Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Expression of CD163 on PB monocytes was measured by FACS after 3 days in no-contact co-cultures with HT-1080-Luc-shPAI1 or HT-1080-Luc-scPAI1 cells, in the absence or presence of doxycycline. The MFIs from 2 independent experiments were normalized to the values obtained in co-cultures with HT1080-Luc-scPAI1 cells in the absence of doxycycline and averaged. Tests of difference in means between groups comprising independent, non-matched observations were based on analysis of variance (*p<0.5); B. MFIs of CD163, CD86 and CD80 on PB monocytes from 4 healthy blood donors cultured in the presence of rPAI-1, rPAI-1 R76E I91L and rPAI-1 T333R A335R (100 nM). The data represent the MFI value obtained for each independent experiment; C. Levels of IL-10 and IL-12 produced by PB monocytes from (B). D. Levels of IL-6 in same conditions as in (C). The data represent the mean (± SD) concentration in technical duplicate samples from four independent experiments. D-E. Analysis of GEO datasets (E) and RNA-seq TCGA datasets (F) for PAI-1 and IL-6 genes for indicated tumors. A two-way t-test was used to establish the significance of the correlation coefficients.

Article Snippet: Cell culture media from the in vitro experiments were analyzed for the expression levels of human IL-6, IL-10 and IL-12 cytokines using DuoSet Elisa Assays (R&D) according to the manufacturer’s protocol.

Techniques: Expressing, Cell Culture, Produced, Concentration Assay, RNA Sequencing Assay

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Phospho-Kinase Array of PB monocytes exposed to rPAI-1 and rPAI-1 T333R A335R (100 nM) for 10 min (left panel). The intensity of the signal was quantified as a fold change in signal intensity in rPAI-1-treated cells over rPAI-1 T333R A335R-treated cells (right panel); B. Top panel: western blot analysis of p38MAPK phosphorylation in lysates of PB monocytes obtained 10 min after exposure to rPAI-1 and rPAI-1 T333R A335R (100 nM). The data are representative of one of 3 independent experiments showing similar results. Lower panel: mean (± 95% CI) fold change in the ratio pp38MAPK:p38MAPK from 3 independent experiments. P values are based on analysis of variance of log10 ratios. C. Western blot analysis of phosphorylation of p38MAPK in lysates of PB monocytes 10 min after exposure to CM from HT1080-Luc-shPAI1 and HT1080-Luc-scPAI1 cultured in the presence or absence of doxycycline. Ratios of pp38MAPK:p38MAPK obtained by scanning densitometry are shown at the bottom of the gel. The data are representative of one of 2 independent experiments showing similar results; D. Western blot analysis of p38MAPK, pSTAT3 and STAT3 in PB monocytes transduced with p38MAPK siRNA and scrambled control siRNA and treated for 7 h with rPAI-1 (100 nM); E. Top panel: western blot analysis of pSTAT3 in PB monocytes exposed for 7 h to 100 nM rPAI-1, DMSO, or SB203580 (20 μM; 1 h preincubation) or their combination. Lower panel: IL-6 protein levels in the medium of PB monocytes under the conditions described in the top panel. The data represent the mean (± SD) of technical duplicates.

Article Snippet: Cell culture media from the in vitro experiments were analyzed for the expression levels of human IL-6, IL-10 and IL-12 cytokines using DuoSet Elisa Assays (R&D) according to the manufacturer’s protocol.

Techniques: Western Blot, Cell Culture, Transduction

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Upper panel: IL-6 mRNA expression level measured by qRT-PCR at indicated times in PB monocytes exposed to rPAI-1 (100 nM). The data represent levels normalized against GAPDH analyzed using 2−ΔCt method. Lower panel: corresponding IL-6 protein levels determined by ELISA on aliquots of the medium collected at indicated times. The data represent the mean (± SD) of technical duplicates; B. Western blot analysis of STAT3 phosphorylation in cell lysates of PB monocytes at indicated times after treatment with 40 nM rPAI-1; C. Western blot analysis of STAT3 phosphorylation (Y705) in cell lysates of PB monocytes co-cultured (no-contact) for 3 days with HT1080-Luc-shPAI1 cells in presence and absence of doxycycline. The bar diagram under each lane represents ratio of pSTAT3:STAT3; D. Western blot analysis of STAT3 phosphorylation of PB monocytes exposed to a PAI-1 blocking antibody (5 μg/mL) or isotype antibody and rPAI-1 (40 nM) for 5 h. Lower panel: IL-6 levels measured by ELISA in the medium of PB monocytes treated as indicated in the top panel. The data represent the mean IL-6 concentrations (± SD) of technical duplicates; E. Western blot analysis of STAT3 phosphorylation in lysates from PB monocytes cultured for 4 days in the presence of rPAI-1 and rPAI-1 mutants (40 nM). When indicated, rPAI-1 was heated for 60 min at 95°C. The numbers under each lane represent ratio of pSTAT3:STAT3; F. Western blot analysis of STAT3 phosphorylation in lysates of PB monocytes obtained after 6 days of treatment with rPAI-1 (40 nM), ruxolitinib (1 μM), tocilizumab (20 μg/mL), IgG (20 μg/mL) or DMSO or their combination. The numbers under each lane represent ratio of pSTAT3:STAT3. Lower panel: corresponding CD163 MFIs of PB monocytes treated as indicated in the upper panel. The data from an independent biological replicate of this experiment is shown in Figure S3; G. CD163 MFIs in PB monocytes after 3 days in no-contact co-cultures with HT1080 cells in the presence of DMSO, IgG, ruxolitinib or tocilizumab. The data represent the mean values of 2 independent experiments. Tests of difference in means between groups comprising independent, non-matched observations were based on analysis of variance.

Article Snippet: Cell culture media from the in vitro experiments were analyzed for the expression levels of human IL-6, IL-10 and IL-12 cytokines using DuoSet Elisa Assays (R&D) according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Blocking Assay

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Immunofluorescence (IF) analysis of PB monocytes treated with 100 nM rPAI-1 and rPAI-1 T333R A335R for 2 h. When indicated PB monocytes were preincubated with SB203580 (20 μM) for 1 h (or DMSO as a control) and treated with rPAI-1 for 2 h. Cells were analyzed by confocal microscopy for NF-κB. NF-κB: green, DAPI: blue. Scale bar: 20 μm. The histogram represents the mean (± SD) % of nuclei staining positive for NF- κB from a total of 450 nuclei per sample in triplicate (*p<0.05; **p<0.01; ***p<0.001). B. Western blot analysis of p38MAPK and NF-κB p65 and their phosphorylated forms in lysates of PB monocytes exposed to 100 nM rPAI-1 or rPAI-1 T333R A335R for the indicated amount of time; C. Western blot analysis of NF-κB p65 and its phosphorylated form in lysates of PB monocytes exposed for 7 h to rPAI-1, DMSO or SB203580 alone or indicated combinations; D. Western blot analysis of p65 NF-κB, and STAT3 and its phosphorylated form in lysates of PB monocytes in which p65 was downregulated with siRNA and exposed for 7 h to 100 nM rPAI-1; E. Top panel: western blot analysis of STAT3 and its phosphorylated form in lysates of PB monocytes exposed for 7 h to rPAI-1, DMSO, BMS-345541 (5 μM; 1 h pre-incubation) or their combination. Lower panel: IL-6 levels in the medium of PB monocytes treated as indicated in the top panel. The data represent the mean concentrations (± SD) of technical duplicates.

