Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL-32β is the most dominant isoform in HepG2+LX-2 spheroids that increases neutral fat content, and IL32 downregulation lowers neutral fat in 2D cultured hepatocytes (A) To test the effect of IL-32 administration on intracellular fat content, immortalized human hepatic cell lines HepG2 and HepaRG were cultured in 2D and incubated with human recombinant IL-32α, IL-32β or IL-32γ isoform for 48 h. Then, intracellular neutral fat content was visualized by Oil Red O staining (ORO). ORO area quantified per DAPI stained nuclei by ImageJ, showed increased intracellular neutral fat content in both (A) HepG2 (top) and HepaRG (bottom). (B) To test the effect of IL32 downregulation on intracellular fat content, 24 h after seeding, cells were transfected with scramble or IL32 siRNA and grown in regular medium without FBS (HepG2) or medium supplemented with 25 μM oleic acid (HepaRG) for an additional 48 h. The average of gene knockdown efficiency was ∼70%–75% as evaluated by real-time qPCR analyzed by the 2 −ΔΔCt method. Intracellular neutral fat content was visualized by Oil Red O staining (ORO). ORO area quantified per DAPI stained nuclei by ImageJ showed lower intracellular neutral fat content in both HepG2 (left) and HepaRG (right). Two-sided p values were calculated by the Mann-Whitney non-parametric t test. Data shown as mean ± SD in all groups for the reported number of experiments. (C) HepG2+LX-2 cells were cultured as spheroids for 96 h exposing them to medium supplemented with 1% BSA or to increasing concentrations of a mixture of fatty acids (PA + OA, 1:2). IL32 gene expression measured after exposure, demonstrated higher IL32 mRNA levels with increasing intra-spheroidal triglyceride levels. The p value was calculated by test for linear trend. Data shown as mean ± SD for the reported number of experiments. (D) Percentage of IL32 gene products versus other IL-32 isoforms was measured from RNA sequencing (RNA-seq) data and IL-32β is the most dominantly expressed IL32 isoform in our spheroid model. The p value was calculated by one-way ANOVA. OA, oleic acid; PA, palmitic acid; RU, relative unit.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Cell Culture, Incubation, Recombinant, Staining, Transfection, Knockdown, MANN-WHITNEY, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Incubation with human recombinant IL-32β increases while IL32 downregulation lowers intracellular triglyceride content in spheroids from immortalized and human primary hepatocytes (A) HepG2+LX-2 cells were cultured as spheroids for a total of 96 h. Initially, 48 h after seeding cells the media was supplemented with 25 nM IL-32β for 48 h. (B) HepG2+LX-2 spheroids were generated by seeding cells along with negative control (SCR) siRNA or 30 nM IL32 siRNA transfection mix for downregulation for total of 96 h. (C) Primary human hepatocytes (PHH) were cultured as spheroids for a total of 7 days. Initially, 48 h after seeding the media was supplemented with 25 nM IL-32β for an additional 5 days with media replacement every 48 h. (D) PHH were cultured as spheroids along with negative control (SCR) siRNA or 30 nM IL32 siRNA transfection mix for downregulation, for a total of 7 days. For both spheroid models, cellular ATP levels (marker of viability) remained stable between the experimental groups. The average of gene knockdown efficiency was ∼70%–75% as evaluated by real-time qPCR analyzed by the 2 −ΔΔCt method, relative to beta-actin. Intracellular neutral fat content measured by Oil Red O staining and AdipoRed assay showed an increase in triglycerides content after incubation with IL-32β while IL32 downregulation lowers triglyceride levels. Two-sided p values were calculated by Mann-Whitney non-parametric t test. Data shown as mean ± SD in all groups for the reported number of experiments. RFU, relative fluorescence unit; RU, relative unit (to beta-actin); SCR, scramble siRNA; untr, untreated.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Incubation, Recombinant, Cell Culture, Generated, Negative Control, Transfection, Marker, Knockdown, Staining, MANN-WHITNEY, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL32 downregulation lowers intracellular COL1A1, increases MMP2 levels, and lowers TIMP2 in primary di-lineage human spheroids Primary human hepatocytes and primary hepatic stellate cells, at the ratio 24:1, were seeded with negative control scramble (SCR) and IL32 siRNA, at 5,000 cells/well in ultra-low attachment 96-well U-bottom ultra-low attachment plates. Fifty percent of the total media was replenished with fresh media every 48 h. (A) After 7 days of formation, spheroids were collected and 8-μM sections were subjected to immunofluorescent staining for COL1A1. Immunofluorescence was quantified by ImageJ, normalized to number of DAPI stained nuclei. The knockdown efficiency was measured by real-time qPCR, relative to beta-actin. (B) MMP2, TIMP-1, TIMP-2, and α-SMA protein levels were measured by western blotting in the cell lysate. Calnexin was used as loading control. Representative images of protein levels are shown. For each panel, data shown as mean ± SD of the reported independent experiments. Two-sided p values were calculated by Mann-Whitney non-parametric test. COL1A1, collagen Iα1; MMP2, matrix metallopeptidase 2; TIMP1, tissue inhibitor of metalloproteinase 1; TIMP2, tissue inhibitor of metalloproteinase 2.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Negative Control, Staining, Immunofluorescence, Knockdown, Western Blot, Control, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Endogenous IL32 downregulation lowers, and incubation with recombinant IL-32 β increases, intracellular triglycerides synthesis For endogenous IL32 downregulation experiments, HepG2+LX-2 spheroids were generated by seeding cells along with negative control (SCR) siRNA or IL32 siRNA transfection mix for a total of 96 h. For IL-32β incubation experiments, initially, 48 h after seeding HepG2+LX2 cells (24:1), the media was supplemented with 25 nM IL-32β for another 48 h. In both conditions, newly synthesized triglycerides were separated by TLC and quantified by scintillation counting after incubation with 6 μCi/mL 3 H-glycerol plus 1.5 mM glycerol for 12 h. (A) Reduction in de novo triglyceride synthesis after IL-32 downregulation. (D) Increase in de novo triglyceride synthesis after 25 nM IL-32β incubation. Cells were incubated with 8.5 μCi/mL 3 H-palmitate +55 μM/L palmitic acid for 6 h, after which palmitate was precipitated with BSA and perchloric acid and quantified by scintillation counting. (B and E) Graph shows no difference in beta oxidation in both experimental groups. APOB-100 synthesis and secretion levels were measured by immunoblotting. (C) Decrease in APOB-100 in cell lysate and cell culture supernatant after IL-32 downregulation. (F) No changes in APOB-100 levels in cell lysate and culture medium, after incubation with 25 nM IL-32β. The reported number of experiments were performed independently. Representative blots are presented. For all experiments, two-sided p value was calculated by Mann-Whitney non-parametric t test. Data shown as mean ± SD. AU, arbitrary units; DPM, disintegrations per minute; TAG, triacylglycerol.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Incubation, Recombinant, Generated, Negative Control, Transfection, Synthesized, Western Blot, Cell Culture, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Differentially expressed genes reveals downregulation of key genes for lipid metabolism in HepG2+LX2 spheroids (A) Key genes of lipid metabolism differentially expressed in spheroids after IL32 downregulation as compared with scramble. (B) Top 100 differentially expressed genes after IL32 downregulation as compared with scramble. Data are presented as log2-fold change in expression and –log 10 of p values adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). VLDL, very low-density lipoprotein.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Co-downregulation of PLA2G2A and IL32 abolished the IL-32-mediated intracellular triglycerides lowering and IL32 downregulation reduces intracellular PI levels in human primary spheroids Primary human hepatocytes were cultured as spheroids and incubated with negative control (SCR) siRNA or 30 nM IL32 , and combination of IL32 and PLA2G2A for a total of 7 days. (A) Cellular ATP levels (marker of viability) were not different within the three groups. There was an 80%^–90% reduction in mRNA levels of IL32 and PLA2G2A , relative to beta-actin. Intracellular neutral lipid content (measured by Oil Red O staining) normalized to nuclei (stained by DAPI) were lower after IL32 downregulation while the co-downregulation of PLA2G2A and IL32 abolished this reduction. (B) IL32 downregulation results in higher PLA2G2A mRNA levels measured by real-time PCR. (C) IL32 and PLA2G2A were downregulated individually or in combination in primary hepatocyte spheroids and PLA2G2A levels were measured by human PLA2G2A ELISA in the culture medium. PLA2G2A levels were higher after IL32 downregulation and lower after co-downregulation of IL32 and PLA2G2A . (D) After 2 days from seeding, hepatocyte spheroids were incubated with 10, 25, and 50 nM human recombinant IL-32β for 5 days. PLA2G2A was measured from cell culture supernatant using human PLA2G2A ELISA and we observed a dose dependent decrease in secreted PLA2G2A levels with increasing concentration of IL-32β. The p values were calculated by test for linear trend. (E) Lipid fingerprint measured by liquid chromatography- quadrupole time-of-flight-mass spectrometry demonstrated lower total PI and triglycerides (top) levels after IL32 downregulation. There was a reduction in all PI species, except 40:5 and 40:6, with the largest effect size in 38:4 (bottom). For each part, data are shown as mean ± SD of the reported independent experiments. Two-sided p values calculated with unpaired t test for n = 3 and Mann-Whitney non-parametric t test for n > 3. Cer, ceramides; CL, cardiolipins; DAG, di-acylglycerides; GalCer, galactosyl ceramides; LPC, lysophosphatidylcholine; ORO, Oil Red O; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; RFU, relative fluorescence units; RU, relative units; PC, phosphatidylcholine; SM, sphingomyelin; TAG, triacylglycerols.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Cell Culture, Incubation, Negative Control, Marker, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Liquid Chromatography, Mass Spectrometry, MANN-WHITNEY, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL-32 rs76580947 minor allele associates with lower IL-32 expression, severe liver steatosis, and lower liver non-invasive tests (A) Regional plots of association between IL32 common genetic variants (minor allele frequency of >0.01) and ALT in the European subset of UK biobank. The x axis shows the variant positions (GRCh37); the y axis shows the –log10 p values. The gray diamond represents rs76580947, with the strongest association in the plotted region (IL-32 ± 50 Kbp), for which its pairwise LD with other variants is color coded as shown on the figure. (B) The association between rs76580947 and hepatic IL-32 mRNA levels was tested in 207 individuals from the MAFALDA cohort adjusting for age, gender, percentage of coding bases (a quality control measure from the Picard toolkit), RNA Integrity Number (RIN), and five surrogate variables detected by surrogate variable analysis. Carriers of the variant have lower IL-32 mRNA levels. Data shown as violin plots and adjusted p values are reported. (C) The association between IL32 rs76580947 stratified by genotype and IL-32 plasma protein level in 365,495 European participants from UK Biobank was tested using a linear regression analysis adjusted for age, gender, body mass index, first 10 genomic principal components, and array batch. Violin plot shows the normalized Protein eXpression (NPX) values that were rank-based inverse normal transformed prior to the analysis. (D) Forest plot of association and meta-analysis for IL32 rs76580947 with steatosis in three independent cohorts: Southern Italy (N = 425), Central Italy (N = 245), and Finnish (N = 745). The plot shows protection against severe liver steatosis (steatosis absence or mild vs. severe; fixed-effect p = 0.027). The association was tested by a binary logistic regression analysis under an additive genetic model adjusted by age, gender, body mass index, and recruitment center (only for the Finnish cohort). Pooled effect estimates were calculated using inverse-variance-weighted fixed effects meta-analysis. (E) The association between IL32 rs76580947 and clinical liver fibrosis scores and APOB levels in 365,495 European participants from UK Biobank. The analysis was performed under an additive model, using linear regression adjusting for age, gender, body mass index, the first 10 genomic principal components, and array batch. All traits were rank-based inverse normal transformed prior to the analysis. CI, confidence interval.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Expressing, Variant Assay, Control, Clinical Proteomics, Transformation Assay

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet:

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Recombinant, Protein Extraction, Staining, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Transfection, Gene Expression, Fluorescence, Software, Cell Counting

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Production of IL-10 by monocytes treated with Tat. (A) Monocytes (106) were incubated in the absence or presence of Tat (1, 10, 100, or 500 nM) or LPS (100 ng/ml) for 24 h. (B) Monocytes were identically treated with LPS (100 ng/ml) or Tat (10 or 100 nM) but for 24, 48, or 72 h. (C) Specificity of Tat. Monocytes (106) were incubated in the absence or presence of native Tat (10 or 100 nM), oxidized Tat (1 h at 25°C in PBS plus 3% H2O2 at 10 and 100 nM), or LPS (100 ng/ml) for 24 h. (D) PBMC were depleted of monocytes by three successive adherence steps (1°, 2°, and 3°) in 24-well plates. After each adherence step, cells remaining in suspension (106) were incubated in the absence or presence of LPS at 100 ng/ml or Tat at 1, 10, or 100 nM. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. For A, B, and D, the values are the means ± standard deviation (SD) of three experiments with cells from one donor. Similar results were obtained with cells from three different donors. For C, the values are from results obtained with three different donors. Ctr, control.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Incubation, Suspension, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Specificity of Tat-induced IL-10 production and mapping of the active domain. (A) Monocytes (106) were incubated with wild-type GST-Tat 1-101 (1 or 10 nM) or with recombinant mutants GST-Tat 1-72, GST-Tat 1-55, GST-Tat 1-45, GST-Tat 20-72, or GST-Tat 30-72 (1 or 10 nM) or with GST as a negative control for 24 h. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. The values are the means ± SD of three experiments with cells from one donor. Similar results were obtained with cells from three different donors. (B) Tat (10 nM) was incubated for 1 h or not with MAbs directed against Tat epitopes 1 to 15, 46 to 60, or 74 to 86 (2.5 or 0.025 μg/ml) or the mixture of the three MAbs. After 24 h, culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. As control, an MAb directed against gp140 of simian immunodeficiency virus was used as a control in the same conditions. Results represent the ratio of production of IL-10 by monocytes stimulated by Tat (10 nM) incubated with MAb and production of IL-10 produced after stimulation by Tat (10 nM) alone (P/P0). On the right, the percent inhibition of IL-10 production induced by Tat (10 nM) is represented. Mouse MAbs were obtained from the Agence Nationale de la Recherche sur le SIDA (Paris, France).

