Journal: The Journal of clinical endocrinology and metabolism

Article Title: β2-Agonist Induces Net Leg Glucose Uptake and Free Fatty Acid Release at Rest but Not During Exercise in Young Men.

doi: 10.1210/jc.2018-01349

Figure Lengend Snippet: Figure 5. Muscle protein phosphorylation and representative Western blots for terbutaline (filled bars) and placebo (open bars) at rest and at cessation of knee extensor exercise. (a) Phosphorylation of PKA substrateSer/Thr. (b) Phosphorylation ratio of p-AktSer473/Akt total. (c) Representative blots. Values are means and upper bound of the 95% CI (n = 9 for rest; n = 8 for exercise). *P , 0.05, for treatment difference at sampling point. EX, cessation of exercise; R, rest.

Article Snippet: Primary antibodies used were p-PKA substrateSer/Thr (dilution, 1:500; no. 9621, Cell Signaling Technology, Leiden, Netherlands), Akt (dilution, 1:1000; no. 2967, Cell Signaling Technology), and p-AktSer473 (dilution, 1:1000; no. 9271, Cell Figure 1.

Techniques: Phospho-proteomics, Western Blot, Sampling

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 1 Overexpression of Tmprss6 in neuro-2a cells. A qRT-PCR detection of Tmprss6 mRNA expression in neuro-2a cells. Western blot detection of FLAG B and Tmprss6 C expression in neuro-2a cells. A representative blot image for each protein and the respective β-actin levels are shown. Quantification of Tmprss6 D expression in neuro-2a cells. Each sample’s relative expression level was calculated by normalizing its specific band to its corresponding β-actin or Histone3 band, and then comparing the mean ratio to that of the control group to get the relative fold change. E Immunofluorescence was used to detect the location of FLAG tags in neuro-2a cells (Scale bar = 20 μm). Data are expressed as the mean ± SD, n = 4. ∗∗∗p < 0.001.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 2 Effect of Tmprss6 overexpression on the cell cycle, iron content, and Bmp-Smad signaling pathway. A The interaction between FLAG (Tmprss6) and HJV was detected by immunoprecipitation. B Western blot analysis of HJV expression in neuro-2a cells (n = 4). C Quantification of HJV expression from the experiment shown in B. D The expression of P-Smad1/5/8, Smad1 and Smad4 proteins in the cytoplasm and nucleus were detected by western blot analysis (n = 4). Quantification of P-Smad1/5/8 / Smad1 E and Smad4 F expression from the experiment shown in D. G Western blot analysis of pro-hepcidin and FPN1 levels in neuro-2a cells (n = 4). H Quantification of pro-hepcidin and FPN1 expression from the experiment shown in G. I Western blot analysis of FtH, FtL, and TfR1 levels in neuro-2a cells (n = 4). J Quantification of FtH, FtL, and TfR1 levels from the experiment shown in I. Each sample’s relative expression level was calculated as described for Fig. 1D. K Total intracellular iron content was determined by ICP-MS (n = 4). L FerroOrange probe was used to detect intracellular Fe2+ content (Scale bar = 100 μm). M Quantitative evaluation of Fe2+ staining. N RNR activity was tested using a kit from Mlbio (n = 5). O The cell cycle was assessed by flow cytometry. P The percentage of cells in the G0/G1, S, and G2/M phases. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Over Expression, Immunoprecipitation, Western Blot, Expressing, Staining, Activity Assay, Cytometry

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 3 Overexpression of Tmprss6 induced apoptosis in neuro-2a cells. A Apoptosis, as measured by flow cytometry. B Proportion of apoptotic cells in the indicated groups. C Apoptosis induced by Tmprss6 overexpression by the TUNEL method, as described in the Methods section (Scale bar = 100 μm). D Levels of Bcl-2, Bax, and cleaved-caspase3, as assessed by western blot analysis (n = 4). E, F Quantification of Bcl-2/Bax E and Cleaved-caspase3 F expression from the experiment shown in D. Each sample’s relative expression level was calculated as described in the legend for Fig. 1D. Data are expressed as the mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Over Expression, Cytometry, TUNEL Assay, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 4 Activation of the ATF3/P38 signaling pathway in Tmprss6-overexpressing neuro-2a cells. A Volcano plot showing the significantly changed mRNAs in Tmprss6-overexpressing cells. B Pathway analysis was performed using GO data bases to identify functional enrichment based on the differentially expressed mRNA. C The ATF3 and Bnip3 expression was detected by qRT-PCR. D Levels of total ATF3, as assessed by western blot analysis (n = 4). E Quantification of total ATF3 expression from the experiment shown in D. F The levels of ATF3 in the cytoplasm and nucleus were detected by western blot analysis. G Quantification of ATF3 levels in the cytoplasm and nucleus from the experiment shown in F. H The subcellular localization of ATF3 in neuro-2a cells was determined by immunofluorescence (Scale bar = 20 μm). I The levels of P-p38 and p38 protein were detected by western blot analysis (n = 4). J Quantification of P-p38/p38 from the experiment shown in I. Each sample’s relative expression level was calculated as described in the legend of Fig. 1D. Data are expressed as the mean ± SD. ∗p < 0.05. ∗∗p < 0.01.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Activation Assay, Functional Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 5 Overexpression of Tmprss6 decreases iron levels to inhibit RNR activity and mediate apoptosis in SH-SY5Y cells. A Western blot analysis of Tmprss6 expression in SH-SY5Y cells. B Quantification of Tmprss6 expression in SH-SY5Y cells. C Western blot analysis of HJV expression in SH-SY5Y cells. D Quantification of HJV expression in SH-SY5Y cells. E Expression of P-Smad1/5/8, Smad1, Smad4 and ATF3 proteins in the cytoplasm and nucleus, as detected by western blot analysis. Quantification of P-Smad1/5/8/Smad1 F, Smad4 G, and ATF3 H expression from the experiment shown in E. I Western blot analysis of pro-hepcidin and FPN1 levels in SH-SY5Y cells. J Quantification of pro- hepcidin and FPN1 expression from the experiment shown in I. K Western blot analysis of FtH, FtL, and TfR1 levels in SH-SY5Y cells. L Quantification of FtH, FtL and TfR1 levels from the experiment shown in K. M RNR activity, as tested using a kit from Mlbio. N Levels of P-p38 and p38 protein, as detected by western blot analysis. O Quantification of P-p38/p38 from the experiment shown in N. P Levels of Bcl-2, Bax and Cleaved-caspase3, as assessed by western blot analysis. Q, R Quantification of Bcl-2/Bax Q and Cleaved-caspase3 R expression from the experiment shown in P. The data are expressed as the mean ± SD, n = 3. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Over Expression, Activity Assay, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 7 Effects of ATF3 expression on Tmprss6-induced apoptosis in neuro-2a cells. A Live ZSgreen1 imaging was performed 72 h after transfection with Scrambled shRNA or ATF3 shRNA plasmids. B Levels of ATF3, as assessed by western blot analysis (n = 4). C Quantification of ATF3 expression from the experiment shown in B. D–I Levels of P-p38 D, p38 D, Bcl-2 F, Bax F, and cleaved-caspase3 H, as assessed by western blot analysis (n = 4), from the first to the seventh lane are the WT group, Vector pcDNA3.1-transfected cells (Vector group), Scrambled shRNA- transfected cells (Scrambled shRNA group), pcDNA3.1 and Scrambled shRNA co-transfection group, Tmprss6-transfected cells (Tmprss6 group), ATF3 shRNA-transfected cells (ATF3 shRNA group), Tmprss6 and ATF3 shRNA co-transfection group. Quantification of P-p38/p38 E, Bcl- 2/Bax G, and Cleaved-caspase3 I expression from the experiments shown in D, F, and H, respectively. Each sample’s relative expression level was calculated as described in the legend of Fig. 1D. J TUNEL method showing the induction of apoptosis in the indicated groups (Scale bar = 100 μm). Data are expressed as the mean ± SD. ∗p < 0.05. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Expressing, Imaging, Transfection, shRNA, Western Blot, Plasmid Preparation, Cotransfection, TUNEL Assay

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 8 Effect of Tmprss6 overexpression on tumor growth and tumor cell apoptosis. A Photo of ex vivo tumors at 5 weeks post cell implantation in nude mice. B Tumor growth curves of nude mice within 5 weeks after cell implantation. C Tumor weights of nude mice at 5 weeks post cell implantation. D–L Levels of FLAG D, Tmprss6 D, ATF3 F, P-p38 F, p38 F, Bcl-2 I, Bax I, and Cleaved-caspase3 K, as assessed by western blot analysis (n = 4). Quantification of Tmprss6 E, ATF3 G, P-p38/p38 H, Bcl-2/Bax J, and Cleaved-caspase3 L expression from the experiments shown in D, F, I, and K, respectively. Each sample’s relative expression level was calculated as described in the legend of Fig. 1D. M TUNEL method showing the induction of apoptosis in the tumors in the indicated groups (Scale bar = 100 μm). Data are expressed as the mean ± SD. ∗p < 0.05. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Over Expression, Ex Vivo, Western Blot, Expressing, TUNEL Assay

Journal: Cell death & disease

Article Title: Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

doi: 10.1038/s41419-024-06442-x

Figure Lengend Snippet: Fig. 9 Schematic diagram of a working model of the inhibition of tumor growth by Tmprss6 overexpression. The overexpression of Tmprss6 reduces HJV levels by cleaving HJV, thereby inhibiting the phosphorylation of Smad1/5/8 and retention of the latter in the cytoplasm. Due to the decreased P-Smad1/5/8 levels, the transport of P-Smad1/5/8 to the nucleus by Smad4 is reduced. Decreased P-Smad1/5/8 in the nucleus inhibits pro-hepcidin expression. The decreased pro-hepcidin levels block the internalization and degradation of FPN1. The increased FPN1 level leads to the export of cellular Fe2+, limiting the activity of RNR, ultimately arresting the cell cycle before the S phase. Additionally, the decreased amount of P-Smad1/5/8 frees up Smad4 to bind to ATF3 and translocate it to the nucleus, thus activating p38 signaling and the expression of Bnip3, leading to apoptosis.

Article Snippet: The following antibodies were used: β-actin (1:10000, cw0096m, CWbio, Beijing, China), FtL (1:5000, ab109373, Abcam, SF, CA, USA), FtH (1:5000, ab183781, Abcam, USA), Hepcidin (1:5000, ab30760, Abcam, USA), Tmprss6 (1:10000, 12950-1-AP, Proteintech, Wuhan, China), FLAG (1:20000, 80010-1- RR, Proteintech, China), HJV (1:5000, 11758-1-AP, Proteintech, China), PSmad1/5/8 (1:1000, #9516, Cell Signaling Technology, St. Louis, MA, USA), Smad1 (1:1000, #6944, Cell Signaling Technology, USA), Smad4 (1:1000, #46535, Cell Signaling Technology, USA), FPN1 (1:5000, MTPP11-S, ADI, San Antonio, Texas, USA), TfR1(1:5000, 13-6800, Invitrogen, USA), Bcl-2 (1:5000, 26593-1-AP, Proteintech, China), Bax (1:5000, 50599-2-Ig, Proteintech, China), Caspase3 (1:3000, #9662 S, Cell Signaling Technology, USA), Cleaved-caspase3 (1:3000, #9664, Cell Signaling Technology, USA), ATF3 (1:1000, ABP55330, Abbkine, Wuhan, China), P-p38 (1:2000, #4511, Cell Signaling Technology, USA), P38 (1:2000, #8690 S, Cell Signaling Technology, USA), ACSL4 (1:5000, ab155282, Abcam, USA), GPX4 (1:5000, ab125066, Abcam, USA), RIP1 (1:5000, 17519-1-AP, Proteintech, China), RIP3 (1:5000, 17563-1-AP, Proteintech, China), protein marker (26617, Thermo, Carlsbad, CA, USA), anti-rabbit IgG (1:20000, RPN4301, Amersham, London, UK), anti-mouse IgG (1:20000, RPN4201, Amersham, UK).

Techniques: Inhibition, Over Expression, Phospho-proteomics, Expressing, Blocking Assay, Activity Assay