Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on whole-cell Ca 2+ -activated K + current ( I K(Ca) ) in mHippoE-14 hippocampal neurons. In these experiments, cells were bathed in normal Tyrode’s solution, the composition of which was described under Section “Materials and Methods.” The recording pipette was filled with K + -containing solution. (A) Superimposed I K(Ca) traces obtained in the control (middle part) and during cell exposure to 10 μM PIO (bottom part). The upper part indicates the voltage protocol applied, and arrowheads are zero current level. (B) Averaged I–V relationships of I K(Ca) obtained in the control ( ), during the exposure ( ) to 10 μM PIO and after washout ( ) of PIO (mean ± SEM; n = 11 for each point). ∗ Significantly different from control groups taken at the same level of voltage pulse. (C) Bar graph showing summary of the effect of PIO, PIO plus TRAM-39, PIO plus apamin, and PIO plus paxilline, and PIO plus tolbutamide on I K(Ca) amplitude (mean ± SEM; n = 10–12 for each bar). Current amplitude was measured at +50 mV. (a) Control; (b) 10 μM PIO; (c) 10 μM PIO plus 3 μM TRAM-39; (d) 10 μM PIO plus 200 nM apamin; (e) 10 μM PIO plus 1 μM paxilline; (f) 10 μM PIO plus 30 μM tolbutamide. ∗ Significantly different from control ( P < 0.05) and ∗∗ significantly different from PIO alone group ( P < 0.05) ( n = 9–10 for each bar).

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on BK Ca channel activity in mHippoE-14 hippocampal neurons. (A) Original current traces of BK Ca channels obtained in the absence (left) and presence (right) of 10 μM pioglitazone (PIO). The examined cells were bathed in symmetrical K + solution (145 mM). Under inside-out current recordings, the potential was held at +60 mV and bath medium contained 0.1 μM Ca 2+ . The upward deflection represents the opening event of the channel. The lower part indicates the expanded trace recorded from the uppermost part in the control and during exposure to PIO. (B) BK Ca -channel trace obtained after washout of PIO. (C) Concentration-dependent increase in channel open probability (mean ± SEM; n = 9–11 for each point). Channel activity measured at +60 mV during the exposure to 100 μM PIO was taken to be 100%. The values for EC 50 , Hill coefficient and maximal percentage increase of BK Ca channels in the presence of PIO were 7.6 μM 1.3 and 100%, respectively.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Activity Assay, Control, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on mean open- (A) and closed-time (B) histograms of BK Ca channels recorded from mHippoE-14 hippocampal neurons. The holding potential was set at +60 mV, and inside-out configuration was performed. In control (left side), the open-time histogram of the channel was fitted by a single exponential function (indicated by red smooth line) with a mean open time of 1.9 ms, while the closed-time histogram was by a sum of a two-exponential function with a mean closed time of 3.5 and 47.5 ms. After addition of 10 μM PIO (right side), the mean open time was increased to 2.7 ms, and the slow component of closed time was shortened to 28.7 ms; however, minimal change in the fast component of closed time (i.e., 3.4 ms) in the presence of this compound. Of note, the abscissa and ordinate in each histogram indicate the logarithm of open or closed time (ms) and the square root of even number, respectively. Data were taken from a measurement of 100 channel openings. The vertical black dashed lines are placed at the values of mean open or closed time for BK Ca channels.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on the I–V relation of BK Ca channels in mHippoE-14 hippocampal neurons. The experiments on BK Ca channels were conducted with symmetrical K + -rich concentration (145 mM). Under inside-out configuration, the potential was held at +60 mV and bath medium medium contained 0.1 μM Ca 2+ . (A) Original current traces obtained in the control and during exposure to 10 μM PIO. The labels in the rightmost side indicate the holding potential applied. Arrowhead in each trace corresponds to zero current level, and the upper deflection indicates the opening event of the channel. In (B) , the single-channel conductance in the absence ( ) and presence ( ) of 10 μM PIO is nearly identical. Each point represents mean ± SEM ( n = 9–10). The dashed red lines obtained with or without addition of PIO are pointed toward the values of the reversal potential (i.e., 0.0 ± 0.1 mV, n = 8). (C) The relationship between relative open probability of BK Ca channels and membrane potential obtained with or without addition of 10 μM PIO. The ramp pulses were applied from 0 to +80 mV with a duration of 1 s. Under inside-out current recordings, PIO (10 μM) was applied to the intracellular surface of the excised patch. The smooth lines represent the best fit to the Boltzmann equation as detailed in Section “Materials and Methods.”

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Concentration Assay, Control, Membrane

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of linopirdine and linopirdine plus flupirtine on the amplitude of M-type K + current [ I K ( M ) ] in mHippoE-14 hippocampal neurons. In this set of experiments, cells were bathed in high K + , Ca 2+ -free solution and the recording pipette was filled with K + -containing solution. (A) Superimposed I K(M) traces obtained in the control (a) and during the exposure to 10 μM linopirdine (b), and 10 μM linopirdine plus 10 μM flupirtine (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of linopirdine and linopirdine plus flupirtine on I K ( M ) amplitude (mean ± SEM; n = 9 for each bar). ∗ Significantly different from control ( P < 0.05). LINO, linopirdine; FLUP, flupirtine.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on I K ( M ) amplitude in mHippoE-14 hippocampal neurons. These experiments were conducted in cells bathed in high K + , Ca 2+ -free solution and the recording pipette was filled with K + -containing solution. (A) Superimposed I K ( M ) traces obtained in the absence (a) and presence of 3 μM (b), and 10 μM PIO (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of PIO, linopirdine, and PIO plus flupirtine on I K ( M ) amplitude (mean ± SEM; n = 9–11 for each bar). The I K ( M ) amplitude elicited by membrane depolarization from –50 to –10 mV was measured. (a) Control; (b) 10 μM PIO; (c) 10 μM linopirdine; (d) 10 μM PIO plus 10 μM flupirtine. ∗ Significantly different from control ( P < 0.05) and ∗∗ significantly different from PIO (10 μM) alone group ( P < 0.05). LINO, linopirdine; FLUP, flupirtine.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Membrane, Control

Journal: bioRxiv

Article Title: RetroCHMP3 Blocks Budding of Enveloped Viruses Without Blocking Cytokinesis

doi: 10.1101/2020.08.30.273656

Figure Lengend Snippet: (A) Schematic of putative regulatory elements upstream of retroCHMP3 ORF in Mus musculus identified by transcription factor binding site prediction tools. Numbers indicate nucleotides relative to retroCHMP3 start codon. LTR – long terminal repeat, MuRRS – murine retrovirus-related sequence, ERV – endogenous retrovirus. (B) RT-PCR detection of retroCHMP3 RNA in mouse cardiac endothelial cells (MCECs) with and without interferon stimulation. RetroCHMP3 bands were excised, cloned, and sequenced to verify a match with the retroCHMP3 sequence. Representative gel from three independent biological repeats. RT – reverse transcriptase. (C) Droplet digital PCR (ddPCR) detection of retroCHMP3 (red) and RNAse L (grey, positive control) RNA in mouse cardiac endothelial cells (MCECs) with and without interferon stimulation. The housekeeping gene succinate dehydrogenase complex, subunit A (SDHA) was used for normalization. Each line represents one independent biological repeat.

Article Snippet: MCEC cells (mouse cardiac endothelial cells, CLU510, sex: unknown) were obtained from Cedarlane.

Techniques: Binding Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Reverse Transcription, Digital PCR, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Varying Glucose Concentrations on ACE2′s Hypothalamic Expression and Its Potential Relation to COVID-19-Associated Neurological Dysfunction

doi: 10.3390/ijms23179645

Figure Lengend Snippet: Cell viability of hypothalamic neurons under various glucose concentrations at different time points. ( A ) Increased glucose concentrations (mg/L) of 5400, 10,800, 16,200, and 21,600 (please check) at 24, 48 and 72 h time points enhanced the viability of cells significantly, with maximum percentage (~200%) observed at the highest glucose concentration of 21,600 mg/L compared to the control condition (4500 mg/L). ( B ) Low glucose concentrations (mg/L) of 2000, 900, 500, and 200 at 24, 48 and 72 h time points affected the viability of cells adversely, with a maximum reduction observed at the lowest glucose concentration of 200 mg/L at longest exposure of 72 h as compared to the control condition (4500 mg/L). The effects of decreasing glucose concentrations on the viability of hypothalamic neurons were published previously by our group. Data is represented as mean ± SEM ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Embryonic mouse hypothalamic cells (mHypoE-N39; Cedarlane, Burlington, ON, Canada) were grown in Dulbecco’s Modified Eagles Medium (DMEM 6429; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) and maintained in a humidified atmosphere at 37 °C with 5% CO 2 .

