Journal: Human Gene Therapy

Article Title: LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor β Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models

doi: 10.1089/hum.2020.078

Figure Lengend Snippet: LyP-1 receptor (p32) and/or TGFβ-1 expression in breast cancer cell lines and patients. (A) LyP-1 receptor (p32) expression in human and mouse breast cancer cells by immunofluorescence staining. In MDA-MB-231 and 4T1 cells, LyP-1 receptors were localized more on plasma membrane (white arrows) and endoplasmic reticulum-like membrane networks (white triangular arrowheads). In MCF-7 cells, they were more enriched in Golgi-like juxtanuclear compartments (white arrows). Scale bar = 25 μm. (B) LyP-1 receptor expression in breast cancer cell lines by Western blot. Note that mouse LyP-1 receptors in 4T1 cells have a slightly different banding pattern from those in human breast cancer cells. All experiments were repeated three times. (C) Representative images of LyP-1 receptor and TGFβ-1 expression in breast cancer patient samples. Yellow arrows point to positive p32 IHC staining in tumor cells; red open arrows point to TGFβ-1 staining in tumor stroma. Magnification is indicated by scale bars. IHC, immunohistochemistry; TGFβ-1, transforming growth factor β-1.

Article Snippet: Human mammary tumor cell lines, MCF-7 (ATCC, Manassas, VA), MDA-MB-231 (ATCC), and MDA-MB-231-luc2, 16 and the mouse mammary tumor cell line, 4T1 (ATCC), were maintained as described earlier.

Techniques: Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Western Blot, Immunohistochemistry

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: CSC/EMT markers are elevated in tumors from DIO mice in association with increased local leptin signaling. (a) Tumor expression of several CSC/EMT-related genes in DIO and control mice identified by RNA sequencing analysis was verified via quantitative RT-PCR. (b) Tumor expression in DIO and control mice of previously established CSC/EMT-related genes (11) not identified by RNA sequencing was measured by quantitative RT-PCR. (c) ALDH activity, another marker of CSC enrichment, was quantified in DIO and control mouse tumors. (d) Immunohistochemical staining for tumor E-cadherin and vimentin expression in DIO and control mice. Representative images shown at x20 magnification. (e) Tumor expression of Lepr as well as mammary fat pad (MFP) expression of Lep in DIO and control mice were measured via quantitative RT-PCR. *P<0.05, **P<0.01

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Expressing, Control, RNA Sequencing, Quantitative RT-PCR, Activity Assay, Marker, Immunohistochemical staining, Staining

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: Leptin stimulates mammosphere formation in triple-negative mammary tumor cells. Mammosphere formation in (a) E-Wnt, (b) M-Wnt, and (c) MDA-MB-231 cells was assessed at the end of propagation 1 (P1), during which the cells were treated for 7 days with leptin, and following propagation 2 (P2), in which the spheres from P1 were dissociated and then replated with the same treatments for another 7 days. Representative images of mammospheres at the end of P2 are shown at x10 magnification. (d) Lepr expression in parental E-Wnt cells (EWnt-P) as well as E-Wnt cells stably transfected with a scrambled shRNA plasmid (EWnt-S) or shRNA to Lepr (EWnt-L1 and EWnt-L2) was measured by quantitative RT-PCR. (e) Mammosphere formation was assessed in EWnt-P, EWnt-S, EWnt-L1, and EWnt-L2 cells after a 7-day incubation in mammosphere media. Socs3 (f) and Foxc2, Twist2, and Vim (g) gene expression was measured by quantitative RT-PCR. Different letters indicate significant differences, P<0.05.

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, Incubation, Gene Expression

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: Obesity-associated circulating factors promote triple-negative mammary tumor cell viability, migration, invasion, and a CSC/EMT genotype. (a) E-Wnt, M-Wnt, and MDA-MB-231 cell viability following a 48-hour exposure to media containing 2% DIO or control mouse serum was assessed by MTT assay. (b) Migration of E-Wnt, M-Wnt, and MDA-MB-231 cells during a 6-hour exposure to media containing 2% DIO or 2% control mouse serum was measured by wound healing assay. Representative images of cells at baseline and 6 hours are shown at x10 magnification. (c) The invasive capacity of E-Wnt, M-Wnt, and MDA-MB-231 cells in response to chemoattraction with media containing 2% DIO or 2% control mouse serum over 24 hours was measured using Matrigel invasion chambers. Representative images of invading cells are shown at x10 magnification. (d) Expression of CSC/EMT-related genes in E-Wnt, M-Wnt, and MDA-MB-231 cells following a 24-hour exposure to media containing 2% DIO or 2% control mouse serum was measured by quantitative RT-PCR. *P<0.05, **P<0.01, ***P<0.001

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Migration, Control, MTT Assay, Wound Healing Assay, Expressing, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: Proposed model illustrating leptin-mediated upregulation in CSC/EMT-related genes and phenotype. Our findings suggest that obesity in MMTV-Wnt-1 mice promotes both an excess of leptin production in the tumor microenvironment (normal mammary tissue) and an upregulation in tumor expression of the leptin receptor and CSC/EMT-related genes. They also indicate that leptin signaling promotes a CSC/EMT-related phenotype, including increased CSC enrichment and cell viability, migration, and invasion, and specifically regulates the expression of Foxc2, Twist2, Vim, Akt3, and Sox2 in triple-negative mammary tumor cells. We hypothesize that these genes may mediate the observed leptin-induced CSC/EMT-related phenotype and that leptin regulates these genes via stimulation of the JAK2/STAT3 and/or PI3K/Akt pathways. Black arrows indicate effects observed in this study, solid blue arrows indicate pathways known from the literature, and dotted blue arrows indicate hypothesized mechanisms.

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Expressing, Migration

Journal: Frontiers in Cellular Neuroscience

Article Title: Hypoxic culture of umbilical cord mesenchymal stem cell-derived sEVs prompts peripheral nerve injury repair

doi: 10.3389/fncel.2022.897224

Figure Lengend Snippet: Cellular localization and uptake of fluorescently labeled sEVs with SCs. (A) Cellular colocalization: SP8 was used to photograph sEVs prelabeled with PKH67 (green fluorescence) with Hoechst 33,342 (blue fluorescence)-stained SC nuclei. (B) Western blot: Detection of ERK1/2, ZEB2, and c-JUN expression levels in NC Schwann cells and after 48-h treatment with hypoxia sEVs. (C) Real-time PCR: Inflammatory and other restoration-related factors (IL-1β, IL-6, TNF-α, etc.) were detected. (D) ELISA test: GDNF NDF and NT-3 in the supernatant of Schwann cell preparation. Statistical significance, * p < 0.05,** p < 0.01, and **** p < 0.0001. *Significant difference.

Article Snippet: The following ELISA kits were: mouse glial cell line-derived neurotrophic factor (GDNF), ELISA kit (CSB-E07341m, CUSABIO), mouse neurotrophin 3 (NT-3), ELISA kit (CSB-E04687m, CUSABIO), and mouse nerve growth factor (NGF), ELISA kit (CSB-E04684m, CUSABIO).

Techniques: Labeling, Fluorescence, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

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