Journal: Frontiers in pharmacology

Article Title: Bilirubin Improves Gap Junction to Alleviate Doxorubicin-Induced Cardiotoxicity by Regulating AMPK-Axl-SOCS3-Cx43 Axis.

doi: 10.3389/fphar.2022.828890

Figure Lengend Snippet: FIGURE 3 | Bilirubin reduces doxorubicin-induced JNK-Cx43 gap junctions’ dysfunction. (A,C) Representative Western blot images showed the levels of p-Cx43, total Cx43, p-JNK and JNK in H9C2 cells in vitro. (B,D) Representative Western blot images showed the levels of p-Cx43, total Cx43, p-JNK and JNK in heart tissues from doxorubicin-mice given different dose of bilirubin (7.5, 15, and 30 mg/kg, i.p.) for 7 days. The western blot samples were collected and analyzed (n = 4 of each group). (E) Apoptosis was assessed by flow cytometric analysis in H9C2 cells. (F) Quantitative data of the apoptosis ratio analyzed by flow cytometry. H9C2 cells were treated with bilirubin (0.1 μM) for 6 h before doxorubicin (1 μM) treatment, and the samples were collected 24 h after doxorubicin treatment (n = 4 of each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Doxorubicin- treated group; Bonferroni post hoc tests).

Article Snippet: Antibodies for phosphorylated c-Jun N-terminal kinase (JNK; Thr183/ Tyr185; Catalog No. 9255), JNK (Catalog No. 9252), Cx43 (Catalog No. 3512); phosphorylated Cx43 (Ser368; Catalog No. 3511) phosphorylated AMPK (Thr172; Catalog No. 2531), AMPK (Catalog No. 2532) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Western Blot, In Vitro, Cytometry, Control

Journal: Frontiers in pharmacology

Article Title: Bilirubin Improves Gap Junction to Alleviate Doxorubicin-Induced Cardiotoxicity by Regulating AMPK-Axl-SOCS3-Cx43 Axis.

doi: 10.3389/fphar.2022.828890

Figure Lengend Snippet: FIGURE 5 | Bilirubin protects against Doxorubicin-induced cardiotoxicity in an AMPK-SOCS3 dependent manner in H9C2 cells. (A) RTCA (xCELLigence platform) was used to record the cell growth curves in H9C2 cells. (B–E) Representative western blot bands showed the levels of p-AMPK, AMPK, SOCS3, p-JNK, JNK, p-Cx43, and total Cx43 in H9C2 cells in vitro. H9C2 cells were pretreated with Compound C (2 μM) for half an hour before bilirubin treatment, and then cells were cultured with bilirubin (0.1 μM), AICAR (20 μM) and metformin (1 mM) for 6 h, followed by doxorubicin treatment (1 μM) for another 24 h. The western blot samples were collected and analyzed (n = 4 of each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, ***p < 0.001 vs. Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Doxorubicin-treated group; $p < 0.05 vs. Doxorubicin and bilirubin-treated group; Bonferroni post hoc tests).

Article Snippet: Antibodies for phosphorylated c-Jun N-terminal kinase (JNK; Thr183/ Tyr185; Catalog No. 9255), JNK (Catalog No. 9252), Cx43 (Catalog No. 3512); phosphorylated Cx43 (Ser368; Catalog No. 3511) phosphorylated AMPK (Thr172; Catalog No. 2531), AMPK (Catalog No. 2532) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Western Blot, In Vitro, Cell Culture, Control

Journal: Frontiers in pharmacology

Article Title: Bilirubin Improves Gap Junction to Alleviate Doxorubicin-Induced Cardiotoxicity by Regulating AMPK-Axl-SOCS3-Cx43 Axis.

doi: 10.3389/fphar.2022.828890

Figure Lengend Snippet: FIGURE 6 | Bilirubin induces SOCS3 expression via activating Axl receptor in H9C2 cells. (A–E) Representative western blot bands showed the levels of p-AMPK, AMPK, Axl, SOCS3, p-JNK, JNK, p-Cx43, and total Cx43 in H9C2 cells in vitro. H9C2 cells were pretreated with R428 (1 μM) for half an hour before bilirubin treatment, and then cells were cultured with bilirubin (0.1 μM) for 6 h, followed by doxorubicin treatment (1 μM) for another 24 h. The western blot samples were collected and analyzed (n = 4 of each group). (F) Representative western blot bands showed the levels of Axl in H9C2 cells. (G) The efficiency of Axl knockdown was assessed by western blot assay. (H–J) Representative western blot bands showed the levels of SOCS3, p-JNK, JNK, p-Cx43, and total Cx43 in H9C2 cells. H9C2 cells were transfected with 100 pM Axl siRNA or control siRNA for 24 h, and then subjected to bilirubin (0.1 μM) for 6 h, followed by exposure to doxorubicin (1 μM) for another 24 h. The western blot samples were collected and analyzed (n ≥3 each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Doxorubicin-treated group; $p < 0.05, $$p < 0.01, $$$p < 0.001 vs. Doxorubicin and bilirubin- treated group; Bonferroni post hoc tests).

Article Snippet: Antibodies for phosphorylated c-Jun N-terminal kinase (JNK; Thr183/ Tyr185; Catalog No. 9255), JNK (Catalog No. 9252), Cx43 (Catalog No. 3512); phosphorylated Cx43 (Ser368; Catalog No. 3511) phosphorylated AMPK (Thr172; Catalog No. 2531), AMPK (Catalog No. 2532) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Expressing, Western Blot, In Vitro, Cell Culture, Knockdown, Transfection, Control

Journal: Frontiers in pharmacology

Article Title: Bilirubin Improves Gap Junction to Alleviate Doxorubicin-Induced Cardiotoxicity by Regulating AMPK-Axl-SOCS3-Cx43 Axis.

doi: 10.3389/fphar.2022.828890

Figure Lengend Snippet: FIGURE 7 | Schematic indicating that moderate increase bilirubin can inhibit doxorubicin-induced gap junction’ dysfunction and conduction abnormalities via regulating AMPK-Axl-SOCS3 signaling axis. Doxorubicin significantly increases the phosphorylation of JNK and Cx43 in mouse cardiomyocytes, leading to gap junction’ dysfunction and conduction abnormalities and causing cardiotoxicity. Bilirubin induces SOCS3 expression in an AMPK-Axl-dependent manner, inhibiting JNK-Cx43 signaling pathway to improve gap junction’ function and conduction abnormalities.

Article Snippet: Antibodies for phosphorylated c-Jun N-terminal kinase (JNK; Thr183/ Tyr185; Catalog No. 9255), JNK (Catalog No. 9252), Cx43 (Catalog No. 3512); phosphorylated Cx43 (Ser368; Catalog No. 3511) phosphorylated AMPK (Thr172; Catalog No. 2531), AMPK (Catalog No. 2532) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Phospho-proteomics, Expressing

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 2. Inhibition of junctional communication by Cx43 siRNA and pharmacological inhibitor TPA. (A) Western blotting probed with anti-Cx43 antibody shows the decrease of the Cx43 level in siRNA-transfected cells. (B and C) Cx43-siRNA and TPA reduce dye coupling through gap junction as measured by parachute dye-coupling assay. Columns, mean for five experiments; bars, SEM. **, Significantly different from control, P<0.01.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Inhibition, Western Blot, Transfection, Control

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 3. Simvastatin attenuates cisplatin toxicity at high cell density and this effect was blocked by Cx43-siRNA. (A) Clonogenic survival of cells pretreated with 10 µM simvastatin for 3 h, and co-incubated with 5 µM cisplatin for 1 h at high and low cell density. (B) Clonogenic survival of cells pretreated with a range of concentrations of simvastatin for the indicated periods, followed by co-incubation with 5 µM cisplatin for 1 h at high cell density. (C) Clonogenic survival of siRNA-transfected cells pretreated with 10 µM simvastatin for 3 h, and co-incubated with 5 µM cisplatin for 1 h at high and low cell density. Columns, mean for five experiments; bars, SEM. *, Significantly different from control, P<0.05; #, significantly different from the cisplatin bar, P<0.05.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Incubation, Transfection, Control

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 4. Effects of simvastatin on gap junction intercellular communication, Cx43 immunostaining. (A) The dye spread of cells exposed to increasing concentrations of simvastatin for 4 h as assayed by parachute dye-coupling assay. (B) Immunostaining for Cx43 in cells with or without 10 µM simvastatin treatment. Magnification, x400.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Immunostaining

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 5. Effects of simvastatin on Cx43 expression and p-Cx43 (ser368) expression. (A) Western blot analysis of Cx43 expression in cells treated with varying concentrations of simvastatin. (B) Western blotting showing p-Cx43 (ser368) levels in cells following treatment with 10 µM simvastatin for varying time periods. Bar graphs are derived from the densitometric scanning of the blots and shows a comparison of the level of Cx to β-tubulin density ratio. Columns, mean from four experiments; bars, SEM. *, Significantly different from control.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Western Blot, Derivative Assay, Comparison, Control

Journal: EBioMedicine

Article Title: Circulating exosomes and gut microbiome induced insulin resistance in mice exposed to intermittent hypoxia: Effects of physical activity

doi: 10.1016/j.ebiom.2021.103208

Figure Lengend Snippet: Effects on insulin sensitivity on naïve adipocytes by plasma exosomes derived from mice exposed to IH or RA for 6 weeks with and without PA. (a) Representative Western blots of pAKT and tAKT after treatment with plasma exosomes followed by 5 nM of exogenous insulin for 30 min. (b) Mean pAKT/tAKT ratios, n = 8 per experimental conditions. (c) Effects of plasma exosomes on CD11c expression by naïve macrophage (RAW 264.7) cells using flow cytometry. An equal number of exosomes (20 × 10 6) ) was used from each condition. * Indicates statistical significance of IH vs. IH+PA, p < 0.05, n = 8/condition. Two-way of variance (ANOVA) for non-repeated measures was used. NS indicates not significant.

Article Snippet: The mouse macrophage cell line RAW cells, RAW 264.7, (ATCC® TIB-7) (40 × 10 4 ) was seeded in 24-well plates in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% 100 mM sodium pyruvate, 1% of 10,000 U/mL penicillin and 10,000 μg/mL streptomycin and maintained in 37 °C humidified incubator containing 5% CO 2 .

Techniques: Clinical Proteomics, Derivative Assay, Western Blot, Expressing, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure

doi: 10.4049/jimmunol.1002060

Figure Lengend Snippet: A and B) Kupffer cells isolated from pair- and ethanol-fed rats were cultured with 1μg/ml globular adiponectin (gAcrp) for 18h. Cells were then stimulated with 100 ng/ml LPS for 4h. The expression of IFN-β and CXCL10 mRNA was normalized to 18S mRNA. Values are expressed relative to Kupffer cells from pair-fed rats not treated with gAcrp. n = 4, * p < 0.05 ethanol-fed compared with pair-fed, +p < 0.05 compared with LPS-stimulated cells not treated with gAcrp. C-F) RAW 264.7 cells were cultured for 18h in the absence or presence of increasing doses of either gAcrp (0–1 μg/ml) or full-length adiponectin (flAcrp, 0–10 μg/ml). Cells were then stimulated with 100 ng/ml LPS for 4h and expression of IFN-β and CXCL10 mRNA was normalized to 18S mRNA. n = 4, + p < 0.05 compared with LPS-stimulated cells not treated with adiponectin. G and H) RAW 264.7 cells were cultured for 18h in the absence or presence of 10 μ/ml gAcrp or full-length adiponectin. Cells were then stimulated with 100 ng/ml LPS for 8h and the concentration of CXCL10 protein accumulated in the media was measured by ELISA. n = 6, + p < 0.05 compared with LPS-stimulated cells not treated with adiponectin.

Article Snippet: Mouse RAW 264.7 cell nucleofection kit was purchased from Lonza (Cologne, Germany).

Techniques: Isolation, Cell Culture, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure

doi: 10.4049/jimmunol.1002060

Figure Lengend Snippet: RAW 264.7 cells were transfected or not with 100 nM of AdipoR1 siRNA, AdipoR2 siRNA or scrambled siRNA and then cultured with or without 1 μg/ml gAcrp or 10 μg/ml flAcrp for 18h. Cells were then stimulated with LPS for 4h. IFN-β and CXCL10 mRNA was normalized to 18S mRNA. n=4, Values with different superscripts are significantly different from each other, p < 0.05.

Article Snippet: Mouse RAW 264.7 cell nucleofection kit was purchased from Lonza (Cologne, Germany).

Techniques: Transfection, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure

doi: 10.4049/jimmunol.1002060

Figure Lengend Snippet: A and B) Kupffer cells from pair- and ethanol-fed rats were isolated and cultured overnight with 0–1000 ng/ml gAcrp for 18 h. Expression of TLR4 and CD14 mRNA was normalized to 18S mRNA. Values are expressed as fold increases relative to Kupffer cells from pair-fed rats not treated with gAcrp. n = 4, * p < 0.05 ethanol-fed compared to pair-fed, +p < 0.05 compared with cells not treated with gAcrp. C) TLR4 promoter activity was measured in RAW 264.7 macrophages transiently transfected with a full-length TLR4 promoter-luciferase reporter construct and a Renilla luciferase control plasmid. After 24 h, cells were treated with or without 1 μg/ml gAcrp for 18 h. Values represent relative luciferase activity. n = 4; + p < 0.05. D) RAW 264.7 macrophages were treated without or with 1 μg/ml gAcrp for 18h and expression of TLR4 mRNA measured and normalized to 18S mRNA. n=4, + p <.05 compared with cells not treated with adiponectin. E) RAW 264.7 macrophages were cultured with or without 1 μg/ml gAcrp for 18h and cell surface expression of TLR4-MD2 (solid line), relative to isotype controls (dotted line), was measured by flow cytometry. Data are representative of four experiments.

Article Snippet: Mouse RAW 264.7 cell nucleofection kit was purchased from Lonza (Cologne, Germany).

Techniques: Isolation, Cell Culture, Expressing, Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure

doi: 10.4049/jimmunol.1002060

Figure Lengend Snippet: A and B) RAW 264.7 macrophages were treated with or without 1 μg/ml gAcrp for 18h. HO-1 mRNA expression was normalized to 18S mRNA. B) HO-1 protein expression was measured by Western blot and normalized to total ERK1/2. n=4, * p < 0.05 compared with cells not treated with adiponectin C-F) RAW 264.7 macrophages were transfected with an empty vector or an HO-1 overexpression plasmid. Twenty four hour after transfection, cells were treated or not with 1 μg/ml gAcrp for 18h. C) TLR4 mRNA was measured and normalized to 18S mRNA. n=4, * p < 0.05 compared to non-treated cells. D and E) Transfected cells were then stimulated with LPS for 4h. IFN-β (D) and CXCL10 (E) mRNA were measured normalized to 18S mRNA. n=4, * p < 0.05 compared with LPS-stimulated cells not treated with gAcrp or HO-1 overexpression. F) Transfected cells were then stimulated with LPS for 2h and Western blot analysis was performed using antibodies to phosphorylated STAT1 and Hsc70 (loading control). Images are representative of four experiments.

Article Snippet: Mouse RAW 264.7 cell nucleofection kit was purchased from Lonza (Cologne, Germany).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Over Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure

doi: 10.4049/jimmunol.1002060

Figure Lengend Snippet: A) RAW 264.7 cells were transfected or not with 100 nM of HO-1 siRNA or scrambled siRNA and then cultured with or without 1 μg/ml gAcrp for 18h. Cells were then stimulated with LPS for 4h. IFN-β and CXCL10 mRNA was normalized to 18S mRNA. n=4, Values with different superscripts are significantly different from each other, p < 0.05. B) RAW264.7 cells were cultured with or without 0.5 μM ZnPP in the presence and absence of 1 μg/ml gAcrp for 18h. Cells were then stimulated with LPS for 4h. IFN-β and CXCL10 mRNA was normalized to 18S mRNA. n=4, Values with different superscripts are significantly different from each other, p < 0.05.

Article Snippet: Mouse RAW 264.7 cell nucleofection kit was purchased from Lonza (Cologne, Germany).

Techniques: Transfection, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure

doi: 10.4049/jimmunol.1002060

Figure Lengend Snippet: A and B) RAW 264.7 cells were cultured for 18h in the absence or presence of either gAcrp (1 μg/ml), CoPP (10μM) or CORM2 (100 μM). Cells were then stimulated without or with 100 ng/ml LPS for 4h and expression of IFN-β and CXCL10 mRNA normalized to 18S mRNA. n = 4, +p < 0.05 compared with LPS-stimulated cells not treated with inducers of HO-1. C and D) Kupffer cells isolated from pair- and ethanol-fed rats were cultured with 10μM CoPP for 18h. Cells were then stimulated with 100 ng/ml LPS for 4h. Expression of IFN-β and CXCL10 mRNA was normalized to 18S mRNA. Values are expressed relative to Kupffer cells from pair-fed rats not treated with gAcrp. n = 4, * p < 0.05 ethanol-fed compared with pair-fed, +p < 0.05 compared with LPS-stimulated cells not treated with CoPP.

Article Snippet: Mouse RAW 264.7 cell nucleofection kit was purchased from Lonza (Cologne, Germany).

Techniques: Cell Culture, Expressing, Isolation

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: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: (A) Total Na + content of RAW264.7 MΦs ± 10 ng/ml LPS under NS or HS (NS + 40 mM NaCl) conditions (mean ± SD; n = 11–12; Student t test ± Welch correction; * p < 0.05). (B) Relative [Na + ] i of RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated with HS, LPS, or both at t = 10 s (mean ± SD; n = 7–9). (C) Relative [Na + ] i of RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated with HS or 80 mM urea at t = 10 s (mean ± SD; n = 5). For numerical raw data, please see . HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; [Na + ] i , intracellular Na + in situ; NS, normal salt.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: In Situ

Journal: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: Lower panel: nitrite levels of RAW264.7 MΦs pretreated with indicated inhibitors and stimulated ± LPS ± HS or 80 mM urea (mean ± SD; n = 9; Student t test or Mann–Whitney test; * p < 0.05). Upper panel: changes of means in nitrite production upon indicated stimulation and/or treatment (Δ nitrite). For numerical raw data, please see . HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; NCX, Na + /Ca 2+ exchanger; NO, nitric oxide; NS, normal salt; n.s., not significant.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: MANN-WHITNEY

Journal: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: (A) A representative real-time amplification plot of Slc8a1 , Slc8a2 , and Slc8a3 in BMDMs, RAW264.7 MΦs, and brain tissue out of two experiments. (B) Shown are the alignments of RNA-seq reads of BMDMs and their coverage with annotated (gray; GENCODE: ENSMUST00000086538.9, ENSMUST00000163123.1, ENSMUST00000163680.8), predicted (blue; RefSeq PREDICTED: XM_006523944), and StringTie-assembled (red; StringTie Assembly version 1, StringTie Assembly version 2) Slc8a1 splice variants. For numerical raw data, please see . The RNA-seq data can be found in the GEO database ( https://www.ncbi.nlm.nih.gov ) under accession number GSE136662. BMDM, bone marrow–derived MΦ; GEO, Gene Expression Omnibus; MΦ, monocyte/macrophage-like cell; RNA-seq, RNA sequencing; Slc8 , solute carrier family 8.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: Amplification, RNA Sequencing Assay, Derivative Assay, Expressing

Journal: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: (A) Relative [Ca 2+ ] i levels of RAW264.7 MΦs. Traces of Fura-2–loaded RAW264.7 MΦs stimulated ± HS at t = 10 s (mean ± SD; n = 6). Where indicated, Tg was added (means ± SD; n = 2). (B) As in (A), but RAW264.7 MΦs were stimulated with LPS ± HS at t = 10 s (mean ± SD; n = 5). Where indicated, Tg was added (means ± SD; n = 2). (C) As in (A), but relative [Ca 2+ ] i levels were assessed in Fluo-3/Fura-Red–loaded MΦs (means ± SD; n = 6). (D) As in (B), but relative [Ca 2+ ] i levels were assessed in Fluo-3/Fura-Red–loaded MΦs (means ± SD; n = 8). For numerical raw data, please see . [Ca2+] i , intracellular Ca 2+ in situ; HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; Tg, thapsigargin.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: In Situ

Journal: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: (A) Relative [Na + ] i levels in RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated ± HS (at t = 10 s) ± KB-R pretreatment (means ± SD; n = 4–8). (B) Relative [Na + ] i levels in RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated with LPS ± HS (at t = 10 s) ± KB-R pretreatment (means ± SD; n = 5–8). (C) As in (A), but relative [Ca 2+ ] i levels were assessed in Fura-2–loaded MΦs (means ± SD; n = 5–8). (D) As in (B), but relative [Ca 2+ ] i levels were assessed in Fura-2–loaded MΦs (means ± SD; n = 6–8). For numerical raw data, please see . [Ca 2+ ] i , intracellular Ca 2+ in situ; HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; KB-R, KB-R7943 mesylate; [Na + ] i , intracellular Na + in situ; NCX, Na + /Ca 2+ exchanger.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: In Situ

Journal: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: (A) Immunoblotting and densitometry of NFAT5 6 h after LPS ± HS in RAW264.7 MΦs ± KB-R pretreatment ( n = 3; paired t test; * p < 0.05). (B) Nos2 levels in RAW264.7 MΦs 4 h after LPS ± HS ± KB-R pretreatment (means ± SD; n = 10; Student t test; * p < 0.05). (C) Immunoblotting and densitometry of NFAT5 in RAW264.7 MΦs 4 h after LPS ± HS ± NiCl 2 pretreatment ( n = 4; paired t tests; * p < 0.05). (D) Nos2 levels in RAW264.7 MΦs 4 h after LPS ± HS ± NiCl 2 pretreatment (means ± SD; n = 6; Student t test + Welch correction; * p < 0.05). (E) Immunoblotting and densitometry of NFAT5 in RAW264.7 MΦs 4 h after LPS ± HS ± SEA pretreatment ( n = 5; paired t test; * p < 0.05). (F) Nos2 in RAW264.7 MΦs 4 h after LPS ± HS ± SEA pretreatment (means ± SD; n = 10–12; Mann–Whitney test; * p < 0.05). (G) RFP-GFP-mLC3 RAW264.7 MΦs were infected with Escherichia coli ± HS ± SEA pretreatment. Representative images 2 h after infection (RFP: red; GFP: green; scale bar: 10 μm). (H) Relative E . coli load at 2 h after infection of RAW264.7 MΦs ± HS ± SEA pretreatment (means ± SD; n = 12; Student t tests; * p < 0.05). For numerical raw data, please see . For raw immunoblots, please see . CFU, colony forming unit; GFP, green fluorescent protein; HS, high salt; KB-R, KB-R7943 mesylate; LPS, lipopolysaccharide; mLC3, microtubule-associated protein 1 light chain 3; MΦ, monocyte/macrophage-like cell; NCX, Na + /Ca 2+ exchanger; NFAT5, nuclear factor of activated T cells 5; Nos2 , nitric oxide synthase 2; NS, normal salt; n.s., not significant; RFP, red fluorescent protein; SEA, SEA 0400.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: Western Blot, MANN-WHITNEY, Infection

Journal: PLoS Biology

Article Title: NCX1 represents an ionic Na + sensing mechanism in macrophages

doi: 10.1371/journal.pbio.3000722

Figure Lengend Snippet: (A) ns siRNA and Slc8a1 siRNA RAW264.7 were treated with LPS ± HS. Representative NFAT5 and ACTIN immunoblot 4 h after stimulation from two experiments. (B) As in (A), but Nos2 in BMDMs 4 h after LPS ± HS (means ± SD; n = 6; Mann–Whitney test; * p < 0.05). (C) As in (B), but Δ nitrite HS LPS-LPS after 24 h (means ± SD; n = 8; Student t test). (D) ns siRNA and Slc8a1 siRNA–treated RFP-GFP-mLC3 RAW264.7 MΦs were infected with E . coli ± HS. Representative images 2 h after infection from three experiments (RFP: red; GFP: green; scale bar: 10 μm). (E) ns siRNA and Slc8a1 siRNA–treated BMDMs were infected with E . coli ± HS. E . coli load at 2 h infection (means ± SD; n = 12; Student t tests; * p < 0.05). For numerical raw data, please see . For raw immunoblots, please see . BMDM, bone marrow–derived MΦ; CFU, colony forming unit; GFP, green fluorescent protein; HS, high salt; LPS, lipopolysaccharide; mLC3, microtubule-associated protein 1 light chain 3; MΦ, monocyte/macrophage-like cell; NFAT5, nuclear factor of activated T cells 5; Nos2 , nitric oxide synthase 2; NS, normal salt; ns, nonsilencing; n.s., not significant; RFP, red fluorescent protein; siRNA, small interfering RNA; Slc8 , solute carrier family 8.

Article Snippet: We seeded RFP-GFP-mLC3 RAW264.7 MΦs (obtained from Invivogen; rawdf-mlc3) on coverslips and infected them with MOI 100 of E . coli HB101 ± HS ± 30 min preincubation with SEA0400.

Techniques: Western Blot, MANN-WHITNEY, Infection, Derivative Assay, Small Interfering RNA