Journal: bioRxiv

Article Title: Live-cell invasive phenotyping uncovers the ALK2/BMP6 iron homeostasis pathway as a therapeutic vulnerability in LKB1-mutant lung cancer

doi: 10.1101/2023.06.14.544941

Figure Lengend Snippet: A) Schematic depicting pre-clinical trial workflow. B) Brightfield images of tumors isolated from vehicle and LDN214117 treated mice. C) Mean tumor volume from mice treated with vehicle ( 6 mice/group) and LDN214117 (7 mice/group). D) Graph depicting mean tumor weight from vehicle and LDN214117 treated mice. E) Western blot to assess BMP6 and hepcidin expression level in tumors from vehicle and LDN214117-treated mice. F) Representative images of immunohistochemistry for BMP6 and Hepcidin on tumor sections from mice treated with vehicle or LDN214117. F) Model depicting mechanism of altered iron homeostasis in LKB1-mutant tumor cells.

Article Snippet: Hepcidin Antimicrobial Peptide antibody (NBP1-59337) was from Novus Biologicals.

Techniques: Isolation, Western Blot, Expressing, Immunohistochemistry, Mutagenesis

Journal: Blood Advances

Article Title: Plasma hepcidin is associated with future risk of venous thromboembolism

doi: 10.1182/bloodadvances.2018018465

Figure Lengend Snippet: Correlation matrix for RDW, hepcidin, FtL, hemoglobin, and CRP

Article Snippet: Plasma samples were thawed, and enzyme-linked immunosorbent assays were used to measure levels of hepcidin (catalog #DY8307-05; R&D Systems, Stillwater, MN), FtL (catalog #H00002512-AP49 and recombinant protein H00002512-P01; Abnova, Taipei, Taiwan), and C-reactive protein (CRP; catalog #DY1707; R&D Systems) in a 384 format using a combination of a CyBi SELMA (CyBio, Jena, Germany), EL406 washer/dispenser (Biotek, Winooski, VT), and Synergy H2 microplate reader (Biotek).

Techniques:

Journal: Blood Advances

Article Title: Plasma hepcidin is associated with future risk of venous thromboembolism

doi: 10.1182/bloodadvances.2018018465

Figure Lengend Snippet: ORs with 95% CIs for VTE by categories and per SD of hepcidin and FtL

Article Snippet: Plasma samples were thawed, and enzyme-linked immunosorbent assays were used to measure levels of hepcidin (catalog #DY8307-05; R&D Systems, Stillwater, MN), FtL (catalog #H00002512-AP49 and recombinant protein H00002512-P01; Abnova, Taipei, Taiwan), and C-reactive protein (CRP; catalog #DY1707; R&D Systems) in a 384 format using a combination of a CyBi SELMA (CyBio, Jena, Germany), EL406 washer/dispenser (Biotek, Winooski, VT), and Synergy H2 microplate reader (Biotek).

Techniques:

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 1. SMILE inhibits hepcidin expression by IL-6 signal. (a) Effect of SMILE on IL-6-induced hepcidin promoter activity. HepG2 cells were transfected with expression vectors encoding hepcidin promoters (hHpeicidn-luc; 100 ng or mHepcidin-luc; 200 ng) and pcDNA3-FLAG-hSMILE (3, 300 ng; 5, 500 ng) and then treated with IL-6 (20 ng/mL) for 12 h. (b) Effect of SMILE on JAK2-mediated hepcidin promoter activity. HepG2 cells were transfected with expression vectors encoding hepcidin promoters, JAK2 (+, 200 ng with hHepcidin-luc or 400 ng with mHepcidin-luc) and pcDNA3-FLAG-hSMILE (1, 100 ng; 3, 300 ng; 5, 500 ng). (c) Effect of SMILE on STAT3-induced hepcidin promoter activity. HepG2 cells were transfected with expression vectors encoding hepcidin promoters, STAT3-c (+, 200 ng with hHepcidin-luc or 500 ng with mHepcidin-luc) and pcDNA3-FLAG-hSMILE (1, 100 ng; 3, 300 ng; 5, 500 ng). (d–e) Inhibitory effect of SMILE on hepcidin mRNA expression. HepG2 cells (d) and AML12 cells (e) were infected with Ad-GFP (50, 100 MOI) or Ad-SMILE (30, 50, 100 MOI) for 36 h and then treated with IL-6 (20 ng/mL) for 12 h. All experiments were performed in duplicate or triplicate and repeated at least three times. The values are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer’s protocols.

Techniques: Expressing, Activity Assay, Transfection, Infection, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 2. SMILE suppresses IL-6-induced hepcidin secretion. (a–f) Huh7 and AML12 cells were transfected with vector encoding SMILE (5 µg) and treated with IL-6 for 12 h after 2 h of serum starvation. Secreted hepcidin was measured using ELISA from culture medium. Ferroportin (FPN) expression was analyzed using western blot. Hepcidin secretion (a), FPN expression (b) and graphical representation (c) in Huh7 cells. Hepcidin secretion (d), FPN expression (e) and graphical representation (f) in AML12 cells. All gels for western blot analysis in (b,e) were run under the same experimental conditions including equal amounts (100 µg) of protein. The independent experiments were repeated at least three times. The values are presented as means ± SD. ns; not significant. * P < 0.05, ** P < 0.01 using two-tailed Student’s t-test.

Article Snippet: Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer’s protocols.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 3. SMILE interacts with and represses STAT3 binding to hepcidin promoter. (a) Subcellular co-localization of SMILE and STAT3. HepG2 cells were co-transfected with vectors encoding GFP- SMILE and FLAG STAT3 and treated with IL-6 for 12 h. Scale bar shows 10 µm. (b) Relative fluorescence of SMILE (left) and FoxO1 (right) between nucleus and cytoplasm. (c) Co-immunoprecipitation analysis showing interaction between SMILE and STAT3. HepG2 cells were transfected with vectors encoding HA-SMILE and FLAG-STAT3 and treated with IL-6 for 12 h. Gels for western blot analysis were run under the same experimental conditions. (d) ChIP assay showing inhibitory effects of SMILE on STAT3 binding activity to hepcidin promoter. HepG2 cells were transfected with vectors encoding hepcidin promoter, HA-SMILE and FLAG-STAT3 and then treated with IL-6 for 12 h. Soluble chromatin was immunoprecipitated using anti-FLAG antibody. ChIP signals were measured using Q-PCR. STAT3-RE, STAT3-response element. The values are presented as means ± SD. ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer’s protocols.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 6. Effects of SMILE knockdown on inhibiting hepcidin expression by EGCG. (a) EGCG effect on IL-6-induced hepcidin expression in mouse primary hepatocytes treated with IL-6 (20 ng/mL) and EGCG (100 µM) for 12 h. (b) Q-PCR analysis showing efficiency of SMILE knockdown. HepG2 cells were transfected with si-Con and si-SMILE for 48 h. (c) Western blot analysis showing efficiency of SMILE knockdown. HepG2 cells were transfected with si-Con and si-SMILE for 66 h. (d) Transient transfection analysis showing the effect of SMILE knockdown on EGCG-reduced hepcidin promoter activity. HepG2 cells were transfected with Hepcidin-luc (200 ng) and si-SMILE and then treated with IL-6 (20 ng/mL) and EGCG (100 µM) for 12 h. (e) Q-PCR analysis showing SMILE knockdown effect on EGCG-mediated inhibition of hepcidin expression. HepG2 cells were transfected with si-SMILE and then treated with IL-6 (20 ng/mL) and EGCG (100 µM) for 12 h. The independent experiments were repeated at least twice. The values are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer’s protocols.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Activity Assay, Inhibition, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 7. EGCG effects on LPS-induced iron metabolism in mice. (a–h) C57BL/6J mice were intraperitoneally injected with LPS (1 mg/kg, n = 7) for 12 h after 2 h of the PBS (n = 5) and EGCG (100 mg/kg, n = 5, EGCG-LPS: n = 6) injection. Serum iron levels (a). Hepcidin mRNA levels in liver (b). Serum hepcidin levels (c). IL-6, FoxO1 and SMILE mRNA levels in liver (d–f). Western blot analysis showing FoxO1 and SMILE expression (g). Graphical representation of FoxO1 (right) and SMILE (left) (h). mRNA levels were measured using Q-PCR. Gels for western blot analysis were run under the same experimental conditions. The values are presented as means ± SEM in (a–f) and ±SD in h. ns; not significant (Con vs. EGCG). * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer’s protocols.

Techniques: Injection, Western Blot, Expressing, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 8. A model for SMILE function in IL-6-mediated hepcidin expression. SMILE expression is increased by EGCG-mediated induction of FoxO1 expression. In addition, SMILE expression inhibits STAT3 binding to hepcidin promoter and subsequently contributes to suppression of hepcidin production and secretion triggered by the IL-6 signal in hepatocytes.

Article Snippet: Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer’s protocols.

Techniques: Expressing, Binding Assay

Journal: Gut Microbes

Article Title: Underlying MASLD-induced gut microbiome dysbiosis and intestinal inflammation are key to poor outcomes in vibriosis infections in a preclinical model

doi: 10.1080/19490976.2026.2652474

Figure Lengend Snippet: Establishment of MASLD pathophysiology in mice by CD-HFD feeding for 20 weeks. (A) Schematic representation of the experimental mouse groups: LEAN [ n = 6; chow diet-fed mice for 20 weeks] and MASLD [ n = 6; CD-HFD fed mice for 20 weeks]. (B) Liver weight (grams), and (C) liver-to-body weight ratio of the LEAN and MASLD mouse groups. Serum levels of (D) ALT (U/L), (E) AST (U/L), (F) hepcidin (ng/mL), and (G) ferritin (μg/mL) in the LEAN and MASLD groups. Formalin-fixed, paraffin-embedded 5 μm liver slices from the LEAN and MASLD groups were used for histopathological analyses. Representative images of (H) hematoxylin and eosin (H&E) staining, picrosirius red (PSR) staining, and immunohistochemistry images depicting α -SMA, and IL-1β immunoreactivity (indicated by black arrowheads) in the liver sections of LEAN and MASLD mouse groups. H&E and immunohistochemistry images were captured at 200× magnification, whereas PSR images were captured at 100× magnification. (I) NAFLD activity score (NAS) and fibrosis score for the LEAN and MASLD groups. Morphometric analyses (calculated as %ROI) of (J) PSR staining, (K) α -SMA, and (L) IL-1β immunoreactivity, where the Y-axis represents % positive immunoreactive area ( n = 3; mean value taken from three separate microscopic fields). The data are presented as mean ± SEM, and statistical significance was tested using unpaired t-test between the two groups, followed by Bonferroni post-hoc corrections (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Serum hepcidin (ng/mL) (Catalog No. NBP2-82129, Novus Biologicals, Centennial, CO, USA), ferritin (μg/mL) (Catalog No. KA1941, Novus Biologicals, Centennial, CO, USA), immunoglobulin-A (IgA) (μg/mL) (Catalog No. ab157717, Abcam, Cambridge, MA, USA) and C-reactive protein (CRP) (μg/mL) (Catalog No. MCRP00, Novus Biological, Centennial, CO, USA) were quantified as per the manufacturers’ instructions using commercially available ELISA kits.

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Activity Assay

Journal: Gut Microbes

Article Title: Underlying MASLD-induced gut microbiome dysbiosis and intestinal inflammation are key to poor outcomes in vibriosis infections in a preclinical model

doi: 10.1080/19490976.2026.2652474

Figure Lengend Snippet: Underlying MASLD conditions in mice caused increased non-cholera vibriosis and affected the hepatic pathophysiology. (A) Schematic representation of the experimental mouse groups: LEAN [ n = 6; chow diet-fed mice for 20 weeks], LEAN + VV [ n = 6; mice fed with chow diet for 20 weeks and received oral VV inoculation for 24 h], MASLD [ n = 6; CD-HFD fed mice for 20 weeks], and MASLD + VV [ n = 6; mice fed with CD-HFD for 20 weeks and received oral VV inoculation for 24 h]. (B) Liver weight (grams), and (C) liver-to-body weight ratio of the LEAN, LEAN + VV, MASLD, and MASLD + VV mouse groups. Serum levels of (D) CRP (μg/mL), (E) ALT (U/L), (F) AST (U/L), (G) hepcidin (ng/mL), and (H) ferritin (μg/mL) in the LEAN, LEAN + VV, MASLD, and MASLD + VV mouse groups. Formalin-fixed, paraffin-embedded 5 μm liver slices from the LEAN, LEAN + VV, MASLD, and MASLD + VV mouse groups were used for histopathological analyses. Representative images of (I) hematoxylin and eosin (H&E) staining and Picrosirius red (PSR) staining of liver sections of LEAN, LEAN + VV, MASLD, and MASLD + VV mouse groups. H&E images were captured at 200× magnification, whereas PSR images were captured at 100× magnification. (J) NAFLD activity score (NAS) and fibrosis score for the LEAN, LEAN + VV, MASLD, and MASLD + VV mouse groups. (K) Morphometric analyses (calculated as %ROI) of PSR staining, where the Y-axis represents % positive immunoreactive area ( n = 3; mean value taken from three separate microscopic fields). Data were represented as mean ± SEM, and statistical significance was tested using one-way ANOVA between all the groups, followed by Bonferroni post-hoc corrections (ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Serum hepcidin (ng/mL) (Catalog No. NBP2-82129, Novus Biologicals, Centennial, CO, USA), ferritin (μg/mL) (Catalog No. KA1941, Novus Biologicals, Centennial, CO, USA), immunoglobulin-A (IgA) (μg/mL) (Catalog No. ab157717, Abcam, Cambridge, MA, USA) and C-reactive protein (CRP) (μg/mL) (Catalog No. MCRP00, Novus Biological, Centennial, CO, USA) were quantified as per the manufacturers’ instructions using commercially available ELISA kits.

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Activity Assay

Journal: Gut Microbes

Article Title: Underlying MASLD-induced gut microbiome dysbiosis and intestinal inflammation are key to poor outcomes in vibriosis infections in a preclinical model

doi: 10.1080/19490976.2026.2652474

Figure Lengend Snippet: FMT in mice with underlying MASLD conditions showed improved pathophysiological outcomes Compared to non-cholera vibriosis infection. (A) Schematic representation of the experimental mouse groups: LEAN + VV [ n = 6; mice fed with chow diet for 20 weeks and received oral VV inoculation for 24 h], LEAN + FMT + VV [ n = 6; chow diet-fed mice, first treated with the ABX cocktail for 15 d followed by FMT for 7 d, and received oral VV inoculation for 24 h], MASLD + VV [ n = 6; mice fed with CD-HFD for 20 weeks and received oral VV inoculation for 24 h], and MASLD + FMT + VV [ n = 6; CD-HFD fed mice, first treated with the ABX cocktail for 15 d followed by FMT for 7 d, and received oral VV inoculation for 24 h]. (B) Liver weight (grams), and (C) liver-to-body weight ratio of the LEAN + VV, LEAN + FMT + VV, MASLD + VV, and MASLD + FMT + VV mouse groups. Serum levels of (D) CRP (μg/mL), (E) ALT (U/L), (F) AST (U/L), (G) Hepcidin (ng/mL), (H) Ferritin (μg/mL) in the LEAN + VV, LEAN + FMT + VV, MASLD + VV, and MASLD + FMT + VV mouse groups. Formalin-fixed, paraffin-embedded 5 μm liver slices were used for histopathological analyses. Representative images of (I) hematoxylin and eosin (H&E) staining and picrosirius red (PSR) staining in the liver sections from the LEAN + VV, LEAN + FMT + VV, MASLD + VV, and MASLD + FMT + VV mouse groups. H&E images were captured at 200× magnification, whereas PSR images were captured at 100× magnification. (J) NAFLD activity score (NAS) and fibrosis score for the LEAN + VV, LEAN + FMT + VV, MASLD + VV, and MASLD + FMT + VV mouse groups. (K) Morphometric analyses (calculated as %ROI) of PSR staining, where the Y-axis represents % positive immunoreactive area ( n = 3; mean value taken from three separate microscopic fields). The data are presented as mean ± SEM, and statistical significance was tested using one-way ANOVA between all the groups, followed by Bonferroni post-hoc corrections (ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Serum hepcidin (ng/mL) (Catalog No. NBP2-82129, Novus Biologicals, Centennial, CO, USA), ferritin (μg/mL) (Catalog No. KA1941, Novus Biologicals, Centennial, CO, USA), immunoglobulin-A (IgA) (μg/mL) (Catalog No. ab157717, Abcam, Cambridge, MA, USA) and C-reactive protein (CRP) (μg/mL) (Catalog No. MCRP00, Novus Biological, Centennial, CO, USA) were quantified as per the manufacturers’ instructions using commercially available ELISA kits.

Techniques: Infection, Formalin-fixed Paraffin-Embedded, Staining, Activity Assay