Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 1 SNL induces elevated S100A4 expression in the SDH, and niclosamide attenuates NP. Western blot analysis revealed the temporal profile of S100A4 expression in both sham and SNL rats (A). SNL induced elevated S100A4 levels in ipsilateral L4/5 spinal cord segments from Days 1 to 21 after surgery (B). The data are presented as the means ± SEMs. ****p < 0.0001 versus the sham group. Real-time PCR (C) revealed similar trends in S100A4 mRNA expression at 3, 5, 7 and 10 days in SNL rats. The data are presented as the means ± SEMs. ****p < 0.0001 versus the sham group. Immunofluorescence assays revealed the distribution of S100A4 (green) protein in the L4/5 SDH after sham or SNL surgery on POD 10 (D1, D2, and D3; scale bar, 500 μm). The fluorescence in tensity significantly increased at 14 days after SNL and remained high at 21 dpi (D4, D5) (scale bar, 500 μm). Data analysis further confirmed these results, as shown in E. Data are presented as the mean ± SEM. **p < 0.01, ****p < 0.0001, ipsilateral SDH versus sham SDH; ####P < 0.0001, ipsilateral SDH versus contralateral SDH. Double immunofluorescence staining images (F1, F2, and F3) revealed extensive coexpression of S100A4 (green) with NeuN (red) and limited coexpression with GFAP (red) but not with iba-1 (scale bar, 500 μm). More details are shown at higher magnification for F1-1, F2-1 and F3-1 (scale bar, 100 μm). Single intrathecal administration of niclosamide (15–45 µg in 20 µl) in the later phase (POD 7) partially suppressed mechanical hypersensi tivity in the ipsilateral hind paw for up to 5 days after SNL, and the effect was observed 12 h after injection. Moreover, 45 µg of niclosamide had a greater effect than 15 µg of niclosamide (G). The data are presented as the means ± SEMs. ****p < 0.0001 for 15 µg of niclosamide versus DMSO. ####p < 0.0001, 45 µg of niclosamide versus DMSO. ψψψp < 0.001, ψψψψp < 0.0001, 15 µg of niclosamide versus 45 µg of niclosamide. Repeated administration of ni closamide in the early phase (POD 1) partially attenuated the development of mechanical hypersensitivity in SNL rats as well (I), which was maintained until POD 12. The data are presented as the means ± SEMs. ****p < 0.0001 for niclosamide versus DMSO. However, niclosamide treatment did not affect mechanical pain sensitivity in the contralateral hind paw (H, J). As shown in K, a single intrathecal injection of the rat S100A4 peptide (200 ng or 800 ng in 20 µl) rapidly decreased the PWT in the bilateral hind paws of naive rats from 2 h to 7 d. Moreover, 800 ng S100A4 induced more severe hyperalgesia than did 200 ng S100A4. On the other hand, saline did not affect the PWT of the hind paw. The data are presented as the means ± SEMs. ****p < 0.0001, 200 ng of S100A4 versus saline. ####p < 0.0001 800 ng S100A4 versus saline. ψψψψp < 0.0001 200 ng S100A4 versus 800 ng S100A4

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Double Immunofluorescence Staining, Injection, Saline

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 3 Exogenous S100A4 significantly contributes to A1 astrocyte activation in vitro. Western blot analysis revealed that GFAP and C3 protein levels were significantly increased after stimulation of 70% fusion primary astrocyte cultures with different concentrations of rat recombinant S100A4 protein (A), whereas 80 ng/ml S100A4 stimulation induced the greatest increase in GFAP and C3 expression (B). The data are presented as the means ± SEMs. *p < 0.05, **p < 0.01, ****p < 0.0001, normal versus S100A4. ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus 80 ng/ml S100A4. RT‒PCR analysis revealed changes in the mRNA levels of A1-specific astrocyte genes after 80 ng/ml S100A4 stimulation, similar to the effects of the commonly known “cytokine cocktail” containing TNF-α, IL-1α and C1q (C). The data are presented as the means ± SEMs. *p < 0.05, ****p < 0.0001 S100A4 versus normal. #p < 0.05, ###p < 0.001, ####p < 0.0001 TNF-α, IL-1α and C1q versus normal. Moreover, incubation with 80 ng/ml S100A4 resulted in higher OD values than those of normal cells and cells incubated with other concentrations of S100A4 (D). The data are presented as the means ± SEMs. ****p < 0.0001 versus 80 ng/ml S100A4. Im munofluorescence revealed that 70% of the fusion primary astrocytes (GFAP, green; DAPI, blue) incubated with TNF-α, IL-1α and c1q and S100A4 (80 ng/ ml) for 24 h presented stronger GFAP (green) and C3 (red) fluorescence intensities and, accordingly, showed higher GFAP+C3+ fluorescence intensities (E) (scale bar, 100 μm). Quantitative analysis further confirmed these results (F). The data are presented as the means ± SEMs. ****p < 0.0001, normal versus S100A4. ####p < 0.0001, normal versus TNF-α, IL-1α and C1q

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Recombinant, Expressing, Incubation, Fluorescence

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 4 S100A4 inhibition reduces A1 astrocyte activation following SNL. Western blot analysis revealed significantly reduced levels of GFAP and C3 at 10 and 14 days post-SNL in rats treated with successive niclosamide injections, starting from POD 1, along with decreased S100A4 expression (A and B). The data are presented as the means ± SEMs. ***p < 0.001, ****p < 0.0001, normal versus SNL + DMSO. #p < 0.05, ##p < 0.01, ####p < 0.0001, SNL + DMSO versus SNL + niclosamide. In addition, fluorescence images confirmed that astrocytes were remarkably activated in the SDH after SNL, and the fluorescence in tensity of GFAP (C3) (green) was much greater than that in the sham group (C1) (scale bar, 500 μm). Successive intrathecal injection of niclosamide begin ning on POD1 for 3 days, but not DMSO, suppressed SNL-induced activation of astrocytes (C3 and C4). However, niclosamide had no effect on sham rats (C2) (scale bar, 500 μm). The data summary further confirmed these results, as shown in D. Moreover, niclosamide treatment reduced the fluorescence intensity of GFAP+C3+ (yellow) in SNL rats (E and F). The data are presented as the means ± SEMs. ****p < 0.0001, sham + DMSO group versus SNL + DMSO group. ###p < 0.001, SNL + DMSO versus SNL + niclosamide. ELISA (G) revealed that the administration of niclosamide also decreased the protein levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), while RT‒PCR data confirmed that the injection of niclosamide decreased the mRNA levels of A1 astrocyte-specific factors, including CCL2, CXCL1, MMP2, and tPA (H). The data are presented as the means ± SEMs. ****p < 0.0001, sham + DMSO versus SNL + DMSO. ##p < 0.01, ###p < 0.001, ####p < 0.0001 SNL + DMSO versus SNL + niclosamide. Finally, pain behavioural assessment revealed that the ad ministration of recombinant S100A4 protein decreased the PWT of the bilateral hind paws from 2 h to 7 days, whereas fluorocitrate partially reversed S100A4-induced mechanical hyperalgesia, which persisted for up to 7 days (I). The data are presented as the means ± SEMs. ****p < 0.0001, saline versus S100A4. ##p < 0.01, ###p < 0.001, ####p < 0.0001 S100A4 versus S100A4 + fluorocitrate

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Fluorescence, Injection, Enzyme-linked Immunosorbent Assay, Recombinant, Saline

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 5 Exogenous S100A4 induces A1 astrocyte activation via the TLR4/NF-κB signalling pathway in vitro. Western blot analysis revealed that TLR4 and p-NF-κB p65 protein levels were also significantly increased after stimulation with 80 ng/ml rat recombinant S100A4 protein in primary astrocyte cultures, whereas total NF-κB p65 expression was not affected (A, B). The data are presented as the means ± SEMs. **p < 0.01, ****p < 0.0001, normal versus S100A4. Pretreatment with PDTC decreased the S100A4-induced upregulation of GFAP and C3, as well as p-NF-κB p65 expression (C). The selective TLR4 inhibitor Resatorvid significantly decreased the S100A4-mediated upregulation of GFAP and C3, along with the protein levels of p-NF-κB p65 and TLR4 (D). Quanti tative analysis confirmed these results, as shown in E and F. Immunofluorescence staining confirmed that, after confluence, adherent astrocytes activated by S100A4 presented significantly greater fluorescence intensities of both GFAP and C3 than did control cells. PDTC ammonium or Resatorvid treatment reduced the fluorescence intensity of GFAP and C3 (G) (scale bar, 100 μm). Quantitative analysis further confirmed these results, as shown in H. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 normal versus S100A4. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 S100A4 versus S100A4 + PDTC ammonium or Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Recombinant, Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 6 SNL upregulates spinal TLR4/NF-κB signalling, whereas S100A4 inhibition attenuates A1 astrocyte activation via this pathway. Western blot show ing the time courses of TLR4 and p-NF-κB p65 expression in sham and SNL rats (A). SNL induced a progressive increase in TLR4 and p-NF-κB p65 levels in the spinal cord, especially during the maintenance period of NP, and then tended to decrease from Days 1 to 21 after surgery (B). The data are presented as the means ± SEMs. For TLR4, ***p < 0.001, ****p < 0.0001 SNL versus sham. For NF-κB, ##p < 0.01, ###p < 0.001, ####p < 0.0001 SNL versus sham. Dual- label fluorescence colocalization analysis revealed that the intensity of GFAP/C3 double-positive fluorescence in the ipsilateral L4/5 SDH was significantly greater on POD 10 after SNL than in the sham group (scale bar, 500 μm). Immunofluorescence images (C1-C, C2-C and C3-C) revealed intense colo calization of p-NF-κB p65 (green) with NeuN (red) and limited coexpression with GFAP (red) but not with iba-1 (red) (scale bar, 500 μm). More details are shown at higher magnification in C1-D, C2-D and C3-D (scale bar, 100 μm). WB analysis revealed that successive intrathecal niclosamide injections beginning at 1 dpi induced significantly lower levels of TLR4 and p-NF-κB p65 expression at 10 and 14 days in SNL rats (D and E). The data are presented as the means ± SEMs. ***p < 0.001, ****p < 0.0001 SNL versus sham. ##p < 0.01, ###p < 0.001 SNL versus SNL + niclosamide. Moreover, the administration of Resatorvid caused a decrease in GFAP and C3 expression, as well as p-NF-κB p65 protein levels, at 10 and 14 days in SNL rats (F and G). Repeated ad ministration of the NF-κB inhibitor PDTC decreased the expression of GFAP and C3 (H and I). The data are presented as the means ± SEMs. ****p < 0.0001 sham versus SNL + DMSO. ###p < 0.001, ####p < 0.0001 SNL + DMSO versus SNL + PDTC ammonium or Resatorvid. Dual-label fluorescence colocalization analysis revealed that the intensity of GFAP/C3 double-positive fluorescence in the ipsilateral L4/5 SDH was significantly greater on POD 10 after SNL than in the sham group. Repeated administration of PDTC ammonium or Resatorvid decreased the intensity of GFAP+C3+ fluorescence (J and K) (scale bar, 500 μm). Quantitative analysis further confirmed these results, as shown in L and M. Data are presented as the mean ± SEM. ****p < 0.0001 sham versus SNL + DMSO. ###p < 0.001 SNL versus SNL + PDTC ammonium or Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Fluorescence, Immunofluorescence

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 7 Blocking TLR4/NF-κB signalling alleviates neuropathic pain after SNL and impacts exogenous S100A4-induced hyperalgesia. Repeated administra tion of PDTC ammonium (50 µg/20 µl) in the early postoperative phase (POD 1) partially attenuated the development of mechanical hypersensitivity in the ipsilateral hind paw of SNL rats (A), which was maintained until POD 14. Moreover, after receiving repeated intrathecal injections of 20 µg Resatorvid, the PWT of the ipsilateral hind paw was significantly greater than that of the saline group from Day 3 to Day 14 after SNL (A). Nonetheless, the treatment did not influence the PWT of the contralateral hind paws (B). The data are presented as the means ± SEMs. ****p < 0.0001 SNL + DMSO versus SNL + PDTC ammonium. ####p < 0.0001 SNL + DMSO versus SNL + Resatorvid. Moreover, pain behavioural assessment revealed that PDTC ammonium and Resator vid could partially reverse S100A4-induced mechanical hyperalgesia in the bilateral hind paws of naive rats, with the effect lasting up to 5 days (left, C; right, D). The data are presented as the means ± SEMs. ****p < 0.0001, saline versus S100A4. ####p < 0.0001 S100A4 versus S100A4 + PDTC ammonium. ѱѱѱp < 0.001, ѱѱѱѱp < 0.0001 S100A4 versus S100A4 + Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Blocking Assay, Saline

Journal: Food Science and Human Wellness

Article Title: Ginsenoside Rb3 prevents myocardial injury in sleep-deprived mice by suppressing ferroptosis through SIRT3/P53/SCL7A11/GPX4 axis

doi: 10.26599/fshw.2024.9250274

Figure Lengend Snippet: Fig. 3. GRb3 ameliorates myocardial ferroptosis-related protein and SIRT3 changes in sleep deprived mice. (A) Expression bands of individual proteins in mice hearts. (B-K) Gray scale analysis of ANP, SIRT3, P53, SLC7A11, GPX4, TFRC, ACSL4, HO-1, FTH1, FSP1. n=5/group. **P < 0.01, ***P < 0.001, contrast to control group. #P < 0.05, ##P < 0.01, ###P < 0.001contrast to the sleep deprived group. n=5/group.

Article Snippet: Antibodies against P53 (A19585), SLC7A11 (A2413), GPX4 (A11243), TFRC (A18083), ACSL4 (A6826), and FSP1 (A1631) were from Abclonal (Wuhan, China).

Techniques: Expressing, Control

Journal: Food Science and Human Wellness

Article Title: Ginsenoside Rb3 prevents myocardial injury in sleep-deprived mice by suppressing ferroptosis through SIRT3/P53/SCL7A11/GPX4 axis

doi: 10.26599/fshw.2024.9250274

Figure Lengend Snippet: Fig. 5. GRb3 inhibits expression of ferroptosis-related proteins in Erastin-induced H9c2 cells. (A) Expression bands of individual proteins in H9c2 cells. (B-K) Gray scale analysis of ANP, SIRT3, P53, SLC7A11, GPX4, TFRC, ACSL4, HO-1, FTH1, FSP1. n=5/group. **P < 0.01, ***P < 0.001, contrast to control group; #P < 0.05, ##P < 0.01, ###P < 0.001, contrast to Erastin group. n=5/group.

Article Snippet: Antibodies against P53 (A19585), SLC7A11 (A2413), GPX4 (A11243), TFRC (A18083), ACSL4 (A6826), and FSP1 (A1631) were from Abclonal (Wuhan, China).

Techniques: Expressing, Control

Journal: Cell death & disease

Article Title: UHRF1-mediated ferroptosis promotes pulmonary fibrosis via epigenetic repression of GPX4 and FSP1 genes.

doi: 10.1038/s41419-022-05515-z

Figure Lengend Snippet: Fig. 3 GPX4 cooperates with FSP1 to regulate SiO2-induced AEC2 ferroptosis. A mRNA levels of GPX4 and FSP1 were measured in 150 µg/ mL SiO2 treated primary AEC and 250 µg/mL SiO2 treated A549 cells (*P < 0.05 versus control group, **P < 0.01 versus control group). B Representative immunoblotting of GPX4 and FSP1 in 0, 50, 100, 150, 200, and 250 µg/mL SiO2 treated primary AEC2 and A549 cells (left panel), means ± SEM of three independent experiments (right) are shown. C Mouse primary AEC2 and A549 cells were treated with 1 μM erastin or DMSO, representative immunoblotting of GPX4 and FSP1 (left), as well as means ± SEM of three independent experiments (right) are shown. Then AEC2 and A549 cells were treated with 150 or 250 µg/mL SiO2 individually, SiO2 with FSP1 plasmid, and SiO2 with GPX4 plasmid. D Flow cytometry representative pictures of primary AEC2 and quantification results of both primary AEC2 and A549 cells. E Representative images of C11-BODIPY in treated-primary AEC cells (red: reduced C11-BODIPY, green: oxidized C11-BODIPY; scale bars, 50 µm; left panel), and quantification of C11-BODIPY fluorescence in primary AEC2 and A549 cells (right panel). F MDA concentration in cell lysates from primary AEC2 and A549 cells was measured using a Lipid Peroxidation MDA Assay Kit. G GSH/GSSG ratio of primary AEC2 and A549 cells. H Intracellular Fe2+ was detected with the FerroOrange probe, and representative images of primary AEC2 cells were shown (red: FerroOrange-stained Fe2+; scale bars, 25 µm; left panel). Fe2+ fluorescence intensity was quantified by ImageJ in both primary AEC2 and A549 cells (right panel). **P < 0.01 versus control group, #P < 0.01 versus SiO2-treated group.

Article Snippet: GPX4 plasmid from Generay Biotech (Shanghai, China), and FSP1 plasmid from Origene, Inc (Origene, Rockville, MD, USA), UHRF1 inhibitor NSC232003, ferroptosis inhibitors ferrostain-1, a pan-caspase inhibitor ZVAD-FMK, a potent necroptosis inhibitor necrostatin-1, and the DNA methyltransferase inhibitor 5-AZA-dC were phased from MedChemExpress.

Techniques: Control, Western Blot, Plasmid Preparation, Flow Cytometry, Concentration Assay, Multiple Displacement Amplification, Staining

Journal: Cell death & disease

Article Title: UHRF1-mediated ferroptosis promotes pulmonary fibrosis via epigenetic repression of GPX4 and FSP1 genes.

doi: 10.1038/s41419-022-05515-z

Figure Lengend Snippet: Fig. 4 DNA-methylation occurs in GPX4 and FSP1 promoter regions in SiO2-stimulated AEC2s. Primary AEC2 and A549 cells were treated with SiO2, SiO2 + 5-Aza, and SiO2 + DMSO. A Protein levels of GPX4 and FSP1 were detected by western blot and qualified (means ± SD, n = 3) (**P < 0.01 versus control group, #P < 0.01 versus SiO2 + DMSO group). B Representative immunofluorescence staining of GPX4 (red) and C FSP1 (green) in primary AEC2 cells were shown, scale bars, 10 μm. D Methylation-specific PCR detected DNA methylation levels of ACE2 cells with or without SiO2. E DNA from control and SiO2-treated AEC2 cells were subjected to bisulfite sequencing of 5′UTR. Methylated CpG sites are shown with black circles and unmethylated sites with open circles. F Intracellular Fe2+ was detected with the FerroOrange probe, and representative images of primary AEC2 cells were shown (red: FerroOrange-stained Fe2+; scale bars, 25 µm; upper panel). Fe2+ fluorescence intensity was quantified by ImageJ in both primary AEC2 and A549 cells (**P < 0.01 versus control group, #P < 0.01 versus SiO2 + DMSO group; lower panel).

Article Snippet: GPX4 plasmid from Generay Biotech (Shanghai, China), and FSP1 plasmid from Origene, Inc (Origene, Rockville, MD, USA), UHRF1 inhibitor NSC232003, ferroptosis inhibitors ferrostain-1, a pan-caspase inhibitor ZVAD-FMK, a potent necroptosis inhibitor necrostatin-1, and the DNA methyltransferase inhibitor 5-AZA-dC were phased from MedChemExpress.

Techniques: DNA Methylation Assay, Western Blot, Control, Staining, Methylation, Sequencing

Journal: Cell death & disease

Article Title: UHRF1-mediated ferroptosis promotes pulmonary fibrosis via epigenetic repression of GPX4 and FSP1 genes.

doi: 10.1038/s41419-022-05515-z

Figure Lengend Snippet: Fig. 6 UHRF1 liposomal siRNA could block PF progression in mouse models. A Schematic diagram of UHRF1 siRNA liposome or scramble liposome together with silica-treated mouse experiment. B Representative HE, Sirius red, UHRF1 IHC, and Masson staining of lung tissues from each group of C57BL/6 mice sacrificed on day 28 (scale bars, 100 µm). C Representative immunofluorescence staining of α-SMA and Collagen I from mouse lung tissues (scale bars, 100 µm). D Ashcroft score of mice from each group. E The labile iron concentration of lung tissues was assessed using an Iron Colorimetric Assay Kit. F The levels of hydroxyproline content were determined at 550 nm and expressed as micrograms per mg of lung tissues, determined by the hydroxyproline content assay kit. G The protein levels of Fibronectin, Collagen I, α-SMA in lung tissues, and UHRF1, FSP1, GPX4 in primary AEC2 cells from each group were detected by western blot and qualified (means ± SD, n = 3). *P < 0.05 versus control group, **P < 0.01 versus control group, #P < 0.01 versus silica+scramble liposomes group.

Article Snippet: GPX4 plasmid from Generay Biotech (Shanghai, China), and FSP1 plasmid from Origene, Inc (Origene, Rockville, MD, USA), UHRF1 inhibitor NSC232003, ferroptosis inhibitors ferrostain-1, a pan-caspase inhibitor ZVAD-FMK, a potent necroptosis inhibitor necrostatin-1, and the DNA methyltransferase inhibitor 5-AZA-dC were phased from MedChemExpress.

Techniques: Blocking Assay, Staining, Concentration Assay, Colorimetric Assay, Western Blot, Control, Liposomes

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: S100A4 stimulates inflammatory signaling pathways in airway smooth muscle (ASM) tissues. A and B: stimulation of tracheal smooth muscle tissues with recombinant His-S100A4 or IL-13 significantly increases Akt phosphorylation (A) (one-way ANOVA with repeated measures, n = 6, *P < 0.01) and NF-κB Ser536 phosphorylation (B) (two-tailed paired Student’s t test, n = 6, *P < 0.0007) relative to unstimulated (US) tissues C: eotaxin secretion was significantly increased by stimulation with S100A4 or IL-13 in ASM tissues (one-way ANOVA with repeated measures, n = 5, *P < 0.05). D: expression of smooth muscle myosin heavy chain (SmMHC) was significantly inhibited in tissues stimulated with S100A4 or IL-13 (one-way ANOVA with repeated measures, n = 8, *P < 0.001).

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Protein-Protein interactions, Recombinant, Phospho-proteomics, Two Tailed Test, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: S100A4 regulates inflammatory signaling pathways by acting through receptors for advanced glycation end products (RAGE) in airway smooth muscle tissues. A: RAGE recombinant protein expression was confirmed by immunoblotting using RAGE antibody. More RAGE protein was observed in tissues treated with plasmids for wild-type (WT)-RAGE or RAGEΔcyto than in sham-treated tissues. B and C: NF-κB Ser536 phosphorylation and Akt Ser473 phosphorylation were significantly increased in response to His-S100A4 stimulation in tissues expressing WT-RAGE and in sham-treated tissues. The expression of RAGEΔcyto significantly inhibited S100A4-stimulated NF-κB Ser536 phosphorylation and Akt Ser473 phosphorylation (n = 6; *P < 0.01). D: expression of RAGEΔcyto prevents the suppression of smooth muscle myosin heavy chain (SmMHC) protein expression in response to S100A4 in airway smooth muscle tissues (n = 5; P > 0.05). Stimulation with His-S100A4 significantly inhibits SmMHC expression in sham-treated tissues (n = 5; *P < 0.001) or tissues expressing WT-RAGE (n = 5; *P < 0.05). All values are means ± SE (one-way ANOVA with repeated measures).

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Protein-Protein interactions, Recombinant, Expressing, Western Blot, Phospho-proteomics

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: The inhibition of receptors for advanced glycation end products (RAGE) suppresses inflammatory responses to S100A4 in airway smooth muscle tissues. Airway smooth muscle tissues were treated for 2 h with 100 μM of the RAGE antagonist, FPS-ZM1 [RAGE inhibitor (Inh)] and their responses to stimulation with His-S100A4 were determined. A and B: stimulation with S100A4 significantly increased NF-κB Ser536 phosphorylation and Akt Ser473 phosphorylation. Treatment with RAGE inhibitor significantly inhibited S100A4-stimulated NF-κB Ser536 phosphorylation (n = 5, *P < 0.01) and Akt Ser473 phosphorylation (n = 5, *P < 0.001). C: smooth muscle myosin heavy chain (SmMHC) protein was significantly inhibited by stimulation with His-S100A4. Treatment of tissues with RAGE Inh prevented the reduction in SmMHC expression induced by stimulation with His-S100A4 (n = 6, *P < 0.001). D: stimulation of tissues with His-S100A4 significantly increased eotaxin secretion as measured by ELISA. Treatment of tissues with RAGE Inh prevented the increase in eotaxin secretion stimulated by His-S100A4 (n = 4, *P < 0.01). All values are means ± SE (one-way ANOVA with repeated measures).

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Inhibition, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: The inflammatory cytokines IL-13 and TNF-α induce the secretion of S100A4 by airway smooth muscle tissues. IL-13 and TNF-α significantly increased the secretion of S100A4 from tissues compared with unstimulated tissues. S100A4 secretion was measured by ELISA (one-way ANOVA, n = 9, *P < 0.05) (A) or by immunoblotting (one-way ANOVA with repeated measures, n = 7, *P < 0.01) (B). C: time course for secreted S100A4 in the incubation medium during stimulation with IL-13 or TNF-α. All values are means ± SE.

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: Stimulation of airway smooth muscle with the inflammatory cytokines IL-13, TNF-α, or S100A4 promotes the expression of S100A4 and receptors for advanced glycation end products (RAGE). A: immunoblot of S100A4 and RAGE in airway smooth muscle tissue extracts after stimulation with IL-13, TNF-α, or His-S100A4 for 20 h. B and C: incubation with IL-13, TNF-α, or His-S100A4 significantly increased the expression of S100A4 and RAGE protein (n = 7, *P < 0.001) (B) and mRNA (n = 6, *P < 0.001) (C) in airway smooth muscle tissues. D: time course for protein expression of RAGE and S100A4 in airway smooth muscle tissues during stimulation with IL-13, TNF-α, or His-S100A4. Values are means ± SE. Statistical analysis performed using one-way ANOVA with repeated measures.

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Expressing, Western Blot, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: S100A4 overexpression in airway smooth muscle tissues increases S100A4 secretion and the activation of inflammatory signaling pathways. A: S100A4 overexpression in tissues expressing recombinant S100A4 (no epitope tag) was confirmed by immunoblotting. More S100A4 was detected in tissues transfected with plasmids compared with sham-treated tissues (n = 5, *P = 0.005). B: S100A4 in the incubation medium was detected by immunoblotting. The overexpression of S100A4 significantly increased the secretion of S100A4 from airway smooth muscle tissues (n = 4, *P = 0.035). C: overexpression of S100A4 significantly increases NF-κB Ser536 phosphorylation (n = 7; *P = 0.006) and Akt Ser473 phosphorylation (n = 6; *P = 0.002) compared with sham-treated tissues. Smooth muscle myosin heavy chain (SmMHC) expression was significantly lower in tissues overexpressing S100A4 compared with sham-treated tissues (n = 4; *P = 0.006). Values are means ± SE. Statistical analysis performed by paired Student’s t test.

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Over Expression, Activation Assay, Protein-Protein interactions, Expressing, Recombinant, Western Blot, Transfection, Incubation, Phospho-proteomics

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products

doi: 10.1152/ajplung.00347.2019

Figure Lengend Snippet: S100A4 plays an autocrine role in promoting inflammation in airway smooth muscle tissues. Extracellular S100A4 acts on receptors for advanced glycation end products (RAGE) in airway smooth muscle to activate proinflammatory pathways mediated by Akt and NF-κB. The activation of Akt and NF-κB promotes eotaxin secretion. Akt suppresses the expression of smooth muscle myosin heavy chain (SmMHC) by inhibiting the translocation of serum response factor (SRF) to the nucleus. Activated NF-κB increases the transcription and expression of S100A4 and RAGE. Increased expression of intracellular S100A4 promotes S100A4 secretion from airway smooth muscle tissues and the activation of inflammatory signaling pathways.

Article Snippet: 2 (CircuLex, MBL Medical & Biological Laboratory Co., Ltd), plasmids encoding human RAGE, pcDNA3-RAGE (Addgene plasmid no. 71435), and pcDNA3-RAGEΔcyto (Addgene plasmid no. 71436) ( 20 ); pCMV6-XL4 human untagged S100A4 (no. SC109749, Origene); and RAGE Antagonist, FPS-ZM1, from Sigma-Aldrich (cat. no. 553030).

Techniques: Activation Assay, Expressing, Translocation Assay, Protein-Protein interactions