Journal: The Journal of Physiology

Article Title: Pannexin 1 and pannexin 3 differentially regulate the cancer cell properties of cutaneous squamous cell carcinoma

doi: 10.1113/JP286172

Figure Lengend Snippet: An MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay was used to assess cell viability with increasing concentrations of probenecid (PBN) ( A ) and spironolactone (SPL) ( B ) treatment of SCC‐13 cells compared to corresponding water or ethanol (EtOH) vehicle controls. Treatment with 1 m m staurosporine was used as a positive control for reduced cell viability. No significant cytotoxic effects occur at 1 m m PBN ( P = 0.6045) or 20 µM SPL ( P = 0.5783) in SCC‐13 cells, indicating findings of growth and migration experiments resulted from PANX1 channel blockade, not from changes in cell viability. Two‐way ANOVA followed by Sidak's and Tukey's multiple comparisons tests. Bars indicate mean ± SD. N = 3.

Article Snippet: Cells were transfected using Lipofectamine 3000 (Invitrogen L3000015, Carlsbad, CA, USA) in Opti‐MEM medium (Life Technologies 51985‐034, Carlsbad, CA, USA) according to the manufacturer's protocol with 1 μg of PANX1 (InvivoGen puno1‐hpanx1, San Diego, CA, USA) or PANX3 plasmid in a six‐well plate.

Techniques: Positive Control, Migration

Journal: The Journal of Physiology

Article Title: Pannexin 1 and pannexin 3 differentially regulate the cancer cell properties of cutaneous squamous cell carcinoma

doi: 10.1113/JP286172

Figure Lengend Snippet: Representative images for haematoxylin and eosin (H&E)–stained patient‐derived cSCC tumour fragment and adjacent skin tissue, with matching immunohistochemistry [brown DAB (3,3′‐diaminobenzidine) stain] indicating PANX1 localization and secondary‐only control (2° Only). Nuclei (blue) counterstained with Harris haematoxylin. N = 8. A , Whole sample view of cSCC tumour and adjacent skin. Objective magnification: 0.35×. Scale bar: 2 mm. B , Border of cSCC tumour (T) and adjacent skin (S), with division between tissue types denoted by a dotted line. Objective magnification: 6.26×. Scale bar: 200 µm. C , Carcinoma cells contained within tumour nests (outlined by dotted line) express PANX1. The nucleus of a carcinoma cell is denoted by an arrow. D , The stromal region of the cSCC tumour contains tumour‐infiltrating lymphocytes (indicated by arrowhead), cancer‐associated fibroblasts (indicated by arrow) and blood vessels (lumen denoted by an asterisk), which also express PANX1. E , In the tumour‐adjacent skin, PANX1 is present throughout all layers of the epidermis. Epidermal layers are bracketed, with asterisk (*) indicating the basal layer of keratinocytes. Objective magnification: 40×, scale bar: 50 µm for C–E .

Article Snippet: Cells were transfected using Lipofectamine 3000 (Invitrogen L3000015, Carlsbad, CA, USA) in Opti‐MEM medium (Life Technologies 51985‐034, Carlsbad, CA, USA) according to the manufacturer's protocol with 1 μg of PANX1 (InvivoGen puno1‐hpanx1, San Diego, CA, USA) or PANX3 plasmid in a six‐well plate.

Techniques: Staining, Derivative Assay, Immunohistochemistry, Control

Journal: The Journal of Physiology

Article Title: Pannexin 1 and pannexin 3 differentially regulate the cancer cell properties of cutaneous squamous cell carcinoma

doi: 10.1113/JP286172

Figure Lengend Snippet: Representative haematoxylin and eosin (H&E) and PANX1 immunohistochemistry [DAB (3,3′‐diaminobenzidine)] stain in brown) images of structures within cSCC tumour and adjacent skin. A , Necrotic core contains mostly keratin and dying or dead cells. B , Arrow indicates keratin pearl within cSCC tumour, which does not stain for PANX1. C , Arrowhead indicates nerve in adjacent skin, which stains strongly for PANX1. Objective magnification: 11.83×. Scale bar: 100 µm. Nuclei shown in blue.

Article Snippet: Cells were transfected using Lipofectamine 3000 (Invitrogen L3000015, Carlsbad, CA, USA) in Opti‐MEM medium (Life Technologies 51985‐034, Carlsbad, CA, USA) according to the manufacturer's protocol with 1 μg of PANX1 (InvivoGen puno1‐hpanx1, San Diego, CA, USA) or PANX3 plasmid in a six‐well plate.

Techniques: Immunohistochemistry, Staining

Journal: PLoS ONE

Article Title: Vaspin Increases Nitric Oxide Bioavailability through the Reduction of Asymmetric Dimethylarginine in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0052346

Figure Lengend Snippet: A–B. Effects of vaspin on STAT3 phosphorylation in HAECs according to the incubation time of vaspin (A) and dose of vaspin (B). HAECs were treated with 100 ng/ml of vaspin for the indicated time (A) and indicated concentrations of vaspin for 6 hr (B). C Effects of vaspin on STAT3-DNA binding activity. Electrophoretic mobility shift analysis (EMSA) was used to determine the DNA-binding activity of STAT3 in HAECs. Cells were treated with STAT3 probe or cold probe in the absence or the presence of 100 ng/ml of vaspin for 6 hr. Nuclear extracts from the treated and untreated control cells were isolated and used in an EMSA with biotin-labeled STAT3 oligonucleotide or cold probe. We used 2 ug and 4 ug of STAT3 Ab to identify the supershifts. The solid arrow and dashed arrow indicate the STAT3 binding complex and supershift, respectively. Data are representative results from three separate experiments. D. Effect of STAT3 siRNA on vaspin-induced eNOS phosphorylation in HAECs. HAECs were treated with control siRNA ± vaspin 100 ng/ml or STAT3 siRNA ± vaspin 100 ng/ml. Relative expression of STAT3 and eNOS were measured at 16 hr after vaspin treatment. Data are shown as means ± SEM of at least three independent experiments.In A: * p <0.05 vs. untreated (Control). In B: * p <0.05 vs. untreated (Control); # p <0.05 vs. 25 ng/ml of vaspin group. In D: * p <0.05 vs. control siRNA alone; # p <0.05 vs. control siRNA+vaspin. STAT3, signal transducer and activator of transcription 3; eNOS, endothelial nitric oxide synthase.

Article Snippet: Cells were transfected with 200 ng of STAT3-expression vector (pUNO1-hSTAT3b, InvivoGen, San Diego, CA, USA), or 200 ng of wild type or mutant type reporter gene using LipofectAMINE 2000 reagent (Invitrogen). pRL-SV40 renilla luciferase control reporter vectors (10 ng, Promega, Madison, WI, USA) were co-transfected as an internal control.

Techniques: Incubation, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Isolation, Labeling, Expressing

Journal: PLoS ONE

Article Title: Vaspin Increases Nitric Oxide Bioavailability through the Reduction of Asymmetric Dimethylarginine in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0052346

Figure Lengend Snippet: The effect of STAT3-expression vector on STAT3 expression. The expressions of STAT3 at the level of protein (A) and mRNA were measured after 24 hr of transfection (B). C. The stimulatory effects of vaspin on STAT3-induced DDAH II expression. HAECs were transfected with 200 ng of STAT3-expression vector or control empty vector and then incubated with or without vaspin (100 ng/ml) for 16 hr. D. The role of STAT3-binding site in vaspin’s stimulatory effect on DDAH II expression. HAECs were transfected with DDAH II promoter-reporter plasmid (wild type or mutant type at putative STAT3-binding site) and stimulated with vehicle or vaspin (100 ng/ml) for 16 hr. Data are shown as means ± SEM of at least three independent experiments. In A and B: * p <0.05 vs. control vector. In C: * p <0.05 vs. untreated (Control); # p <0.05 vs. vaspin+control vector group; † p <0.05 vs. STAT3 vector alone group. In D: * p <0.05 vs. wild promoter only group; # p <0.05 vs. wild promoter+vaspin group. STAT3, signal transducer and activator of transcription 3; DDAH II, dimethylarginine dimethylaminohydrolase II.

Article Snippet: Cells were transfected with 200 ng of STAT3-expression vector (pUNO1-hSTAT3b, InvivoGen, San Diego, CA, USA), or 200 ng of wild type or mutant type reporter gene using LipofectAMINE 2000 reagent (Invitrogen). pRL-SV40 renilla luciferase control reporter vectors (10 ng, Promega, Madison, WI, USA) were co-transfected as an internal control.

Techniques: Expressing, Plasmid Preparation, Transfection, Incubation, Binding Assay, Mutagenesis

Journal: Theranostics

Article Title: Evaluation of cancer immunotherapy using mini-tumor chips

doi: 10.7150/thno.71761

Figure Lengend Snippet: Profiling of tumor responses to anti-PD1 blockade using the mini-tumor chip. (A) Treatment scheme of EO771 tumor-bearing mice. (B) Orthotopic EO771 tumor growth curve with representative responder (RS) and non-responder (NR) tumor. (C) Live/dead staining images of RS and NR tumors with on-chip anti-PD1 treatment for 12 h and quantitative analysis. Scale bar: 500 µm. (D) Viability of RS and NR tumors on-chip. Data points from anti-PD1 treated groups were compared with corresponding controls at the same timepoint by student's t-test (n = 20, **p < 0.01, ****p < 0.001), viability data points of representative images shown in Fig C were highlighted in red color. (E) Flow analysis of CD4+ and CD8+ T cell composition of EO771 dissociated primary tumors and on-chip cell components pre-treatment with both RS and NR tumors.

Article Snippet: To establish PD-L1 overexpression EO771 cell line, EO771 cells were transfected pUNO1-mCD274 vector expressing mouse PD-L1 (Invivogen #puno1-mcd274).

Techniques: Staining

Journal: Theranostics

Article Title: Evaluation of cancer immunotherapy using mini-tumor chips

doi: 10.7150/thno.71761

Figure Lengend Snippet: Prediction of tumors' responses to anti-PD1 treatment using the mini-tumor chip. (A) Experimental scheme test on-chip prediction of primary tumors' responses to anti-PD1 treatment. (B) Dissociated tumor cells on-chip responses to anti-PD1 at day 10, with orthotopic tumors formed by EO771 wild type (WT), PD-L1 overexpression (OE), and PD-L1 knockdown (KD) cells. (C) Tumor growth curve of orthotopic EO771 tumors with wild type, PD-L1 overexpression, and knock-down. Data points from anti-PD1 treated groups were compared with corresponding controls at the same timepoint by student's t-test (n = 20, *p < 0.05, ****p < 0.001).

Article Snippet: To establish PD-L1 overexpression EO771 cell line, EO771 cells were transfected pUNO1-mCD274 vector expressing mouse PD-L1 (Invivogen #puno1-mcd274).

Techniques: Over Expression