gluc on promoter reporter vector  (Genecopoeia)

Journal: International Journal of Molecular Sciences

Article Title: Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms23010071

Figure Lengend Snippet: Effects of extracellular Mg 2+ concentration on HAS2/3 expressions in HaCaT cells. ( A , B ) Cells were incubated in the presence of 0, 0.8, and 5.8 mM MgCl 2 for 6 h. The mRNA levels of HAS2/3 were measured by real-time PCR analysis and represented as a percentage of 0.8 mM MgCl 2 . ( C , D ) Cells were incubated in the presence of 0, 0.8, and 5.8 mM MgCl 2 for 24 h. The protein levels of HAS2/3 were measured by Western blotting analysis and represented as a percentage of 0.8 mM MgCl 2 . HA contents in the media were measured using a Hyaluronan Quantification kit. n = 3–4. ** p < 0.01 and * p < 0.05 significantly different from 0.8 mM MgCl 2 . NS p > 0.05.

Article Snippet: The GLuc-ON Promoter Reporter vectors for human HAS2 and HAS3 were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan).

Techniques: Concentration Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms23010071

Figure Lengend Snippet: Effect of NIPAL4 siRNA on HAS2/3 expressions. Cells were transfected with negative (Neg) or NIAPL4 (N4) siRNA. ( A ) After transfection, the cells were incubated in the presence of 0.8 mM MgCl 2 for 72 h. The mRNA levels of N4 and HAS2/3 were measured by real-time PCR analysis and represented as a percentage of negative siRNA. ( B ) After 66 h of transfection, the cells were incubated in the presence of 0.8 and 5.8 mM MgCl 2 for 6 h. The mRNA levels of HAS2/3 were measured by real-time PCR analysis and represented as a percentage of 0.8 mM MgCl 2 . ( C , D ) After 48 h of transfection, the cells were incubated in the presence of 0.8 and 5.8 mM MgCl 2 for 24 h. The protein levels of NIPA4 and HAS2/3 were measured by Western blotting analysis and represented as a percentage of 0.8 mM MgCl 2 . HA contents in the media were measured using a Hyaluronan Quantification kit. n = 3–4. ** p < 0.01 significantly different from negative siRNA or 0.8 mM MgCl 2 . NS p > 0.05. ## p < 0.01 significantly different from negative siRNA plus 5.8 mM MgCl 2 .

Article Snippet: The GLuc-ON Promoter Reporter vectors for human HAS2 and HAS3 were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan).

Techniques: Transfection, Incubation, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms23010071

Figure Lengend Snippet: Effects of extracellular Mg 2+ concentration and NIPAL4 siRNA on reporter activities of HAS2/3. Cells were transfected with negative (Neg) or NIAPL4 (N4) siRNA. After 66 h of transfection, the cells were incubated in the presence of 0.8 and 5.8 mM MgCl 2 for 6 h. The activities of secreted luciferase and SEAP were measured using a Ready-To-Glow Dual Secreted Reporter Assay kit. The reporter activity is represented as a percentage of 0.8 mM MgCl 2 . n = 4. ** p < 0.01 and * p < 0.05 significantly different from 0.8 mM MgCl 2 . ## p < 0.01 significantly different from negative siRNA plus 5.8 mM MgCl 2 .

Article Snippet: The GLuc-ON Promoter Reporter vectors for human HAS2 and HAS3 were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan).

Techniques: Concentration Assay, Transfection, Incubation, Luciferase, Reporter Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms23010071

Figure Lengend Snippet: Effects of MgCl 2 supplementation and signaling inhibitors on HAS2/3 expressions and HA production. ( A ) Cells transfected with promoter reporter vectors for HAS2/3 were incubated in the absence (Veh) and presence of 10 μM CHIR-99021 (CHIR) or 10 μM naphthol AS-E (Naph) for 6 h. The reporter activities of HAS2/3 are represented as a percentage of 0.8 mM MgCl 2 . ( B ) Cells were incubated in the absence (Veh) and presence of 0.8 mM MgCl 2 , 5.8 mM MgCl 2 , 10 μM CHIR-99021 (CHIR) or 10 μM naphthol AS-E (Naph) for 6 h. The mRNA levels of HAS2/3 are represented as a percentage of 0.8 mM MgCl 2 . ( C ) Cells were incubated in the absence (Veh) and presence of 0.8 mM MgCl 2 , 5.8 mM MgCl 2 , 10 μM CHIR-99021 (CHIR) or 10 μM naphthol AS-E (Naph) for 24 h. After collecting the media, HA contents were measured using a Hyaluronan Quantification kit. n = 4–6. ** p < 0.01 significantly different from 0.8 mM MgCl 2 . ## p < 0.01 and # p < 0.05 significantly different from 5.8 mM MgCl 2 plus 5.8 mM MgCl 2 .