Techniques: Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Varying Glucose Concentrations on ACE2′s Hypothalamic Expression and Its Potential Relation to COVID-19-Associated Neurological Dysfunction

doi: 10.3390/ijms23179645

Figure Lengend Snippet: Gene expression of ACE2 in hypothalamic neurons under various glucose concentrations at different time points. ( A ) Increase in glucose concentrations (mg/L) of 5400, 10,800, 16,200, and 21600 at 24, 48 and 72 h time points showed an increase in ACE2 ′s gene expression, with significant fold changes observed at the higher concentrations of 10,800, 16,200, and 21,600 mg/L after longest exposure of 72 h as compared to the control condition (4500 mg/L). ( B ) Decreasing glucose concentrations (mg/L) of 2000, 900, 500, and 200 at 24, 48 and 72 h time points also showed an increase in gene expression of ACE2 , with significant fold changes observed at the lower concentrations of 500 and 200 mg/L at 72 h time point, as compared to the control condition (4500 mg/L). Data is represented as mean ± SEM ( n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Embryonic mouse hypothalamic cells (mHypoE-N39; Cedarlane, Burlington, ON, Canada) were grown in Dulbecco’s Modified Eagles Medium (DMEM 6429; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) and maintained in a humidified atmosphere at 37 °C with 5% CO 2 .

Techniques: Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Varying Glucose Concentrations on ACE2′s Hypothalamic Expression and Its Potential Relation to COVID-19-Associated Neurological Dysfunction

doi: 10.3390/ijms23179645

Figure Lengend Snippet: Protein expression of ACE2 in hypothalamic neurons under various glucose concentrations at different time points. ( A ) With increasing glucose concentrations (mg/L) of 5400, 10,800, 16,200, and 21,600, ACE2 (120 kDa) showed an upward trend in protein expression, with significant upregulation ( p < 0.05) observed at the highest concentration of 21,600 mg/L after 72 h exposure as compared to the control condition (4500 mg/L). ( B ) Histogram with relative fold change for ACE2′s protein expression ( n = 3) with increasing glucose concentrations compared to loading control (β-actin; 40 kDa). ( C ) With decreasing glucose concentrations (mg/L) of 2000, 900, 500, and 200, ACE2′s protein expression increased significantly ( p < 0.05) at longest exposure of 72 h and at the lowest concentrations of 500 and 200 mg/L glucose, as compared to the control condition (4500 mg/L). ( D ) Histogram with relative fold change for ACE2′s protein expression ( n = 3) with decreasing glucose concentrations compared to loading control (β-actin). Data is represented as mean ± SEM (* p < 0.05). Full blot images are provided in .

Article Snippet: Embryonic mouse hypothalamic cells (mHypoE-N39; Cedarlane, Burlington, ON, Canada) were grown in Dulbecco’s Modified Eagles Medium (DMEM 6429; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) and maintained in a humidified atmosphere at 37 °C with 5% CO 2 .

Techniques: Expressing, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Varying Glucose Concentrations on ACE2′s Hypothalamic Expression and Its Potential Relation to COVID-19-Associated Neurological Dysfunction

doi: 10.3390/ijms23179645

Figure Lengend Snippet: Gene expression of TMPRSS2 in hypothalamic neurons under various glucose concentrations at different time points. ( A ) Increase in glucose concentrations (mg/L) of 5400, 10,800, 16,200, and 21,600 at 24, 48 and 72 h time points showed that TMPRSS2 expression increased slightly at 24 h and significantly after 48 h but decreased after 72 h, as compared to the control condition (4500 mg/L). ( B ) With decreasing glucose concentrations (mg/L) of 2000, 900, 500, and 200, at the lowest concentrations of 900, 500, and 200 mg/L, TMPRSS2 expression was observed to decrease significantly compared to the control condition (4500 mg/L). Data is represented as mean ± SEM ( n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Embryonic mouse hypothalamic cells (mHypoE-N39; Cedarlane, Burlington, ON, Canada) were grown in Dulbecco’s Modified Eagles Medium (DMEM 6429; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) and maintained in a humidified atmosphere at 37 °C with 5% CO 2 .

