Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 1 HFD feeding induces insulin resistance and disruptes BCAAs catabolism in APP/PS1 mice. A Body weight of APP/PS1 mice during experiment period (n = 11 to 12). B Blood glucose levels in the glucose tolerance test (OGTT) and the area under the curve (AUC) of APP/ PS1 mice (n = 8). C Fasting serum glucose and insulin levels, and corresponding HOMA-IR index of APP/PS1 mice (n = 8). D Heatmaps of the amino acid levels in serum of APP/PS1 mice in the indicated groups (n = 8). E Comparison of the levels of BCAAs and BCKAs in cerebral cortex (n = 8). F Heatmap analysis of the Spearman correlation of circulating and cortical levels of BCAAs and BCKAs. Western blot analysis of the levels of BCAAs catabolism-related protein in liver (G), skeletal muscle (H), and cerebral cortex (I), and the corresponding quantification results (n = 4–5). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, two-way or one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Comparison, Western Blot

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 2 BCAAs addition and restriction affect the development of cognitive deficits and AD-related pathology in HFD-fed APP/PS1 mice. A Escape latency to the platform during the training trails in a Morris water maze (n = 11 to 12). B Target (platform) entries in the probe test (n = 11 to 12). C Target quadrant entries in the probe test (n = 11 to 12). D Mean swimming speed of mice (n = 11 to 12). E Representative swimming tracks of mice in the probe test (n = 11 to 12). (F) Heatmap analysis of the Spearman correlation of cognitive function-related indexes and cortical levels of BCAAs and BCKAs (n = 8). G Western blot analysis of the protein levels of postsynaptic (PSD95) and presynaptic (synaptophysin) markers in cerebral cortex of mice, and the corresponding quantification results (n = 4). (H) Western blot analysis of the protein levels of Aβ secretase (BACE1) in cerebral cortex and the corresponding quantification results (n = 4). I Immunostainings of Aβ plaques by using specific antibody (6E10) in cortex and hippocampus (Scale bars, 200 μm), and quantitation of positive area rate (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, two-way or one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Western Blot, Quantitation Assay

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 3 BCAAs and BCKAs contribute to microglial phagocytosis deficiency in HFD-fed APP/PS1 mice and BV2 cell. A Representative images of microglia (IBA1) staining in cortex and hippocampus of APP/PS1 mice (Scale bars, 100 μm), and corresponding quantitation of positive cell number (n = 3). B Western blot analysis of IBA1 levels in cortex, and the corresponding quantification results (n = 4). C Representative images of Aβ plaques (6E10) and microglia (IBA1) co-staining in cortex and hippocampus of APP/PS1 mice (Scale bars, 20 μm), and corresponding quantitation of positive cell number around plaque (n = 3). Microglial phagocytosis D and degradation E of fluorescein labeled-Aβ for the indicated time in BV2 cells in the presence or absence of BCAAs/ BCKAs (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Staining, Quantitation Assay, Western Blot, Labeling

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 4 BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Western Blot

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 5 BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine-13C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D, E, F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 6 BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Expressing, Western Blot, Labeling

Journal: PLoS ONE

Article Title: Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal

doi: 10.1371/journal.pone.0001384

Figure Lengend Snippet: (A) Samples of KOSR were filtered using Amicon Ultra filters (Millipore) of three different molecular weight cutoffs (MWCO), so that only molecules with molecular weights (MW) below the filters' MWCOs could go through them. For each filter, two fractions were obtained corresponding to the low and high MW fractions. The latter fractions were obtained by resuspending the molecules retained by the filters in a volume of D-PBS equal to the original KOSR volume. (B) The six fractions obtained in this manner were analyzed by SDS-PAGE to ensure that filters were working properly. Coomassie blue staining of the gel revealed that filters were indeed preventing high MW components to go through them. Lipid-rich BSA is the most abundant protein component in KOSR by a factor of about 1000 times and this is why no other proteins are seen in the gel. (C) HUES7 cells were cultured for five passages in N2/B27-CDM+15% (V∶V) of each of the six fractions. After this period of time cells were fixed and stained for ALP activity. As seen in the top panel for the >50 kDa fraction, all high MW fractions were able to maintain widespread ALP expression whereas, as shown in the bottom panel for the <50 kDa fraction, all low MW fractions failed to maintain hESC pluripotency, as assessed by ALP expression.