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Incubation, Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay, Virus, Produced, Inhibition

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Production of IL-10 by monocytes stimulated by Tat immobilized in wells. (A) Tat was immobilized in wells by incubation for 2 h at 37°C. After two washes to eliminate nonfixed Tat, monocytes (106) were added and cultured for 24 h with different concentrations of Tat (5, 50, or 500 nM). Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. (B) HeLa P4 cells were incubated with soluble (solid bars) or immobilized (shaded bars) Tat, and a Tat-dependent-transactivating test was done as described in Materials and Methods. The values are the means ± SD of three experiments. For A, similar results were obtained with cells from two different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Effect of PLC inhibitor U73122 on Tat-induced production of IL-10. Monocytes (106) were treated or not with U73122 (2.5 or 7.5 μM) for 30 min. Tat (10 nM) was then added for 24 h. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. The values are the means ± SD of three experiments. Similar results were obtained with cells from three different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Involvement of the calcium pathway. (A) Effect of calcineurin inhibitor cyclosporin A (Cs A) and of the chelator of intracellular calcium BAPTA/AM on the Tat-induced production of IL-10. Monocytes were treated or not for 30 min as indicated and then treated or not with Tat at 10 nM. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. (B) Positive control of inhibition by cyclosporin A and BAPTA/AM on the ionomycin-induced production of TNF-α. Monocytes were treated or not for 30 min as indicated and then treated or not with ionomycin (1 μM) for 24 h. Culture supernatants were then collected, and the presence of TNF-α was determined by ELISA. These results are representative of three independent experiments done on cells from two donors. (C and D) Variations in intracellular calcium concentrations determined by microspectrofluorimetry using fluo-3AM as the probe. Cells were incubated for 30 min in the presence of fluo-3 (5 μM) and observed microscopically after two washes. Monocytes were stimulated by 100 nM Tat (C) or 1 μM ionomycin (D). The curves are the results obtained from the means for 11 different cells from a single donor. Similar results were obtained with cells from two different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Inhibition, Incubation

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Involvement of PKC in Tat-induced production of IL-10. (A) Monocytes (106) were treated or not with the PKC inhibitor RO318220 (RO) at the concentrations indicated (2.5 and 5 μM) or by the PKA inhibitor H89 (50 and 100 μM) for 30 min. Tat was then added for 24 h. These results are representative of three independent experiments done on cells from three donors. (B) Control of the specificity of the kinase inhibitors H89 and RO318220. Monocytes (106) were treated or not with the PKC inhibitor RO318220 (2.5 and 5 μM) or by the PKA inhibitor H89 (50 and 100 μM) for 30 min. Rolipram was then added for 24 h. Culture supernatants were then collected, and the presence of IL-10 was determined by ELISA. (C and D) Monocytes were treated or not with PMA (50 or 100 ng/ml), a PKC activator, for 24 h (C) or 48 h (D), and Tat (10 nM) was then added. After 24 h, culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. The values in C are the means ± SD of three experiments. The results in C and D are representative of three independent experiments done on cells from three donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Role of NF-κB in Tat-induced IL-10 production. (A) Effect of the serine protease inhibitor TLCK (50 and 100 μM) on NF-κB nuclear translocation induced by Tat (10 nM) (lanes 7 and 8). Lanes 3 and 4 correspond to the activation of NF-κB with Tat (10 and 1 nM, respectively). Ctr, control. (B) Effect of treatment with TLCK (TL) at 50 and 100 μM on IL-10 production by monocytes (106) treated with 10 nM Tat (T). The values are the means ± SD of three experiments. Similar results were obtained with cells from three different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Protease Inhibitor, Translocation Assay, Activation Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Involvement of MAP kinases ERK1/2 in the Tat-induced production of IL-10. (A) Western blot analysis of the activation of MAP kinases ERK1/2 by Tat. Cytoplasmic protein extracts were prepared from monocytes (2 × 106) treated with PMA at 50 ng/ml (lanes 2 and 6) or 10 nM (lanes 3 and 7) or 100 nM (lanes 4 and 8) Tat for 15 or 30 min. Visualization was done with an antibody recognizing total ERK1/2 or only phosphorylated ERK1/2 (pERK1/2). These results are representative of two independent experiments done on cells from two different donors. Ctr, control. (B) Effect of PD 98 059 (PD) at 10 or 100 μM, an inhibitor of MAP kinases ERK1/2, on monocytes (106) treated with 10 nM Tat (T). Similar results were obtained with cells from three different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Western Blot, Activation Assay