Article Snippet: Cell culture media from the in vitro experiments were analyzed for the expression levels of human IL-6, IL-10 and IL-12 cytokines using DuoSet Elisa Assays (R&D) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Western Blot, Incubation

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Experimental design: Rag1−/− PAI-1−/− mice (n=39) were injected with HT-1080-Luc-shPAI1. The mice received doxycycline (DOX) in drinking water for 30 days after which 5 mice were sacrificed and doxycycline was stopped in 10 of the mice (STOP DOX). In each group, mice were sacrificed at day 37 (n=9), 44 (n=8) and 51 (n=5) post-implantation, and tumors were examined for the presence of M2 macrophages (F4/80) and total PAI-1 expression. As controls, we used PAI-1−/− mice, injected subcutaneously with HT-1080-Luc-scrambled cells and receiving doxycycline (n=5) which were sacrificed at day 44; B. percent of necrotic tumors in each 3 experimental groups. Inset: photograph of a necrotic tumor C. Quantified average mean vascular density (CD31+ area) in necrotic and non-necrotic tumors; D. Representative IHC analysis of tumor sections at indicated times for F4/80 expression; E. Spearman correlation analysis of PAI-1 expression level (PAI-1/actin) vs average per cent of F4/80 positive area from IHC analysis. Each data-point represents an average of 5 sections per tumor and 5 fields per section. Red dots – mice receiving doxycycline, blue dots – mice in which doxycycline was discontinued; F. Experimental design: Rag1−/− PAI-1−/− mice were injected with HT-1080-Luc-shPAI1 and Rag1−/− PAI-1+/+ mice were injected with HT-1080-Luc-scPAI1 tumor cells. Half of the mice received doxycycline in drinking water for 35 days (+DOX). After 35 days, mice were sacrificed and tumors were examined for the presence of macrophages and concentrations of cytokines as indicated in Methods (see also Figures S4 and S5); G-K: Quantitative analysis of pSTAT3+ in F4/80+ cells (G), CD206+ cells (H), CD163+ cells (I), Arginase+ (J) and iNOS+ (K) cells. The data represent the Log 10 average of number of positive cells. Bars indicate the mean log10 value in each group. * = p<0.05; ** = p<0.1; *** = p<0.01. L: Experimental design: Rag1−/− PAI-1−/− mice were injected with HT-1080-shPAI1 and with HT-1080-scPAI1 tumor cells. After 40 days, mice were sacrificed and tumors were examined for cytokine expression; M-N. Each data-point represents a technical duplicate and has been expressed as pg of cytokine per μg of protein in tumor lysate.

Article Snippet: Cell culture media from the in vitro experiments were analyzed for the expression levels of human IL-6, IL-10 and IL-12 cytokines using DuoSet Elisa Assays (R&D) according to the manufacturer’s protocol.

Techniques: Injection, Expressing

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell culture media from the in vitro experiments were analyzed for the expression levels of human IL-6, IL-10 and IL-12 cytokines using DuoSet Elisa Assays (R&D) according to the manufacturer’s protocol.

Techniques: Produced, Plasmid Preparation, Purification, Recombinant, Modification, Mutagenesis, Binding Assay, Blocking Assay, Staining, Enzyme-linked Immunosorbent Assay, Detection Assay, Software

Journal: Scientific Reports

Article Title: Dysregulated JAK2 expression by TrkC promotes metastasis potential, and EMT program of metastatic breast cancer

doi: 10.1038/srep33899

Figure Lengend Snippet: ( a ) ELISA assay of IL-6 secretion by Hs578T control-shRNA or TrkC-shRNA cells (n = 3). Data are presented as mean ± standard error of the mean (SEM). ( b ) Expression levels of mRNA encoding IL-6 in basal-like breast cancer cells (MDA-MB-231, MDA-MB-435, Hs578T, and SUM149) and normal or nontransformed cells (HMLE, MCF7, or MCF10A breast cells). 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). ( c ) Expression levels of mRNA encoding IL-6 in Hs578T control-shRNA or TrkC-shRNA cells. 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). ( d ) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. β-actin was used as a loading control. ( e ) Immunofluorescence staining of the nuclear translocation of STAT3 in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. The green signal represents staining of the corresponding protein, while the blue signal represents DAPI nuclear DNA staining. ( f ) Expression levels of mRNA encoding Twist-1 and Twist-2 in Hs578T control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). ( g ) Luciferase reporter assay of Twist-1 and Twist-2 in Hs578T control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. Each bar represents the mean ± SEM of three experiments. Data are presented as mean ± standard error of the mean (SEM). Some of the bar graphs do not have visible error bars due to low values of standard error of the mean (SEM).

