI214 (MedChemExpress, HY-12501A), which was replaced with fresh assay media containing 1 μM ITI214 at the time of glycerol or cGMP induction.

Techniques: Expressing, Control, Sequencing, Incubation, Two Tailed Test