Journal: International Journal of Molecular Sciences

Article Title: Effects of Long-Term Physical Activity and BCAA Availability on the Subcellular Associations between Intramyocellular Lipids, Perilipins and PGC-1 α

doi: 10.3390/ijms24054282

Figure Lengend Snippet: Marker signal comparison between compartments; ( A ) proportion of nuclear signal (blue bars) in relation to cytosolic signal (full bars). Data normalized to the cytosolic reference and measured from the control group (Normal BCAA|Rest). Differences between nuclear fractions of IMCL versus remaining markers * ( p < 0.05 ) and ** ( p < 0.001 ). Whiskers signify standard deviation; ( B ) representative images of respective markers. Gray is signal, blue are limits of segmented nuclei, magenta are limits of segmented myotubes. Bar = 5 μm.

Article Snippet: For the C2C12 samples, a quintuple staining was performed, using 3 antibody markers: differentiated myotubes (5 μ g · mL −1 , MF-20, DSHB), PLIN5 (1:200 dilution, GP31, Progen), plus either PLIN2 (5 μ g · mL −1 , ab52356, Abcam) or PGC-1 α (5 μ g · mL −1 , ab191838, Abcam).

Techniques: Marker, Comparison, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Effects of Long-Term Physical Activity and BCAA Availability on the Subcellular Associations between Intramyocellular Lipids, Perilipins and PGC-1 α

doi: 10.3390/ijms24054282

Figure Lengend Snippet: Compartmental association and distribution of PLIN5, IMCL and PGC-1 α after EPS and BCAA deprivation. ( A ) representative image. Note more abundant PLIN5 in nuclei after EPS, with stronger association with PGC-1 α (orange arrows) and IMCL (cyan arrow). Gray is signal, blue are limits of segmented nuclei, magenta are limits of segmented myotubes. Bar = 3 μm; ( B ) PLIN5 signal intensity in different compartments; ( C ) PGC-1 α signal intensity in different compartments; ( D ) ICA between IMCL and PLIN5 in different compartments; ( E ) ICA between PGC-1 α and PLIN5 in different compartments. Main effect differences denoted with # ( p < 0.05 ) and ## ( p < 0.01 ). Combined group differences denoted with ** ( p < 0.01 ). Dots in B–E represent averaged coverslip values; data normalized to the control group reference (Normal BCAA|Rest).

Article Snippet: For the C2C12 samples, a quintuple staining was performed, using 3 antibody markers: differentiated myotubes (5 μ g · mL −1 , MF-20, DSHB), PLIN5 (1:200 dilution, GP31, Progen), plus either PLIN2 (5 μ g · mL −1 , ab52356, Abcam) or PGC-1 α (5 μ g · mL −1 , ab191838, Abcam).

Techniques: Control

Journal: Science Advances

Article Title: Mitochondrial respiratory complex III sustains IL-10 production in activated macrophages and promotes tumor-mediated immune evasion

doi: 10.1126/sciadv.adq7307

Figure Lengend Snippet: ( A to D ) BMDMs were pretreated with DMSO or S3QEL 1.2 (0.1 to 10 μM) for 3 hours before LPS (100 ng/ml) stimulation for 4 hours, and cell lysates and supernatants were harvested. (A) and (B) Quantification of IL-10 mRNA (A) and released protein (B) by RT-qPCR, relative to the Rps18 housekeeping gene, and ELISA. (C) and (D) Quantification of TNF- α mRNA (C) and released protein (D) by RT-qPCR, relative to the Rps18 housekeeping gene, and ELISA. FC, fold change. ( E to H ) BMDMs were pretreated with DMSO and MYX (500 nM) for 3 hours before LPS (100 ng/ml) stimulation for 4 hours, and cell lysates and supernatants were harvested. (E) and (F) Quantification of IL-10 mRNA (E) and released protein (F) by RT-qPCR, relative to the Rps18 housekeeping gene, and ELISA. (G) and (H) Quantification of TNF- α mRNA (G) and released protein (H) by RT-qPCR, relative to the Rps18 housekeeping gene, and ELISA. ( I ) Schematic diagram of in vivo procedure and sample collection. Mice were intraperitoneally (ip) injected with PBS or S3QEL 1.2 (1 mg/kg) for 2 hours, followed by LPS (2.5 mg/kg) for 2 hours. Blood (then serum) was collected. hrs, hours. ( J ) Quantification of IL-10 by ELISA in the serum. Data from (A) to (H) are expressed as means ± SEM for n = 5 to 9 from three independent experiments. Data from (J) are expressed as means ± SEM ( n = 10 per group within two independent in vivo experiments). P values were calculated using one-way ANOVA for multiple comparisons. Differences were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For in vivo samples, a Quantikine ELISA kit for mouse IL-10 (R&D Systems) was used according to the manufacturer’s instructions with appropriately diluted serum added to each plate.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, In Vivo, Injection

Journal: Science Advances

Article Title: Mitochondrial respiratory complex III sustains IL-10 production in activated macrophages and promotes tumor-mediated immune evasion

doi: 10.1126/sciadv.adq7307

Figure Lengend Snippet: ( A ) BMDMs were pretreated with DMSO, MYX (500 nM), or S3QEL 1.2 (10 μM) for 3 hours, stimulated with CpG (1 μg/ml) for 4 hours, and cell lysates and supernatants were harvested. Quantification of IL-10 mRNA by RT-qPCR, relative to the Rps18 housekeeping gene, and of IL-10 levels by ELISA. ( B ) Schematic diagram of the melanoma in vivo model. Mice were challenged with 5 × 10 6 B16F10 cells subcutaneously and administered with PBS, S3QEL 1.2 (1 mg/kg), and/or CpG (2.5 mg/kg). Tumors were measured daily. D, day. ( C ) Mean initial tumor growth curve relative to tumor volume from day −1 to day 8. ( D ) Kaplan-Meier survival graph up to day 65 (end of experiment). ( E ) Tumor growth rate curve relative to tumor area from day 0 to day 17. ( F to H ) Immune cells infiltrating the tumor were analyzed by flow cytometry. Cells expressing the surface markers Ly6C, F4/80, and CD11b showing the percentage of MHCII + macrophages and mean fluorescence intensity (MFI) of MHCII (F), the percentage of CD206 + macrophages and MFI of CD206 (G), and the percentage of IL-10 + macrophages and MFI of IL-10 (H). Data from (A) are means ± SEM for n = 5 from three independent experiments. Data from (C) and (D) are means ± SEM for n = 5 for the PBS group, n = 6 for the S3QEL 1.2 group, n = 7 for the CpG group, and n = 6 for the S3QEL 1.2 + CpG group. Statistical significance for survival analysis was determined by Mantel-Cox test. Statistical significance for tumor growth analysis was determined by two-way ANOVA; asterisks are for P < 0.05 in the group’s comparison. Data from (E) to (H) are means ± SD for n = 9 from one independent in vivo experiment. P values were calculated using two-tailed unpaired Student’s t test. Differences were statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For in vivo samples, a Quantikine ELISA kit for mouse IL-10 (R&D Systems) was used according to the manufacturer’s instructions with appropriately diluted serum added to each plate.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, In Vivo, Flow Cytometry, Expressing, Fluorescence, Comparison, Two Tailed Test

Journal: Applied Sciences

Article Title: Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids

doi: 10.3390/app15116170

Figure Lengend Snippet: Figure 9. Comparative analysis of CD63 expression in exosomes isolated from various types of biological samples using Western blot and ELISA: (A) urine, (B) plasma, (C) serum, and (D) saliva.

Article Snippet: For urine and saliva samples, a human CD63 ELISA kit (Cusabio, Wuhan, China) was used following the manufacturer’s instructions.

Techniques: Expressing, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Endocrinology

Article Title: Evidence for Adipocyte-Derived Extracellular Vesicles in the Human Circulation

doi: 10.1210/en.2018-00266

Figure Lengend Snippet: Major adipokines were present in post–magnetic-bead and post–solid-phase depletion samples. (A) Inverted raw data of dot blots predepletion, post–magnetic-bead depletion, and post–solid-phase depletion. (B) Major adipokines are highlighted with corresponding pixel densities. Representative dot blots of n = 3.

Article Snippet: Detection of an array of adipokines in plasma EV samples A commercially available Proteome Profiler Human Adipokine Array Kit (R&D Systems, Bio-Techne, Abingdon, United Kingdom) was used to analyze 58 adipocyte-related molecules in predepletion, post–magnetic-bead depletion, and post–solid-phase depletion EV samples.

Techniques: