Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: mRNA quantitative expression versus immunohistochemistry. (A) DCSIGN, CD163 M2, α‐smooth muscle actin (α‐SMA), fibroblast‐specific protein 1 (FSP1) and fibroblast activation protein (FAP) immunohistochemistry. Representative pictures of high and low protein expression levels of DCSIGN, CD163 M2 macrophage and α‐SMA, FSP1 and FAP cancer‐associated fibroblast (CAF) markers in human tumor samples. Arrows indicate positive cells. Original magnification, ×20. (B) Association between protein and mRNA expression levels. Statistical association between protein and mRNA expression levels of FSP1 and FAP, CAF markers. In addition, a clear trend toward statistical association is observed between protein and mRNA expression levels of DCSIGN and CD163, M2 macrophage markers and the α‐SMA, CAF marker. Bar charts show the average (±confidence interval) of mRNA expression levels of each gene in the low and high protein expression groups.

Article Snippet: Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Immunohistochemistry, Activation Assay, Marker

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and disease‐free survival (DFS) in colorectal cancer patients. Association between CD163, a M2 macrophage marker (A), and α‐smooth muscle actin (α‐SMA) (B), fibroblast‐specific protein 1 (FSP1) (C) and fibroblast activation protein (FAP) (D) CAF markers with DFS. “Low expression” refers to a low‐expression tertile for α‐SMA and low‐ and medium‐expression tertiles for CD163, FSP1 and FAP.

Article Snippet: Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and overall survival in colorectal cancer patients. Association of CD163, a M2 macrophage marker (A), and fibroblast‐specific protein 1 (FSP1) (C), a CAF marker, with overall survival. α‐Smooth muscle actin (α‐SMA) (B) and fibroblast activation protein (FAP) (D) expression levels, CAF markers, showed a trend towards an association with overall survival.

Article Snippet: Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 1. Pharmacological block or genetic knockdown of ANO1 produces a similar inhi- bition of the contraction of mouse pulmonary artery to 5-HT as blocking VGCC or emptying Ca2+ stores from the SR. (A) Typical isometric force recordings in response to high K+ Krebs (85.4 mM) and increasing cumulative concen- trations of 5-HT ranging from 0.01 to 30 μM as indicated by the bars above the traces, in the absence (left) or presence (right) of the ANO1 inhibitor CaCCInh-A01, also indicated by a hori- zontal bar above the trace. (B) Mean cumulative dose–response curves to 5-HT in mouse pul- monary arteries from wild-type C57/BL6 mice in the absence (black circles, Control; n = 14), or presence (blue squares; n = 5) of 1 μM nifedipine to block VGCC, 10 μM CPA to deplete SR Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Blocking Assay, Knockdown, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 2. The ANO1 blocker CaCCInh-A01 produced no effect on the high K+-mediated contraction of the mouse pulmonary artery. (A) Typical contractile force experiment showing that increasing the concentration of CaCCInh-A01 from 1 to 30 μM (progressively thickening black bar shown over the trace) produced no notice- able effect on the contraction (blue trace) eli- cited by 85.4 mM K+–Krebs solution (K+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Produced, Concentration Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 3. Ca2+ oscillations triggered by 5-HT in individual smooth muscle cells from an intact mouse endothelium-denuded PA are potently inhibited by the inhibition of ANO1. All data were collected from the same PA from a conditional smooth muscle–specific and inducible GCaMP3 mouse injected with tamoxifen to induce Cre expression. (A) Ca2+ imaging was performed in the absence of an agonist (Control). The left panel shows one image from a video from which a ST map (middle colored image) was created in the area spanned by the diagonal white line. Fluorescence intensity was measured under the three white lines on the ST map (corresponding to two different cells) and plotted as a function of time as shown on the right. There was no detectable activity in these two cells as well as across the entire field of view of the movie. (B) Same nomenclature as in A except that the preparation was exposed to 1 μM 5-HT for 5 min. A ST map created in the same manner as that in A shows clear evidence of asynchronous Ca2+ transients. This is more evident from examining the fluorescence intensity profile of the same two cells analyzed in A, which displayed repetitive Ca2+ transient of distinct magnitude and frequency. (C) The nomenclature of this panel is identical to that of B and C, with the exception that the PA was exposed to 10 μM CaCCInh-A01 for 10 min while still being incubated with 5-HT. Examination of the ST map reveals little, if any, Ca2+ oscillations in the presence of the ANO1 inhibitor; Ca2+ transients were no longer apparent in the same two cells analyzed in A and B.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Inhibition, Injection, Expressing, Imaging, Control, Fluorescence, Activity Assay, Incubation