Article Snippet: The GLuc-ON Promoter Reporter vectors for human HAS2 and HAS3 were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan).

Techniques: Transfection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms23010071

Figure Lengend Snippet: Involvement of CREB in the elevation of HAS2/3 expressions by MgCl 2 supplementation. ( A ) Cells were incubated in the absence (Veh) and presence of 0.8 mM MgCl 2 , 5.8 mM MgCl 2 , or 10 μM naphthol AS-E (Naph) for 2 h. After isolation of nuclear fraction, the protein levels of p-CREB and nucleoporin p62 (p62), an internal control, were measured by Western blotting analysis and represented as a percentage of 0.8 mM MgCl 2 . ( B ) Cells were transfected with reporter vectors of HAS2/3 containing the mutation in CREB-binding site (HAS2m and HAS3m). Then, the cells were incubated in the presence of 0.8 and 5.8 mM MgCl 2 for 6 h. The reporter activity is represented as a percentage of 0.8 mM MgCl 2 . n = 3–4. ** p < 0.01 significantly different from 0.8 mM MgCl 2 . NS p > 0.05. ## p < 0.01 significantly different from 5.8 mM MgCl 2 plus 5.8 mM MgCl 2 .

Article Snippet: The GLuc-ON Promoter Reporter vectors for human HAS2 and HAS3 were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan).

Techniques: Incubation, Isolation, Control, Western Blot, Transfection, Mutagenesis, Binding Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms23010071

Figure Lengend Snippet: Primer pairs for real-time PCR.

Article Snippet: The GLuc-ON Promoter Reporter vectors for human HAS2 and HAS3 were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan).

Techniques: Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

doi: 10.1007/s00018-023-05095-x

Figure Lengend Snippet: SND1 bound with SRF-CArG complex to promote VSMC proliferation- and migration-related gene transcription. A EMSA assays were performed using purified SRF1(1–266 aa, 0.1 μg), or SND1 (1–660 aa, 0.1 μg) protein and Biotin-labeled probes targeting the CArG motif (–CCATATTAGG–, 20 fmoL) of the Fos gene. Unlabeled- or unspecific probes were used for competition experiments. B , C 100 μg of cell nuclear lysate were collected from 1.5 × 10 7 primary Snd1-F/F and Snd1-F/F Tagln-Cre VSMCs. SRF enrichments at Cdk2 , Fos , Cdk6 , Rock1 , Rock2 , and Iqgap1 promoter were analyzed by ChIP assay. D , G The Snd1-F/F and Snd1-F/F Tagln-Cre VSMCs were allowed to grow until the cells were 70% confluent (2 × 10 5 per well in 6-well plates) and infected with the GLuc- Cdk2 , GLuc- Fos , GLuc- Cdk6 , GLuc- Rock1 , GLuc- Rock2 , and GLuc- Iqgap1 promoter plasmids, empty GLuc-Vector as the negative control (NC). Relative luciferase activity was evaluated by the ratio of GLuc / SEAP activity. 2 × 10 6 primary VSMCs were obtained in a 6 cm dish. E , H The mRNA levels of Cdk2 , Fos , Cdk6, Rock1 , Rock2 , and Iqgap1 were detected by RT-qPCR. F , I The protein levels of SND1, CDK2, FOS, CDK6, ROCK1, ROCK2, IQGAP1 and ACTB were detected by western blotting. Western blotting results were analyzed by ImageJ (2 ×) software. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed by unpaired two-tailed Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns no significance

Article Snippet: The wild type and ELK1 binding sites (–CCGGAAGT–) mutant promoter sequences of Snd1 were cloned into the Gluc-ON™ promoter-reporter vector (GeneCopoeia, EZX-LvPG04).

Techniques: Migration, Purification, Labeling, Infection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Software, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

