embryonic mouse hypothalamic cell line n39  (Cedarlane)

Journal: Clinical and Translational Science

Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

doi: 10.1111/cts.13749

Figure Lengend Snippet: Induction of autophagy in hypothalamic neurons in response to hypoglycemic conditions. N39 cells were exposed to glucose concentrations of 4500, 2000, 900, 500, and 200 mg/L for 24, 48, and 72 h. The relative fold change in the gene expression levels of (a) LC3B and (c) LAMP2 in N39 cells as compared with the gene expression levels at the glucose concentration of 4500 mg/L, which is considered as the reference with a value of 1, and determined by using the quantitative real‐time polymerase chain reaction (qPCR) process. (b) Shows the representative estern blot indicating the protein expression of LC3BI (16KDa) and LC3BII (18KDa) at 24, 48, and 72 h across decreasing glucose concentrations # and the corresponding densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI as well as LC3BII normalized to β‐actin. (d) Shows the representative Western blot indicating the protein expression of LAMP2A (120 KDa) at 24, 48, and 72 h across decreasing glucose concentrations # and the corresponding densitometric analysis of LAMP2A expression. The protein expression of β‐actin (42 KDa) at 24, 48, and 72 h at decreasing glucose concentrations # was used as loading control. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, **** p < 0.0001 determined using two‐way ANOVA. # Glucose concentrations: L1 = 4500 mg/L, L2 = 2000 mg/L, L3 = 900 mg/L, L4 = 500 mg/L, L5 = 200 mg/L.

Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Gene Expression, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: Clinical and Translational Science

Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

doi: 10.1111/cts.13749

Figure Lengend Snippet: Fluorescence microscopy images of live N39 cells exposed to various glucose concentrations for 24 and 48 h depicting the levels of autophagosomes (green) and nuclei (blue with DAPI stain). Scale bar 200 μM.

Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Fluorescence, Microscopy, Staining

Journal: Clinical and Translational Science

Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

doi: 10.1111/cts.13749

Figure Lengend Snippet: Effects of low glucose conditions on FOS and BECN1 gene expression in N39 cells with FOS and BECN1 gene knockdowns. N39 cells were treated with siRNA negative control, FOS siRNA, and BECN1 siRNA, and were exposed to various concentrations of glucose (4500, 900, and 200 mg/L) for 48 h post‐siRNA transfection. The relative fold change in the mRNA expression levels of (a) FOS and (b) BECN1 in FOS knockdown (FOS KD) and BECN1 knockdown (BECN1 KD) cells were compared with the mRNA expression of negative control cells at the corresponding glucose concentrations of 4500, 900, and 200 mg/L, as determined using the quantitative real‐time polymerase chain reaction (qPCR) process. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 determined using two‐way ANOVA.

Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Gene Expression, Negative Control, Transfection, Expressing, Knockdown, Real-time Polymerase Chain Reaction

Journal: Clinical and Translational Science

Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

doi: 10.1111/cts.13749

Figure Lengend Snippet: Effects of low glucose conditions on the expression of LC3B and LAMP2A in N39 cells with FOS and BECN1 gene knockdowns. N39 cells (Ctrl), N39 + siRNA negative control (−ve Ctrl), N39 + FOS siRNA (FOS KD), and N39 + BECN1 siRNA (BECN1 KD) cells were exposed to various concentrations of glucose (4500, 900, and 200 mg/L) for 48 h post‐siRNAs transfection. The relative fold change in the mRNA expression levels of (a) LC3B and (c) LAMP2 in these cells were compared with the mRNA expression of Ctrl cells at the glucose concentration of 4500 mg/L, which is considered as the reference with a value of 1, and determined using the quantitative real‐time polymerase chain (qPCR) reaction. (b) Representative Western blot indicating the protein expression of LC3BI (16 KDa) and LC3BII (18 KDa) in these cells at glucose concentrations of 4500 and 200 mg/L and the corresponding densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI. (d) Representative Western blots indicating the protein expression of LAMP2A (120 KDa) in these cells at glucose concentrations of 4500 and 200 mg/L and the corresponding densitometric analysis of LAMP2A expression. The protein expression of β‐actin (42KDa) was used as loading control. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, **** p < 0.0001 determined using two‐way ANOVA. Representation of lanes in images for Western blots: L1 = control (4500 mg/L glucose), L2 = control (200 mg/L glucose), L3 = negative control (4500 mg/L glucose), L4 = negative control (200 mg/L glucose), L5 = FOS KD (4500 mg/L glucose), L6 = FOS KD (200 mg/L glucose), L7 = BECN1 KD (4500 mg/L glucose), L8 = BECN1 KD (200 mg/L glucose). Glucose concentrations are represented in milligrams/liter (mg/L).

Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Expressing, Negative Control, Transfection, Concentration Assay, Western Blot, Control

Journal: Clinical and Translational Science

Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

doi: 10.1111/cts.13749

Figure Lengend Snippet: Fluorescence microscopy images of (a) N39 cells without siRNA treatment (positive control), (b) N39 with siRNA negative control, (c) FOS knockdown (KD) N39 cells, and (d) BECN1 KD N39 cells at different glucose conditions (4500, 2000, 900, 500, and 200 mg/L). The levels of autophagosomes are depicted in green and nuclei in blue with DAPI stain. Scale bar 200 μM.

Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Fluorescence, Microscopy, Positive Control, Negative Control, Knockdown, Staining

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: Figure 1. 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% CO2.

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: Figure 2. 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% CO2.

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: Figure 3. 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 Supplementary Materials (Figures S1 and S2).

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% CO2.

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: Figure 4. Gene expression of TMPRSS2 in hypothalamic neurons under various glucose concentra- tions 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% CO2.

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: Figure 5. 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 Supplementary Materials (Figures S3 and S4).

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% CO2.

Techniques: Expressing, Control