Article Snippet: Subsequently, conditioned media from these cell cultures were collected and analyzed by the Human IL-6 Quantikine ELIZA kit (R&D systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, shRNA, Expressing, Western Blot, Immunofluorescence, Staining, Translocation Assay, Luciferase, Reporter Assay

Journal: Scientific Reports

Article Title: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

doi: 10.1038/s41598-017-10694-3

Figure Lengend Snippet: Inflammatory responses in H69 cells induced by LPS-derived EVs. ( a ) Analysis of inflammatory responses. H69 cells were incubated with PBS- or LPS- derived EVs for 48 hours. Total RNAs were harvested and two-step RT-PCR was performed (n = 5). For ELISA (n = 10) and cell proliferation assay (n = 10), H69 cells were cultured in 10 cm dishes or 96-well plates, respectively and incubated with EVs for 48 hours. Analyses were performed using commercial kits according to manufacturers’ instructions. ( b ) EV disruption abolished the effects of EVs. EVs were boiled at 95 °C for 15 min and incubated with H69 cells. After 48 hours, RT-PCR analysis for IL-1β and IL-6 (n = 3) and proliferation assay (n = 10) were performed. ( c ) H69-derived EVs did not induce inflammatory responses in primary hepatocytes. Human primary hepatocytes were incubated with PBS- or LPS-derived EVs isolated from H69 cells for 48 hours. RT-PCR was performed to analyze cytokine production (n = 10).

Article Snippet: IL-6 in culture media was detected using Human IL-6 ELISA Kit II (BD Biosciences, San Jose, CA).

Techniques: Derivative Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Cell Culture, Isolation

Journal: Scientific Reports

Article Title: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

doi: 10.1038/s41598-017-10694-3

Figure Lengend Snippet: Inflammatory cell-cell communication between large cholangiocytes. ( a ) Small and large cholangiocytes were incubated with PBS- or LPS-derived EVs isolated from small cholangiocytes for 48 hours. RT-PCR for proinflammatory cytokine expression (n = 6) and cell proliferation assay (n = 10) were performed. ( b ) Small and large cholangiocytes were incubated with EVs isolated from large cholangiocytes for 48 hours. RT-PCR (n = 6) and cell proliferation assay (n = 10) were performed.

Article Snippet: IL-6 in culture media was detected using Human IL-6 ELISA Kit II (BD Biosciences, San Jose, CA).

Techniques: Incubation, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Proliferation Assay

Journal: Scientific Reports

Article Title: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

doi: 10.1038/s41598-017-10694-3

Figure Lengend Snippet: Secretion of EVs and responses against LPS in large cholangiocytes with SCT or SR knockdown. ( a ) The morphological analysis by transmission electron microscope for EVs isolated from control large cholangiocytes (left), with SCT knockdown (middle), and with SR knockdown (right). Scale bar: 100 nm. ( b ) Nanoparticle tracking analysis for isolated EVs. An example for PBS- (solid lines) and LPS-derived (dashed lines) EVs isolated from control (blue lines), SCT knockdown (red lines), and SR knockdown (green lines) cholangiocytes is shown (top). Total EV concentrations are shown (bottom). * P < 0.05, ** P < 0.001 vs. control cholangiocytes with same stimulation (n = 5). ( c ) Knockdown efficiencies for SCT and SR. Control, SCT or SR knockdown cholangiocytes were incubated with 50 ng/mL LPS for 72 hours in 6-well plates. Culture media were harvested for ELISA for SCT, and total RNAs were harvested from cells for RT-PCR for SR. * P < 0.05, ** P < 0.001 vs. control cholangiocytes (n = 3). ( d ) For RT-PCR for proinflammatory cytokine expression, cholangiocytes were incubated with 1× PBS or 200 ng/mL LPS for 3 hours. Total RNAs were harvested and two-step RT-PCR was performed (n = 6). For cell proliferation assay, cholangiocytes were incubated with 1× PBS or 200 ng/mL LPS for 48 hours (n = 10).

Article Snippet: IL-6 in culture media was detected using Human IL-6 ELISA Kit II (BD Biosciences, San Jose, CA).

