Journal: Biochemical pharmacology

Article Title: Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

doi: 10.1016/j.bcp.2017.11.012

Figure Lengend Snippet: (A) Transcriptional expression of TLR4 mRNA in RAW-Blue cells by RT-qPCR. Primary peritoneal macrophages (pM) isolated from TLR4-WT and TLR4-KO mice were used as positive and negative control, respectively. B) Comparative TEB gel electrophoresis of TLR4 real time PCR products. (C) TLR4 expression in RAW-Blue cells and pM from TLR4-KO mice following subjection of cell lysates to Western blot analysis.

Article Snippet: 2.1 Chemicals and Materials RAW-Blue selection antibiotic Zeocin, Quanti-Blue detection reagent (alkaline phosphatase detection medium), LPS-EK from E. coli K12 (LPS-EK Ultrapure), and TLR4 signaling inhibitor CLI-095 were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Negative Control, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biochemical pharmacology

Article Title: Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

doi: 10.1016/j.bcp.2017.11.012

Figure Lengend Snippet: RAW-Blue cells were plated at 5 × 105 cells/well in 24-well plates. Cells were treated with oxidants or LPS-EK at the indicated concentrations for 2 h. Media was collected and cells rinsed in fresh media followed by re-incubation with fresh medium in the absence of oxidants or LPS-EK for the following 16 h. The cytotoxicity resulting from treatment with PPC, SIN-1 or LPS-EK at indicated concentrations were determined by quantification of LDH released into culture medium at the end of 2 h (A) and 16 h incubation (B). Cell viabilities upon different treatments were also determined by live cell counting with trypan blue staining at 2 h (C) and 16 h (D) for cell proliferation assay. Cell # was counted at the end of 16 h treatment. The white (blank) bars represent vehicle control, while the black bars represent the initial cell seeding number of 5 × 105 cells. The data represent 3 independent experiments conducted in duplicate at different times. # p ≤ 0.01 vs initial seeding number; * p ≤ 0.01 vs untreated cells. 1-way ANOVA followed Tukey’s post hoc tests.

Article Snippet: 2.1 Chemicals and Materials RAW-Blue selection antibiotic Zeocin, Quanti-Blue detection reagent (alkaline phosphatase detection medium), LPS-EK from E. coli K12 (LPS-EK Ultrapure), and TLR4 signaling inhibitor CLI-095 were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Incubation, Cell Counting, Staining, Proliferation Assay

Journal: Biochemical pharmacology

Article Title: Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

doi: 10.1016/j.bcp.2017.11.012

Figure Lengend Snippet: Cells were exposed to oxidants or LPS-EK at the indicated concentrations for 2 h followed by incubation with complete growth medium (oxidants or LPS-EK free) for next 16 h. Anti-TLR4/MD2 pAb (20 μg/ml) or CLI-095 (5 μM) was added 3 h before oxidants or LPS-EK stimulation. SEAP released in the culture medium at the end of 16 h incubation was determined using Quanti-Blue assay as per the manufacturer’s instructions, and the absorbance was read at 650 nm. (A) PPC concentration-dependent SEAP release. The data represent 6 independent experiments carried out in triplicate. *p ≤ 0.005 vs control; +p ≤ 0.05 vs control. (B) SIN-1 stimulated SEAP release in a dose-dependent manner. The data represent 6 independent experiments carried out in triplicate. +p ≤ 0.05 vs control, *p ≤ 0.005 vs control. (C) Pretreatment with anti-oxidant reagent EUK-134 (4 μM) inhibited PPC or SIN-1 induced SEAP release. The data represent 4 independent experiments carried out in triplicate. *p ≤ 0.005 vs treatment with PPC or SIN-1 alone. (D) Pretreatment with Anti-TLR4 pAb or CLI-095 reduced oxidants or LPS-EK induced SEAP release in RAW-Blue cells. The data represent 4 independent experiments carried out in triplicate. *p ≤ 0.05, **p ≤ 0.005 vs treatment with PPC alone; # p ≤ 0.05 vs SIN-1 treatment alone; +p ≤ 0.05 vs LPS-EK treatment alone, ++p ≤ 0.005 vs LPS-EK treatment alone, 1-way ANOVA in all cases followed by Tukey’s post-hoc tests.

