Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 5. FGF19 induced NET formation by facilitating complement C5a and IL-1𝛽production in iCAFs. A) Cytokine array of the media of LX-2 cells pretreated with rhFGF19 (50 ng mL−1) for 24 h. Seven factors were upregulated in the supernatant of LX-2 cells stimulated with rhFGF19. B) Quantification of NETs formed by neutrophils stimulated with complement C5a, CXCL11, IL-1𝛼, IL-1𝛽, IL-18, MIF, or PAI-1 (n = 18 RMFs from 6 experimental replicates per group). C) Intracellular and D) extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with rhFGF19 or FGF19-containing CM (n = 3 for WB, or 6 for ELISA). E) Quantification of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with FGF19-containing CM in the presence of C5a neutralizing antibody (50 ng mL−1) or IL-1𝛽neutralizing antibody (1 μg mL−1) (n = 18 RMFs from 6 experimental replicates per group). F) Representative IHC staining of C5a or IL-1𝛽expression in paired primary CRC and CRLM tissues. Scale bar: 50 μm. G) Expression of complement C5a and IL-1𝛽were higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). H) Correlation between IHC scores of FGF19 and C5a or IL-1𝛽in human CRLM tissues (n = 30). I) ChIP‒qPCR assays showed the recruitment of STAT3 to the promoter regions of C5 and IL1B (n = 3). J,K) Intracellular and L) extracellular expression of C5a and IL-1𝛽in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM and treated with or without fisogatinib (100 nм), fedratinib (10 μм), C188-9 (5 μg mL−1), and anakinra (20 mg mL−1) (n = 3 for WB, or 6 for ELISA). M) Extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected to regular medium, regular medium with rhIL-1𝛼(1 ng mL−1), FGF19-free CM, fresh FGF19-containing CM, or anakinra for 24 h (n = 6). ** or ##p < 0.01; *** or ###p < 0.001; n.s., nonsignificant. In (B), (D), (I), (L), and (M), data were subjected to Student’s t-test. In (E), data were subjected to Mann–Whitney test. In (G), data were subjected to paired Student’s t-test. See also related Figure S6, Supporting Information.

Article Snippet: Human FGF19 neutralizing antibody (AF969, R&D Systems), human IL-1β neutralizing antibody (AF-201-NA, R&D Systems), and human complement C5a neutralizing antibody (MAB2037, R&D Systems) were used in this study.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 levels were increased in the serum of patients with myocardial fibrosis, in the hearts of mice after CFPMI and in cardiac fibroblasts stimulated with fibrotic factors. A . ADAMTS1 levels in clinical serum samples were evaluated via ELISA. N = 30. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. B . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. C . Survival rate of mice in each group. D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E and F . ADAMTS1 expression in mouse heart tissue was determined through Western blot and IHC. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 12, 24, or 48 h to construct in vitro models. The cells were divided into four groups: the Control, TGF-β1/Ang II (12 h), TGF-β1/Ang II (24 h), and TGF-β1/Ang II (48 h) groups. G and H . Western blot detection of the expression of ADAMTS1, Collagen I and FN. I. The expression levels of ADAMTS1, Collagen I and FN were measured via qRT‒PCR. After the optimal time point (48 h) was determined, the mice were further divided into the control, TGF-β1 and Ang II groups. J . ADAMTS1 expression in cells was assessed by IF. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 15, 30, 60, 90, and 120 min. K. qRT-PCR detection of the mRNA expression of ADAMTS1, Collagen I and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Staining, Expressing, Western Blot, In Vitro, Control, Quantitative RT-PCR

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: SMAD2 regulated ADAMTS1 expression in human and mouse cardiac fibroblasts induced by TGF-β1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 15, 30, 60, 90, and 120 min. A and B . Western blot analysis of the expression of SMAD2, p-SMAD2 and ADAMTS1. Human and mouse cardiac fibroblasts were treated with 10 ng/mL TGF-β1 for 48 h, and the cells were divided into Control and TGF-β1 groups. C . Human and mouse cardiac fibroblasts were induced with 10 ng/mL TGF-β1 for 12 h, 24 h, 48 h, and 72 h. qRT‒PCR was performed to detect the mRNA levels of ADAMTS1, Collagen I, and FN. D . IF staining was conducted to determine the expression of p-SMAD2 and ADAMTS1 at 48 h. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. E . p-SMAD2 expression in mouse heart tissue was measured by IHC. F . ChIP verification of the interaction between SMAD2 and ADAMTS1. Then, we interfered with SMAD2 expression. The groups were the si-NC, si-SMAD2-1, and si-SMAD2-2 groups. G . SMAD2 expression was assessed via qRT‒PCR. After the best si-SMAD2 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-SMAD2, and TGF-β1 + si-SMAD2 groups. H . Western blot detection of ADAMTS1, Collagen I, and FN expression in cells. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Staining, Construct

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Overexpression of ADAMTS1 enhanced the production of collagen fiber proteins in human and mouse cardiac fibroblasts induced by TGF-β1. We overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1 (48 h), oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. A . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. B-D . IF staining of Collagen I and FN expression. Additionally, we interfered with ADAMTS1; the cells were grouped as follows: si-NC, si-ADAMTS1-1, and si-ADAMTS1-2. E . ADAMTS1 expression was determined through qRT‒PCR. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. F and G . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. H and I . IF staining of Collagen I and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: Over Expression, Western Blot, Expressing, Staining

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Knockdown of ADAMTS1 alleviated TGF-β1-induced fibrosis by downregulating HDAC6 protein expression. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. A . HDAC6 expression in mouse heart tissue was determined through IHC. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC and si-ADAMTS1 groups. B . ADAMTS1 and HDAC6 expression was assessed via qRT‒PCR. C . Western blot analysis of ADAMTS1 and HDAC6 expression. Furthermore, we overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1, oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. D . Western blot detection of HDAC6 expression. After the si-ADAMTS1 group was selected, the cells were further divided into the si-NC, TGF-β1, si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. E. Western blot detection of HDAC6 expression. Moreover, cells were treated with the HDAC6 inhibitor ACY1215 (5 μM) for 48 h and then divided into the si-NC, si-ADAMTS1, si-NC + ACY1215, and si-ADAMTS1 + ACY1215 groups. F. Western blot detection of TGF-β1, Collagen I, FN, and ADAMTS1 expression. Subsequently, experiments were performed in human and mouse cardiac fibroblasts with ADAMTS1 overexpression followed by treatment with the HDAC6 inhibitor ACY1215. G. Western blot analysis was conducted to detect the expression levels of ADAMTS1 and HDAC6. H. Western blot analysis was performed to determine the expression levels of TGF-β1, Collagen I, and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: Knockdown, Expressing, Construct, Western Blot, Over Expression

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 interacted with HDAC6 during fibrosis. A . Co-IP verification of the interaction of ADAMTS1 with HDAC6, with ADAMTS1 as the bait protein. In the experiment, we first captured the ADAMTS1 protein using a specific antibody and then used Co-IP technology to detect its interaction with HDAC6. B . Co-IP verification of the interaction of HDAC6 with ADAMTS1, with HDAC6 as the bait protein. In the experiment, we captured the HDAC6 protein using a specific antibody and then used Co-IP technology to detect its interaction with ADAMTS1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 48 h, and the cells were further divided into control and TGF-β1 groups. C . IF staining was performed to evaluate the colocalization of ADAMTS1 and HDAC6 in TGF-β1-treated human and mouse cardiac fibroblasts. D . Changes in the level of ubiquitinated HDAC6 protein in human and mouse cardiac fibroblasts transfected with oe-ADAMTS1/si-ADAMTS1 or oe-NC/si-NC in the presence of 10 μM MG132. E. In si-ADAMTS1-treated human and mouse cardiac fibroblasts, MG132 (10 μM) was added to detect the ubiquitination levels of ADAMTS1 protein. F. Human and mouse cardiac fibroblasts were treated with TGF-β1 and subjected to SMAD2 knockdown to determine the ubiquitination levels of ADAMTS1 protein. N = 3. *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Control, Staining, Transfection, Ubiquitin Proteomics, Knockdown

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-ADAMTS1 treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, and CFPMI + sh-ADAMTS1 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. D . HDAC6 expression was determined through qRT‒PCR. E. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. F. Western blot detection of Collagen I, and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: shRNA, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-HDAC6 transfection combined with ADAMTS1 inhibitor treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, CFPMI + anti-ADAMTS1, CFPMI + sh-HDAC6, and CFPMI + anti-ADAMTS1 + sh-HDAC6 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C and D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E. HDAC6 expression was determined through qRT‒PCR. F. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. G. Western blot detection of Collagen I and FN expression. N = 3. ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: shRNA, Transfection, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: TGF-β1/SMAD2 regulated ADAMTS1 by mediating CFPMI through HDAC6 ubiquitination

Article Snippet: Primary antibodies against VIM (10,366–1-AP, 1:200, Proteintech), HDAC6 (12,834–1-AP, 1:50, Proteintech), ADAMTS1 (12,749–1-AP, 1:50, Proteintech), FN ( AWA10327 , 1:50, Abiowell), Collagen I (ab138492, 1:200, Abcam), and p-SMAD2 (ab280888, 1:50, Abcam) were incubated at 4 °C overnight.

Techniques: Ubiquitin Proteomics

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: Figure 4. Chalcone 21-21 inhibits the expression of melanogenesis-related proteins. (A,B) B16F10 cells were incubated for 24 h with vehicle (0.1% DMSO) alone or various concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). The levels of TRP1, TRP2, and MITF were determined by Western blot or RT-PCR analyses. GAPDH served as a loading control. (C) B16F10 cells were transiently transfected with a reporter construct of MITF together with the pRL-TK. The cells were incubated for 24 h with vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). Luciferase activity was measured and firefly luciferase activities were normalized to that of Renilla luciferase. Results are represented as mean ±SD from three independent experiments (n = 3). p-values of # p < 0.005 compared to the vehicle-treated group and * p < 0.05 and ** p < 0.005 compared to the α-MSH-treated group were considered statistically significant.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Construct, Luciferase, Activity Assay

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: Figure 6. A schematic diagram illustrating the proposed action mechanisms of chalcone 21-21. Chalcone 21-21 specifically targets the ERK/CREB signaling, thereby inhibiting MITF and MITF-regulated target genes including those encoding tyrosinase, TRP1, and TRP2. AC, adenylate cyclase; α-MSH, alpha-melanocyte stimulating hormone; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MC1R, melanocortin receptor type 1; MITF, microphthalmia-associated transcription factor; PKA, protein kinase A; ROS, reactive oxygen species; TRP, tyrosinase-related protein; TYR, tyrosinase.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (A) Downregulation of SASH1 was induced by the Y551D SASH1 mutation in homozygous mice of the F2 generation compared to that of wild-type mice. GNAS and Mitf expression was attenuated by the downregulation of SASH1; however, phospho-ERK1/2 expression was increased (left panel). The total densitometry values of these proteins were also compared collectively (lower right panel). Upper right panel: # P<0.01 vs. all 3 wild-type mice, ** P<0.001 vs. all 3 wild-type mice; ns, not significant vs. all 3 wild-type mice. Lower right panel: # P<0.01, homozygous mice vs. wild-type mice, ** P<0.001 vs. all 3 wild-type mice. (B) SASH1, GNAS and Mitf expression was not attenuated by the Y551D-SASH1 mutation in the F3 generation, as indicated by western blot and statistical analyses. ns: No significance vs. all four wild-type mice. Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (C-E) Tail biopsies of wild-type mice and heterozygous mice of the F2 generation were lysed, ultrasonicated and subjected to western blot analysis. The results of western blot analysis indicated that the expression of SASH1, GNAS, phospho-ERK1/2 and Mitf was enhanced in the heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice of the F2 generation compared to wild-type mice. (D) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 3 wild-type mice were compared, and (E) the total densitometry values of these proteins were also compared collectively. (D) * P<0.05 vs. all 3 wild-type mice, # P<0.01 vs. all 3 wild-type mice, ** P<0.001 vs. all 3 wild-type mice. (E) * P<0.05 vs. wild-type mice; # P<0.01 vs. wild-type mice, ** P<0.001 vs. wild-type mice. Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (F-H) Western blot analysis revealed that the expression of Mitf was enhanced in heterozygous human Y551D SASH1 gene knock-in mice of the F3 generation compared with wild-type mice. (G) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 10 heterozygous and 4 wild-type mice were compared, and (H) the total densitometry values of these proteins were also compared collectively. (G) * P<0.05 vs. all 3 wild-type mice, # P<0.01 vs. all 3 wild-type mice; ** P<0.001 vs. all 3 wild-type mice. (H) ** P<0.001 vs. wild-type mice. For all panels, ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Gene Knock-In, Mutagenesis, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf, but not GNAS, and phospho-ERK1/2 expression is increased in heterozygous human Y551D SASH1 knock-in mice compared to homozygous mice. (A) Western blot analysis indicated that SASH1 was downregulated in heterozygous human Y551D-SASH1- knock-in mice of the F2 generation compared to homozygous mice. However, the protein levels of phospho-ERK1/2 and Mitf were upregulated in heterozygous mice compared to those of homozygous mice. (B and C) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 2 homozygous mice of the F2 generation were compared, and the total densitometry values of these proteins were also compared collectively, respectively. (B) * P<0.05 vs. both homozygous mice, # P<0.01 vs. both homozygous mice; ** P<0.001 vs. both homozygous mice. (C) # P<0.01 vs. homozygous mice, ** P<0.001 vs. homozygous mice, * P<0.05 vs. homozygous mice. (D) Western blot analysis demonstrated that the expression of Mitf and GNAS was enhanced in heterozygous human Y551D SASH1 knock-in mice of the F3 generation compared to homozygous mice. Mitf, but not GNAS, and phospho-ERK1/2 expression is increased in heterozygous human Y551D SASH1 knock-in mice compared to homozygous mice. (E and F) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 1 homozygous F3 generation mice were compared. (F) The total gray values of these proteins were also compared collectively. (E) * P<0.05 vs. one homozygous mouse, respectively, # P<0.01 vs. one homozygous mouse, respectively, ** P<0.001 vs. one homozygous mouse, respectively. (F) ** P<0.001 vs. homozygous mice. (G) The densitometry values of SASH1, Mitf, GNAS and the ratio of p-ERK/ERK in 11 heterozygous mice of the F2 generation and 10 heterozygous mice of the F3 generation were compared. For all panels, ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Expressing, Knock-In, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: In the F2 generation, the number of Mitf- and Mitf-positive epithelial cells was enhanced in heterozygous human Y551D SASH1 knock-in mice. (A) Representative images (magnification, ×10) of Mitf in 3 wild-type mice, 2 homozygous mice and 11 heterozygous mice. (B) A total of 5 random visual fields in each section of 16 mice, including wild-type, homozygous and heterozygous mice, were photographed. The Mitf-positive epithelial cells in the tail tissues of wild-type, homozygous and heterozygous mice were counted and analyzed statistically. Representative Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. * P<0.01. (C) The staining intensity and percentage of Mitf-positive cells per mouse were calculated, scored and analyzed statistically. * P<0.05, *** P<0.001; ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Knock-In, Staining

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: In the F3 generation, the number of Mitf- and Mitf-positive epithelial cells was augmented in heterozygous human Y551D SASH1 knock-in mice. (A) Representative images (magnification, ×10) of Mitf in 4 wild-type mice, 1 homozygous mouse and 11 heterozygous mice. (B) A total of 51 visual fields in each section of 15 mice, including wild-type, homozygous and heterozygous mice, were photographed. The Mitf-positively stained epithelial cells in the tail tissues of wild-type, homozygous and heterozygous mice were calculated in 3 visual fields and analyzed statistically. Representative Mitf-positive cells, which were stained yellowish-brown in the nucleus, are indicated by red arrows. * P<0.01. (C) Staining intensity and percentage of Mitf-positive cells per mouse were calculated, scored and analyzed statistically. ** P<0.01 *** P<0.001; ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Knock-In, Staining

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf expression is promoted by mutated SASH1 in vitro and in epithelial tissues affected by the Y551D-SASH1 mutation. (A) Exogenous SASH1 is not associated with endogenous Mitf in 293T cells. GFP-SASH1 was transfected into 293T cells. At 48 h following transfection, transfected cells were lysed, GFP-SASH1 was immunoprecipitated, and the associated endogenous Mitf was analyzed by IP-WB analyses. (B) Expression of endogenous Mitf was induced by Y551D SASH1. Exogenous Y551D SASH1 and a wild-type SASH1 were introduced into 293T cells. Following transfection, transfected cells were lysed and subjected to western blot analyses. ** P<0.001 vs. wild-type SASH1 and *** P<0.001 vs. blank control. (C) In the lesional epithelial tissues of Y551D SASH1 -affected individuals, SASH1- and Mitf-positive cells were demonstrated in different epithelial layers of the affected epithelial tissues and calculated and analyzed statistically. ** P<0.01, *** P<0.001. Upregulation of SASH1 and enhanced Mitf were also induced in the affected tissues. Magnification, ×40. Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. (D) More melanin was synthesized and present in different epithelial layers of the affected skin epithelial tissues. Magnification, ×40.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Expressing, In Vitro, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Control, Staining, Synthesized