Techniques: Gene Expression, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Varying Glucose Concentrations on ACE2′s Hypothalamic Expression and Its Potential Relation to COVID-19-Associated Neurological Dysfunction

doi: 10.3390/ijms23179645

Figure Lengend Snippet: Protein expression of TMPRSS2 in hypothalamic neurons under various concentrations of glucose at different time points. ( A ) With increasing glucose concentrations (mg/L) of 5400, 10,800, 16,200, and 21,600, protein expression of TMPRSS2 (54 kDa) showed an increase after 24h, which with longer exposures of 48 and 72 h, decreased as compared to the control condition (4500 mg/L) which was not significant. ( B ) Histogram with relative fold change for TMPRSS2′s protein expression ( n = 3) with increasing glucose concentrations compared to loading control (β-actin; 40 kDa). ( C ) With decreasing glucose concentrations (mg/L) of 2000, 900, 500, and 200, there was an increase in TMPRSS2′s protein expression after 24 h, whereas the opposite trend was observed after 48 h. However, after more prolonged exposure to 72 h, its expression first increased (at 2000 and 900 mg/L) and then decreased (at 500 and 200 mg/L concentrations), as compared to the control condition (4500 mg/L) but was not significant. ( D ) Histogram with relative fold change for TMPRSS2′s protein expression ( n = 3) with decreasing glucose concentrations compared to loading control (β-actin). Data are represented as mean ± SEM. Full blot images are provided in .

Article Snippet: Embryonic mouse hypothalamic cells (mHypoE-N39; Cedarlane, Burlington, ON, Canada) were grown in Dulbecco’s Modified Eagles Medium (DMEM 6429; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) and maintained in a humidified atmosphere at 37 °C with 5% CO 2 .

Techniques: Expressing, Control

Journal: Journal of neuroscience research

Article Title: Klf10 Regulates the Emergence of Glial Phenotypes During Hypothalamic Development.

doi: 10.1002/jnr.70020

Figure Lengend Snippet: FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic hypothalamic cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.

Article Snippet: Mice hypothalamic embryonic cells N1 (mHypoE- N1) (CEDARLANE, Ontario, CA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco- BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (FBS) (Biowest International, Coinsins, Switzerland), 0.5 mM glutamine (Sigma), and 1% antibiotic–antimycotic (Gibco- BRL, Gaithersburg, MD, USA).

Techniques: Activity Assay, Clone Assay, Construct, Binding Assay, Transfection, Plasmid Preparation, Concentration Assay, Luciferase, Over Expression, In Vivo, In Vitro, Chromatin Immunoprecipitation, Derivative Assay, Immunoprecipitation

Journal: Journal of neuroscience research

Article Title: Klf10 Regulates the Emergence of Glial Phenotypes During Hypothalamic Development.

doi: 10.1002/jnr.70020

Figure Lengend Snippet: FIGURE 3 | Klf10 expression is upregulated by BDNF in embryonic hypothalamic cells. (A) Left panel. Expression of the Trk neurotrophin re- ceptors at 17.5-day-old embryos from wild-type mice hypothalamus (Hyp-E17.5) and in mHypoE-N1 cells was measured by RT-PCR assays. Right panel. Normalized densitometry values of Trk receptors (NTRs) expression (NTRs cDNA/actin cDNA) in the Hyp-E17.5 and mHypoE-N1 cells. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977, pKlf10-516, or the mutated version p1977, p516. After transfection, cells were cultured for 12 h in DMEM with 5% FBS and then were cultured for 12 h in DMEM with 0.5% FBS. Subsequently, the p38 MAP kinase inhibitor (SB203580) or CREB inhibitor (666–15) was added at a final concentration of 10 μM for 30 min or 5 μM for 60 min, respectively, before the addition of 50 ng/mL BDNF; cells were then cultured for 24 h. Firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induc- tion was calculated relative to untreated or BDNF-treated conditions. (C) mHypoE-N1 cells cultured with 0.5% fetal bovine serum were treated with 50 ng/mL of BDNF for 24 h. The p38 (SB203580, 10 μM) or CREB (666–15, 5 μM) inhibitors were added to the culture 30 or 60 min, respectively, be- fore BDNF treatment. Klf10 mRNA levels were determined by qPCR and calculated as the ratio of Klf10 cDNA/Ubc cDNA signal. Results are plotted and expressed as relative fold change compared to untreated cells. The solid line represents the mean ± SEM of three replicates in three independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Mice hypothalamic embryonic cells N1 (mHypoE- N1) (CEDARLANE, Ontario, CA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco- BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (FBS) (Biowest International, Coinsins, Switzerland), 0.5 mM glutamine (Sigma), and 1% antibiotic–antimycotic (Gibco- BRL, Gaithersburg, MD, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Concentration Assay, Luciferase, Activity Assay