sgc cbp30  (BPS Bioscience)

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

doi: 10.3389/fcell.2021.740203

Figure Lengend Snippet: Modulation of Hif3α expression regulates the adipocyte differentiation toward WAT phenotype. (A) Schematic representation of experimental design. 3T3-L1 cells were plated in presence and absence of pro-inflammatory cytokines the day before the differentiation induction until the appearance of mature adipocytes (T0 to T10). Representative Oil Red O-staining by light microscopy in our experimental condition. (B) Quantification of Oil Red O-staining at 540 nm ( n = 3) using a TECAN robotic station in mature adipocytes at day 7. *** p ≤ 0.001, ** p ≤ 0.01 vs . control. (C) Schematic representation of transfection design and gene expression analysis ( Pparg, C/ebpα, Clec10a, Cd36, Fasn ) in WT, si Hif3α and HIF3A TO adipocytes. Heatmap represents expression profile of WAT genes differentially expressed during adipogenesis. Values represent the means of the fold change vs. T0WT ( n = 3). T0WT is calculated as 1, thus >1 means upregulation, <1 means downregulation. All values are significant ( p ≤ 0.01) except those underlined.

Article Snippet: Mouse monoclonal anti Sirt1 (Abcam #110304), anti Gapdh (sc #47724, Santa Cruz Biotech) anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 (cell signalling #CD19C6), anti Acc (cell signalling #C83B10), anti PDH (cell signalling #2784), anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting.

Techniques: Expressing, Staining, Light Microscopy, Control, Transfection, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

doi: 10.3389/fcell.2021.740203

Figure Lengend Snippet: Hif3α inhibition redirects adipocyte differentiation toward BAT phenotype (A,B) 3T3-L1 cells were plated and transfected with si Hif3α , and exposed to pro-inflammatory cytokines and differentiation medium until the appearance of mature adipocytes (T0 to T10). Representative Oil Red O-staining by light microscopy in our experimental condition. Quantification of Oil Red O-staining at 540 nm ( n = 3) using a TECAN robotic station in mature adipocytes at day 7. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 vs . control. (C) Analysis of BAT gene regulation ( Ucp1, Elovl3, Prdm16, Dio2, Ppargc1a ) in WT, si Hif3α and HIF3A TO adipocytes. Heatmap represents expression profile of BAT genes differentially expressed across the different experimental conditions. Values represent the means of the fold change vs. T0WT ( n = 3). T0WT is calculated as 1, thus >1 means upregulation, <1 means downregulation. All values are significant ( p ≤ 0.01) except those underlined.

Article Snippet: Mouse monoclonal anti Sirt1 (Abcam #110304), anti Gapdh (sc #47724, Santa Cruz Biotech) anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 (cell signalling #CD19C6), anti Acc (cell signalling #C83B10), anti PDH (cell signalling #2784), anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting.

Techniques: Inhibition, Transfection, Staining, Light Microscopy, Control, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

doi: 10.3389/fcell.2021.740203

Figure Lengend Snippet: Hif3α silencing enhances lipid catabolism. (A) Expression levels of Angptl4 by qPCR in WT, si Hif3α , HIF3A TO cultured in differentiated medium (T0-T7) in presence or absence of cytokines. Relative gene expression data are reported as 2-ΔΔCt method, normalized to housekeeping gene (b-actin and 18S mRNA) vs. T0 WT. Data are expressed as means ± SEM ( n = 3; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). (B) Western blot analysis of LC3B-II protein in siHif3α adipocytes cultured in differentiation medium in presence of inflammatory cytokines (T7). The ratio of Lc3B-II/Lc3B-I/Gapdh was measured using ImageJ Analysis tool ( n = 3; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 vs . control cells).

Article Snippet: Mouse monoclonal anti Sirt1 (Abcam #110304), anti Gapdh (sc #47724, Santa Cruz Biotech) anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 (cell signalling #CD19C6), anti Acc (cell signalling #C83B10), anti PDH (cell signalling #2784), anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting.

Techniques: Expressing, Cell Culture, Gene Expression, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

doi: 10.3389/fcell.2021.740203

Figure Lengend Snippet: Hif3α silencing increases mitochondrial respiration. 3T3-L1 cells were transfected with si Hif3α and induced to differentiate (A) Seahorse analysis showed the Oxygen consumption rate (OCR) expressed in ug protein. (B) Proton Leak. (C) Oxygen Consumption Rate. (D) Extracellular Acidification Rate. (E) Baseline OCR/ECAR ratio (F) Mitochondrial respiration vs. glycolysis. Values are mean ± SD ( n = 3; * p < 0.05 vs . WT or control cells).

Article Snippet: Mouse monoclonal anti Sirt1 (Abcam #110304), anti Gapdh (sc #47724, Santa Cruz Biotech) anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 (cell signalling #CD19C6), anti Acc (cell signalling #C83B10), anti PDH (cell signalling #2784), anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting.