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 5. Sample experiment illustrating how ANO1 knockdown exerted a strong inhibition of 5-HT-induced Ca2+ oscillations in a PA from a tamoxifen-injected SMC-ANO1-KO-ΔEx12-GCaMP3 mouse. The top left panel is an image from a video stack recorded in a pulmonary artery from a con- ditional smooth muscle cell-specific and inducible ANO1 knockout mouse expressing GCaMP3 specifically in smooth muscle cells, which was exposed to 1 μM 5- HT for 5 min. One ST map constructed from the white line crossing the image is shown in the lower left corner and reveals very little activity. The fluorescence intensity profile as a function of time of two cells from the ST map labeled with the letters a and b are shown on the right. Cell 1 displayed no significant Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Knockdown, Inhibition, Injection, Knock-Out, Expressing, Construct, Activity Assay, Fluorescence, Labeling

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 6. Asynchronous Ca2+ oscillations evoked by 5-HT require both functional ANO1 and VGCC. Mean data for each of four parameters measured from Ca2+ transients elicited by 1 μM 5-HT (5 min) in PA from control SMC-GCaMP3 (light blue bars) or SMC-ANO1-KO-ΔEx12-GCaMP3 (light gray bars) mice. (A–D) The frequency of Ca2+ oscillations (A), peak Ca2+ transient amplitude (F/F0; B), integrated area under the curve (C), and FWHM (D) were measured as shown in the upper right corner. For each dataset, the mean is indicated by a filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells. SMC-GCaMP3 + 5-HT: N = 7, n = 114 for peak, area under the curve, and FWHM, and n = 116 for frequency; SMC-GCaMP3 + 5-HT + CaCCInh-A01 (CaCCInh): N = 7, n = 15 for peak, area under the curve, and FWHM, and n = 76 for frequency; SMC-GCaMP3 + 5-HT + nifedipine (Nif): N = 2, n = 32 for peak, area under the curve, and FWHM, and n = 47 for frequency; GCaMP3 + 5-HT + CPA: N = 2, n = 29; SMC-ANO1-KO-ΔEx12-GCaMP3; 5-HT: N = 7, n = 39 for peak, area under the curve, and FWHM, and n = 137 for frequency. For all panels, ***, **, and * indicate a significant difference between means with P < 0.001, P < 0.01, and P < 0.05, respectively.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Functional Assay, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 7. Blocking ANO1 or CaV1.2 depletes SR Ca2+ stores. (A) Typical isometric force re- cording obtained under control conditions showing the effect of depleting the SR Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Blocking Assay, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 8. ANO1, CaV1.2, and IP3R colocalize in peripheral coupling sites to form signaling complexes. (A and B) Co-IP of CaV1.2 or IP3R with ANO1 from lysates of the pulmonary artery from wild-type mice. Pulldown was carried out with anti-ANO1 antibody and then probed by Western blot with anti-CaV1.2, anti-IP3R, or anti- ANO1 antibodies. Five to six mouse tissues per experiment, each ran in triplicates. (C and D) Freshly isolated PASMCs from wild-type mice were immunolabeled for ANO1 and CaV1.2 (C) or ANO1 and IP3R (D). All three proteins were preferentially localized to the periphery of the cells. (D and F) Line profiles of the areas indi- cated by the white dashed lines in C and E. The fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the loca- tion of the PM. ANO1 and CaV1.2 show strong immunolabeling at the PM (D). (E) IP3R shows some intracellular immunolabeling, with moder- ate peaks present at the periphery showing an enhancement of protein localization to periphe- ral coupling sites. Source data are available for this figure: SourceData F8.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Isolation, Immunolabeling, Fluorescence

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 9. Superresolution imaging of ANO1, CaV1.2, and IP3R at the PM of PASMCs from wild-type mice. (A and B) Superresolution images of PASMCs labeled for ANO1 and CaV1.2 (A) or ANO1 and IP3R (B) were imaged using GSDIM in epifluorescence mode. Epifluorescence images are shown in the inset for

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Imaging, Labeling

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 10. Membrane cholesterol depletion with MβCD causes the internalization of ANO1 and CaV1.2 proteins. (A and C) Freshly isolated PASMCs from wild-type mice were im- munolabeled for ANO1 and CaV1.2 before (A) or after (C) a 30-min exposure to MβCD (3 mg/ml; MβCD) to deplete membrane cholesterol and disrupt lipid rafts. The two ion channel proteins were preferentially localized to the periphery of the cells in control conditions as similarly shown in Fig. 8. (B–D) Line profiles of the areas indi- cated by the white dashed lines in A and C are respectively displayed in B and D. For these plots, the fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the location of the PM. ANO1 and CaV1.2 show strong immunolabeling at the PM in control condition (C) and translocation toward the cen- ter core of the cell after exposure to MβCD (D). The cells from A and C were isolated from the same mouse. (E and F) Graphs summarizing the effects of exposing PASMCs to MβCD on the distribution of ANO1 (magenta bars) and CaV1.2 (green bars), respectively. Measurements were performed as described in the text and consisted in normalizing membrane fluorescence to total cell fluorescence. For each dataset, the mean is indicated by a large, filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells; for the control group (E): ANO1 and CaV1.2: N = 3, n = 43; for the MβCD group (F): ANO1 and CaV1.2: N = 3, n = 35. *** indicates a significant difference between means with P < 0.001.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Membrane, Isolation, Control, Fluorescence, Immunolabeling, Translocation Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 12. Hypothetical models of EC coupling involving ANO1, CaV1.2, and IP3R during agonist-mediated contraction of mouse pulmonary ar- terial smooth muscle cells. (A) General uniform model depicting the acti- vation of ANO1 by both Ca2+ release from IP3-sensitive SR Ca2+ stores and Ca2+ entry through CaV1.2. In this model, the three ion transporters are evenly distributed in the membrane and are not physically coupled. The depolari- zation is maintained by the positive feedback loop established by CaV1.2- mediated activation of Cl−efflux through ANO1 and its impact on the state of activation of CaV1.2 through regulation of membrane potential. (B) Schematic diagram illustrating the local interaction of ANO1, CaV1.2 with IP3R and their impact on membrane potential, Ca2+ entry, and contraction. In this model, the three ion channels are physically coupled in a restricted number of sites (Super Cluster) distributed across the long axis of the cell (shown as red boxes in the bottom diagram) and are organized for compartmentalized Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Membrane, Activation Assay

Journal: Scientific Reports

Article Title: A unique olfactory bulb microcircuit driven by neurons expressing the precursor to glucagon-like peptide 1

doi: 10.1038/s41598-019-51880-9

Figure Lengend Snippet: PPG neurons express glutamic acid decarboxylase 67 but not 65. ( a ) Representative photomicrograph of the GCL in PPG-Cre-RFP transgenic mice (red channel; left) whereby glutamic acid decarboxylases (GAD) GAD65 and ( b ) GAD67 are visualized using immunocytochemistry (green channel; middle). ( c ) Representative photomicrograph of the GCL in progeny resulting from a cross between PPG-Cre-RFP x GAD67-GFP mice. Arrow = PPG neurons that co-express RFP and GAD67. PPG neurons largely did not express GAD65 as evidenced by a lack of RFP and GAD65 co-labeled neurons. ( d ) Representative photomicrograph of the GCL in PPG-Cre-RFP transgenic mice (red channel; left) where the vesicular GABA transporter (VGAT) is visualized by immunohistochemistry (green channel; middle).

