Journal: Molecular Metabolism

Article Title: Long-chain acyl-CoA synthetase-4 regulates endometrial decidualization through a fatty acid β-oxidation pathway rather than lipid droplet accumulation

doi: 10.1016/j.molmet.2024.101953

Figure Lengend Snippet: Inhibition of FA β-oxidation promotes lipid droplet accumulation. After MPA and db-cAMP-induced ESCs were treated with 10 μM, 20 μM FP-06424439 for 24 or 48 h, WB (A) was performed to detect the expression of CPT1A, CPT2, and ATP assay kit (B) was used to detect ATP level, ∗vs. NC. After MPA and db-cAMP-induced ESCs were transfected with DGAT2 siRNA, and WB (C) was performed to detect the expression of CPT1A, CPT2, and ATP assay kit (D) was used to detect ATP level, ∗vs. NC. E. The OCR was detected after MPA and db-cAMP-induced ESCs were treated with DGAT2 inhibitor PF-06424439 (20 μM) or/and DGAT1 inhibitor PF-04620110 (5 μM). F. Basal respiration, Basal respiration was calculated as the three OCR measurements before the addition of oligomycin minus the three OCR measurements after the addition of rotenone and antimycin A. G. ATP production, ATP production was calculated as the three OCR measurements before oligomycin injection minus the three OCR measurements after oligomycin injection. After MPA and db-cAMP-induced ESCs were treated with 25 μM etomoxir for 48 h, BIODIPY 493/503 staining (H, scale bar = 10 μm) was used to detect lipid droplet levels, and qPCR (I, normalized by GAPDH) was performed to detect the DGAT2 expression, ∗vs. NC, #vs. MPA + db-cAMP. After MPA and db-cAMP-induced ESCs were transfected with CPT1A siRNA, BIODIPY 493/503 staining (J, scale bar = 10 μm) was used to detect lipid droplet levels, and qPCR (K, normalized by GAPDH) was performed to detect the DGAT2 expression, ∗vs. NC, #vs. MPA + db-cAMP + NC siRNA. After MPA and db-cAMP-induced ESCs were transfected with OverACSL4 plasmid and treated with CPT1A siRNA or 25 μM etomoxir for 48 h, qPCR (L, normalized by GAPDH) or WB (M) was performed to detect the expression of PRL, IGFBP1, HOXA10, FOXO1, and FITC-phalloidin staining (N, scale bar = 50 μm) was performed to detect the F-actin expression, ∗vs. MPA + db-cAMP, #vs. MPA + db-cAMP + OverACSL4. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗ p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: Endometrial stromal cells induced to decidualization in vitro were treated with the carnitine palmitoyl transferase (CPT)1 inhibitor, etomoxir (25 μM, 50 μM; #HY-50202; MedChemExpress, Monmouth Junction, NJ, USA) for 24 h or 48 h; the diacylglycerol acyltransferase 2 (DGAT2) inhibitor, PF-06424439 (10 μM, 20 μM; #HY-108341A; MedChemExpress) for 24 h or 48 h; the DGAT1 inhibitor, PF-04620110 (5uM; #HY-13009; MedChemExpress) for 24 h; and CPT1 agonists, baicalin (25 μM, 50 μM; #HY-N0197; MedChemExpress), and C75 (1 μM, 4 μM; HY-12364, MedChemExpress) for 24 h, respectively.

Techniques: Inhibition, Expressing, ATP Assay, Transfection, Injection, Staining, Plasmid Preparation

Journal: Medical Microbiology and Immunology

Article Title: Inhibiting lipid droplet biogenesis enhances host protection against hypervirulent Klebsiella pneumoniae infections

doi: 10.1007/s00430-024-00807-x

Figure Lengend Snippet: LDs formation contributes to proinflammatory cytokine production during hvKp infections. A , B RAW264.7 cells were incubated with or without A922500 (10 µM; A ) or CAY10650 (15 nM; B ) for 1 h and then infected with hvKp strains (MOI 40) for 2 h. The mRNA expression levels of Tnfa and Il6 were measured using qRT-PCR. C RAW264.7 cells were incubated with or without A922500 (10 µM) or CAY10650 (15 nM) for 1 h and then infected with hvKp strains (MOI 40) for 4 h. TNF-α levels were determined using ELISA. D RAW264.7 cells were transfected with siRNA specific for DGAT1 (siDGAT1), cPLA 2 (sicPLA 2 ), or a non-specific scrambled siRNA (S.C.). At 24 h after transfection, cells were infected with hvKp strains (MOI 40) for 4 h. Supernatants were then assessed by ELISA for TNF-α levels. Data are expressed as the mean ± standard deviation of three independent experiments. TNF, tumor necrosis factor; siRNA, small interfering RNA. * P < 0.05, ** P < 0.01, and *** P < 0.001 according to analysis of variance

Article Snippet: The anti- fatty acid synthase (FAS, #3180, Cell Signaling Technology, Beverly, MA, USA), anti-perilipin 1 (PLIN1, NB100-449, Novus Biologicals, Centennial, CO, USA), anti-acetyl-CoA carboxylase 2 (ACC2, #3676, Cell Signaling Technology), anti- phospho- ACC2 (Ser79) (#3661, Cell Signaling Technology), anti- ACC1 (#4190, Cell Signaling Technology), anti-stearoyl-CoA desaturase-1 (SCD1, #2794, Cell Signaling Technology), anti-phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), anti-phospho-p70S6K (Thr421/Ser424) (#9204, Cell Signaling Technology), and phospho-S6 Ribosomal Protein (Ser235/236) (#2211, Cell Signaling Technology), anti-DGAT1 (#NB110-41487, Novus Biologicals) and anti-cPLA 2 (#sc-454, Santa Cruz Biotechnology, Inc.) were used as primary antibodies.

Techniques: Incubation, Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Standard Deviation, Small Interfering RNA

Journal: Medical Microbiology and Immunology

Article Title: Inhibiting lipid droplet biogenesis enhances host protection against hypervirulent Klebsiella pneumoniae infections

doi: 10.1007/s00430-024-00807-x

Figure Lengend Snippet: Inhibiting LDs formation and knocking down of lipogenesis regulators promotes antimicrobial responses during hvKp infections. A , B RAW264.7 cells were pretreated with A922500 (10 µM) or CAY10650 (15 nM) for 1 h. Cells were then infected with hvKp strains (MOI 40) for 4 h. A Representative confocal images of LDs and nuclei were stained with BODIPY 493/503 (Green) and DAPI (Blue), respectively. Scale bar, 20 μm. B BODIPY 493/503 area quantification. C RAW264.7 cells were pretreated with A922500 (10 µM) or CAY10650 (15 nM) for 1 h, then infected with hvKp strains (MOI 40) for 6 h. Intracellular survival of hvKp was determined using colony forming units (CFU) assay. D RAW264.7 cells were transfected with siRNA against S.C., DGAT1, or cPLA 2 for 24 h and infected with the hvKp strains for 6 h. Intracellular survival of hvKp assessed by CFU assay. E RAW264.7 cells were pretreated with rapamycin (200 nM) for 30 min, followed by hvKp strain (MOI 40) infection for 6 h. Macrophage bacterial loads were detected using CFU assay. Data are expressed as the mean ± standard deviation of three independent experiments. Un, uninfected; LDs, lipid droplets; Rapa, rapamycin; hpi, hours post infection. * P < 0.05, ** P < 0.01, and *** P < 0.001 according to analysis of variance

Article Snippet: The anti- fatty acid synthase (FAS, #3180, Cell Signaling Technology, Beverly, MA, USA), anti-perilipin 1 (PLIN1, NB100-449, Novus Biologicals, Centennial, CO, USA), anti-acetyl-CoA carboxylase 2 (ACC2, #3676, Cell Signaling Technology), anti- phospho- ACC2 (Ser79) (#3661, Cell Signaling Technology), anti- ACC1 (#4190, Cell Signaling Technology), anti-stearoyl-CoA desaturase-1 (SCD1, #2794, Cell Signaling Technology), anti-phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), anti-phospho-p70S6K (Thr421/Ser424) (#9204, Cell Signaling Technology), and phospho-S6 Ribosomal Protein (Ser235/236) (#2211, Cell Signaling Technology), anti-DGAT1 (#NB110-41487, Novus Biologicals) and anti-cPLA 2 (#sc-454, Santa Cruz Biotechnology, Inc.) were used as primary antibodies.

Techniques: Infection, Staining, Colony-forming Unit Assay, Transfection, Standard Deviation

Journal: Medical Microbiology and Immunology

Article Title: Inhibiting lipid droplet biogenesis enhances host protection against hypervirulent Klebsiella pneumoniae infections

doi: 10.1007/s00430-024-00807-x

Figure Lengend Snippet: LDs formation contributes to proinflammatory cytokine production during hvKp infections. A , B RAW264.7 cells were incubated with or without A922500 (10 µM; A ) or CAY10650 (15 nM; B ) for 1 h and then infected with hvKp strains (MOI 40) for 2 h. The mRNA expression levels of Tnfa and Il6 were measured using qRT-PCR. C RAW264.7 cells were incubated with or without A922500 (10 µM) or CAY10650 (15 nM) for 1 h and then infected with hvKp strains (MOI 40) for 4 h. TNF-α levels were determined using ELISA. D RAW264.7 cells were transfected with siRNA specific for DGAT1 (siDGAT1), cPLA 2 (sicPLA 2 ), or a non-specific scrambled siRNA (S.C.). At 24 h after transfection, cells were infected with hvKp strains (MOI 40) for 4 h. Supernatants were then assessed by ELISA for TNF-α levels. Data are expressed as the mean ± standard deviation of three independent experiments. TNF, tumor necrosis factor; siRNA, small interfering RNA. * P < 0.05, ** P < 0.01, and *** P < 0.001 according to analysis of variance

Article Snippet: For transient silencing of DGAT1 and cPLA 2 in RAW264.7 murine macrophage cells, DGAT1 siRNA (#sc-40488), cPLA 2 siRNA (#sc-35098), and a scrambled siRNA (#sc-37007) were obtained from Santa Cruz Biotechnology, Inc. All siRNAs were transfected using Lipofectamine ® 2000 Reagent (11668027, Invitrogen, Carlsbad, CA, USA).

Techniques: Incubation, Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Standard Deviation, Small Interfering RNA

Journal: Medical Microbiology and Immunology

Article Title: Inhibiting lipid droplet biogenesis enhances host protection against hypervirulent Klebsiella pneumoniae infections

doi: 10.1007/s00430-024-00807-x

Figure Lengend Snippet: Inhibiting LDs formation and knocking down of lipogenesis regulators promotes antimicrobial responses during hvKp infections. A , B RAW264.7 cells were pretreated with A922500 (10 µM) or CAY10650 (15 nM) for 1 h. Cells were then infected with hvKp strains (MOI 40) for 4 h. A Representative confocal images of LDs and nuclei were stained with BODIPY 493/503 (Green) and DAPI (Blue), respectively. Scale bar, 20 μm. B BODIPY 493/503 area quantification. C RAW264.7 cells were pretreated with A922500 (10 µM) or CAY10650 (15 nM) for 1 h, then infected with hvKp strains (MOI 40) for 6 h. Intracellular survival of hvKp was determined using colony forming units (CFU) assay. D RAW264.7 cells were transfected with siRNA against S.C., DGAT1, or cPLA 2 for 24 h and infected with the hvKp strains for 6 h. Intracellular survival of hvKp assessed by CFU assay. E RAW264.7 cells were pretreated with rapamycin (200 nM) for 30 min, followed by hvKp strain (MOI 40) infection for 6 h. Macrophage bacterial loads were detected using CFU assay. Data are expressed as the mean ± standard deviation of three independent experiments. Un, uninfected; LDs, lipid droplets; Rapa, rapamycin; hpi, hours post infection. * P < 0.05, ** P < 0.01, and *** P < 0.001 according to analysis of variance

Article Snippet: For transient silencing of DGAT1 and cPLA 2 in RAW264.7 murine macrophage cells, DGAT1 siRNA (#sc-40488), cPLA 2 siRNA (#sc-35098), and a scrambled siRNA (#sc-37007) were obtained from Santa Cruz Biotechnology, Inc. All siRNAs were transfected using Lipofectamine ® 2000 Reagent (11668027, Invitrogen, Carlsbad, CA, USA).

Techniques: Infection, Staining, Colony-forming Unit Assay, Transfection, Standard Deviation

Journal: Biomolecules

Article Title: Inhibition of Ghrelin Activity by Receptor Antagonist [ d -Lys-3] GHRP-6 Attenuates Alcohol-Induced Hepatic Steatosis by Regulating Hepatic Lipid Metabolism

doi: 10.3390/biom9100517

Figure Lengend Snippet: Quantitative analysis of hepatic fatty acid uptake and synthesis. ( A ) qRT–PCR analysis of hepatic gene expression related to fatty acid synthesis-acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and diacylglyceride acyltransferase (DGAT1). ( B ) Hepatic gene expression related to fatty acid transport—FATP2 (fatty acid transport protein 2) and fatty acid translocase CD36. Values expressed as a relative to the control values. Values are means ± SEM, n = 8. Values not sharing a common letter are statistically different, p ≤ 0.05.

Article Snippet: Quantitative PCR (qPCR) was performed using a TaqMan Gene Expression assay specific for rat DGAT1 (Rn00584870) and TaqMan Fast Universal PCR Master Mix (Applied Biosystems).

Techniques: Quantitative RT-PCR, Gene Expression, Control