Article Snippet: 2.1 Chemicals and Materials RAW-Blue selection antibiotic Zeocin, Quanti-Blue detection reagent (alkaline phosphatase detection medium), LPS-EK from E. coli K12 (LPS-EK Ultrapure), and TLR4 signaling inhibitor CLI-095 were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Incubation, Concentration Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations.

doi: 10.1016/j.biopha.2019.109684

Figure Lengend Snippet: Fig. 5. Increase in urinary transferrin excretion during the predisposition stage in different animal models: Representative Western blot images of urinary transferrin and the corre- sponding densitometry quantification. “Increase in urinary transferrin” reflects the difference between the selected time point and baseline values (i.e. day zero or week 11, de- pending on the model). Both time points pre- ceded the administration of the second (trig- gering) insult (i.e. subnephrotoxic gentamicin or CsA). Control animals received saline or vehicle. Values are expressed as the mean ± SEM. **, p < 0.01 vs. Control group. AU, arbitrary units; d, day; w, week; CP, cis- platin; CsA, cyclosporine; G, gentamicin; UN, uranyl nitrate.

Article Snippet: Immediately, proteins were electrically transferred to an Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with anti-transferrin antibody (sc-22597, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescent detection (Clarity Western ECL Substrate, Bio-Rad Laboratories, Hercules, CA, USA) with photographic films (Fujifilm, Tokyo, Japan).

Techniques: Western Blot, Control, Saline

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations.

doi: 10.1016/j.biopha.2019.109684

Figure Lengend Snippet: Fig. 6. Correlation between urinary transferrin excretion during the predisposition stage and the increase in plasma creatinine and urea during the AKI stage. AU: arbitrary units; R Pearson: Pearson's correlation coefficient.

Article Snippet: Immediately, proteins were electrically transferred to an Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with anti-transferrin antibody (sc-22597, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescent detection (Clarity Western ECL Substrate, Bio-Rad Laboratories, Hercules, CA, USA) with photographic films (Fujifilm, Tokyo, Japan).

Techniques: Clinical Proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations.

doi: 10.1016/j.biopha.2019.109684

Figure Lengend Snippet: Fig. 7. Representative images of Western blot analysis and the corresponding densitometry quantification of urinary transferrin excretion, a) during the phase of auto-perfusion with blood and during the Krebs-dextran perfusion phase; and b) before (-30 and -15 min) and after (15 min) administration of sodium maleate or saline vehicle. Values are expressed as the mean ± SEM. ***, p < 0.001 vs. 1. AU, arbitrary units.

Article Snippet: Immediately, proteins were electrically transferred to an Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with anti-transferrin antibody (sc-22597, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescent detection (Clarity Western ECL Substrate, Bio-Rad Laboratories, Hercules, CA, USA) with photographic films (Fujifilm, Tokyo, Japan).

Techniques: Western Blot, Saline

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations.

doi: 10.1016/j.biopha.2019.109684

Figure Lengend Snippet: Fig. 8. a) Basal and pre-injury urinary levels of transferrin in samples from Cases and Controls patients in a) oncology patients treated with platinated antineoplastics (Study 1); and b) basal urinary levels of transferrin in samples from Cases and Controls in cardiology patients subject to iodi- nated contrast media (Study 2). Representative images of Western blot analysis and its corresponding densitometry quantification; and evaluation of diagnostic performance of urinary transferrin through ROC curves and area under the curves values (95 % CI). Data are expressed as the mean ± SEM. AU: arbitrary units; AUC, area under the curve; B, basal; Cr, creatinine; PeI, pre-injury. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. Controls group.

Article Snippet: Immediately, proteins were electrically transferred to an Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with anti-transferrin antibody (sc-22597, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescent detection (Clarity Western ECL Substrate, Bio-Rad Laboratories, Hercules, CA, USA) with photographic films (Fujifilm, Tokyo, Japan).

Techniques: Western Blot, Diagnostic Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations.

doi: 10.1016/j.biopha.2019.109684

Figure Lengend Snippet: Fig. 9. Urinary levels of NAG and NGAL in basal samples from Case and Control patients, and evaluation of diagnostic performance of urinary transferrin through ROC curves in a) oncology patients treated with platinated antineoplastics (Study 1); and b) cardiology patients subject to iodinated contrast media (Study 2). Area under the curve (AUC) values (95 % CI). In left panels, data are expressed as the mean ± SEM. Cr, creatinine; NAG, N-acetyl-β-D-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin.

Article Snippet: Immediately, proteins were electrically transferred to an Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with anti-transferrin antibody (sc-22597, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescent detection (Clarity Western ECL Substrate, Bio-Rad Laboratories, Hercules, CA, USA) with photographic films (Fujifilm, Tokyo, Japan).

Techniques: Control, Diagnostic Assay

Journal: Journal of Translational Medicine

Article Title: Complement C3a promotes the formation of osteoclasts by inhibiting Sirt1 to activate the PI3K/PDK1/SGK3 pathway in patients with multiple myeloma

doi: 10.1186/s12967-025-06319-3

Figure Lengend Snippet: Sirt1 is involved in osteoclast formation in NDMM and is associated with C3a. A Bone marrow single nucleated cells from NDMM were collected by density gradient centrifugation. Recombinant human RANKL (150 ng/ml) recombinant human macrophage colony-stimulating factor M-CSF (50 ng/ml) was subsequently added, and mature adherent osteoclasts were obtained after 14 days. B We employed a mainstream hierarchical clustering approach to analyze the FPKM values of genes, applying normalization to the rows (genes) through Z-score standardization. In the heatmap, genes or samples with similar expression patterns are clustered together. The color in each cell represents the normalized value obtained after standardizing the rows of Sirt1-7 expression data, typically ranging from -3 to 3. C Sirt1 expression was decreased in the presence of C3a, (P = 0.0134) but not Sirt2-7. D The expression of Sirt1 measured by PCR of OCs in NDMM (0.3789 ± 0.2835) was significantly lower than HD (0.9509 ± 0.5485). (P < 0.0001, t = 4.577). E Sirt1 expression of OCs in NDMM was negatively correlated with serum levels of C3a. F Sirt1 expression of OCs in NDMM was negatively correlated with the number of OCPs and the osteolysis-related markers serum CTX/TRACP-5b. G Sirt1 expression of OCs in NDMM was negatively correlated with Osteoclast-related genes.The measures taken are from serum samples that are matched with the PCR results of Sirt1 gene expression. Results are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Isolated bone marrow mononuclear cells (BMMCs) or RAW264.7 cells were incubated in a-MEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in the presence of recombinant RANKL (PBMCs, recombinant human 150 ng/mL, Miltenyi Biotec, USA; RAW264.7 cell line, recombinant mouse 100 ng/mL, Miltenyi Biotec, USA), recombinant macrophage colony-stimulating factor (M-CSF; PBMCs, recombinant human 50 ng/mL, Miltenyi Biotec, USA; RAW264.7 cell line, recombinant mouse 50 ng/mL, Miltenyi Biotec, USA) and recombinant human/mouse complement C3a (Human complement C3a, Emd Millipore, Burlington, MA, USA; Mouse complement C3a, R&D System, USA) at 1 μg/mL, followed by seeding in a 24-well plate at 1 × 10 6 cells/well, according to a previously described protocol [ , , ].

Techniques: Gradient Centrifugation, Recombinant, Expressing, Gene Expression

Journal: Journal of Translational Medicine

Article Title: Complement C3a promotes the formation of osteoclasts by inhibiting Sirt1 to activate the PI3K/PDK1/SGK3 pathway in patients with multiple myeloma

doi: 10.1186/s12967-025-06319-3

Figure Lengend Snippet: Sirt1 inhibits the formation of osteoclasts, and complement C3a reverse this inhibitory function of Sirt1 to activate osteoclasts in NDMM patients. A SRT1720 led to a reduction in osteoclasts from NDMM, but this was reversed to some extent with the addition of C3a. Mean ± SEM for Control, SRT1720and SRT1720&C3a are (16.04 ± 0.7062), (8.381 ± 0.5928) and (12.64 ± 1.091) respectively. Control-SRT1720 (P < 0.0001, t = 8.310) Control-SRT1720&C3a (P = 0.0127, t = 2.615) SRT1720-SRT1720&C3a (P = 0.0015, t = 3.433). C In the presence of EX527, additional addition of C3a increased the area of osteoclast fusion from NDMM. Mean ± SEM for Control, EX527and EX527&C3a are (18.84 ± 0.1135), (27.13 ± 0.1.693) and (36.63 ± 2.179) respectively. Control-EX527 (P = 0.0006, t = 4.118) Control-EX527&C3a (P < 0.0001, t = 2.238) EX527-EX527&C3a (P = 0.0014, t = 3.443). B , D The same experiment was performed in osteoclasts from RAW264.7 cell. Mean ± SEM for Control, SRT1720and SRT1720&C3a are (16.86 ± 0.6166), (7.637 ± 0.8073) and (12.70 ± 0.5629) respectively. Control-SRT1720 (P = 0.0019, t = 7.290) Control-SRT1720&C3a (P = 0.0216, t = 3.659) SRT1720-SRT1720&C3a (P = 0.0102, t = 4,579). Mean ± SEM for Control, EX527 and EX527&C3a are (15.65 ± 0.5771), (30.27 ± 3.614) and (48.19 ± 2.692) respectively. Control-EX527 (P = 0.0187, t = 3.823) Contral-EX527&C3a (P = 0.0003, t = 11.82) EX527-EX527&C3a (P = 0.0148, t = 4.104). E , G The relative expressions of mRNAs of genes TRAP/RANKL/OSCAR/cathepsin K in osteoclasts from NDMM were performed by PCR. SRT1720 (0.8135 ± 0.1826) (1.050 ± 0.2876) (1.437 ± 0.3486) (2.233 ± 0.5925) -Control (1.861 ± 0.2466) (2.749 ± 0.6223) (4.828 ± 0.8233) (5.747 ± 1.154) (P = 0.0016, P = 0.0177, P = 0.0005, P = 0.0101, respectively) SRT1720&C3a (1.761 ± 0.2467) (2.767 ± 0.4317) (4.261 ± 0.8570) (5.802 ± 0.9964) -SRT1720 (P = 0.0038, P = 0.0020, P = 0.0041, P = 0.0038, respectively) SRT1720&C3a- Control (P = 0.7743, P = 0.9809, P = 0.6366, P = 0.9713, respectively). EX527 (1.557 ± 0.1260) (1.977 ± 0.1731) (1.656 ± 0.1077) (1.451 ± 0.0541) -Control(0.9855 ± 0.0152) (0.8966 ± 0.0200) (0.9398 ± 0.0249) (0.9394 ± 0.0087) (P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, respectively) EX527&C3a (2.830 ± 0.4010) (3.094 ± 0.4052) (2.109 ± 0.1475) (1.778 ± 0.1650) -EX527 (P = 0.0026, P = 0.0124, P = 0.0227, P = 0.0770, respectively) EX527&C3a-Control (P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, respectively). F , H The same experiment was performed in osteoclasts from RAW264.7cell SRT1720 (0.6750 ± 0.2504) (0.6579 ± 0.2532) (1.887 ± 0.1129) (2.173 ± 0.7950) -Control (2.812 ± 0.3697) (2.716 ± 0.3641) (7.629 ± 1.172) (10.27 ± 2.651) (P = 0.0087, P = 0.0097, P = 0.0082, P = 0.0430, respectively) SRT1720&C3a (2.921 ± 0.0903) (2.651 ± 0.4693) (5.480 ± 0.8143) (9.843 ± 1.028) -SRT1720 (P = 0.0011, P = 0.0200, P = 0.0120, P = 0.0041, respectively) SRT1720&C3a-Control (P = 0.7880, P = 0.9183, P = 0.2065, P = 0.8882, respectively). EX527 (1.371 ± 0.0402) (1.415 ± 0.2014) (0.5591 ± 0.1881) (1.070 ± 0.0298) -Control (0.9202 ± 0.0927) (1.040 ± 0.2130) (0.4765 ± 0.1351) (0.7507 ± 0.3158) (P = 0.0111, P = 0.2693, P = 0.7391, P = 0.0018, respectively) EX527&C3a (1.294 ± 0.0509) (1.661 ± 0.0933) (1.764 ± 0.3600) (1.599 ± 0.0940) -EX527 (P = 0.2989, P = 0.3310, P = 0.0413, P = 0.0038, respectively). Results are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Isolated bone marrow mononuclear cells (BMMCs) or RAW264.7 cells were incubated in a-MEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in the presence of recombinant RANKL (PBMCs, recombinant human 150 ng/mL, Miltenyi Biotec, USA; RAW264.7 cell line, recombinant mouse 100 ng/mL, Miltenyi Biotec, USA), recombinant macrophage colony-stimulating factor (M-CSF; PBMCs, recombinant human 50 ng/mL, Miltenyi Biotec, USA; RAW264.7 cell line, recombinant mouse 50 ng/mL, Miltenyi Biotec, USA) and recombinant human/mouse complement C3a (Human complement C3a, Emd Millipore, Burlington, MA, USA; Mouse complement C3a, R&D System, USA) at 1 μg/mL, followed by seeding in a 24-well plate at 1 × 10 6 cells/well, according to a previously described protocol [ , , ].