Article Snippet: Vesicular GABA transporter (VGAT) was labelled using a rabbit polyclonal serum (1:1000, BOSTER Biological Technology Pleasanton, USA).

Techniques: Transgenic Assay, Immunocytochemistry, Labeling, Immunohistochemistry

Journal: Scientific Reports

Article Title: A unique olfactory bulb microcircuit driven by neurons expressing the precursor to glucagon-like peptide 1

doi: 10.1038/s41598-019-51880-9

Figure Lengend Snippet: Light-stimulation of PPG neurons in the GCL induces inhibitory- and excitatory-postsynaptic currents in mitral cells. ( a ) (top) Schematic representation as in Fig. with electrode placement on the M/TCs and optical stimulation of the GCL (blue ellipse). (bottom) photomicrograph of electrode placement on a mitral cell in the GCL of a 300 µm slice of OB. ( b ) Light-induced inhibitory- (IPSC, V hold = + 0 mV) and excitatory- (EPSC, V hold = –70 mV) postsynaptic currents recorded in response to light stimulation (blue vertical bar) of PPG neurons using cesium-methane-sulfonate (CsMeSO 3 ) internal solution or, ( c ) potassium-gluconate (KGluc) internal solution. ( d , d’ ) (top) Light-induced EPSCs in a single M/TC under control ACSF conditions (Control), (middle) following bath application of the GABA B receptor antagonist gabazine (10 µM GBZ), (bottom) and then with the addition of the glutamate blockers (100 µM AP V , and 20 µM NBQX), ( e , e’ ) (top) Light-evoked EPSCs in a single M/TC under control ACSF conditions (Control), (middle) following bath application of 1 µM TTX, (bottom) and then with the addition of 1 mM 4-AP. ( f , f’ ) (top) Light-induced IPSCs in a single M/TC under control ACSF conditions (Control), (bottom) and following bath application of GBZ, ( f , f’ ), (top) light-induced IPSCs in a single M/TC under control ACSF conditions (Control), (bottom), and following bath application of AP V and NBQX. ( b – g ) Vertical blue bars (light stimulation) = 5 ms, 75% intensity. Red traces = mean of 8–10 recordings. ( d’ – g’ ) Values noted with the same letter are not significantly different. Non-parametric Friedman test followed with a Dunn’s multiple comparison test ( d’ , e’ ) or Wilcoxon matched-pairs signed rank test ( f’ , g’ ).

Article Snippet: Vesicular GABA transporter (VGAT) was labelled using a rabbit polyclonal serum (1:1000, BOSTER Biological Technology Pleasanton, USA).

Techniques: Control, Comparison

Journal: Life

Article Title: Association of Metabolically Healthy and Unhealthy Obesity Phenotype with Markers Related to Obesity, Diabetes among Young, Healthy Adult Men. Analysis of MAGNETIC Study

doi: 10.3390/life11121350

Figure Lengend Snippet: Levels of selected markers related to diabesity and obesity among subjects according to metabolic status.

Article Snippet: Plasma level of angiopoietin-like 3 protein (ANGPTL3) and angiopoietin-like 4 protein (ANGPTL4) were measured using magnetic bead-based multiplex reagents from the R&D System (Minneapolis, MN, USA).

Techniques:

Journal: Life

Article Title: Association of Metabolically Healthy and Unhealthy Obesity Phenotype with Markers Related to Obesity, Diabetes among Young, Healthy Adult Men. Analysis of MAGNETIC Study

doi: 10.3390/life11121350

Figure Lengend Snippet: The connection between visceral adiposity index and markers related to obesity and diabesity—differences between men with different metabolic status. MHNW: metabolically healthy normal weight; MHO: metabolically healthy obese; MUO: metabolically unhealthy obese; VAI: visceral adiposity index; GIP: gastric inhibitory peptide; GLP-1: glucagon-like peptide-1; PAI-1: plasminogen activator inhibitor-1; ANGPTL3: angiopoietin-like 3 protein; ANGPTL4: angiopoietin-like 4 protein. Data are presented in plots where the y-axis shows the concentration of the selected marker, and the x-axis shows the visceral adiposity index value. ( A ) adiponectin; ( B ) adipsin; ( C ) ghrelin; ( D ) GIP; ( E ) GLP-1; ( F ) glucagon; ( G ) insulin; ( H ) PAI-1; ( I ) resistin; ( J ) visfatin; ( K ) leptin/adiponectin index; ( L ) ANGPTL3; ( M ) ANGPTL4; ( N ) leptin; and ( O ) c-peptide.