Techniques: Transmission Assay, Microscopy, Isolation, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Proliferation Assay

Journal: International Journal of Endocrinology

Article Title: Heat Shock Protein 70 Mediates the Protective Effect of Naringenin on High-Glucose-Induced Alterations of Endothelial Function

doi: 10.1155/2022/7275765

Figure Lengend Snippet: Effect of Nar on HG-induced alterations of endothelial function. HUVECs were exposed to NG (5.5 mM glucose) or HG (30 mM glucose) media with or without the 50 mM of Nar for 36 h. To observe the effect of Nar on insulin signaling, the cells were treated with or without 100 nM of insulin for 10 min. (a) Effect of Nar on ROS formation by DHE staining. (b) Quantification of relative DHE fluorescence in (a). (c) Effect of Nar on intracellular MDA concentration. (d) Effect of Nar on NF- κ B activity. (e) Effect of Nar on IL-6 concentration in the culture media. (f) Effect of Nar on mRNA levels of cell adhesion molecules ICAM-1 and VCAM-1. (g) Effect of Nar on insulin-stimulated phosphorylation of Akt and its downstream AS160. (h) Quantification of phosphorylated Akt and AS160 in (g). (i) Effect of Nar on insulin-stimulated 2-DG uptake. Data are expressed as means ± SD ( n = 3). ∗∗ P < 0.01 and ∗∗∗ P < 0.001 versus control (NG) group or the indicated group. ## P < 0.01 and ### P < 0.001 versus high-glucose (HG) group.

Article Snippet: IL-6 concentration in the culture media was measured using a Human IL-6 ELISA Kit (ab178013, Abcam) according to the manufacturer's protocol.

Techniques: Staining, Fluorescence, Concentration Assay, Activity Assay

Journal: International Journal of Endocrinology

Article Title: Heat Shock Protein 70 Mediates the Protective Effect of Naringenin on High-Glucose-Induced Alterations of Endothelial Function

doi: 10.1155/2022/7275765

Figure Lengend Snippet: Effect of HSP70 knockdown (KD) on endothelial function. The siRNA technique was used to silence HSP70 in HUVECs. The cells were then grown in NG (5.5 mM glucose) media for 36 h. To observe the effect of Nar on insulin signaling, the cells were treated with or without 100 nM of insulin for 10 min. (a) Effect of HSP70 KD on ROS formation by DHE staining. (b) Quantification of relative DHE fluorescence in (a). (c) Effect of HSP70 KD on intracellular MDA concentration. (d) Effect of HSP70 KD on NF- κ B activity. (e) Effect of HSP70 KD on IL-6 concentration in the culture media. (f) Effect of HSP70 KD on mRNA levels of cell adhesion molecules ICAM-1 and VCAM-1. (g) Effect of HSP70 KD on insulin-stimulated phosphorylation of Akt and its downstream AS160. (h) Quantification of phosphorylated Akt and AS160 in (g). (i) Effect of HSP70 KD on insulin-stimulated 2-DG uptake. Data are expressed as means ± SD ( n = 3). ∗∗ P < 0.01 and ∗∗∗ P < 0.001 versus siRNA control group or the indicated group.

Article Snippet: IL-6 concentration in the culture media was measured using a Human IL-6 ELISA Kit (ab178013, Abcam) according to the manufacturer's protocol.

Techniques: Staining, Fluorescence, Concentration Assay, Activity Assay

Journal: International Journal of Endocrinology

Article Title: Heat Shock Protein 70 Mediates the Protective Effect of Naringenin on High-Glucose-Induced Alterations of Endothelial Function