doi: 10.1007/s00018-023-05095-x

Figure Lengend Snippet: ELK1 promoted transcriptional activation of Snd1 in VSMC proliferation. A Venn diagram showed the upstream transcription factors of Snd1 predicted by JASPAR, EPD, GeneCard, GTRD, and PROMO bioinformatics software. B ELK1 binding motif (–CCGGAAGT–) in Snd1 promoter region across vertebrates was analyzed by the JASPAR database. C EMSA assays were performed using purified ELK1 (0.1 μg) protein and Biotin-labeled probes targeting motif (–CCGGAAGT–, 20 fmoL) of the Snd1 , unlabeled- or unspecific probes were used for competition experiments. D Primers were designed for ChIP based on the promoter regions containing the ELK1 binding motif (Region a), the region located outside the promoter region (Region b) as the negative control. E The mice femoral arteries were obtained after the injury ( n = 8 /group), ChIP analysis of ELK1 enrichment at different regions of Snd1 promoter. F 100 μg of cell nuclear lysate were collected by 1.5 × 10 7 primary cells for ChIP assay. ChIP analysis of ELK1 enrichment at different regions of Snd1 promoter in VSMCs that infected with pLVX-IRES-ELK1 or pLVX-IRES-Vector and stimulated with or without PDGF (40 ng/mL) for 24 h. G , J The primary VSMCs were allowed to grow until the cells were 70% confluent (2 × 10 5 per well in 6-well plates). Then primary VSMCs were infected with the GLuc plasmids carrying wild-type Snd1 promoter (− 2500 to + 500 bp) or Snd1 promoter with ELK1 binding site mutant ( Snd1 -mut), empty GLuc-Vector as the negative control (NC), together with pLVX-IRES-ELK1, or pLVX-IRES-Vector plasmids, or TRC2-pLKO-shElk1-1, TRC2-pLKO-shElk1-2, or TRC2-pLKO-Vector, with or without PDGF treatment for 24 h. Relative luciferase activity was evaluated by the ratio of GLuc / SEAP activity. H , I The primary VSMCs were infected with pLVX-IRES-ELK1 or pLVX-IRES-Vector and then were stimulated with or without PDGF (40 ng/mL) for 24 h, 2 × 10 6 primary VSMCs were obtained in a 6 cm dish. The mRNA levels of Snd1 were detected by RT-qPCR. The protein levels of SND1, ELK1, pELK1 and ACTB in were detected by western blotting. K – L The primary VSMCs were infected with TRC2-pLKO-Vector, shElk1-1 or shElk1-2 and then stimulated with or without PDGF (40 ng/mL) for 24 h, 2 × 10 6 primary VSMCs were obtained in a 6 cm dish. The mRNA levels of Snd1 were detected by RT-qPCR. The protein levels of SND1, ELK1, pELK1 and ACTB were detected by western blotting. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed by one-way ANOVA (from E, F, H, I, K, and L), or three-way ANOVA (G, J) followed by Bonferroni post-hoc correction. ** P < 0.01; *** P < 0.001; ns no significance

Article Snippet: The wild type and ELK1 binding sites (–CCGGAAGT–) mutant promoter sequences of Snd1 were cloned into the Gluc-ON™ promoter-reporter vector (GeneCopoeia, EZX-LvPG04).

Techniques: Activation Assay, Software, Binding Assay, Purification, Labeling, Negative Control, Infection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

doi: 10.1007/s00018-023-05095-x

Figure Lengend Snippet: Inhibition of the phosphorylation of ELK1 by U0126 retards injury-induced SND1 upregulation and neointima hyperplasia. A Wire-induced mouse femoral artery injury models were constructed, and U0126 was injected intraperitoneally every 3 days (1 mg/kg) after wire injury. The femoral arteries were collected ( n = 4 /group). Representative HE staining of sham-operated and wire-injured femoral arteries from DMSO and U0126 treated mice. ( N neointima; M media) B Averaged data of the neointima area, neointima/media ratio, lumen area, media area, peripheral vessel circumference, lumen vessel circumference of sham-operated or wire-injured femoral arteries ( n = 4 /group). C 100 μg of cell nuclear lysate were collected by 1.5 × 10 7 primary cells for ChIP assay. ChIP analysis of ELK1 enrichment at different regions of Snd1 promoter in primary VSMCs with PDGF in the presence or absence of U0126 (10 mM). D The primary VSMCs were allowed to grow until the cells were 70% confluent (2 × 10 5 per well in 6-well plates). Then cells were infected with the GLuc plasmids carrying wild-type Snd1 promoter (− 2500 to + 500 bp) or Snd1 promoter with ELK1 binding site mutant ( Snd1 -mut), empty GLuc-Vector as the negative control (NC), together with PDGF in the presence or absence of U0126 (10 mM). Relative luciferase activity was evaluated by the ratio of GLuc/SEAP activity. E , F The primary VSMCs were allowed to grow until the cells were 70% confluent and were stimulated with or without PDGF for 24 h in the presence or absence of U0126 (10 mM) for 24 h. The mRNA levels of Snd1 were detected by RT-qPCR. The protein levels of SND1, ELK1, pELK1 and ACTB were detected by western blotting. All western blotting results were analyzed by ImageJ (2 ×) software. Scale bar, 100 μm. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed by unpaired two-tailed Student’s t -test (A), one-way ANOVA (C, E, and F) or three-way ANOVA (A and D) followed by Bonferroni post-hoc correction. * P < 0.05; ** P < 0.01; *** P < 0.001; ns no significance

Article Snippet: The wild type and ELK1 binding sites (–CCGGAAGT–) mutant promoter sequences of Snd1 were cloned into the Gluc-ON™ promoter-reporter vector (GeneCopoeia, EZX-LvPG04).

Techniques: Inhibition, Construct, Injection, Staining, Infection, Binding Assay, Mutagenesis, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Software, Two Tailed Test