Journal: Redox biology

Article Title: The E3 ubiquitin ligase TRIM31 is involved in cerebral ischemic injury by promoting degradation of TIGAR.

doi: 10.1016/j.redox.2021.102058

Figure Lengend Snippet: Fig. 4. TRIM31 deficiency maintained mitochondria homeostasis in brain of mice subjected to middle cerebral artery occlusion (MCAO). (A) TRIM31 deficiency improved ultrastructural defects of neuronal mitochondria from mice subjected to MCAO. (B–D) The effect of TRIM31 on the expression of protein related to mitochondrial dynamics, PGC-1α, MFN1, MEN2 and DRP1, in the mice brain after 24 h of MCAO. Scale bars: 10 μm. Data were presented as the mean ± SEM of 3 independent experiments. *p < 0.05 vs. indicated group.

Article Snippet: Primary antibodies against TRIM31 (12543-1-AP), G6PD (66 373-1-1 g), DRP1 (12957-1-AP), MFN1 (13798-1-AP), MFN2 (12186-1-AP) and PGC-1α (66 369-1-1 g) were from Proteintech (Wuhan, China).

Techniques: Expressing

Journal: Journal of lipid research

Article Title: Disruption of nucleotide biosynthesis reprograms mitochondrial metabolism to inhibit adipogenesis.

doi: 10.1016/j.jlr.2024.100641

Figure Lengend Snippet: Fig. 4. PPARγ overexpression rescues mitochondrial function induced by the loss of de novo nucleotide biosynthesis. A: OXPHOS protein levels were measured in primary SVF cells that were differentiated and treated with PPARγ inhibitor SR 16832 (2 μM or 10 μM) for 6 days (B) 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM 5FU or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. C: 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM MIZ or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. D: Live cell imaging with MTG and TMRE staining was used to assess changes in mito- chondrial membrane potential relative to mitochondrial mass in the presence or absence of 25 μM MIZ. E: ImageJ was used to measure the ratio of MTG and TMRE. All experiments were repeated two or three times with two or three biological replicates. Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ****P < 0.0001.

Article Snippet: PPARγ2 plasmid (Addgene #8859) or pBabe control was transfected into PLAT-E cells.

Techniques: Over Expression, Stable Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Live Cell Imaging, Staining, Membrane

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Treatment effects of γ -tocotrienol, PPAR γ agonists, and PPAR γ antagonists on +SA cells. +SA cells were plated at a density of 5 × 10 4 (6 wells per group) in 24-well culture plates and exposed to treatment media for a 4-day period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. * P < 0.05 as compared with vehicle-treated controls.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Colorimetric Assay, Cell Counting

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Treatment effects of γ -tocotrienol, (a) PPAR γ agonists and (b) PPAR γ antagonists in combination in +SA cells. +SA cells were plated at a density of 5 × 10 4 (6 wells per group) in 24-well plates and exposed to treatment media for a 4-day period. Afterwards, viable cell number was determined using MTT colorimetric assay. Vertical bars indicate the mean cell count ± SEM in each treatment group. * P < 0.05 as compared with vehicle-treated controls and # P < 0.05 as compared to their corresponding control treated with γ -tocotrienol alone.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Colorimetric Assay, Cell Counting, Control

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Isobologram analysis of treatment of γ -tocotrienol with PPAR γ agonists and antagonists in +SA cells. Individual IC 50 doses for γ -tocotrienol, (a) PPAR γ agonists (rosiglitazone and troglitazone) and (b) PPAR γ antagonists (GW9662 and T0070907) were calculated and then plotted on the x -axes and y -axes, respectively. The data point on the isobologram represents the actual doses of combined γ -tocotrienol and PPAR γ agonists and antagonists. Combined treatment of γ -tocotrienol with PPAR γ agonists rosiglitazone and troglitazone was found to be antagonistic, as evidenced by the location of the data point in the isobologram being well above the line defining additive effect. In contrast, the growth inhibitory effect of combined treatment of γ -tocotrienol with PPAR γ antagonists GW9662 and T0070907 was found to be synergistic, as evidenced by the location of the data point in the isobologram being well below the line defining additive effect for both cell lines.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques:

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Western blot analysis to determine expression of PPAR γ in mammary cancer cells. (a) PPAR γ levels were determined in untreated +SA, MCF-7, and MDA-MB-231 cells and (b) PPAR γ levels were determined after treatment with γ -tocotrienol, rosiglitazone, and GW9662 alone or in combination in +SA cells. +SA, MCF-7, and MDA-MB-231 cells were initially plated at 1 × 10 6 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μ g/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β -actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit).