Article Snippet: Plasma level of angiopoietin-like 3 protein (ANGPTL3) and angiopoietin-like 4 protein (ANGPTL4) were measured using magnetic bead-based multiplex reagents from the R&D System (Minneapolis, MN, USA).

Techniques: Metabolic Labelling, Concentration Assay, Marker

Journal: Life

Article Title: Association of Metabolically Healthy and Unhealthy Obesity Phenotype with Markers Related to Obesity, Diabetes among Young, Healthy Adult Men. Analysis of MAGNETIC Study

doi: 10.3390/life11121350

Figure Lengend Snippet: Univariate linear regression analyses of the effects of markers related to obesity and diabesity on the metabolic health status.

Article Snippet: Plasma level of angiopoietin-like 3 protein (ANGPTL3) and angiopoietin-like 4 protein (ANGPTL4) were measured using magnetic bead-based multiplex reagents from the R&D System (Minneapolis, MN, USA).

Techniques:

Journal: Cell cycle (Georgetown, Tex.)

Article Title: SIRT1 is upregulated in cutaneous T-cell lymphoma, and its inhibition induces growth arrest and apoptosis.

doi: 10.4161/cc.27523

Figure Lengend Snippet: Figure 3. Chemically induced SIRT1 inhibition causes cell death in association with reduced SIRT1 enzymatic activity, increased PARP cleavage, and increased FoxO3. (A) Tenovin-1 mediated reduction in SIRT enzymatic activity. Y-axis represents percent SIRT normalized luminescent activity compared with untreated control. (B) Tenovin-1 treatment reduces the viability of CTCL cells in a dose-dependent fashion that is maximal at 24 or 48 h depending on the specific CTCL line. White bars represent untreated controls and light and dark gray bars represent tenovin-1 at 10 and 25 μM doses, respectively. Data represented as mean ± standard deviation of 3 experiments with similar results (*P < 0.05). (C) Immunoblot analysis shows tenovin-1 mediated downregulation of SIRT1 expression, at 48 h post-treatment, is associated with PARP cleavage. (D) It is also associated with increased nuclear FoxO3. Numbers represent densitometric analysis of protein bands. For each cell line, the levels of endogenous SIRT1, cleaved PARP, and FoxO3 expression in untreated controls were considered as 1.0. Densitometric analysis of protein bands was performed relative to GAPDH/TATA loading control.

Article Snippet: Loading control antibodies were GAPDH and TATA binding protein (TBP) (Novus Biologicals and Abcam).

Techniques: Inhibition, Activity Assay, Control, Standard Deviation, Western Blot, Expressing

Journal: Cell cycle (Georgetown, Tex.)

Article Title: SIRT1 is upregulated in cutaneous T-cell lymphoma, and its inhibition induces growth arrest and apoptosis.

doi: 10.4161/cc.27523

Figure Lengend Snippet: Figure 4. Tenovin-1 enhances p53 pathway signaling in wtp53 MyLa cells. (A) Immunoblot shows baseline expression levels of p53 using anti- body (FL393) that detects full-length p53 of human origin. Densitometric analysis of protein bands was performed relative to GAPDH loading con- trol. (B) Immunoblot using another antibody (7F5; 2527) specific for the N terminus of p53 shows that p53 is upregulated only in the cell line bear- ing wtp53 (MyLa). (C) Tenovin-1 treatment upregulates activated (acety- lated) p53 expression only in wtp53 MyLa nuclear lysates. Densitometric analysis of protein bands was performed relative to TATA-binding pro- tein (TATA) loading control. (D) Tenovin-1 treatment upregulates p53 promoter activity in wtp53 MyLa cells. Data represented as mean ± stan- dard deviation of 3 experiments (6 replicates per experiment) with simi- lar results (*P < 0.05). (E) Immunoblot shows that the upregulated p53 in MyLa cells is associated with upregulation of p21, a downstream target of p53. Densitometric analysis of protein bands was performed relative to GAPDH loading control.

