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 the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice

doi: 10.1161/JAHA.124.037460

Figure Lengend Snippet: MCECs were cultured in low glucose DMEM with 5% FBS and treated with 7‐ketocholesterol for the indicated times. A and D , Representative immunofluorescence images and quantification show mitochondrial superoxide levels. Representative immunofluorescence images and quantification show the expression of proinflammatory proteins VCAM‐1 ( B and E ) and CCL2 ( C and F ). G , Representative immunofluorescence images and quantification show the nuclear TFEB positive percentage. Nuclei were stained with DAPI. H , Representative immunoblots and summarized data show the effects of 7‐ketocholesterol on the protein expression levels of microtubule‐associated proteins light chain 3‐II. I , Real‐time reverse transcription polymerase chain reaction analyses of TFEB, LAMP‐1, LAMP‐2A, beclin‐1, microtubule‐associated proteins light chain 3, and p62/SQSTM1 mRNA levels after treatment with 0 or 40 μM 7‐ketocholesterol for 24 hour. Scale bar=20 μm. * vs 0, P <0.05 (n=4–5). CCL2 indicates C‐C motif chemokine ligand 2; LAMP‐1, lysosomal‐associated membrane protein; MCECs, mouse cardiac endothelial cells; TFEB, transcriptional factor EB; and VCAM‐1, vascular cell adhesion molecule 1.

Article Snippet: MCECs (Cedarlane, CLU510) were cultured in low glucose DMEM with 5% FBS, 1% penicillin/streptomycin, and 1 mmol/L of HEPES at 37 °C with 5% CO 2 .

Techniques: Cell Culture, Immunofluorescence, Expressing, Staining, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Membrane

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice

doi: 10.1161/JAHA.124.037460

Figure Lengend Snippet: MCECs were cultured and treated in low glucose DMEM with 5% FBS, pretreated with or without 50 nM BAF for 1 hour, and then cotreated with or without 40 μM of 7‐ketocholesterol for 6 hour. A , Representative immunofluorescence images and quantification show the nuclear TFEB‐positive percentage. Nuclei were stained with DAPI. B through G , MCECs are treated in low‐glucose DMEM with 1% FBS for 2 hour before pretreatment with or without 50 nM of BAF for 1 hour, and then the cells are cotreated with or without 40 μM of 7‐ketocholesterol for 24 hour. B , Representative images of VCAM‐1 and summarized data. C through G , Representative images of FLICA/PI staining and summarized data. H , Cell numbers were detected by using CCK8 kit. Scale bar=20 μm. * vs 0, # vs BAF or 7‐ketocholesterol, P <0.05 (n=4). BAF indicates bafilomycin A1; CCK8, Cell‐Counting Kit 8; MCECs, mouse cardiac endothelial cells; PI, propidium iodide; TFEB, transcriptional factor EB; and VCAM‐1, vascular cell adhesion molecule 1.

Article Snippet: MCECs (Cedarlane, CLU510) were cultured in low glucose DMEM with 5% FBS, 1% penicillin/streptomycin, and 1 mmol/L of HEPES at 37 °C with 5% CO 2 .

Techniques: Cell Culture, Immunofluorescence, Staining, Cell Counting

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice

doi: 10.1161/JAHA.124.037460

Figure Lengend Snippet: MCECs were cultured in low glucose DMEM with 5% FBS, then treated with EZE with or without 7‐ketocholesterol for the indicated time. A , Representative immunofluorescence images and quantification show the effect of ezetimibe on nuclear TFEB positive percentage. B , Representative immunofluorescence images and quantification show the effect of ezetimibe and 7‐ketocholesterol on nuclear TFEB positive percentage. Representative immunofluorescence images and quantification of mitochondrial superoxide ( C ), proinflammatory proteins vascular cell adhesion molecule 1 (VCAM‐1) ( D ) and CCL2 ( E ), and monocyte adhesion ( F ). Scale bar=20 μm. * vs 0, # vs 7‐keto, P <0.05 (n=4). CCL2 indicates C‐C motif chemokine ligand 2; EZE, ezetimibe; MCECs, mouse cardiac endothelial cells; TFEB, transcriptional factor EB; and VCAM‐1, vascular cell adhesion molecule 1.

Article Snippet: MCECs (Cedarlane, CLU510) were cultured in low glucose DMEM with 5% FBS, 1% penicillin/streptomycin, and 1 mmol/L of HEPES at 37 °C with 5% CO 2 .

Techniques: Cell Culture, Immunofluorescence

Journal: Molecular metabolism

Article Title: Targeting Clic1 for the treatment of obesity: A novel therapeutic strategy to reduce food intake and body weight.

doi: 10.1016/j.molmet.2023.101794

Figure Lengend Snippet: Figure 1: Hypothalamic Expression of Clic1. A. Hypothalamic expression of clic1 in control, OLZ and OLZ þ minocycline (MINO) treated mice (n ¼ 4/group) determined from RNA seq studies (GSE119772) [4]. B. Hypothalamic expression of Clic1 in ad libitum fed, 24 h fasted and 23 h fasted mice and then re-fed for 1 h (n ¼ 6e8 per group) determined by quantitative PCR. C. Expression of Clic1 in Agrp and Pomc neurons, using RNA seq data from Henry et al., 2015, (GSE93374) [48]. D. Clic1, Npy and Agrp expression in non- fasted, 8-h fasted, and 8-h fasted þ IAA94 treated hypothalamic cells (HypoA-59, n ¼ 3/group). EeF. Immunoblots and densinometric quantification of Clic1 membrane (Mem) and cytosolic (Cyto) localization in the hypothalamus of lean and obese mice (n ¼ 6/group) and G. ratio of membrane and cytosolic Clic1. A, B, D, *p < 0.05 One-way ANOVA followed by Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with 0.05 FDR. F. *p < 0.05 Two-way ANOVA followed by Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with 0.05 FDR. G. ***p < 0.01 Student’s t-test.

Article Snippet: In vitro studies in hypothalamic cells Adult mouse hypothalamic cell line (mHypoA-59, CLU468 cells, Cedarlane) were cultured as previously described.

Techniques: Expressing, Control, RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Membrane

Journal: Molecular metabolism

Article Title: Targeting Clic1 for the treatment of obesity: A novel therapeutic strategy to reduce food intake and body weight.

doi: 10.1016/j.molmet.2023.101794

Figure Lengend Snippet: Figure 7: Hypothalamic RNA seq in Clic1 KO and WT mice. A. Volcano plot and B. Heat map of differentially expressed genes between the hypothalamus of Clic1 WT and KO mice. C. All cell-types and D. Specific neuronal population co-expression of Clic1 hypothalamic DEGs.

Article Snippet: In vitro studies in hypothalamic cells Adult mouse hypothalamic cell line (mHypoA-59, CLU468 cells, Cedarlane) were cultured as previously described.

Techniques: RNA Sequencing, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Modulation of Sirt1 and FoxO1 on Hypothalamic Leptin‐Mediated Sympathetic Activation and Inflammation in Diet‐Induced Obese Rats

doi: 10.1161/JAHA.120.020667

Figure Lengend Snippet: A , Immunofluorescent photomicrographs stained for Sirt1 (in green), FoxO1 (in green) with leptin receptor (in red) in mHypoA‐POMC cells (top panel), and microglial‐SIM‐A9 cells (bottom panel). Blue: DAPI. Scale bar=20 µm. B , Representative gel and mean protein expressions of IL‐6, TNF‐α, IL‐1β in mHypoA‐POMC cells (left panel), and microglial‐SIM‐A9 cells (right panel) treated with leptin (50–400 ng/mL). * P <0.05 vs control without leptin treatment. C , Mean level of lipoxin A4 in mHypoA‐POMC cells and microglial‐SIM‐A9 cells treated with leptin (50–400 ng/mL). * P <0.05 vs control without leptin treatment.

Article Snippet: Adult mouse hypothalamic POMC cell line (mHypoA‐POMC/GFP) was purchased from CEDARLANE Cellutions Biosystems (CLU500, Burlington, Ontario, Canada).

Techniques: Staining, Control