Journal: Molecular Metabolism

Article Title: The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

doi: 10.1016/j.molmet.2024.101889

Figure Lengend Snippet: SerpinB3, lipid accumulation and development of murine NASH. Representative examples of morphological findings at sacrifice of the liver of a BALB/c wild-type mouse (WT) and of a mouse deficient of the reactive site loop of Serpinb3a (KO), fed with CDAA diet. A) liver macroscopic features, B) liver histology after hematoxylin eosin staining, E) collagen deposition assessed by Sirius red staining. The pictures provide evidence of the lower extent of steatosis in KO mouse, confirmed by the lower levels of steatosis scores detected by liver histology in KO mice (7 animals) compared to WT mice (7 animals) (C). Similar findings are reported for the profile of inflammatory genes (D), including IL-1β, CCL2 and TNF-α, and of fibrosis genes (G), including TGF−β, α-SMA and collagen 1A1. Densitometric analysis of collagen deposition measured after Sirius red staining (F) shows the lower extent of collagen deposition detected in KO mice, compared to WT mice. The results are reported as mean ± SEM (Unpaired t test with Welch's correction). Levels of mRNA gene expression are reported as 2 - ΔΔCT . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For Western blot in cellular extracts, the following antibodies were used: mouse anti-SerpinB3 monoclonal antibody (1:200, Origene, Rockville, MD), rabbit anti-C/EBP-β monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK), rabbit anti-PAR2 monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK).

Techniques: Staining, Gene Expression

Journal: Molecular Metabolism

Article Title: The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

doi: 10.1016/j.molmet.2024.101889

Figure Lengend Snippet: Dysregulation of fat distribution in mice with different expression of SerpinB3. A) Representative examples of subcutaneous or visceral white fat and of brown fat distribution are shown after skin removal in BALB/c mice lacking the Serpinb3a reactive site loop (KO) and in the corresponding BALB/c controls fed with normal diet. B) Values of relative adipose tissue in interscapular brown adipose tissue (BAT), subcutaneous adipose tissue (SAT) and in visceral adipose tissue (VAT), expressed as percentage of fat on total body weight (grams) in the WT and KO groups of mice. The results are reported as mean ± SEM (Unpaired t test with Welch's correction). C) Representative examples of subcutaneous or visceral white fat and of brown fat distribution in SerpinB3 transgenic (TG) mice and in the corresponding C57BL/6 controls fed with normal diet. D) Values of relative adipose tissue in interscapular brown adipose tissue (BAT), subcutaneous adipose tissue (SAT) and in visceral adipose tissue (VAT), expressed as percentage of fat on total body weight (grams) in the WT and TG groups of mice. The results are reported as mean ± SEM (Unpaired t test with Welch's correction). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For Western blot in cellular extracts, the following antibodies were used: mouse anti-SerpinB3 monoclonal antibody (1:200, Origene, Rockville, MD), rabbit anti-C/EBP-β monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK), rabbit anti-PAR2 monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK).

Techniques: Expressing, Transgenic Assay

Journal: Molecular Metabolism

Article Title: The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

doi: 10.1016/j.molmet.2024.101889

Figure Lengend Snippet: Inhibitory activity of 1-Piperidin Propionic acid on inflammatory response in mice fed with CDAA diet. A) Representative examples of macrophage infiltration, detected by F4/80 immunostaining, in the liver of control mice (WT) and of SerpinB3 transgenic mice (TG) injected or not with 1-PPA. B) Densitometric analysis of F4/80 immunostaining in the liver of the corresponding groups of mice (7 animals/group). C) mRNA levels of inflammatory genes, including TNF-α and IL-1β, in control mice (WT) or in SerpinB3 transgenic mice (TG), fed or not with CDAA diet and injected or not with 1-PPA. D) mRNA levels of markers of NAMs, Galectin 3 (GAL3), CD9 and TREM2, in control mice (WT) or in SerpinB3 transgenic mice (TG), fed or not with CDAA diet and injected or not with 1-PPA. E) Representative results of SerpinB3 immunostaining in the liver of control mice (WT) and of SerpinB3 transgenic mice (TG) fed with CDAA and injected or not with 1-PPA. Cytoplasmic and or nuclear brown color refers to SerpinB3 immune reactivity. The results are reported as mean ± SEM (Unpaired t test with Welch's correction). mRNA levels are expressed as 2 −ΔΔCT . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For Western blot in cellular extracts, the following antibodies were used: mouse anti-SerpinB3 monoclonal antibody (1:200, Origene, Rockville, MD), rabbit anti-C/EBP-β monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK), rabbit anti-PAR2 monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK).

Techniques: Activity Assay, Immunostaining, Control, Transgenic Assay, Injection

Journal: Molecular Metabolism

Article Title: The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

doi: 10.1016/j.molmet.2024.101889

Figure Lengend Snippet: Inhibitory effect of 1-Piperidin Propionic acid on fibrogenesis and inflammatory response in vitro . A) Profile of fibrosis genes (collagen 1A1, α-SMA, CCL-2 and VEGF-A) in LX2 cells in presence of SerpinB3 (SB3, 200 ng/ml) alone or mixed with 1-PPA at 100 ng/ml concentration (SB3 + 1-PPA) after 24 h incubation. B) Profile of inflammatory genes (TGF-β, CCL-15, TNF-α, IL-1β, IL-13, CCL-2) in THP1 cells exposed to human recombinant SerpinB3 (SB3, 100 ng/ml) alone or mixed with 1-PPA at 100 ng/ml concentration (SB3 + 1-PPA) after 24 h incubation. The results are reported as mean + SEM of at least three experiments (Unpaired t test with Welch's correction). mRNA levels are expressed as 2 - ΔΔCT .

Article Snippet: For Western blot in cellular extracts, the following antibodies were used: mouse anti-SerpinB3 monoclonal antibody (1:200, Origene, Rockville, MD), rabbit anti-C/EBP-β monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK), rabbit anti-PAR2 monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK).

Techniques: In Vitro, Concentration Assay, Incubation, Recombinant

Journal: Molecular Metabolism

Article Title: The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

doi: 10.1016/j.molmet.2024.101889

Figure Lengend Snippet: 1-Piperidin Propionic acid reduces CEBP-β transcription. A) CEBP-β expression in HepG2 cells transfected with the plasmid vector alone (HepG2/CTR) and in HepG2 cells transfected to overexpress SerpinB3 (HepG2/SB3) in presence of increasing concentrations of 1-PPA or of Medium alone. B) CEBP-β expression profile in the THP-1 cell line. This transcription factor, not detectable in THP-1 cells, is markedly induced by SerpinB3 and the effect is efficiently reverted by the addition of 1-PPA. C) Example of Western blot of the isoforms Liver Activating Protein (LAP) and Liver Inhibitory Protein (LIP) of CEPB-β in control HepG2 (empty vector), in HepG2 cells overexpressing SerpinB3 wild type (SB3/WT) and in HepG2 cells overexpressing SerpinB3 lacking the antiprotease activity (SB3/Δ7). D) Densitometric analysis of protein expression of Liver Activating Protein (LAP) and of Liver Inhibitory Protein (LIP) of CEPB-β, normalized to GAPDH, in control HepG2 cells (Empty vector), in HepG2 cells overexpressing SerpinB3 wild type (SB3/WT) and in HepG2 cells lacking the antiprotease activity (SB3/Δ7). E) CEBP-β expression profile in C57BL6/J and BALB/c wild type mice (WT mice), SerpinB3 transgenic mice (SB3/TG) and mice deficient of the reactive site loop of Serpinb3a (SB3/KO).

Article Snippet: For Western blot in cellular extracts, the following antibodies were used: mouse anti-SerpinB3 monoclonal antibody (1:200, Origene, Rockville, MD), rabbit anti-C/EBP-β monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK), rabbit anti-PAR2 monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Activity Assay, Transgenic Assay

Journal: Molecular Metabolism

Article Title: The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

doi: 10.1016/j.molmet.2024.101889

Figure Lengend Snippet: C/EBP-β and PAR2 in relation to different extent of expression of SerpinB3. Immunofluorescence results for C/EBP-β (Α) and for PAR2 (B) in HepG2 cells transfected with the plasmid vector alone (HepG2/CTR), in HepG2 cells transfected to overexpress SerpinB3 (HepG2/SB3), in HepG2 cells transfected with a plasmid vector carrying the SerpinB3 sequence deleted of 7aa in the reactive site loop (HepG2/SB3Δ7) and in the HA22T/VGH cells constitutively expressing SerpinB3. C) Effect of C/EBP-β silencing on the expression of C/EBP-β and of SerpinB3 (D) in the HA22T/HGV cell line. E) Representative results of immunostaining for PAR2 the liver in a wild type mouse (TG) and in SerpinB3 transgenic mice (TG/SB3) fed with CDAA and injected or not with 1-PPA. F) Densitometric analysis of PAR2 immunostaining in the liver of the corresponding groups of mice.

Article Snippet: For Western blot in cellular extracts, the following antibodies were used: mouse anti-SerpinB3 monoclonal antibody (1:200, Origene, Rockville, MD), rabbit anti-C/EBP-β monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK), rabbit anti-PAR2 monoclonal antibody (1:1000 dilution, ABCAM, Cambridge, UK).

Techniques: Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Sequencing, Immunostaining, Transgenic Assay, Injection

Journal: Cancer cell international

Article Title: Therapeutic effect of a MUC1-specific monoclonal antibody-drug conjugates against pancreatic cancer model.

doi: 10.1186/s12935-022-02839-w

Figure Lengend Snippet: Fig. 1 HzMUC1 antibody binds to MUC1 on the cell surface of pancreatic cancer cells. A Lysates from SW1990, Mia-PaCa-2, PATU-8988, PANC-1, CFPAC-1, and Capan-2 pancreatic cancer cell lines, and hTERT-HPNE (a normal epithelial pancreatic cell line) were resolved by SDS-PAGE and immunoblotted with the commercially available anti-MUC1 N-terminal (NT) antibody and reprobed with anti-β-actin antibody as loading control. B Cell lysates from Capan-2, CFPAC-1, and SW1990 cells were immunoprecipitated with human IgG or HzMUC1 antibody and immunoblotted with the anti-MUC1-NT antibody or anti-MUC1-CT antibody. C HzMUC1 antibody binds to MUC1 protein on the cell surface. Capan-2, CFPAC-1 and SW1990 cells grown on coverslips were washed with PBS, incubated with HzMUC1 antibody (1 µg/well) on ice, and subsequently with Alexa 488-conjugated secondary antibody, and examined with fluorescent (upper panels) and phase contrast (lower panels) confocal microscopy. Scale bars, 20 μm

Article Snippet: Anti-MUC1 mouse monoclonal antibody to MUC1-N (clone GP1.4; Catalog No. AM32842PU-S at 1:2000) was obtained from OriGene (Rockville, USA).

Techniques: SDS Page, Control, Immunoprecipitation, Incubation, Confocal Microscopy