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Western Blot, Expressing, Control, Incubation, Polyacrylamide Gel Electrophoresis

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: (a) Western blot analysis and (b) luciferase assay to determine expression and activity of PPAR γ in PPAR γ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot, cells were plated at a density of 2 × 10 5 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic free media. Transfections were performed using 5 μ L lipofectamine 2000 according to the manufacturer's protocol. For western blot analysis, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μ g/lane). Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β -actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For luciferase assay, cells were plated at a density of 2 × 10 4 cells/well (3 replicates per group) in 96-well plates. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPAR γ siRNAs using 0.8 μ L of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μ L of control media. Afterwards, cells were lysed with 75 μ L of passive lysis buffer and treated according to manufacturer's instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit). * P < 0.05 as compared with vehicle-treated controls.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Western Blot, Luciferase, Expressing, Activity Assay, Transfection, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Control, Lysis

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Western blot analysis and luciferase assay to determine effect of γ -tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PPAR γ expression and activity in PPAR γ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot analysis, (a) MCF-7 and (b) MDA-MB-231 cells were plated at a density of 2 × 10 5 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media and allowed to adhere overnight. Transfections were performed using 5 μ L lipofectamine 2000 according to the manufacturer's protocol. Afterwards whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μ g/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β -actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For luciferase assay, (c) MCF-7 and (d) MDA-MB-231 cells were plated at a density of 2 × 10 4 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPAR γ siRNAs using 0.8 μ L of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μ L of control or treatment media. Afterwards, cells were lysed with 75 μ L of passive lysis buffer and treated according to manufacturer's instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit) in each treatment group. * P < 0.05 as compared with vehicle-treated controls.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Western Blot, Luciferase, Expressing, Activity Assay, Transfection, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Control, Lysis

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Western blot was performed to determine effect of treatment of γ -tocotrienol and rosiglitazone alone or in combination in PPAR γ siRNA transfected (a) MCF-7 and (b) MDA-MB-231 cells. In addition, Western blot was performed to determine effect of treatment of γ -tocotrienol and GW9662 alone or in combination in PPAR γ siRNA transfected (c) MCF-7 and (d) MDA-MB-231 cells. Cells were plated at a density of 2 × 10 5 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μ L lipofectamine 2000 according to the manufacturer's protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPAR γ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture period. Afterwards whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μ g/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β -actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). * P < 0.05 as compared with vehicle-treated controls.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Western Blot, Transfection, Cell Culture, Control, Polyacrylamide Gel Electrophoresis

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: (a) Effect of γ -tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PGD 2 synthesis and (b) treatment effect of 15d-PGJ 2 in PPAR γ positive MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were plated at a density of 2 × 10 5 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μ L lipofectamine 2000 according to the manufacturer's protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPAR γ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture period. Afterwards whole cell lysates were assayed for PGD 2 according to the manufacturer's protocol. Vertical bars indicate the amount of PGD 2 synthesized (pg/mL) ± SEM in each treatment group. For 15d-PGJ 2 effect, MCF-7 and MDA-MB-231 cells were plated at a density of 2 × 10 4 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. Transfections were performed using 0.25 μ L lipofectamine 2000. For each well of cells to be transfected, 5 pmol of the scrambled or PPAR γ siRNAs was diluted with 100 μ L of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% fetal bovine serum and cells were cultured for 18 h. Cells were then exposed to 100 μ L of control or treatment media for a 4-day culture period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. * P < 0.05 as compared with vehicle-treated controls.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Transfection, Cell Culture, Control, Synthesized, Colorimetric Assay, Cell Counting

Journal: PPAR Research

Article Title: Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells

doi: 10.1155/2014/439146

Figure Lengend Snippet: Apoptotic effects of γ -tocotrienol and PPAR γ antagonists alone or in combination on (a) cleaved caspase-3, cleaved PARP levels and (b) viable cell number on +SA cells. For Western blot studies, +SA cells were initially plated at 1 × 10 6 cells/100 mm culture dish and maintained on control media for a 3-day culture period. Afterwards, cells were divided into the various treatment groups, media were removed, and cells were exposed to their respective treatment media for a 24 h treatment period. In addition, cells were exposed to their respective treatment media for a 96 h treatment period, where fresh media were added every other day. +SA cells were exposed to treatment media containing 2 μ M γ -tocotrienol and 3.2 μ M GW9662 or T0070907 alone or in combination. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μ g/lane) followed by western blot analysis. In parallel studies, (b) +SA cells were plated at a density of 5 × 10 4 (6 wells per group) in 24-well culture plates and exposed to the same treatments as described above. After a 24 h treatment exposure, viable cell number in all treatment groups was determined using MTT assay. Vertical barsindicate the mean cell count ± SEM in each treatment group. * P < 0.05 as compared with vehicle-treated controls.

Article Snippet: Antibodies, β -actin (Cell Signaling 4970), PPAR γ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPAR γ siRNAs (Santa Cruz sc-29455) were used in the present study.

Techniques: Western Blot, Control, Polyacrylamide Gel Electrophoresis, MTT Assay, Cell Counting