Techniques: Transfection, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

doi: 10.3389/fcell.2021.740203

Figure Lengend Snippet: The inhibition of Hif3α induces fatty acids synthesis enzymes (A) Western blot analysis of Acc, AceCS1 and PDH in si Hif3α after 7 days of differentiation induction. SIRTs are activated in absence of Hif3α (B) Western blot analysis of Hif3α, Sirt1, Sirt3 and Lc3B-I/II detected in si Hif3α adipocytes at T3. Densitometry values are mean ± SEM of biological triplicates and are graphed as normalized for relative housekeeping (Actin or Gapdh) using the ImageJ Gel Analysis tool and expressed as fold change. Gapdh was showed as representative housekeeping. ( n = 3; *** p ≤ 0.001, ** p ≤ 0.01 vs . control cells).

Article Snippet: Mouse monoclonal anti Sirt1 (Abcam #110304), anti Gapdh (sc #47724, Santa Cruz Biotech) anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 (cell signalling #CD19C6), anti Acc (cell signalling #C83B10), anti PDH (cell signalling #2784), anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting.

Techniques: Inhibition, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

doi: 10.3389/fcell.2021.740203

Figure Lengend Snippet: The causal Hif3α/Ucp1 relation in thermogenesis-induced model. (A) Animal genotyping by PCR analysis of genomic DNA. (B) Animal and fat depots weights (C) Schematic representation of the distribution of adipose tissue depots collected: visceral white fat (vFAT) and inguinal/subcutaneous white fat (iFAT). (D) Representative haematoxylin and eosin-stained of vFAT and iFAT sections (upper panel) and fat cell density (lower panel). (E) Expression levels of Hif3α isoforms gene by qPCR in vFAT and iFAT of WT and CB1KO mice. Relative gene expression data are reported as 2-ΔΔCt, normalized to housekeeping genes (b-actin and 18S mRNA). Data are expressed as means ± SEM ( n = 3 animals/genotype, *** p ≤ 0.001, * p ≤ 0.05 vs. WT mouse). (F) Heatmap represents expression profile of WAT-specific ( Pparg, C/ebpα, Fasn ) and BAT-specific genes ( Ucp1, Elovl3, Prdm16, Dio2, Ppargc1a ) in vFAT and iFAT differentially expressed in CB1KO compared to WT. Values represent the means of the fold change vs. WT ( n = 3 animals/genotype). All values are significant ( p < 0.05), except those underlined.

Article Snippet: Mouse monoclonal anti Sirt1 (Abcam #110304), anti Gapdh (sc #47724, Santa Cruz Biotech) anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 (cell signalling #CD19C6), anti Acc (cell signalling #C83B10), anti PDH (cell signalling #2784), anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting.

Techniques: Staining, Expressing, Gene Expression

Journal: Molecular Metabolism

Article Title: Genome-wide association study of adipocyte lipolysis in the GENetics of adipocyte lipolysis (GENiAL) cohort

doi: 10.1016/j.molmet.2020.01.009

Figure Lengend Snippet: Functional validation of HIF3A function in human adipocytes. A) HIF3A gene expression during hMSC differentiation in vitro . Data retrieved from FANTOM5 dataset ( http://fantom.gsc.riken.jp/5/ ). B) and D-E) Expression of HIF3A was knocked down using siRNA in hMSCs 24 h after induction of differentiation and followed until differentiation days 6, 10, and 13, upon which B) the expression of HIF3A, LIPE, PLIN1 , and PNPLA2 was monitored, D) glycerol amount in medium was measured, E) accumulation of neutral lipids was evaluated, and F) the expression of ADIPOQ and FABP4 was measured. C) The expression of HIF3A was knocked down using siRNA in hMSCs 24 h before induction of adipogenic differentiation of hMSCs; 72 h post-transfection, the cells were collected, fractionated to the cytosolic and nuclear fraction, and the proteins were analyzed by Western blotting. The expression of HIF3A was normalized to the total protein amount. Results in B-E are based on four biological/independent experiments. The expression of genes was normalized to the reference gene 18s. The results were analyzed using t-tests and presented as fold change ± SD relative to negative control of a corresponding time point (Neg C). Results in C) were analyzed using one-sided t-tests and presented as fold change ± SD relative to negative control (Neg C). ∗∗∗P < 0.005, ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Antibodies against HIF3A were obtained from Proteintech (Rosemont, IL, USA) and Prosci (Poway, CA, USA), both used at 1:1000 dilution.

Techniques: Functional Assay, Biomarker Discovery, Gene Expression, In Vitro, Expressing, Transfection, Western Blot, Negative Control

Journal: Molecular Metabolism

Article Title: Genome-wide association study of adipocyte lipolysis in the GENetics of adipocyte lipolysis (GENiAL) cohort

doi: 10.1016/j.molmet.2020.01.009

Figure Lengend Snippet: A) Genotype-specific gene expression for HIF3A , with higher levels observed with 2 copies of rs73048031-C. Gene expression expressed relative to 18 S. Genotype groups were compared with Student's t-test. P = 0.076 (two-sided), P = 0.038 (one-sided). B) Relationship between abdominal SAT HIF3A expression and spontaneous lipolysis.

Article Snippet: Antibodies against HIF3A were obtained from Proteintech (Rosemont, IL, USA) and Prosci (Poway, CA, USA), both used at 1:1000 dilution.

Techniques: Gene Expression, Expressing