stem pro adipocyte differentiation media  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: UVA irradiation inhibits adipocyte differentiation. Two-day post-confluent hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® adipocyte differentiation (DIF) media for 3 days. The medium was then replaced with STEM PRO® adipocyte differentiation media every 3 days until the end of the experiment at day 14. The assays were performed on fully differentiated adipocytes (day 14). A, intracellular lipids were stained with Oil Red O. The results were confirmed by three independent experiments, which were each conducted in duplicate. B, triglyceride content was measured using a triglyceride assay kit (Cayman Chemical, Ann Arbor, MI). Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by three repetitions of the experiments, each of which was conducted in triplicate. C, GPDH activity was measured using GPDH activity assay kits (Takara Bio Inc., Japan). Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by four repetitions of the experiments. D, 2-day post-confluent 3T3-L1 preadipocytes (day 0) were treated with the indicated concentrations of UVA irradiation and then stimulated with differentiation medium for 3 days. The medium was then replaced with maintenance media every 3 days until the end of the experiment on day 9. The assays were performed on fully differentiated adipocytes (day 9). Intracellular lipids were stained with Oil Red O. The results were confirmed by three independent experiments, which were each conducted in duplicate. E, general viability of cultured cells was determined by the reduction of WST-8 to a highly water-soluble formazan dye. The results were verified by four repetitions of the experiments. F, DNA-damaging effects of UVA irradiation were determined using a DNA Damage ELISA kit. The results were verified by four repetitions of the experiments. G, apoptotic effects of UVA irradiation were determined by Hoechst 33332 staining. The results were verified by four repetitions of the experiments. DIF/MDI, differentiation media.

Article Snippet: Immunoblotting Two-day post-confluent hAMSCs were irradiated with the indicated doses of UVA and then stimulated with STEM PRO® adipocyte differentiation media (Invitrogen) for 3 days.

Techniques: Irradiation, Staining, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: Effects of UVA irradiation on the expression of the adipogenic transcription factors. hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® adipocyte differentiation media for 3 days. The medium was then replaced with STEM PRO® adipocyte differentiation media every 3 days until the end of the experiment on day 14. A and D--G, at 1 day or 14 days after the induction of differentiation, the total RNA was isolated, and the mRNA levels of the PPAR γ (A), C/EBP α (D), C/EBP β and C/EBP δ (E), aP2 and LPL (F), and CD36 and LXR α and (G) genes were measured by real time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against the 18 S rRNA. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by four repetitions of the experiments, each of which was conducted in triplicate. B, at day 14 after the induction of differentiation, the cell lysates were analyzed by Western blot analysis using the indicated antibodies. The results were verified by three repetitions of the experiments, each of which was conducted in duplicate. C, hAMSCs were cotransfected with luciferase constructs that contained PPRE×3-Luc reporter and PPAR γ cDNA plasmid. After 16 h, the transfected cells were incubated for 20 h with the indicated doses of UVA irradiation. The cells were then harvested and lysed. Luciferase activity is expressed as the ratio of PPRE×3 promoter-dependent firefly luciferase activity divided by control thymidine kinase Renilla luciferase activity (relative luciferase units). Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The experiments were repeated eight times, with each experiment being conducted in triplicate. DIF, differentiation media.

Article Snippet: Immunoblotting Two-day post-confluent hAMSCs were irradiated with the indicated doses of UVA and then stimulated with STEM PRO® adipocyte differentiation media (Invitrogen) for 3 days.

Techniques: Irradiation, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Luciferase, Construct, Plasmid Preparation, Transfection, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: Effects of UVA irradiation on the production of anti-adipogenic cytokines. hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® adipocyte differentiation media for 3 days. The medium was then replaced with STEM PRO® adipocyte differentiation media every 3 days until the end of the experiment on day 14. At day 14 after the induction of differentiation, the supernatants were harvested for TNF-α (A), IL-1β (B), VEGF (C), TGF-β1 (D), and MIF (E and G) measurements. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by three repetitions of the experiments, each of which was conducted in duplicate. F, at 14 days after the induction of differentiation, the total RNA was isolated, and the mRNA levels of the PPAR γ gene were measured by real time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against 18 S rRNA. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by four repetitions of the experiments, each of which was conducted in triplicate. DMI, differentiation media; PD, PD98059 (a p42/44 MAPK inhibitor); SB, SB203580 (a p38 MAPK inhibitor); SP, SP600125 (JNK inhibitor); PDTC, pyrrolidine dithiocarbamate.

Article Snippet: Immunoblotting Two-day post-confluent hAMSCs were irradiated with the indicated doses of UVA and then stimulated with STEM PRO® adipocyte differentiation media (Invitrogen) for 3 days.

Techniques: Irradiation, Isolation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: UVA irradiation has no effects on osteoblast differentiation in hAMSCs. Two-day post-confluent hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® osteogenesis differentiation media for 3 days. The medium was then replaced with STEM PRO® osteogenesis differentiation media every 3 days until the end of the experiment at day 14. A, alkaline phosphatase staining. Osteogenesis was confirmed with alkaline phosphatase staining. The results were confirmed by three independent experiments, which were each conducted in duplicate. B, alkaline phosphatase activity was measured in cell lysates for 14 days, using a colorimetric assay, and then normalized to the level of total cellular protein. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were confirmed by three independent experiments, which were each conducted in duplicate. ODIF, osteogenic differentiation media.

Article Snippet: Immunoblotting Two-day post-confluent hAMSCs were irradiated with the indicated doses of UVA and then stimulated with STEM PRO® adipocyte differentiation media (Invitrogen) for 3 days.

