Journal: Frontiers in immunology

Article Title: Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

doi: 10.3389/fimmu.2023.1251120

Figure Lengend Snippet: FIGURE 2 Hypercapnia increases Pseudo-SARS-CoV-2 entry into epithelial cells. HBE (A), BEAS-2B (B), and VERO (D) cells were pre-exposed to NC or HC for 2 days, pseudo-SARS-CoV-2 virus (p-SARS-CoV-2, PV) was added, cells were cultured for an additional day in NC or HC, respectively, the fixed. Entry of PV into cells was assessed by IF (representative images in A, B, D) and quantified as the percentage of PV-positive cells in BEAS-2B (C) and VERO (E) cultures. In (A), ciliated cells were labeled with acetylated-tubulin (Ac-tub). Nuclei were labeled with DAPI. Scale bars = 50 µM. All data are means ± SD. P values for comparisons between groups are shown (C, E).

Article Snippet: BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS.

Techniques: Virus, Cell Culture, Labeling

Journal: Frontiers in immunology

Article Title: Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

doi: 10.3389/fimmu.2023.1251120

Figure Lengend Snippet: FIGURE 1 Hypercapnia increases ACE2 protein expression in murine bronchial epithelium, human bronchial epithelial cells, and VERO cells. ACE2 protein expression was assessed by immunofluorescence microscopy (IF) in lungs of mice breathing ambient air or normoxic hypercapnia (HC) for 7 days (A), in differentiated human bronchial epithelial (HBE) cells (B) and BEAS-2B cells (C) cultured in normocapnia (NC) or HC for 2 (D2) or 4 (D4) days, and by IF in VERO cells cultured in NC or HC for 3 days (D). ACE2 expression in VERO cells was quantified as relative fluorescence intensity per cell and expressed as arbitrary units (AU)/cell (E). Nuclei were stained with DAPI. In (A, D), scale bars = 50 µM. For immunoblots, b-actin was used as loading control. All data are means ± SD. P values for comparisons between groups are shown (B, C, E).

Article Snippet: BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS.

Techniques: Expressing, Microscopy, Cell Culture, Staining, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

doi: 10.3389/fimmu.2023.1251120

Figure Lengend Snippet: FIGURE 3 Hypercapnia decreases cholesterol efflux transporters and increases expression and activation of SREBP2, expression of HGMCS1, and cholesterol accumulation in epithelial cells. HBE cells were exposed to NC or HC for 24 h, after which global gene expression was analyzed using Affymetrix GeneChip Hybridization. Heat map shows k-means clustering of cholesterol gene expression profiles, each column represents one sample, and each row represents one transcript (A). Diagram of the major metabolic intermediates in the pathways for synthesis of cholesterol regulated by the transcription factor SREBP2, created with BioRender.com (B). HGMCS1 (C), ABCA1 (D), and SREBP2 (E) protein expression was assessed by IF in the bronchi of mice exposed to air or normoxic HC for 7 days. SREBP2 protein expression was assessed by IF (F), and cleavage of pre-SREBP2 (p) to its active form (a) was assessed by immunoblot (G) in BEAS-2B cells cultured in NC or HC for 3 days. Cholesterol accumulation was assessed by labeling cholesterol with filipin in BEAS-2B cells (H) and with GM1 specifically in lipid rafts in BEAS-2B (I) and VERO (J) cells after culture in NC or HC for 3 days. Nuclei were stained with DAPI. b-actin was used as loading control in immunoblots. Scale bars = 50 µM, except in (H) = 20 µM. .

Article Snippet: BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS.

Techniques: Expressing, Activation Assay, Gene Expression, Hybridization, Western Blot, Cell Culture, Labeling, Staining, Control

Journal: Frontiers in immunology

Article Title: Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

doi: 10.3389/fimmu.2023.1251120

Figure Lengend Snippet: FIGURE 4 Inhibition of cholesterol synthesis or depletion of cholesterol blocks the hypercapnia-induced increases in ACE2 and Pseudo-SARS-CoV-2 entry into epithelial cells. Mice were exposed to normoxic HC for 7 days, or room air as control. Betulin (5 mg/kg body weight) or vehicle were administrated 3 days before and at the start of HC exposure, and ACE2 protein expression in mouse bronchi was assessed by IF (A). BEAS-2B and VERO cells were treated with the SREBP2 inhibitors betulin (bet, 7.5 µM) or AM580 (20 µM), the statins fluvastatin (F, 50 nM) or rosuvastatin (R, 0.5 µM), or the cholesterol depleting agent methyl-b-cycle odextrin (MbCD, 100 µM) and incubated in NC or HC for 2 days. Pseudo-SARS-CoV-2 virus (PV) or vehicle were then added, and cells were cultured for an additional day in NC or HC, respectively, then fixed and stained. ACE2 expression was quantified as relative fluorescence intensity per cell, expressed in arbitrary units (AU) (B, E, I). Viral entry was quantified as the percentage of PV- positive cells in BEAS-2B (C, F, J) and VERO (D, G, K) cultures. Nuclei were stained with DAPI. All data are means ± SD. P values for comparisons between groups are shown.

Article Snippet: BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS.

