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

Journal: Nature communications

Article Title: Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERBα.

doi: 10.1038/s41467-022-29653-2

Figure Lengend Snippet: Fig. 10 Chlorpromazine recovers REV-ERBα level by rectifying hypermethylation of the Nr1d1 promoter through the inhibition of PI3K/AKT1 pathway in Psen2 N141I KI/+ microglia. a Methylation-specific PCR analysis on Nr1d1 promoter of KI/+ microglia after CPZ (0.5 μM) treatment for 12 h. 5-Aza (10 μM) as a positive control was treated for 24 h. Genomic DNA extracted from microglia was conversed using bisulfite administration. The agarose gel image is representative of three independent experiments with similar results. b Comparison of relative mRNA expression levels of DNA (de)methylases between WT and KI/+ primary microglia (n = 3) (WT vs KI/+: #p = 0.0161, #p = 0.0447, and #p = 0.0459; unpaired t-test). c, d Relative Dnmt3a mRNA (c; n = 3) and protein levels (d; n = 3) after CPZ treatment for 12 h in WT and KI/+ microglia (untreated WT vs untreated KI/+: ##p = 0.0013 and ##p = 0.0083; untreated KI/+ vs CPZ-treated KI/+: †††p < 0.0001; one-way ANOVA). e Western blotting analyses of the phosphorylation levels of AKT1 at S473, MAPK1/3 at T202/Y204, and GSK3β at S9 in WT and KI/+ microglia. f CPZ inhibits PI3K/AKT pathway, but not MAPK1/3 or GSK3β. Cells were analyzed after CPZ (0.5 μM) treatment for 12 h. g LY (LY294002, 20 μM) inhibits PI3K/AKT pathway and downregulates DNMT3A leading to the restoration of REV-ERBα levels. The blots shown are representative of three experiments with similar results. h Relative Nr1d1 mRNA expression levels after LY treatment for 12 h in WT and KI/+ microglia (n = 4). mRNA levels were normalized to 18s rRNA (b, c, h). i Schematic diagram suggesting hyperactive immune responses caused by the Psen2 N141I mutation. The mutation activates PI3K/AKT pathway and upregulates DNMTs, which induces the epigenetic suppression of REV-ERBα and hyperactive immune response in innate immune cells. An antipsychotic, CPZ, rescues REV-ERBα expression by DNA demethylation through the inhibition of PI3K/AKT1 pathway, and prevents hyperimmunity and cognitive decline in Psen2N141I/+ mice. Data are mean ± SEM. †††p < 0.001 vs untreated KI/+ control. #p < 0.05, ##p < 0.01, and ###p < 0.001 for the indicated comparisons. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: horseradish peroxidase–conjugated β-Actin (sc47778; 1:5000) from Santa Cruz Biotechnology; PSEN1 (5643; 1:1000), REV-ERBα (13418; 1:1000), DNMT3A (3598; 1:1000), AKT1 (2938; 1:2000), phosphorylated AKT1 (S473, 9271; 1:1000), MAPK1/3 (4969; 1:1000), phosphorylated MAPK1/3 (T203/Y205, 4370; 1:2000), GSK3β (9315; 1:1000) and phosphorylated GSK3β (9336; 1:1000) from Cell Signaling Technology; IBA-1 (019-19741; 1:250) from Wako Chemicals; N-cadherin (610920; 1:1000) from BD Bioscience; PSEN2 (ab51249; 1:2000) from Abcam; REV-ERBα (PA5-29865; 1:1000) from Thermo Fisher Scientific.

Techniques: Inhibition, Methylation, Positive Control, Agarose Gel Electrophoresis, Comparison, Expressing, Western Blot, Phospho-proteomics, Mutagenesis, Control

Journal: BMC Cancer

Article Title: Ibuprofen regulates the expression and function of membrane-associated serine proteases prostasin and matriptase

doi: 10.1186/s12885-015-2039-6

Figure Lengend Snippet: Effect of COX inhibitors and prostasin on matriptase expression. a & b RT-qPCR ( n = 3). The UROtsa cells were treated with different dosages of ibuprofen for 24 h or at 2 mM ibuprofen for different time periods. c RT-qPCR ( n = 3). The UROtsa cells were treated with COX inhibitors for 24 h. IBU: ibuprofen, 2 mM; COX-1(−): COX-1 inhibitor, 10 μM; COX-2(−): COX-2 inhibitor, 10 μM. a , b , and c The numbers above each bar indicate the fold change of the expression as compared to the DMSO control. ANOVA: p > 0.05. Note: the bars of DMSO and IBU in ( c ) are the same of those in ( a ). d Western blot analysis of matriptase in UROtsa treated with different dosages of ibuprofen for 24 h as indicated. Note the samples on the membrane were blotted with the anti-matriptase antibody after blotting with anti-COX-2 and anti-tubulin antibodies shown in Fig. . The previously blotted COX-2 protein bands is shown along with the matriptase protein bands. e Western blot analysis of prostasin, matriptase, HAI-1 expression in stable or transient prostasin over-expressing cell lines as indicated. Vec: vector control; Pro: prostasin over-expressing lines

Article Snippet: The primary antibodies used were human prostasin (1:4,000, Ref. 3), tubulin (1:5,000, Sigma-Aldrich), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:5,000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), matriptase (mouse monoclonal, 1:4,000, Santa Cruz Biotechnology), matriptase (rabbit polyclonal, 1:4,000, Bethyl Laboratories, Inc., Montgomery, TX) and COX-2 (1:1,000, R&D System, Inc., Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Membrane, Plasmid Preparation

Journal: BMC Cancer

Article Title: Ibuprofen regulates the expression and function of membrane-associated serine proteases prostasin and matriptase

doi: 10.1186/s12885-015-2039-6

Figure Lengend Snippet: Western blot analysis of prostasin and matriptase expression in UROtsa and human bladder transitional cell carcinoma cell lines (UC-9 and UC-5). The UROtsa cells were treated with 2 mM ibuprofen and cancer cells were treated with 0.5 mM ibuprofen for 24 h. Twenty-five micrograms of total protein from each sample were loaded in each lane. Samples from two separated experiments were shown. The numbers under each lane indicated the fold change of the expression as compared to the DMSO control. IBU: ibuprofen. GAPDH was used as a loading control

Article Snippet: The primary antibodies used were human prostasin (1:4,000, Ref. 3), tubulin (1:5,000, Sigma-Aldrich), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:5,000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), matriptase (mouse monoclonal, 1:4,000, Santa Cruz Biotechnology), matriptase (rabbit polyclonal, 1:4,000, Bethyl Laboratories, Inc., Montgomery, TX) and COX-2 (1:1,000, R&D System, Inc., Minneapolis, MN).

Techniques: Western Blot, Expressing, Control

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: PAR2 functional expression in human and mouse cells relevant to migraine . Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. ( A , D and G ) PAR2 agonists concentration response curve in ( A ) HDuF, ( D ) HDuMEC and ( G ) bEnd.3 cells. ( B , E and F ) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in ( B ) HDuF, ( E ) HDuMEC and ( H ) bEnd.3 cells. ( C , F and I ) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in ( C ) HDuF, ( F ) HDuMEC and ( I ) bEnd.3 cells. ( J and K ) Single-cell calcium imaging from mouse trigeminal neuron cultures. ( J ) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. ( K ) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Functional Assay, Expressing, Imaging, Concentration Assay, Control, Inhibition, Activity Assay, Comparison

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: ST14 expression in different datasets from patients with ovarian cancer. ( A ) Oncomine analysis of the mRNA expression levels of ST14 genes in different cancers. The differences in expression levels of genes between cancer and normal tissues are concluded. The thresholds are indicated in the colored cells. P < 0.05, fold-change > 2 and gene rank = 10% were considered statistically significant. Red cells represent overexpression of the target gene in tumor tissues compared to normal tissues, while blue cells indicate downregulation of the gene. Gene rank is depicted by the color depth in the cells. ( B ) UALCAN analysis of the mRNA expression levels of ST14 genes in different cancers. ( C ) GEPIA analysis of the mRNA expression levels of ST14 genes in different cancers. ( D ) ST14 DNA copy numbers based on chips for ovarian cancer research in TCGA Ovary. * P < 0.05. ( E ) Levels of ST14 mRNA in ovarian cancer based on research in the GEPIA websites (red for tumor, black for normal). The boxplot analysis showed the expression level by log2 (TPM + 1) on a log-scale. * P < 0.05. ( F ) Correlation between ST14 and TMEFF1 expression in ovarian cancer based on the GEPIA website. R = 0.32, *** P < 0.001. ( G ) The expression of ST14 and TMEFF1 in ovarian malignant tumor tissues and normal tissues detected by western blot. ( H ) Quantification of TMEFF1 normalized to GAPDH. Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing, Over Expression, Western Blot

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Expression and co-localization of ST14 and TMEFF1 in different ovarian tissues. ( A ) Immunohistochemical staining of ovarian malignant tumors (i, v), borderline tumors (ii, vi), benign tumors (iii, vii), and normal ovarian tissues (vi, viii). ST14 (i-iv) and TMEFF1 (v-viii) staining is shown (original magnification, ×400). ( B ) Dual-labeled immunofluorescence technology was used to detect the co-localization of ST14 and TMEFF1 in different ovarian tissues. Blue represents the nucleus, red represents ST14, green represents TMEFF1, orange represents the co-localization of ST14 and TMEFF1 (original magnification, ×400). Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) of the co-localization images: malignant tumors (Rr:0.64, R:0.87), borderline tumors (Rr:0.76,R:0.79), benign tumors (Rr:0.63,R:0.68), and normal ovarian tissues (Rr:0.76,R:0.92)

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing, Immunohistochemical staining, Staining, Labeling, Immunofluorescence

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Expression of TMEFF1 in different ovarian tissues

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Association between TMEFF1 expression and pathological features in ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Univariate Kaplan-Meier prognostic analysis of ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of patients with ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Relevance of ST14 and TMEFF1 expression in ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Co-localization and interaction of ST14 and TMEFF1 in ovarian cancer cells, and ST14 regulates the expression of TMEFF1. ( A ) Dual-labeled immunofluorescence technology was used to detect the co-localization of ST14 and TMEFF1 in ovarian cancer cell. Blue represents the nucleus, red represents ST14, green represents TMEFF1, and orange represents the co-localization of ST14 and TMEFF1 (original magnification, ×600). Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) of the co-localization images are Rr:0.73, R:0.61. (B-C) The cell lysates of CAOV3, OVCAR3, and SKOV3 cells were immunoprecipitated with an anti-TMEFF1 antibody ( B ) and anti-ST14 antibody ( C ), and then, western blotting was performed with an anti-ST14 antibody and anti-TMEFF1 antibody. “Input” is the total cell lysate of CAOV3 cells. “IgG” is the negative control. ( D ) In the ovarian cancer cell lines CAOV3 and SKOV3, the expression of TMEFF1 decreased after knocking down the ST14 gene. ( E ) Quantification of ST14 and TMEFF1 normalized to GAPDH. Representative images and accompanying statistical plots are presented. Blank, blank control group, untreated original cells; siST14, ST14 gene knockdown group (through siRNA); NC, negative control group, negative gene (no sequence homology with ST14) knockdown group (through siRNA). Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing, Labeling, Immunofluorescence, Immunoprecipitation, Western Blot, Negative Control, Control, Knockdown, Sequencing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Interaction between ST14 and TMEFF1 promotes proliferation, invasion and migration of ovarian cancer. ( A , B , E , F ) The invasion capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by Transwell assay. Number 1,2,3,4 respectively represents CAOV3-NC, CAOV3-siST14, CAOV3-NC + TMEFF1 and CAOV3-siST14 + TMEFF1; Number 5,6,7,8 respectively represents SKOV3-NC, SKOV3-siST14, SKOV3-NC + TMEFF1 and SKOV3-siST14 + TMEFF1. ( C , D , G , H ) The migration capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by Wound healing assay. Number 1,2,3,4 respectively represents CAOV3-NC, CAOV3-siST14, CAOV3-NC + TMEFF1 and CAOV3-siST14 + TMEFF1; Number 5,6,7,8 respectively represents SKOV3-NC, SKOV3-siST14, SKOV3-NC + TMEFF1 and SKOV3-siST14 + TMEFF1. ( I - J ) The proliferation capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by MTT assay. Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Migration, Recombinant, Transwell Assay, Wound Healing Assay, MTT Assay