Techniques: Irradiation, Staining, Activity Assay, Colorimetric Assay

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: UVA irradiation inhibits adipocyte differentiation. Two-day post-confluent hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® adipocyte differentiation (DIF) media for 3 days. The medium was then replaced with STEM PRO® adipocyte differentiation media every 3 days until the end of the experiment at day 14. The assays were performed on fully differentiated adipocytes (day 14). A, intracellular lipids were stained with Oil Red O. The results were confirmed by three independent experiments, which were each conducted in duplicate. B, triglyceride content was measured using a triglyceride assay kit (Cayman Chemical, Ann Arbor, MI). Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by three repetitions of the experiments, each of which was conducted in triplicate. C, GPDH activity was measured using GPDH activity assay kits (Takara Bio Inc., Japan). Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by four repetitions of the experiments. D, 2-day post-confluent 3T3-L1 preadipocytes (day 0) were treated with the indicated concentrations of UVA irradiation and then stimulated with differentiation medium for 3 days. The medium was then replaced with maintenance media every 3 days until the end of the experiment on day 9. The assays were performed on fully differentiated adipocytes (day 9). Intracellular lipids were stained with Oil Red O. The results were confirmed by three independent experiments, which were each conducted in duplicate. E, general viability of cultured cells was determined by the reduction of WST-8 to a highly water-soluble formazan dye. The results were verified by four repetitions of the experiments. F, DNA-damaging effects of UVA irradiation were determined using a DNA Damage ELISA kit. The results were verified by four repetitions of the experiments. G, apoptotic effects of UVA irradiation were determined by Hoechst 33332 staining. The results were verified by four repetitions of the experiments. DIF/MDI, differentiation media.

Article Snippet: The medium was then replaced with STEM PRO® adipocyte differentiation media (Invitrogen) every 3 days until the end of the experiment on day 14.

Techniques: Irradiation, Staining, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: Effects of UVA irradiation on the expression of the adipogenic transcription factors. hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® adipocyte differentiation media for 3 days. The medium was then replaced with STEM PRO® adipocyte differentiation media every 3 days until the end of the experiment on day 14. A and D--G, at 1 day or 14 days after the induction of differentiation, the total RNA was isolated, and the mRNA levels of the PPAR γ (A), C/EBP α (D), C/EBP β and C/EBP δ (E), aP2 and LPL (F), and CD36 and LXR α and (G) genes were measured by real time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against the 18 S rRNA. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by four repetitions of the experiments, each of which was conducted in triplicate. B, at day 14 after the induction of differentiation, the cell lysates were analyzed by Western blot analysis using the indicated antibodies. The results were verified by three repetitions of the experiments, each of which was conducted in duplicate. C, hAMSCs were cotransfected with luciferase constructs that contained PPRE×3-Luc reporter and PPAR γ cDNA plasmid. After 16 h, the transfected cells were incubated for 20 h with the indicated doses of UVA irradiation. The cells were then harvested and lysed. Luciferase activity is expressed as the ratio of PPRE×3 promoter-dependent firefly luciferase activity divided by control thymidine kinase Renilla luciferase activity (relative luciferase units). Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The experiments were repeated eight times, with each experiment being conducted in triplicate. DIF, differentiation media.

Article Snippet: The medium was then replaced with STEM PRO® adipocyte differentiation media (Invitrogen) every 3 days until the end of the experiment on day 14.

Techniques: Irradiation, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Luciferase, Construct, Plasmid Preparation, Transfection, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: Effects of UVA irradiation on the production of anti-adipogenic cytokines. hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® adipocyte differentiation media for 3 days. The medium was then replaced with STEM PRO® adipocyte differentiation media every 3 days until the end of the experiment on day 14. At day 14 after the induction of differentiation, the supernatants were harvested for TNF-α (A), IL-1β (B), VEGF (C), TGF-β1 (D), and MIF (E and G) measurements. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by three repetitions of the experiments, each of which was conducted in duplicate. F, at 14 days after the induction of differentiation, the total RNA was isolated, and the mRNA levels of the PPAR γ gene were measured by real time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against 18 S rRNA. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were verified by four repetitions of the experiments, each of which was conducted in triplicate. DMI, differentiation media; PD, PD98059 (a p42/44 MAPK inhibitor); SB, SB203580 (a p38 MAPK inhibitor); SP, SP600125 (JNK inhibitor); PDTC, pyrrolidine dithiocarbamate.

Article Snippet: The medium was then replaced with STEM PRO® adipocyte differentiation media (Invitrogen) every 3 days until the end of the experiment on day 14.

Techniques: Irradiation, Isolation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

doi: 10.1074/jbc.M110.135830

Figure Lengend Snippet: UVA irradiation has no effects on osteoblast differentiation in hAMSCs. Two-day post-confluent hAMSCs (day 0) were treated with the indicated doses of UVA irradiation and then stimulated with STEM PRO® osteogenesis differentiation media for 3 days. The medium was then replaced with STEM PRO® osteogenesis differentiation media every 3 days until the end of the experiment at day 14. A, alkaline phosphatase staining. Osteogenesis was confirmed with alkaline phosphatase staining. The results were confirmed by three independent experiments, which were each conducted in duplicate. B, alkaline phosphatase activity was measured in cell lysates for 14 days, using a colorimetric assay, and then normalized to the level of total cellular protein. Data are expressed as the means ± S.D. *, p < 0.05 versus controls. The results were confirmed by three independent experiments, which were each conducted in duplicate. ODIF, osteogenic differentiation media.

Article Snippet: The medium was then replaced with STEM PRO® adipocyte differentiation media (Invitrogen) every 3 days until the end of the experiment on day 14.

Techniques: Irradiation, Staining, Activity Assay, Colorimetric Assay