Techniques: Inhibition, Control, Expressing, Incubation, Virus, Cell Culture, Staining

Journal: Frontiers in immunology

Article Title: Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

doi: 10.3389/fimmu.2023.1251120

Figure Lengend Snippet: FIGURE 5 Cigarette smoke extract (CSE) increases ACE2 expression, Pseudo-SARS-CoV-2 entry, and cholesterol accumulation in epithelial cells in a manner not additive to the effects of hypercapnia. HBE, BEAS-2B and VERO cells were exposed to cigarette smoke CSE, (1 mg/ml) during culture in NC or HC for 2 days, pseudo-SARS-CoV-2 virus (PV) or vehicle were added, and cells were cultured for an additional day in NC or HC, respectively, then fixed and stained. ACE2 protein expression was assessed by IF in HBE (A) and VERO cells (B) and quantified in the latter as relative fluorescence intensity per cells, expressed in arbitrary units (AU) (B). ACE2 mRNA expression was assessed by qPCR after exposure of BEAS-2B cells to CSE in NC or HC for 1 day (C). Viral entry was assessed by IF and quantified as the percentage of PV-positive cells in BEAS-2B (D) and VERO (E) cultures. Cholesterol accumulation was assessed by Amplex red assay in BEAS-2B (F) and VERO (G) cell lysates, and lipid droplets (LD) in VERO cells were labeled with LipidSpot 488 (H). Ciliated cells were labeled with acetylated-tubulin (Ac-tub) (A). Nuclei were labeled with DAPI. Scale bars = 50 µM. All data are means ± SD. P values for comparisons between groups are shown.

Article Snippet: BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS.

Techniques: Expressing, Virus, Cell Culture, Staining, Amplex Red Assay, Labeling

Journal: Frontiers in immunology

Article Title: Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

doi: 10.3389/fimmu.2023.1251120

Figure Lengend Snippet: FIGURE 6 Statins block CSE-induced increases in lipid raft cholesterol, ACE2 protein expression and Pseudo-SARS-CoV-2 entry into epithelial cells. VERO and BEAS-2B cells were treated with fluvastatin (F, 50 nM) or rosuvastatin (R, 0.5 µM) for 1 day prior to and during exposure to CSE (1 µg/ml) for 2 days, pseudo-SARS-CoV-2 virus (PV) or vehicle were added, and cells were cultured for an additional day, in NC throughout, then fixed and stained. Cholesterol accumulation in lipid rats was assessed by labeling GM1 (A). ACE2 protein was assessed by IF and quantified as relative fluorescence intensity per cell, expressed in arbitrary units (AU) (B). Viral entry was assessed by IF and quantified as the percentage of PV-positive cells in VERO (C) and BEAS-2B cells (D). Nuclei were labeled DAPI. Scale bars = 50 µM. All data are means ± SD. P values for comparisons between groups are shown.

Article Snippet: BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS.

Techniques: Blocking Assay, Expressing, Virus, Cell Culture, Staining, Labeling

Journal: Frontiers in Pharmacology

Article Title: Notoginsenoside R1 Regulates Ischemic Myocardial Lipid Metabolism by Activating the AKT/mTOR Signaling Pathway

doi: 10.3389/fphar.2022.905092

Figure Lengend Snippet: Validation of key differential protein expression in the lipid metabolism–associated network. (A) Heatmap of differential proteins in the lipid metabolism–associated network. (B) Expression of COX2, CPT2, ACADVL, and CD36 by Western blots. Values expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the MI group; # p < 0.05, ## p < 0.01, and #### p < 0.001 vs. the sham group ( n = 3 for sham, MI, NGR1-L, and NGR1-H groups).

Article Snippet: The primary antibodies against very long-chain specific acyl-CoA dehydrogenase (ACADVL) (Cat# PB1076) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat# BA2913) were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Biomarker Discovery, Expressing, Western Blot

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: The KISS1 metastasis suppressor appears to reverse the Warburg effect by shifting from glycolysis to mitochondrial beta-oxidation

doi: 10.1007/s00109-017-1552-2

Figure Lengend Snippet: KISS1 upregulates beta-oxidation-related gene expression. A PCR Array was used to probe for various gene expressions in C1861.9Vector (V), C8161.9KFM59 (KFM), C8161.9KFM59/shKISS1 (K/shK), and C8161.9KFMΔSS6 (ΔSS) cells and selected upregulated genes are shown in (a & b). (c) mRNA expression of beta-oxidation related genes, PPARα, ACADVL, ACADS, and FABP4 in C1861.9Vector, C8161.9KFM59, C8161.9KFM59/shKISS1, and C8161.9KFMΔSS6 cells were quantified by qRT-PCR. N ≥ 3; error bars, SEM; * P , <0.05

Article Snippet: The following Taqman probes were used: ACADVL (Hs00825606_g1), ACADS (Hs00163506_m1), ACC1 (ACACA, Hs01046047_m1), ACC2 (ACACB, Hs00153715_m1), ACLY (Hs00982738_m1), FABP4 (Hs01086177_m1), FASN (Hs01005622_m1), HKII (Hs00606086_m1), LDHA (Hs00855332_g1), LDHB (Hs00929956_m1), PFKM (Hs01075411_m1), PKM1 (Hs00987255_m1), PKM2 (Hs00987261_g1), PPARα (Hs00947539_m1), SLC2A1 (Hs00892681_m1), and RNA18S5 (Hs03928985_g1).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR