Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Relative p‐SMAD2/3 (left) and p‐SMAD1/5/8 (right) response, evaluated by AlphaLISA signal, of HEK293T cells after 1‐h exposure to recombinant myostatin (Mstn), activin A, GDF11, TGFβ, and BMP2. Relative p‐SMAD2/3 response of C2C12 myoblasts (left) and myotubes (right) after 1‐h exposure to Mstn, activin A, GDF11, and TGFβ. Phosphorylation of SMAD2 and SMAD3 in differentiated C2C12 myotubes following stimulation with recombinant Mstn or GDF11 for 30 and 60 min, as detected by immunoblotting. Equal loading is verified by Ponceau Red staining. EC 50 values (in nM) for p‐SMAD2/3 and p‐SMAD1/5/8 responses of HEK293T, C2C12 myoblasts, and C2C12 myotubes to the above listed ligands, as well as p‐SMAD1/5/8 response to BMP4, BMP6, and BMP7. Data information: Values are displayed as mean ± SEM; n = 4 for all data points.

Article Snippet: To assess the phosphorylation of SMAD in response to TGFβ superfamily members in undifferentiated cells, C2C12 myoblasts (ATTC CRL‐1772; passage 14) and HEK293T cells (ATCC CRL‐11268; passage 13) were plated on 96‐well tissue culture‐treated plates (Greiner‐Bio‐One 655098) in growth medium [high glucose Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 1% penicillin streptomycin (PS)], grown to confluency, and were treated with various doses of the following recombinant ligands for 1 h ( n = 4): Mstn (R&D Systems #788‐G8), GDF11 (R&D Systems # 1958‐GD), TGFβ (R&D Systems #240‐B), activin A (R&D Systems # 338‐AC), BMP‐2 (R&D Systems # 355‐BM), BMP‐4 (R&D Systems # 314‐BP), BMP‐6 (R&D Systems # 507‐BP), or BMP‐7 (R&D Systems # 354‐BP).

Techniques: Recombinant, Phospho-proteomics, Western Blot, Staining

Journal: Experimental & Molecular Medicine

Article Title: Hepatocyte toll-like receptor 4 mediates lipopolysaccharide-induced hepcidin expression

doi: 10.1038/emm.2017.207

Figure Lengend Snippet: LPS induces hepcidin expression by hepatocytes. ( a ) AML12 cells were treated with different concentrations of LPS for 24 h, and the expression of mouse hepcidin (Hamp1) mRNA was measured by quantitative real time polymerase chain reaction (qRT-PCR). ( b ) AML12 cells were treated with LPS (1 μg ml −1 ) for the designated times. ( c ) Expression of mRNA encoding inducible nitric oxide synthase (iNOS) and Hamp1 in AML12 cells treated with 1 μg ml −1 LPS for 24 h. ( d ) AML12 cells were treated for 12 h with BMP6 (20 ng ml −1 ), IL-6 (20 ng ml −1 ) or LPS (1 μg ml −1 ), and the expression of Hamp1 mRNA was measured by qRT-PCR. ( e ) Hepcidin concentration in cell culture medium from LPS (1 μg ml −1 )-treated AML12 cells. ( f ) Iron concentration in AML12 cells treated with LPS (1 μg ml −1 ) for 24 h.

Article Snippet: LPS ( Escherichia coli 026:B6, L2654, Sigma Aldrich, St Louis, MO, USA), BMP6 (6325-BM, R&D Systems, Minneapolis, MN, USA) and IL-6 (CYT-213, PROSPEC, Ness-Ziona, Israel) were dissolved in manufacturer-recommended solvents.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Cell Culture

Journal: Neurochemical Research

Article Title: IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

doi: 10.1007/s11064-021-03322-0

Figure Lengend Snippet: Effects of LPS and LTA on the IL-6 secretion of BV2 cells. IL-6 content of cell culture supernatants was determined with IL-6 Mouse ELISA Kit. a IL-6 secretion of LPS treated BV2 cells. b IL-6 production of LTA treated BV2 cells. The columns represent mean values and error bars represent standard deviation (SD) of three independent determinations ( n = 3). The asterisk marks p < 0.05 compared to the control. Data was analysed by one-way ANOVA followed by Tukey’s HSD post hoc test

Article Snippet: The secreted mature hepcidin content of the samples was determined with Mouse Hepcidin 25 ELISA Kit (Abbexa Ltd., Cambridge, UK) and the secreted BMP6 protein concentration was determined with Mouse BMP6 ELISA Kit (Cusabio Technology LLC, Houston, TX, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Neurochemical Research

Article Title: IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

doi: 10.1007/s11064-021-03322-0

Figure Lengend Snippet: Real-time PCR analysis of HAMP and ELISA measurements of secreted hepcidin of LPS and LTA treated BV2 cells. Quantitative Real-Time PCR analysis was performed using gene specific primers and SYBR Green method. The expression level of the gene of interest was compared to the level of the β-actin in each sample. The mRNA expressions of the treated cells were compared to the appropriate untreated controls (9 h or 24 h). The relative expression of the controls was regarded as 1. Hepcidin content of cell culture supernatants was determined with Mouse hepcidin-25 ELISA Kit. a , b mRNA levels of HAMP. c , d Secreted hepcidin concentrations of LPS and LTA treated BV2 cells. The columns represent mean values and error bars represent standard deviation (SD) of three independent determinations ( n = 3). The asterisk indicates p < 0.05 compared to the untreated controls. Data was analysed by two-way ANOVA followed by Tukey’s HSD post hoc test

Article Snippet: The secreted mature hepcidin content of the samples was determined with Mouse Hepcidin 25 ELISA Kit (Abbexa Ltd., Cambridge, UK) and the secreted BMP6 protein concentration was determined with Mouse BMP6 ELISA Kit (Cusabio Technology LLC, Houston, TX, USA).

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Expressing, Cell Culture, Standard Deviation

Journal: Neurochemical Research

Article Title: IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

doi: 10.1007/s11064-021-03322-0

Figure Lengend Snippet: ELISA measurement of secreted hepcidin of IL-6 neutralized, LPS or LTA treated BV2 cells. Hepcidin content of cell culture supernatants was determined with Mouse hepcidin-25 ELISA Kit. The columns represent mean values and error bars represent standard deviation (SD) of three independent measurements ( n = 3). Ctrl untreated control, NCtrl control + neutralizing IL-6 antibody, NLPS neutralizing IL-6 antibody + LPS, NLTA neutralizing antibody + LTA. The asterisk indicates p < 0.05 compared to the controls. The cross marks the significant difference, p < 0.05 between neutralized and non-neutralized samples. Data was analysed by two-way ANOVA followed by Tukey’s HSD post hoc test

Article Snippet: The secreted mature hepcidin content of the samples was determined with Mouse Hepcidin 25 ELISA Kit (Abbexa Ltd., Cambridge, UK) and the secreted BMP6 protein concentration was determined with Mouse BMP6 ELISA Kit (Cusabio Technology LLC, Houston, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Standard Deviation, Control

Journal: Neurochemical Research

Article Title: IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

doi: 10.1007/s11064-021-03322-0

Figure Lengend Snippet: ELISA measurement of secreted BMP6 of IL-6 neutralized and LPS or LTA treated BV2 cells. BMP6 content of cell culture supernatants was determined with Mouse BMP6 ELISA Kit according to the instructions of the manufacturer. The columns represent mean values and error bars represent standard deviation (SD) of three independent measurements ( n = 3). Ctrl untreated control, NCtrl control + neutralizing IL-6 antibody, NLPS neutralizing IL-6 antibody + LPS, NLTA neutralizing antibody + LTA. The asterisk indicates p < 0.05 compared to the controls. The cross marks the significant difference, p < 0.05 between neutralized and non-neutralized samples. Data was analysed by two-way ANOVA followed by Tukey’s HSD post hoc test

Article Snippet: The secreted mature hepcidin content of the samples was determined with Mouse Hepcidin 25 ELISA Kit (Abbexa Ltd., Cambridge, UK) and the secreted BMP6 protein concentration was determined with Mouse BMP6 ELISA Kit (Cusabio Technology LLC, Houston, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Standard Deviation, Control

Journal: Neurochemical Research

Article Title: IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

doi: 10.1007/s11064-021-03322-0

Figure Lengend Snippet: ELISA measurement of secreted TNFα of IL-6 neutralized and LPS or LTA treated BV2 cells. TNFα content of cell culture supernatants was determined with Mouse TNFα ELISA Kit according to the instructions of the manufacturer. The columns represent mean values and error bars represent standard deviation (SD) of three independent measurements ( n = 3). Ctrl untreated control, NCtrl control + neutralizing IL-6 antibody, NLPS neutralizing IL-6 antibody + LPS, NLTA neutralizing antibody + LTA. The asterisk indicates p < 0.05 compared to the controls Ctrl and NCtrl, respectively. Data was analysed by two-way ANOVA followed by Tukey’s HSD post hoc test

Article Snippet: The secreted mature hepcidin content of the samples was determined with Mouse Hepcidin 25 ELISA Kit (Abbexa Ltd., Cambridge, UK) and the secreted BMP6 protein concentration was determined with Mouse BMP6 ELISA Kit (Cusabio Technology LLC, Houston, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Standard Deviation, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naotaifang formula attenuates OGD/R-induced inflammation and ferroptosis by regulating microglial M1/M2 polarization through BMP6/SMADs signaling pathway.

doi: 10.1016/j.biopha.2023.115465

Figure Lengend Snippet: Fig. 5. The immunofluorescence of BMP6/SMADs signaling pathway-related proteins in BV2 microglia following LND-193189 treatment. The fluorescence intensity of BMP6 (a), Hepcidin (c), FPN (d), GPX4 (e), and the positive cell rate of p-SMADs. (b). Relative quantitative analysis of BMP6 (f), p-SMADs (g), Hepcidin (h), FPN (i), and GPX4 (j). Each experiment was repeated at least three times (*P < 0.05, **P < 0.01 compared to CON group. #P < 0.05, ##P < 0.01 compared to OGD/R group #P < 0.05, ##P < 0.01 compared to OGD/R group).

Article Snippet: The fixed BV2 cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with a blocking solution for 30 min, and separately incubated with the primary antibodies CD16/32 (1:50; CST, USA), CD206 (1:400; CST, USA), FPN (1:50; Affinity, USA), hepcidin (1:50; Boster, China), GPX4 (1:50; Boster, China), BMP6 (1:50; Boster, China), and p-SMADs (1:50; CST, USA) overnight at 4 °C.

Techniques: Immunofluorescence, Fluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naotaifang formula attenuates OGD/R-induced inflammation and ferroptosis by regulating microglial M1/M2 polarization through BMP6/SMADs signaling pathway.

doi: 10.1016/j.biopha.2023.115465

Figure Lengend Snippet: Fig. 6. The expression of BMP6/SMADs and STAT3 signaling pathway-related proteins in BV2 microglia as determined by western blot. Proteins expression of BMP6 (a), p-SMADs, SMADs (b), Hepcidin, FPN (c) p-STAT3, STAT3 (d), and GPX4 (e). Relative quantitative analysis of BMP6 (f), p-SMADs (g), SMAD (h), hepcidin (i), FPN (j), p-STAT3 (k), STAT3 (l), and GPX4 (m). Each experiment was repeated at least three times (*P < 0.05, **P < 0.01 compared to the CON group. #P < 0.05, ##P < 0.01 compared to the OGD/R group).

Article Snippet: The fixed BV2 cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with a blocking solution for 30 min, and separately incubated with the primary antibodies CD16/32 (1:50; CST, USA), CD206 (1:400; CST, USA), FPN (1:50; Affinity, USA), hepcidin (1:50; Boster, China), GPX4 (1:50; Boster, China), BMP6 (1:50; Boster, China), and p-SMADs (1:50; CST, USA) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naotaifang formula attenuates OGD/R-induced inflammation and ferroptosis by regulating microglial M1/M2 polarization through BMP6/SMADs signaling pathway.

doi: 10.1016/j.biopha.2023.115465

Figure Lengend Snippet: Fig. 9. NTF ameliorates iron metabolism via the BMP6/SMADs pathway in BV2 microglia after OGD/R. The fluorescence intensity of BMP6 (a), Hepcidin (c), FPN (d), GPX4 (e), and the positive cell rate of p-SMADs (b). Relative quantitative analysis of BMP6 (f), p-SMADs (g), Hepcidin (h), FPN (i), and GPX4 (j). Each experiment was repeated at least three times (*P < 0.05, **P < 0.01 compared to CON group. #P < 0.05, ##P < 0.01 compared to OGD/R group. ▴P < 0.05, ▴▴P < 0.01 compared to Vehicle group. ◆P < 0.05, ◆◆P < 0.01 compared to DFP group).

Article Snippet: The fixed BV2 cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with a blocking solution for 30 min, and separately incubated with the primary antibodies CD16/32 (1:50; CST, USA), CD206 (1:400; CST, USA), FPN (1:50; Affinity, USA), hepcidin (1:50; Boster, China), GPX4 (1:50; Boster, China), BMP6 (1:50; Boster, China), and p-SMADs (1:50; CST, USA) overnight at 4 °C.

Techniques: Fluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naotaifang formula attenuates OGD/R-induced inflammation and ferroptosis by regulating microglial M1/M2 polarization through BMP6/SMADs signaling pathway.

doi: 10.1016/j.biopha.2023.115465

Figure Lengend Snippet: Fig. 10. The expression of BMP6/SMADs and STAT3 signaling pathway-related proteins in BV2 microglia after NTF treatment. Protein expression levels of BMP6 (a), p-SMADs, SMADs, p-STAT3, and STAT3 (b), Hepcidin and FPN (c), and GPX4 (d). Relative quantitative analysis of BMP6 (e), p-SMADs (f), Hepcidin (g), FPN (h), p- STAT3 (i), STAT3 (j), and GPX4 (k). Each experiment was repeated at least three times (*P < 0.05, **P < 0.01 compared to CON group. #P < 0.05, ##P < 0.01 compared to OGD/R group. ▴P < 0.05, ▴▴P < 0.01 compared to Vehicle group. ◆P < 0.05, ◆◆P < 0.01 compared to DFP group).

Article Snippet: The fixed BV2 cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with a blocking solution for 30 min, and separately incubated with the primary antibodies CD16/32 (1:50; CST, USA), CD206 (1:400; CST, USA), FPN (1:50; Affinity, USA), hepcidin (1:50; Boster, China), GPX4 (1:50; Boster, China), BMP6 (1:50; Boster, China), and p-SMADs (1:50; CST, USA) overnight at 4 °C.

Techniques: Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naotaifang formula attenuates OGD/R-induced inflammation and ferroptosis by regulating microglial M1/M2 polarization through BMP6/SMADs signaling pathway.

doi: 10.1016/j.biopha.2023.115465

Figure Lengend Snippet: Fig. 11. NTF alleviated ferroptosis and inflammation after OGD/R in microglia thought BMP6/SMADs pathway. OGD/R resulted in the imbalance of the pro portion of M1 and M2 microglia, activation of IL-6/STAT3 inflammatory pathway, and production of inflammatory factors to worsen the inflammatory response. BMP6 can stimulate p-SMADs in cytoplasm to enter the nucleus after OGD/R, which can promote transcription of hepcidin and inhibit FPN to efflux of Fe2+, thus initiating the Fenton reaction, and promoting iron death. NTF can balance M1 and M2 type microglia and reduce the release of inflammatory factors. NTF simultaneously regulated hepcidin, reduced Fe2+ deposition, and reduced iron death.

Article Snippet: The fixed BV2 cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with a blocking solution for 30 min, and separately incubated with the primary antibodies CD16/32 (1:50; CST, USA), CD206 (1:400; CST, USA), FPN (1:50; Affinity, USA), hepcidin (1:50; Boster, China), GPX4 (1:50; Boster, China), BMP6 (1:50; Boster, China), and p-SMADs (1:50; CST, USA) overnight at 4 °C.

Techniques: Activation Assay