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: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Expression of BCL6 in breast cancer and benign breast disease tissues

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Expressing

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Association of BCL6 protein expression with clinicopathological parameters from breast cancer patients

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Expressing

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Expression of BCL6 mRNA and protein in human breast cancer cell lines and tissue specimens. (a) qRT-PCR. Level of BCL6 mRNA expression in eight human mammary cell lines was analyzed by qRT-PCR. (b) qRT-PCR. Levels of BCL6 mRNA expression were examined in 30 breast cancer (BC) and 25 breast benign disease tissue specimens (BD) by qRT-PCR. (c) Representative imagines of BCL6 expression analyzed by in situ hybridization and immunohistochemistry (Magnification: ×400). (d) Kaplan-Meier curve of the relapse-free survival (RFS) or overall survival (OS) according to BCL6 expression.

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Expressing, Quantitative RT-PCR, In Situ Hybridization, Immunohistochemistry

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Effects of BCL6 on regulation of breast cancer cell phenotype. (a) Cell viability MTT assay. Cells were transiently transfected with BCL6 siRNA vs. negative control (NC) or BCL6 cDNA vs. control vector (VEC), respectively and then seeded in 96-well plates (3 × 10 3 per well) and grown for 4 days for MTT assay. (b) Wound healing assay. T47D cells were grown and transiently transfected with BCL6 siRNA or negative control (NC), the wounded monolayers were cultured in the absence (left) or presence (right) of mitomycin C. (c) Flow cytometric analysis of cell cycle distribution in MCF-7 cells after gene transfection. (d) Flow cytometric analysis of apoptosis in MCF-7 cells after gene transfection. The average of apoptosis rate is presented as mean ± SD. All experiments were repeated at least three times. ** P < 0.01.

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: MTT Assay, Transfection, Negative Control, Control, Plasmid Preparation, Wound Healing Assay, Cell Culture

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Effects of BCL6 expression on regulation of breast cancer colony formation and migration and invasion capacity. (a) Soft agar assay. After gene transfection, cells were seeded in 0.35% top agarose and 10% FBS in six-well plates in triplicate. The number of colonies was counted after 14 days incubation. (b) Tumor cell migration and invasion assay. MDA-MB-453 cells were grown and transiently transfected with BCL6 siRNA or negative control (NC) for 72 h. MCF-7 cells were grown and transiently transfected with BCL6 cDNA or vector-only (VEC) for 48 h. Cells in the upper chamber were removed and those cells migrated to the lower layer of the inner chamber were stained and counted. **, P < 0.01.

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Expressing, Migration, Soft Agar Assay, Transfection, Incubation, Invasion Assay, Negative Control, Plasmid Preparation, Staining

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Effects of BCL6 expression on regulation of MCF- 7 xenograft growth in nude mice. MCF7-VEC and MCF7-BCL6 cells were transplanted into the mammary fat pad of female BALB/c-nu, respectively. The volume of xenografts was measured twice a week and calculated. (a) Xenograft growth curve of MCF7-VEC and MCF7-BCL6-derived tumors over 27 days. (b) Hematoxylin and eosin staining of tumor xenograft sections. More aggressive behavior was observed in the margin of tumor nodule of MCF-7-BCL6 cells (red arrow) compared to that of MCF-7-VEC cells (blue arrow). Tumor embolus (red arrow head) was visualized in blood vessel (Magnification: ×200). *, P < 0.05; **, P < 0.01.

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Expressing, Derivative Assay, Staining

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: Effects of BCL6 expression on CXCR4 and cyclinD1 expression. (a) qRT-PCR. MCF-7 cells were transiently transfected with BCL6 cDNA or negative control vector and grown for 2 days. (b) Western blot. MCF-7 and T74D cells were transiently transfected with BCL6 cDNA, BCL6 siRNA, or negative control vector and grown for 2 days and subjected to Western blot. *, P < 0.05.

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Expressing, Quantitative RT-PCR, Transfection, Negative Control, Plasmid Preparation, Western Blot

Journal: BMC Cancer

Article Title: B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

doi: 10.1186/1471-2407-14-418

Figure Lengend Snippet: BCL6 as the direct target gene of miR - 339 - 5p in breast cancer cells. (a) qRT-PCR and Western blot. miR-339-5p mimics or miR-339-5p ASO was transiently transfected into T47D cells and subjected to analysis of BCL6 expression. (b) The binding site of BCL6 3′-UTR and miR-339-5p. (c) Luciferase reporter assay. T47D cells were transfected with psiCHECK-2-BCL6 3′-UTR or psiCHECK-2-BCL6 mutated 3′-UTR plus either miR-339-5p mimics or negative control and subjected to luciferase reporter assay. (d) Tumor cell migration and invasion assay and Western blot. MCF-7 cells were grown and transiently transfected with miR-339-5p ASO, miR-339-5p ASO plus BCL6 siRNA or scrambled sequence oligonucleotides as negative control for 2 days and subjected to migration, invasion and western blot assays. * P < 0.05; ** P < 0.01.

Article Snippet: For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [ , ].

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Expressing, Binding Assay, Luciferase, Reporter Assay, Negative Control, Migration, Invasion Assay, Sequencing