Article Snippet: Based on previous reports (see ) and on the HPLC analysis just described, we decided to test the following lipids for effects on hESC self-renewal: lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), prostaglandin E2 (PGE 2 ) and a chemically defined mix of unsaturated and saturated free fatty acids (FFA) known commercially as chemically defined lipid concentrate (Invitrogen).

Techniques: Molecular Weight, SDS Page, Staining, Cell Culture, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal

doi: 10.1371/journal.pone.0001384

Figure Lengend Snippet: Lipids present in AlbuMAX

Article Snippet: Based on previous reports (see ) and on the HPLC analysis just described, we decided to test the following lipids for effects on hESC self-renewal: lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), prostaglandin E2 (PGE 2 ) and a chemically defined mix of unsaturated and saturated free fatty acids (FFA) known commercially as chemically defined lipid concentrate (Invitrogen).

Techniques:

Journal: PLoS ONE

Article Title: Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal

doi: 10.1371/journal.pone.0001384

Figure Lengend Snippet: HUES9 cells were grown in N2/B27-CDM alone, with 1% AlbuMAX or with the indicated lipids for six passages. At the end of this period cells were (A) stained for ALP activity and their DNA visualized with propidium iodide or (B) RNA was extracted from cells and the expression of Oct4 and Nanog mRNAs was analyzed by quantitative RT-PCR. Lipids used are: lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), prostaglandin E2 (PGE2) and a chemically-defined mixture of saturated and unsaturated free fatty acids (FFA) known commercially as chemically defined lipid concentrate (Invitrogen).

Article Snippet: Based on previous reports (see ) and on the HPLC analysis just described, we decided to test the following lipids for effects on hESC self-renewal: lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), prostaglandin E2 (PGE 2 ) and a chemically defined mix of unsaturated and saturated free fatty acids (FFA) known commercially as chemically defined lipid concentrate (Invitrogen).

Techniques: Staining, Activity Assay, Expressing, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Induction of autophagy and autophagy‐dependent apoptosis in diffuse large B‐cell lymphoma by a new antimalarial artemisinin derivative, SM 1044

doi: 10.1002/cam4.1276

Figure Lengend Snippet: SM 1044 induces Ca MKK 2– AMPK – ULK 1 pathway‐mediated autophagy through promoting de novo synthesis of ceramide. (A) SU ‐ DHL ‐4 cells were treated with SM 1044 for the indicated time courses. The active ULK 1, AMPK and mTOR were detected by western blot. (B) SU ‐ DHL ‐4 cells were pretreated with AMPK inhibitor compound C for 1 h, followed by SM 1044 treatment for another 24 h. The levels of LC 3, Survivin, and p‐ AMPK were detected by western blot. (C) SU ‐ DHL ‐4 cells were pretreated with Ca MKK 2 inhibitor STO ‐609 for 1 h, followed by SM 1044 treatment for another 24 h. The levels of p‐ AMPK , Survivin, caspase‐8, ‐9, ‐3, and PARP were detected by western blot. (D) SU ‐ DHL ‐4 cells were treated with SM 1044 for 3 h and the level of ceramide was detected by HPLC ‐ MS / MS . C24: Control versus SM 1044, P = 0.011. C26: Control versus SM 1044, P = 0.023. (E) SU ‐ DHL ‐4 cells were pretreated with ceramide inhibitor S1P for 1 h, followed by SM 1044 treatment for another 24 h. The levels of p‐ AMPK and Survivin were detected by western blot. (F) SU ‐ DHL ‐4 cells were pretreated with ceramide de novo synthesis inhibitor l ‐cycloserine for 1 h, followed by SM 1044 treatment for another 24 h. Cell viability was detected by CCK ‐8 (upper panel, mean ± SEM, n = 3) and apoptosis was measured by flow cytometry (lower panel, mean ± SEM, n = 3). Cell viability: SM 1044 versus control, P < 0.001, SM 1044 versus l ‐cycloserine plus SM 1044, P < 0.001. Percent apoptosis: SM 1044 versus control, P < 0.001, SM 1044 versus l ‐cycloserine plus SM 1044, P < 0.001. (G) SU ‐ DHL ‐4 cells were pretreated with l ‐cycloserine for 1 h, followed by SM 1044 treatment for another 24 h. The levels of p‐ AMPK , LC 3, Survivin, caspase‐8, ‐9, ‐3, and PARP were detected by western blot. * P < 0.05, *** P < 0.001.

Article Snippet: Sphingosine‐1‐phosphate (S1P) was obtained from LKT Laboratories (St. Paul, MN).

Techniques: Western Blot, Tandem Mass Spectroscopy, Control, CCK-8 Assay, Flow Cytometry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor Signaling

doi: 10.1161/atvbaha.117.309571

Figure Lengend Snippet: Figure 2. Loss of growth differentiation factor 6 (GDF6) function results in impaired vascular integrity in vitro. A, Reverse transcription polymerase chain reaction (PCR) of GDF6 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; loading control) from human umbili- cal vein endothelial cell (HUVECs) that were treated with either control small interfering RNA (siRNA; Control, column 1) or 1 of 2 different siRNAs targeting GDF6 (GDF6 #1 and GDF6 #2, columns 2 and 3). Both GDF6-targeting siRNAs knocked down GDF6 transcript levels, but siRNA #2 was much more effective. Observation noted from 3 independent experiments. B, TaqMan assay quantification of GDF6 transcript levels in control or GDF6 siRNA-treated HUVEC cells (both siRNAs were used in tandem to ensure maximal gene suppression efficiency for all experiments), showing >90% reduction in GDF6 transcript levels in GDF6 siRNA-treated HUVEC. C, Schematic diagram illustrating the HUVEC monolayer permeability assay. Briefly, HUVECs are seeded in the inner well of a transwell chamber in complete growth medium (i). The cells attach to the membrane and form cell–cell adhesions, assembling a confluent monolayer on the membrane before dye addition (ii). Fluorescein isothiocyanate (FITC)-dextran is added to the inner well of the transwell chamber, remaining largely contained within the inner chamber when cell–cell adhesion is intact (iii). Defects in endothelial cell–cell adhesion result in increased per- meability across the monolayer and increased FITC-dextran leakage into the outer, plate well. The intensity of fluorescence in the outer well is proportional to the extent of monolayer permeability (iv). D, Quantification of the amount of FITC transferred through a HUVEC monolayer, expressed as a percentage of the amount of dye that transfers across the membrane in the absence of any added HUVEC cells (ie, no cell control). A control siRNA-treated HUVEC monolayer reduces FITC transfer to ≈10% of the no cell condition, but treating HUVEC with GDF6 siRNA results in a 3.5-fold increase in permeability. Treatment with either soluble GDF6 protein or anti–vascular endo- thelial growth factor (VEGF) A antibody reduces the increased permeability caused by GDF6 siRNA knockdown. Equivalent results noted in 3 separate experiments. E, Quantification of the amount of FITC transferred through the HUVEC monolayer, expressed as a percentage of the amount of dye that transfers across the membrane with a control HUVEC monolayer. VEGFA treatment results in increased mono- layer permeability while sphingosine-1-phosphate (S1P) treatment results in decreased monolayer permeability. Quantitative PCR data (B) were collected from at least 3 biological repeats with 3 technical repeats per probe. Permeability assays (D and E) were done as 3 biologi- cal replicates with at least 3 technical repeats per each experimental condition. Data were shown from a single biological replicate.

Article Snippet: Recombinant VEGFA (R&D Systems, 293-VE/CF) and GDF6 (Fitzgerald, 30R-AB021) were used at 40 ng/ml and S1P (Tocris, 1370) was used at 1 uM concentrations.

Techniques: In Vitro, Reverse Transcription, Polymerase Chain Reaction, Control, Small Interfering RNA, TaqMan Assay, Permeability, Membrane, Fluorescence, Knockdown, Real-time Polymerase Chain Reaction