doi: 10.1155/2022/7275765

Figure Lengend Snippet: HSP70 knockdown (KD) attenuated the protective effect of Nar on HG-induced alterations of endothelial function. The siRNA technique was used to silence HSP70 in HG-treated HUVECs. The cells were then exposed to NG (5.5 mM glucose) or HG (30 mM glucose) media with or without the 50 mM of Nar for 36 h. To observe the effect of Nar on insulin signaling, the cells were treated with or without 100 nM of insulin for 10 min. (a) Effect of HSP70 KD on ROS formation by DHE staining. (b) Quantification of relative DHE fluorescence in (a). (c) Effect of HSP70 KD on intracellular MDA concentration. (d) Effect of HSP70 KD on NF- κ B activity. (e) Effect of HSP70 KD on IL-6 concentration in the culture media. (f) Effect of HSP70 KD on mRNA levels of cell adhesion molecules ICAM-1 and VCAM-1. (g) Effect of HSP70 KD on insulin-stimulated phosphorylation of Akt and its downstream AS160. (h) Quantification of phosphorylated Akt and AS160 in (g). (i) Effect of HSP70 KD on insulin-stimulated 2-DG uptake. Data are expressed as means ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus the indicated group.

Article Snippet: IL-6 concentration in the culture media was measured using a Human IL-6 ELISA Kit (ab178013, Abcam) according to the manufacturer's protocol.

Techniques: Staining, Fluorescence, Concentration Assay, Activity Assay

Journal: Aging (Albany NY)

Article Title: Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer

doi: 10.18632/aging.202289

Figure Lengend Snippet: Pharmacological blockade of FASN activity modifies autocrine prolactin secretion in a PR-dependent manner. ( A ). Top. Autocrine prolactin secretion levels in the extracellular milieu of estradiol-depleted cells cultured in the absence or presence of graded concentrations of C75 in 0.5% CCS for 48 h were determined by a commercially available EASIA kit. Data are means ( columns ) ± S.D. ( bars ) from three independent experiments performed in duplicate. Secreted amounts of prolactin in C75-treated cells were compared with those in vehicle-treated control cells (* P < 0.05; ** P < 0.005). Bottom. Cell lysates strictly obtained from the same experimental replicates employed in A were subjected to immunoblotting for PRLR protein expression. β-actin was used to control for protein loading and transfer. Figure shows a representative immunoblot analysis. Similar results were obtained in 3 independent experiments. ( B ). Autocrine IL-6 levels in the extracellular milieu of estradiol-depleted cells cultured in the absence or presence of 10 μg/mL C75 in 0.5% CCS for 48 h were determined by a commercially available ELISA kit. Secreted amounts of IL-6 in C75-treated cells were compared with those in vehicle-treated control cells (** P < 0.005).

Article Snippet: The amount of IL-6 conditioned media was determined with the Human IL-6 Quantikine ELISA Kit (catalog #D6050; R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Cell Culture, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Excessive ENaC-mediated sodium influx drives NLRP3 inflammasome-dependent autoinflammation in cystic fibrosis

doi: 10.1101/458208

Figure Lengend Snippet: A-F ELISA assays were used to detect IL-18 (HC n=21, CF n=36, SAID n=12, NCFB=4) (A), IL-1β (HC n=10, CF n=31, SAID n=13, NCFB=4) (B), IL-1Ra (HC n=20, CF n=37, SAID n=7, NCFB=4) (C), TNF (E) and IL-6 (HC n=24, CF n=40, SAID n=8, NCFB=4) (F) in patient serum. D Flow cytometry was used to detect ASC specks in sera of patients with CF, SAID, NCFB and HCs (HC n=10, CF n=10, SAID n=10, NCFB=4). G A colorimetric assay to detect caspase-1 activity in sera of patients with CF, SAID and NCFB as a percentage of the average caspase-1 activity from HC (n=10) with a mean absorbance of 0.20845 nm (HC n=10, CF n=21, SAID n=4, NCFB=4). Data Information: The Mann-Whitney non-parametric test was performed (p values * = 0.05, **= 0.01, ***= 0.001 and ****= 0.0001).

Article Snippet: Cytokines from patient sera and cell cultured media were detected by ELISAs (Human IL-1β CytoSet TM , human IL-18 matched antibody pairs, human IL-1Ra CytoSet TM , human TNF-α antibody pair and human IL-6 CytoSet TM ) (Invitrogen), as per the manufactures recommendations.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Colorimetric Assay, Activity Assay, MANN-WHITNEY