Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Sulfur compounds inhibit LPS-induced inflammation and iron/heme metabolism. (A) Flow cytometry showing Fe2+ levels in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. Western blot analysis of (B) transferrin receptor, ferroportin, DMT1 and STEAP3; and (C) MFRN, ABCB6, ABCB10, ALAS1, HO-1, FECH and FLVCR proteins in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ALAS1, 5′-aminolevulinate synthase 1; DMT1, divalent metal transporter 1; Fe2+, ferrous ion; FECH, ferrochelatase; FPN, ferroportin; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; MFRN, mitoferrin; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TfR, transferrin receptor; TLR, Toll-like receptor.

Article Snippet: The following primary antibodies were purchased from Santa Cruz Biotechnology, Inc.: Ferrochelatase (FECH; cat. no. sc-377377), phosphorylated (p)-IκBα (cat. no. sc-8404), TLR2 (cat. no. sc-21759), TLR4 (cat. no. sc-293072), COX-1 (cat. no. sc-19998), COX-2 (cat. no. sc-19999, IKKα/β (cat. no. sc-7607), and β-actin (cat. no. sc-47778); from Cell Signaling Technology, Inc.: IL-1β (cat. no. 12703), p-ERK (cat. no. 9101), ERK (cat. no. 9102), IκBα (cat. no. 9242), p-IKKα/β (cat. no. 2697), NF-κB (cat. no. 8242), p-p38 (cat. no. 4511), p38 (cat. no. 8690), ATR (cat. no. 2790), ATM (cat. no. 2873), Chk2 (cat. no. 2662), BRCA1 (cat. no. 9010), p53 (cat. no. 9282), p-MDM2 (cat. no. 3521), MDM2 (cat. no. 86934) and the DNA Damage Antibody Sampler Kit (cat. no. 9947; containing p-ATM, p-ATR, p-Chk2, p-BRCA1 and p-p53 antibodies); from Abcam: Divalent metal transporter (DMT)1 (cat. no. ab55735), 5′-aminolevulinate synthase (ALAS)1 (cat. no. ab84962), STEAP3 (cat. no. ab151566), HO-1 (cat. no. ab137749), transferrin receptor (TfR; cat. no. ab84036), PKCα (cat. no. ab179523), p-PKCα (cat. no. ab59411), TNF-α (cat. no. ab183218), IL-6 (cat. no. ab6672) and TATA-binding protein (TBP; cat. no. ab818); from LifeSpan BioSciences, Inc.: ABCB10 (cat. no. LS-C381841) and FLVCR1 (cat. no. LS-C750126); from LS Bio; Vector Laboratories, Inc.: FPN (cat. no. NBP1-21502) and iNOS (cat. no. NB300-605); from Novus Biologicals; Bio-Techne: Mitoferrin (MFRN; cat. no. MBS6013473); and from Boster Biological Technology: ABCB6 (cat. no. PA1723).

Techniques: Flow Cytometry, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 4. Expression of the 3GalT4 gene in TGF--, CoCl2-, and hypoxia-treated cells. RNAs were prepared from control, TGF-, CoCl2-, or hypoxia-treated cells, as described in Mate- rials and Methods. cDNA was prepared using SuperScript III First-Strand Super Mix and digested with DNase, and quanti- tative real-time PCR was performed as described in Materials and Methods. Data were analyzed using the 2 Ct method as described previously (44) and represent fold change in gene expression relative to the nontreated control. *P 0.05; **P 0.01.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Gene Expression

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 5. Inhibition of CoCl2-induced EMT by overexpressed 3GalT4 or exogenous Gg4. To study the effect of ganglioside GM1 or Gg4 on the CoCl2-induced EMT process, cells were preincubated with 50 M GM1 or Gg4 for 24 h and then were cultured in medium with 100 M CoCl2 in the continued presence of 50 M Gg4 or GM2 for 24 h. For study of the effect of overexpressed 3GalT4 on the CoCl2-induced EMT process, cells were treated with CoCl2 for 24 h and then were transfected with mouse 3GalT4 gene/pIRES-puro3 or vector only, as described in Materials and Methods. These cells were used for analysis of Gg4 and 3GalT4 expression, EMT marker molecules, and cell motility. Trans., transfection. A) For Gg4 analysis, cells (4 105/dish in a 6-cm dish) were treated with CoCl2 and transfected. GSLs were extracted and analyzed by HPTLC: a) orcinol/sulfuric acid spray; b) stained with TKH7 for Gg4. Experiments were performed in triplicate; representative HPTLC-immunostaining results are shown. c) Gg4 expression detected with TKH7 was calculated based on density analysis from 3 experiments; relative expression is shown as percentage of the control value. **P 0.01. B) For 3GalT4 expression analysis, cells (5 104/well in a 12-well plate) were treated and transfected as described above. Cells were harvested, lysed in RIPA buffer, and subjected (10 g of protein/slot) to SDS-PAGE. 3GalT4 expression in transfected cells was determined by Western blot as described in Materials and Methods. Experiments were performed in triplicate; representative Western blot results are shown (bottom). Normalized values with each loading control are shown as mean sd relative intensity on the ordinate (top). C) For the phagokinetic cell motility assay, NMuMG cells (5 104 cells/well in a 12-well plate) were treated as described above. Cells were detached with trypsin/EDTA, and 5 103 cells in complete culture medium were added onto each gold sol-coated well and incubated for 8 h, as described in Materials and Methods. Photos of track areas of 30 cells were taken. Cleared areas on gold sol were measured; values are means sd (squared pixels) from the Scion Image program. *P 0.05; ***P 0.001. D) For EMT marker protein analysis, cells were treated, and SDS-PAGE and Western blot were performed as described above. Experiments were performed in triplicate, and representative Western blot results are shown (top). Normalized values with each loading control are shown as mean sd relative intensity on the ordinate (bottom). *P 0.01–0.05; **P 0.001–0.005; NS, not significant.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Inhibition, Cell Culture, Transfection, Plasmid Preparation, Expressing, Marker, High Performance Thin Layer Chromatography, Staining, Immunostaining, Control, SDS Page, Western Blot, Motility Assay, Incubation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 6. Inhibition of hypoxia-induced EMT by overexpressed 3GalT4 or exogenous Gg4. Cells were grown and treated with the hypoxia condition as described in Materials and Methods. GM1 or Gg4 was added, and transfection procedures were performed as described for Fig. 5. Expression of Gg4 (A) and 3GalT4 (B) in transfected cells, cell motility (C), and expression of EMT marker protein (D) affected by overexpressed 3GalT4 or exogenous Gg4 are shown as described for Fig. 5. Trans., transfection. *P 0.01–0.05; **P 0.001–0.005; ***P 0.001; NS, not significant.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Inhibition, Transfection, Expressing, Marker

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 7. Inhibition of TGF--induced EMT by overexpressed 3GalT4. Cells were cultured in culture medium overnight; then medium was replaced with fresh medium alone (control) or with fresh medium containing 1 ng/ml TGF- for 18 h. Transfection was performed as described in Materials and Methods and for Fig. 5. Cells were analyzed for Gg4 expression (A), 3GalT4 expression (B), cell motility (C), and EMT marker molecules (D), as described for Fig. 5. Trans., transfection. A) For Gg4 analysis, cells (4 105/dish in a 6-cm dish) were treated with TGF- and transfected. GSLs were extracted, and analyzed by HPTLC: a) orcinol/sulfuric acid spray; b) stained with TKH7 for Gg4. Experi- ments were performed in triplicate, and representative HPTLC immunostaining results are shown. c) Gg4 expression detected with TKH7 was calculated based on density analysis from 3 experiments and is shown as relative expression, as described for Fig. 5. **P 0.01. B) For 3GalT4 expression analysis, cells (5 104/well in a 12-well plate) were treated and transfected as described above. Cells were harvested, lysed in RIPA buffer, and subjected (10 g of protein/slot) to SDS-PAGE. 3GalT4 expression in transfected cells was determined by Western blot as described in Materials and Methods. Experiments were per- formed in triplicate; representative Western blot results are shown (bottom). Normalized values with each loading control are shown as mean sd relative intensity on the ordinate (top). *P 0.01–0.05. C) NMuMG cells were treated as described above. Phagokinetic cell motility assay was performed as described for Fig. 5. Values are means sd (squared pixels) from Scion Image. ***P 0.001. D) For EMT marker protein analysis, cells were treated as described above. Experiments were performed in triplicate. Representative Western blot results are shown (top); normalized values with each loading control are shown as mean sd relative intensity on the ordinate (bottom). *P 0.01–0.05; **P 0.001–0.005; ***P 0.001; NS, not significant.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Inhibition, Cell Culture, Control, Transfection, Expressing, Marker, High Performance Thin Layer Chromatography, Staining, Immunostaining, SDS Page, Western Blot, Motility Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 8. Expression of Ecad, -catenin, 3GalT4, and Gg4 at various times after TGF- treatment. Cells were treated with 2 ng/ml TGF- for various periods of time as indicated. A) Cells were harvested, and cell lysates prepared using RIPA buffer were subjected to Western blot analysis with antibodies against Ecad, -catenin, 3GalT4, or -tubulin. B) Cells treated as above were harvested and extracted with isopropa- nol/hexane/water (55:25:20) for Gg4 detection with mAb TKH7. as described in Materials and Methods.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Expressing, Western Blot

Journal: Blood Advances

Article Title: Proteome alterations in erythrocytes with PIEZO1 gain-of-function mutations

doi: 10.1182/bloodadvances.2022008673

Figure Lengend Snippet: Band 3 and ABCB6 overexpression in plasma membrane protein of RBCs from patients with DHS and PIEZO1–band 3 colocalization. (A) Left: representative immunoblot of RBC membrane proteins showing band 3 and ABCB6 protein expression normalized to β-actin in the patients DHS1, DHS2, DHS3, DHS4, DHS5, and DHS6 compared with HCs (2 pools of HCs, each of n = 3). Right: densitometric analysis of 3 representative western blotting is shown for band 3 and ABCB6 proteins. Data are presented as mean ± standard deviation; ∗ P < .05; ∗∗ P < .01 (Student t test). (B) Representative confocal imaging by ZEISS LSM 980 Airyscan 2 of HC RBCs treated with vehicle (upper panel) compared with HC RBCs treated with 2.5 μM Yoda1 (10 minutes) (middle panel) and HC RBCs treated with 2.5 μM Yoda1 (20 minutes) (lower panel). Rabbit anti-PIEZO1 antibody is shown in green, mouse anti–band 3 is shown in red. The overlapping of both signals (MERGE) is shown on the right (yellow). (C) Quantification by Pearson correlation of PIEZO1–band 3 colocalization in untreated HC RBCs, HC RBCs + 2.5 μM Yoda1 (10 minutes), and HC RBCs + 2.5 μM Yoda1 (20 minutes) is shown. Data shown are the means of 12 independent acquisitions ± standard error of the mean; P < .05 for trend (ANOVA test), ∗ P < .05 (post hoc correction by Tukey multiple comparisons test).

Article Snippet: Erythrocyte membrane extracts (80 μg protein) were loaded on sodium dodecyl sulfate polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (BioRad, Milan, Italy), and incubated with mouse anti-BAND3 glycoprotein antibody (dilution, 1:5000; GTX11012; GeneTex) and mouse anti-ABCB6 antibody (dilution, 1:1000; 101-10003; Raybiotech, Inc).

Techniques: Over Expression, Membrane, Western Blot, Expressing, Standard Deviation, Imaging