Journal: The Journal of Biological Chemistry

Article Title: Matriptase-2- and Proprotein Convertase-cleaved Forms of Hemojuvelin Have Different Roles in the Down-regulation of Hepcidin Expression *

doi: 10.1074/jbc.M110.183160

Figure Lengend Snippet: sHJV generated by proprotein convertase cleavage suppresses BMP6-induced hepcidin expression, but sHJV produced by matriptase-2 does not. A, sHJV/BMP6 binding assay is shown. BMP6 (500 ng) alone, fHJV/PC (500 ng) alone, fHJV/M2 (500 ng) alone, or BMP6 (500 ng) in combination with fHJV/PC (500 ng) or fHJV/M2 (500 ng) was incubated at 4 °C overnight in 250 μl of 50 mm Tris-HCl, 150 mm NaCl, 0.2% Tween 20, pH 7.4. sHJV was pulled down with an anti-FLAG-M2 affinity gel (Sigma). sHJV and BMP6 in 2% of input, sHJV in 10% of eluate, and BMP6 in 90% of eluate were immunodetected using the corresponding antibodies. This experiment was performed three times with consistent results. B, immunoblot analysis of sHJV in the concentrated conditioned medium from HEK293, HEK293-HJV, and HEK293-HJV/matriptase-2 cells is shown. 10 μl of the media described in C was subjected to SDS-PAGE followed by immunodetection of HJV using chemiluminescence. C, sHJV produced by matriptase-2 does not suppress BMP6-induced hepcidin expression in HepG2 cells. HepG2 cells were subcultured into 12-well plates. After 48 h of culture, medium was switched to MEM/1% FBS and incubated for 6 h to serum-starve the cells. The medium was then changed to MEM/1% FBS with 10% concentrated conditioned medium in volume from HEK293 (C), HEK293-HJV (HJV) and HEK293-HJV/matriptase-2 cells (HJV/M2), respectively, as well as 2.5 ng/ml BMP6. The estimated final concentrations of sHJV in HJV and HJV/M2 groups were about 2 μg/ml. After 18 h of incubation, total RNA was isolated, and cDNA was prepared for quantitative RT-PCR analysis of hepcidin and GAPDH mRNA. The hepcidin mRNA levels are expressed as the amount relative to that of GAPDH in each specific sample. The results are from three separate experiments and the mean values ± S.D. (error bars) are presented.

Article Snippet: Immunoblot analyses on cell lysates, conditioned medium, and binding eluate were performed as described previously ( 22 ) using an affinity-purified rabbit anti-HJV 18745 antibody generated against residues 1–401 of human HJV (0.22 μg/ml) ( 21 ), a mouse anti-β-actin antibody (1:10,000; Chemicon International, Billerica, MA), a rabbit anti-matriptase-2 antibody (1 μg/ml), a goat anti-BMP6 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), and the corresponding horseradish peroxidase-conjugated (1:5,000) or Alexa Fluor 680-conjugated secondary antibodies (1:10,000) (Chemicon International).

Techniques: Generated, Expressing, Produced, Binding Assay, Incubation, Western Blot, SDS Page, Immunodetection, Isolation, Quantitative RT-PCR

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