Article Snippet: Loading control antibodies were GAPDH and TATA binding protein (TBP) (Novus Biologicals and Abcam).

Techniques: Western Blot, Expressing, Binding Assay, Control, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Metformin upregulates circadian gene PER2 to inhibit growth and enhance the sensitivity of glioblastoma cell lines to radiotherapy via SIRT2/G6PD pathway

doi: 10.3389/fphar.2025.1563865

Figure Lengend Snippet: Verify PER2 -KD and PER2 -OE cell models of the U87 and U251. (A–D) The relative mRNA levels of PER2 in U87- PER2 -KD, U87- PER2 -OE, U251- PER2 -KD and U251- PER2 -OE cells. (E–H) Western blotting analysis and densitometric quantification of the expression of PER2 proteins in U87- PER2 -KD, U87- PER2 -OE, U251- PER2 -KD and U251- PER2- OE cells. Data are expressed as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. KD, knock down; OE, overexpression.

Article Snippet: PER2 knock-down and overexpression lentivirus with green fluorescence protein (GFP) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China), and the extent of GFP expression was observed with a fluorescence microscope.

Techniques: Western Blot, Expressing, Knockdown, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Metformin upregulates circadian gene PER2 to inhibit growth and enhance the sensitivity of glioblastoma cell lines to radiotherapy via SIRT2/G6PD pathway

doi: 10.3389/fphar.2025.1563865

Figure Lengend Snippet: Validate the expression level of G6PD, SIRT2, PER2 in GBM cell lines. (A) The activity of G6PDH was measured in U87- PER2 -KD, U87- PER2 -OE cells. (B) The activity of G6PDH was measured in U251- PER2 -KD, U251- PER2 -OE cells. (C) Western blotting analysis of the expression of G6PD and SIRT2 proteins in U87- PER2 -OE cells. (D) Western blotting analysis of the expression of G6PD and SIRT2 proteins in U87- PER2 -OE cells. (E) Western blotting analysis of the expression of G6PD and SIRT2 proteins in U251- PER2 -KD cells. (F) Western blotting analysis of the expression of G6PD and SIRT2 proteins in U251- PER2- OE cells. Data are expressed as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. KD: knock down, OE: overexpression.

Article Snippet: PER2 knock-down and overexpression lentivirus with green fluorescence protein (GFP) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China), and the extent of GFP expression was observed with a fluorescence microscope.

Techniques: Expressing, Activity Assay, Western Blot, Knockdown, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Metformin upregulates circadian gene PER2 to inhibit growth and enhance the sensitivity of glioblastoma cell lines to radiotherapy via SIRT2/G6PD pathway

doi: 10.3389/fphar.2025.1563865

Figure Lengend Snippet: Clarify the influence of metformin on PER2 and SIRT2. (A) Representative fluorescence and densitometric quantification intensity of SIRT2 in Ctrl, U251- PER2 -KD, U251- PER2 -OE cells treated with/without metformin. (B) . Western blotting analysis of the expression of G6PD and SIRT2 proteins in U87- PER2 -KD GBM cells treated with/without metformin. (C) . Western blotting analysis of the expression of G6PD and SIRT2 proteins in U87- PER2 -OE GBM cells treated with/without metformin. Data are expressed as the means ± SD. * P < 0.05, **P < 0.01, ** *P < 0.001. KD: knock down, OE: overexpression, Ctrl: control, Met: metformin.

Article Snippet: PER2 knock-down and overexpression lentivirus with green fluorescence protein (GFP) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China), and the extent of GFP expression was observed with a fluorescence microscope.

Techniques: Fluorescence, Western Blot, Expressing, Knockdown, Over Expression, Control