Journal: eLife

Article Title: Dual separable feedback systems govern firing rate homeostasis

doi: 10.7554/eLife.45717

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , XE-991 dihydrochloride , Tocris , 2000; CAS 122955-13-9 , .

Techniques: CRISPR, Mutagenesis, Sequencing, Amplification, Recombinant, Gene Expression, Software

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Functional characteristics of K V 7.3 channel in MG-63 cells during osteoblast differentiation. ( A ) Representative current responses to depolarizing steps observed at day 0 of undifferentiated MG-63 cells. XE991-sensitive currents were obtained by the subtraction of the currents in controls from in the presence of XE991 (50 μM); ( B ) Current-voltage relationships in controls and in the presence of XE991 at day 0 ( n = 3); ( C ) Representative current responses to depolarizing steps observed at day 2 of osteoblastic differentiation; ( D ) Current-voltage relationships in controls and in the presence of XE991 at day 2 ( n = 4); ( E ) XE991-sensitive currents at days 0–4 were normalized by mean current value at day 0. At day 1 of osteoblast differentiation, normalized XE991-sensitive currents had no significant change. At days 2 and 3, the K V 7.3 currents were increased, but not statistically significant. Data are presented as mean ± SEM. * p < 0.05.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Effect of flupirtine, linopirdine, and XE991 on MG-63 cell viability. The MTT assay was performed on MG-63 cells. ( A ) MG-63 cells were incubated in growth medium (GM) with 30 μM of flupirtine, 30 μM of linopirdine, and 10 μM of XE991. At 72 h, linopirdine, XE991, and flupirtine caused notable decreases in cell viability ( n = 16); ( B ) MG-63 cells were cultured in osteoblast-induction medium (OM) with 30 μM of flupirtine, 30 μM of linopirdine, and 10 μM of XE991. The cell viability was not significantly influenced by OM containing linopirdine, XE991, or flupirtine for 72 h of treatment ( n = 16). The values are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: MTT Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Regulation of osteoblastic differentiation by K V 7 channel in MG-63 and Saos-2 cells. ( A ) Alizarin Red S staining data showed that 30 μM of linopirdine and 10 μM of XE991 augmented mineralization in the extracellular matrix in MG-63 cells ( n = 10). There was no significant change in the mineralized matrix with 30 μM of flupirtine ( n = 3); ( B ) The OD values demonstrated that linopirdine and XE991 increased the amount of calcium deposits; ( C ) Alizarin Red S staining illustrated that mineralization of Saos-2 cells was increased with 30 μM of linopirdine and 10 μM of XE991 ( n = 8), while 30 μM of flupirtine reduced the amount of calcium deposits ( n = 7); ( D ) The OD values are shown parallel to Alizarin Red S staining results. Data are presented as mean ± SEM. *** p < 0.005. Scale bar represents 100 μm. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991; OD: optical density.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: mRNA expression of osteoblastic differentiation markers in MG-63 cells. The relative mRNA expression levels of osteoblastic differentiation markers, ALP, OSC, Runx2, and osterix, were measured with qRT-PCR and normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. ( A ) While linopirdine (30 μM) and XE991 (10 μM) increased ALP mRNA expression at days 7 and 10 of osteoblastic induction, flupirtine (30 μM) decreased ALP mRNA expression at days 7 and 10 ( n = 3–7); ( B ) The mRNA expression level of OSC was increased by linopirdine or XE991 at days 7 and 10, respectively ( n = 3–7); ( C ) mRNA expression of Runx2 was decreased by flupirtine at day 4 and by linopirdine at day 10 ( n = 3); ( D ) Osterix gene expression was increased by linopirdine and by XE991 at day 10 of osteoblastic induction ( n = 3–7). The values are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005. ALP: alkaline phosphatase; OSC: osteocalcin; CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Regulation of synaptic vesicle-related protein, synapsin, by K V 7.3 channel in MG-63 cells. Western blot analysis showed that K V 7.3 blockade by linopirdine (30 μM) or XE991 (10 μM) increased synapsin expression during osteoblast differentiation. However, K V 7 activation by flupirtine (30 μM) had no significant effect on the protein expression of synapsin ( n = 3). Vinculin is used as a loading control for Western blot analysis. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Activation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Alterations of ERK1/2 phosphorylation by the K V 7 opener or K V 7.3 blockers in MG-63 cells. Western blot analysis showed that, while linopirdine (30 μM) and XE991 (10 μM) increased the expression level of ERK1/2 phosphorylation at days 4 and 7 of osteoblast induction, flupirtine (30 μM) had no significant effect on the level of ERK1/2 phosphorylation. Treatment with linopirdine or XE991 showed no increase of ERK1/2 phosphorylation at day 14 of osteoblast differentiation ( n = 3). Flupirtine augmented the expression of ERK1/2 phosphorylation at day 14 ( n = 3). Vinculin is used as a loading control for Western blot analysis. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991; ERK1/2: extracellular-signal-regulated kinase 1/2; p-ERK: ERK1/2 phosphorylation.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Effect of K V 7 channel on glutamate release during osteoblastic differentiation in MG-63 cells. ( A ) On day 2, flupirtine (30 μM) significantly decreased glutamate release after inducing osteoblastic differentiation ( n = 6). However, linopirdine (30 μM) or XE991 (10 μM) caused significantly increased glutamate release ( n = 5); ( B ) On day 4, flupirtine also caused decreased glutamate release ( n = 5), whereas XE991 notably increased the extracellular glutamate ( n = 7). Linopirdine augmented the amount of glutamate, but not statistically significantly ( n = 8). The values are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Suppressive effect of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainite receptor antagonist, on osteoblastic differentiation promoted by K V 7.3 blockers. ( A ) Alizarin Red S staining showed that CNQX (50 μM) co-applied with flupirtine (30 μM), linopirdine (30 μM), or XE991 (10 μM) produced amounts ofcalcium deposits similar to those of the controls ( n = 3). ( B ) The OD values are shown parallel to Alizarin Red S staining results. The relative mRNA expression levels of osteoblastic differentiation markers, including ALP and OSC, were measured with qRT-PCR and normalized against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression ( n = 3). ( C ) At day 4, CNQX treatment with XE991 significantly reduced the ALP mRNA expression. At day 7, CNQX with flupirtine, linopirdine, or XE991 attenuated the ALP level. ( D ) CNQX treatment with flupirtine, linopirdine, or XE991 showed no significant changes in the OSC levels. Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. Scale bar represents 100 μm. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991; ALP: alkaline phosphatase; OSC: osteocalcin; OD: optical density.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Staining, Produced, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Suppressive effect of MK801, an NMDA (N-methyl-D-aspartate) receptor antagonist, on osteoblastic differentiation promoted by K V 7.3 blockers. ( A ) Alizarin Red S staining showed that MK801 (50 μM) treatment co-applied with flupirtine (30 μM), linopirdine (30 μM), or XE991 (10 μM) produced amounts of calcium deposits similar to those of the controls ( n = 3); ( B ) The OD values are shown parallel to Alizarin Red S staining results. The relative mRNA expression levels of osteoblastic differentiation markers, including ALP and OSC, were measured with qRT-PCR and normalized against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression ( n = 3); ( C ) At days 4 and 7, MK801 treatment with linopirdine or XE991 attenuated ALP mRNA expression; ( D ) At day 7, MK801 treatment with flupirtine or linopirdine significantly reduced OSC mRNA expression. Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. Scale bar represents 100 μm. CONT: non-treated controls; MK: MK801; FLU: flupirtine; LINO: linopirdine; XE: XE991; ALP: alkaline phosphatase; OSC: osteocalcin; OD: optical density.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Staining, Produced, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Counter-effect of riluzole, a glutamate release inhibitor, on osteoblastic differentiation promoted by K V 7.3 blockers. ( A ) Alizarin Red S staining showed that riluzole (30 μM) co-applied with flupirtine (30 μM), linopirdine (30 μM), or XE991 (10 μM) produced mineralization levels similar to those of the controls ( n = 3); ( B ) The OD values are shown parallel to Alizarin Red S staining results. The relative mRNA expression levels of osteoblastic differentiation markers, including ALP and OSC, were measured with qRT-PCR and normalized against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression ( n = 3); ( C ) At day 4, riluzole treatment with linopirdine or XE991 reduced the mRNA expression of ALP. At day 7, there was no significant change in ALP levels, although riluzole treatment with flupirtine had a tendency to increase these levels; ( D ) At day 7, riluzole treatment with linopirdine or XE991 reduced the OSC mRNA expression. Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. Scale bar represents 100 μm. CONT: non-treated controls; RLZ: riluzole; FLU: flupirtine; LINO: linopirdine; XE: XE991; ALP: alkaline phosphatase; OSC: osteocalcin; OD: optical density.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Staining, Produced, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: The Role of K V 7.3 in Regulating Osteoblast Maturation and Mineralization

doi: 10.3390/ijms17030407

Figure Lengend Snippet: Induction of intracellular type 1 collagen by K V 7 channel during osteoblast differentiation in MG-63 cells. Western blot analysis demonstrated that 30 μM of linopirdine or 10 μM of XE991 considerably increased the expression of type 1 collagen on day 7 of osteoblastic induction, while 30 μM of flupirtine attenuated type 1 collagens. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Article Snippet: K V 7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing