rat anti notch1  (Cell Signaling Technology Inc)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Unraveling the Oncogenic Characteristics of the Cytolinker, Plectin, in Esophageal Squamous Cell Carcinoma

doi: 10.1016/j.jcmgh.2025.101549

Figure Lengend Snippet: Plectin expression during the Ca 2+ -dependent keratinocyte proliferation-differentiation process. ( A ) Schematic diagram depicting Ca 2+ -dependent keratinocyte proliferation-differentiation in vitro. D3 cells were cultured in the complete medium (1.16 mM/L Ca 2+ ). Then, D3 cells were treated with the conditioned medium (without Ca 2+ ) for 48 hours. Third, D3 cells were induced to differentiation by switching the conditioned medium to the differentiation medium (0.6 mM/L Ca 2+ ) for 48 or 72 hours. ( B ) Representative bright field images of cell morphology during the Ca 2+ -dependent proliferation-differentiation process and quantification of the thickness of cells in the z-axis direction with confocal microscopy (n = 5 random microscope fields; 63×). W/O Ca 2+ 48 hours, without Ca 2+ 48 hours; W/ Ca 2+ 48 hours, with Ca 2+ 48 hours. ∗∗ P < .01; ∗∗∗ P < .001. ( C ) Representative IF images of plectin/plectin-associated proteins in D3 cells during the Ca 2+ -dependent proliferation-differentiation process in vitro. ( D ) Quantification of mean fluorescence intensities of plectin/plectin-associated proteins (n = 5 random microscope fields; 40×). ns, not significant; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. ( E ) Protein expressions of plectin, p63, Col17A1, CK14, FL-Notch1, NICD1, IVL, CK13, and HES1 in D3 cells during the Ca 2+ -dependent proliferation-differentiation process by Western blotting assay. GAPDH was used as an endogenous control. The ratios were obtained by the comparison of the band intensities of target proteins with GAPDH. ( F ) Western blotting to examine the protein levels of plectin and Notch signaling after GSI treatment (0 μM, 0.2 μM, 1 μM, and 5 μM) in D3 cells. GSI is a γ-secretase inhibitor. β-actin was used as an endogenous control. The ratios were obtained by the comparison of the band intensities of target proteins with β-actin. ( G ) Cell cycle profiles of D3 cells were detected during the Ca 2+ -dependent proliferation-differentiation process using FACS (n = 3 experiments). ∗∗∗ P < .001.

Article Snippet: The primary antibodies were used against plectin (1:5000, Abcam; ab32528), TP63 (1:1000, Abcam, ab124762), Col17A1 (1:1000, Abcam, ab184996), Notch1 (1:1000, Cell Signaling Technology; #3447), HES1 (1:500, Signalway Antibody; 49016), CK13 (1:5000, Proteintech; 66684), IVL (1:1000, Proteintech, 55328), DSP (1:1000, Abcam, ab16434), and β-actin (1:5000, ZSGB-BIO; TA-09), respectively.

Techniques: Expressing, In Vitro, Cell Culture, Confocal Microscopy, Microscopy, Fluorescence, Western Blot, Control, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Unraveling the Oncogenic Characteristics of the Cytolinker, Plectin, in Esophageal Squamous Cell Carcinoma

doi: 10.1016/j.jcmgh.2025.101549

Figure Lengend Snippet: Plectin expression and localization in D3-organoids. ( A ) Schematic illustration of D3 organoid formation (day 1, 3), development (day 5, 7), and maturation (day 10) trajectory and representative bright field and HE images. Scale bars, 100 μm. ( B ) Expression and localization of plectin in D3-organoid cultured trajectory by IF assay. Scale bars, 50 μm. ( C ) Super-resolution IF image showing the localization of plectin at the basal and suprabasal layers of D3 organoid at the nanoscale level using N-STORM system in super-resolution microscopy. B, basal layer; SB, suprabasal layer. Scale bars, 5 μm. ( D ) IF images of CK14, Col17A1, ITGβ4, plectin, p63, CK13, DSP, and Notch1 in D3 organoid cultured trajectory. Scale bars, 50 μm.

Article Snippet: The primary antibodies were used against plectin (1:5000, Abcam; ab32528), TP63 (1:1000, Abcam, ab124762), Col17A1 (1:1000, Abcam, ab184996), Notch1 (1:1000, Cell Signaling Technology; #3447), HES1 (1:500, Signalway Antibody; 49016), CK13 (1:5000, Proteintech; 66684), IVL (1:1000, Proteintech, 55328), DSP (1:1000, Abcam, ab16434), and β-actin (1:5000, ZSGB-BIO; TA-09), respectively.

Techniques: Expressing, Cell Culture, Super-Resolution Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Unraveling the Oncogenic Characteristics of the Cytolinker, Plectin, in Esophageal Squamous Cell Carcinoma

doi: 10.1016/j.jcmgh.2025.101549

Figure Lengend Snippet: Plectin regulation by Notch signaling in esophageal squamous epithelial cells. ( A ) Heatmaps of the Notch1 CUT&Tag signal density at promoter (+767∼−4233 bp) of PLEC. ( B ) Binding motif of the transcription factor NICD-RBPJ according to the CUT&Tag data. ( C ) Location of predicted NICD-RBPJ binding sites at PLEC promoter (−1∼−4100 bp) by the Jaspar database. T refers to the top 10 NICD-RBPJ binding sites. C represents the conserved NICD-RBPJ binding sites. ( D ) The tables list the top 10 NICD-RBPJ binding sites according to the relative scores and the conserved NICD-RBPJ binding sites by the Jaspar database. ( E ) Sequence homology of 4 conserved NICD-RBPJ binding sites in Rattus norvegicus (Norway rat), Mus musculus (house mouse), and Homo sapiens (human). ( F ) Notch signaling regulates the expression of PLEC. The image on the left shows the wild-type (WT) sequence of putative RBPJ binding site (T4) in the PLEC promoter (−741∼−1990bp) and the corresponding mutant (Mut) sequence. Western blotting was used to examine the level of NICD1 in D3 cells after transfection of pcDNA3.1-NICD1 plasmid for 48 hours (intermediate image). β-actin serves as an endogenous control. The ratios were obtained by the comparison of the band intensities of target proteins with β-actin. The dual luciferase reporter assay was conducted to detect the luciferase activity of D3 cells transfected with pcDNA3.1-NICD1 and pGL3- PLEC-wild or pGL3- PLEC-mutant plasmids for 48 hours ( the right image ). Relative luciferase activity is calculated as the ratio of firefly luciferase activity to renilla luciferase activity. ∗∗∗ P <.001.

Article Snippet: The primary antibodies were used against plectin (1:5000, Abcam; ab32528), TP63 (1:1000, Abcam, ab124762), Col17A1 (1:1000, Abcam, ab184996), Notch1 (1:1000, Cell Signaling Technology; #3447), HES1 (1:500, Signalway Antibody; 49016), CK13 (1:5000, Proteintech; 66684), IVL (1:1000, Proteintech, 55328), DSP (1:1000, Abcam, ab16434), and β-actin (1:5000, ZSGB-BIO; TA-09), respectively.

Techniques: Binding Assay, Sequencing, Expressing, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Control, Comparison, Luciferase, Reporter Assay, Activity Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Unraveling the Oncogenic Characteristics of the Cytolinker, Plectin, in Esophageal Squamous Cell Carcinoma

doi: 10.1016/j.jcmgh.2025.101549

Figure Lengend Snippet: Aberrant expression and localization of plectin in human ESCC tissues. ( A ) Expression and localization of plectin, p63, Col17A1, DSP, and Notch1 in human ESCC tissues by IHC assay. ( B ) Representative IHC images of misexpression and mislocalization of plectin in ESCC. Scale bars, 100 μm. Arrowheads show the localization of plectin in human ESCC tissues. ( C ) Expression of Notch1/p63 in ESCC samples with plectin mutations. ( D ) Distribution of plectin, Notch1, p63 expressions in 171 human ESCC samples. ( E ) Cluster and correlation analysis of the expressions of plectin, Notch1, and p63 in 171 human ESCC samples by Pearson’s test. ( F ) Correlation analysis of PLEC with TP63 and Notch1 in esophageal carcinoma using GEPIA database. TPM, transcripts per million.

Article Snippet: The primary antibodies were used against plectin (1:5000, Abcam; ab32528), TP63 (1:1000, Abcam, ab124762), Col17A1 (1:1000, Abcam, ab184996), Notch1 (1:1000, Cell Signaling Technology; #3447), HES1 (1:500, Signalway Antibody; 49016), CK13 (1:5000, Proteintech; 66684), IVL (1:1000, Proteintech, 55328), DSP (1:1000, Abcam, ab16434), and β-actin (1:5000, ZSGB-BIO; TA-09), respectively.

Techniques: Expressing