Techniques: Control

Journal: PLoS Pathogens

Article Title: Toxoplasma GRA15 and GRA24 are important activators of the host innate immune response in the absence of TLR11

doi: 10.1371/journal.ppat.1008586

Figure Lengend Snippet: THP1 monocyte-derived macrophages were infected with indicated Toxoplasma strains for 24 h, after which IL12/IL23p40 ( a ), IL1β ( b ) and TNFα were measured. Relative parasite growth was measured by luciferase growth assay ( d ). Nuclear translocation of the NFκB p65 ( e ), p-p38 MAPK ( f ), and NFκB cREL ( g ) subunits were quantified from infected THP1 macrophages 18 h p.i with indicated strains. In each experiment at least 15 cells were quantified. THP1 macrophages were treated with indicated inhibitors 2 h prior to infection and subsequently infected for an additional 20 h. IL12/IL23p40 ( h ), IL1β ( i ) and growth ( j ) were measured. Each dot represents a technical mean value from a single experiment, and each experiment was done 3 times. Statistical analysis was done by One-way ANOVA followed with Tukey’s multiple comparison test. Data are represented as mean ± standard error of the mean (SEM).

Article Snippet: Immunofluorescence to detect nuclear translocation of p65 (NFκB), p-p38 MAPK and c-REL was done in HFFs, MEFs, RAW 264.7 macrophages and THP1-derived macrophages using the following antibodies: rabbit anti p65 (1:200 dilution, sc-109, Santacruz Biotechnology, CA, USA), rabbit anti p-p38 MAPK (1:800 dilution, #4511, Cell Signaling Technology, MA, USA), rabbit anti cREL (1:500 dilution, #4727, Cell Signaling Technology, MA, USA) and mouse anti cREL (1:200 dilution, NBP2-37593, Novus Biologicals, CO, USA for RAW 264.7 macrophages).

Techniques: Derivative Assay, Infection, Luciferase, Growth Assay, Translocation Assay

Journal: PLoS Pathogens

Article Title: Toxoplasma GRA15 and GRA24 are important activators of the host innate immune response in the absence of TLR11

doi: 10.1371/journal.ppat.1008586

Figure Lengend Snippet: BMDMs were infected with indicated Toxoplasma strains for 24 h and IL12/IL23p40 ( a ), IL1β ( b ) and TNFα ( c ) were measured in the supernatant. Experiments were done 3 times. Arginase activity was measured ( d ) from RAW 264.7 macrophages infected with indicated strains 24 h p.i. Nuclear translocation of the cREL subunit of NFκB ( e ) was quantified from infected RAW 264.7 macrophages 18 h p.i. with indicated strains. In each experiment at least 15 cells were quantified as shown in the graph (left) and experiment was done 3 times (right panel graph). A representative image for each group is shown in the middle panel. Scale bar is 10 μm. Each dot represents the mean of 3 technical replicates from individual experiments, except for the scatter diagram in (e). Statistical analysis was done by two sample student’s t test for figures a-e. Data are represented as mean ± standard error of the mean (SEM).

Article Snippet: Immunofluorescence to detect nuclear translocation of p65 (NFκB), p-p38 MAPK and c-REL was done in HFFs, MEFs, RAW 264.7 macrophages and THP1-derived macrophages using the following antibodies: rabbit anti p65 (1:200 dilution, sc-109, Santacruz Biotechnology, CA, USA), rabbit anti p-p38 MAPK (1:800 dilution, #4511, Cell Signaling Technology, MA, USA), rabbit anti cREL (1:500 dilution, #4727, Cell Signaling Technology, MA, USA) and mouse anti cREL (1:200 dilution, NBP2-37593, Novus Biologicals, CO, USA for RAW 264.7 macrophages).

Techniques: Infection, Activity Assay, Translocation Assay