Journal: eBioMedicine

Article Title: Chronic lung inflammation and CK14+ basal cell proliferation induce persistent alveolar-bronchiolization in SARS-CoV-2-infected hamsters

doi: 10.1016/j.ebiom.2024.105363

Figure Lengend Snippet: Highly elevated N otch signaling long after SARS-CoV-2 infection in hamster lung . a. Relative mRNA expression levels of Notch signaling related genes in the lung tissues in SARS-CoV-2 and IAV infected hamsters. n = 5 for IAV 42&120dpi, 6 for SARS-CoV-2 42dpi, 4 for SARS-CoV-2 120dpi. Data represents mean ± SD. b. Representative images of immunofluorescence stained Notch3 (green) in mock and SARS-CoV-2 infected hamster lung at 7, 14, 42, 84 and 120dpi. Scale bar = 100 μm or 20 μm. White triangles indicate Notch3 positive cells. White arrows indicate Notch3 signals inside nuclei. c. Representative magnified images of immunofluorescence stained Notch3 (green) captured by confocal microscopy in SARS-CoV-2 infected hamster lung at 14, 42, 84 and 120dpi. Scale bar = 5 μm or 10 μm. White arrows indicate Notch3 signals inside nuclei. d. Representative images of immunofluorescence stained Hes1 (green) in mock and SARS-CoV-2 infected hamster lung at 42 and 120dpi. Scale bar = 100 μm, 50 μm or 20 μm. White arrows indicate Notch3 positive cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by Two-way ANOVA with Tukey's multiple comparisons test (a).

Article Snippet: Viral antigens and cellular proteins were detected by immunohistochemistry or immunofluorescence with specific antibodies: home-made rabbit/mouse anti-SARS-CoV-2 nucleocapsid protein antibody, home-made mouse anti-H1N1 nucleocapsid protein antibody, anti-Ki67 antibody (556003, BD; ab15580, Abcam), anti-β tubulin-IV antibody (T7941, Sigma), anti-CK14 antibody (PA516722, Invitrogen; MA5-11599, Invitrogen), anti-SCGB1A1 antibody (10490, Proteintec), anti-SPC antibody (ab3786, Millipore), anti-Iba1 antibody (ab178846, Abcam), anti-Notch3 antibody (MBS242006, MyBioSource), anti-Hes1 antibody (LS-B2211 LSBio), anti-Sox2 antibody (11064-1-AP, Proteintech) and anti-Mlf1 antibody (MBS9605647, MyBioSource).

Techniques: Infection, Expressing, Immunofluorescence, Staining, Confocal Microscopy

Journal: Journal of Cancer

Article Title: Comprehensive analysis based on glycolytic and glutaminolytic pathways signature for predicting prognosis and immunotherapy in ovarian cancer

doi: 10.7150/jca.88359

Figure Lengend Snippet: AAK1 promotes OV progression via Notch3 pathway in vitro . ( A ) Representative immunohistochemical staining images of AAK1 in normal ovary and OV tissue from The Human Protein Atlas (THPA) database. ( B-C ) Relative expression of AAK1 between IOSE-80 cell lines and five OV line were tested by RT-qPCR ( B ) (n = 3) and western blot ( C ) (n = 3), respectively. ( D ) Immunofluorescence staining showing the subcellular localization of AAK1 protein in A2780 and OVCAR3 cells (Scale bar: 10 µm). ( E-G ) CCK8 assay ( E ), Edu assays ( F, Scale bar: 100 µm), and invasion assays ( G ) revealing the cell viability, proliferation, and invasion of A2780 and OVCAR3 cells stably transfected as indicated, respectively (n = 3). ( H ) Western blotting showing the protein levels of AAK1, Notch3, MMP-2, and MMP-9 in A2780 and OVCAR3 cells stably transfected as indicated, respectively (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Western blotting assay was executed as previously reported , with rabbit anti-human antibodies for AAK1 (1:2000, PA5408, Abmart), Notch3 (1:1000, PS08936, Abmart), GLS (1:2000, T55719, Abmart), MMP-2 (1:1000, PA1748, Abmart), MMP-9 (1:2000, TA5228, Abmart), and β-actin (1:2000, GB11001, Servicebio).

Techniques: In Vitro, Immunohistochemical staining, Staining, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, CCK-8 Assay, Stable Transfection, Transfection

Journal: Journal of Cancer

Article Title: Comprehensive analysis based on glycolytic and glutaminolytic pathways signature for predicting prognosis and immunotherapy in ovarian cancer

doi: 10.7150/jca.88359

Figure Lengend Snippet: AAK1 promotes glutamine metabolism of OV via Notch pathway in A2780 cells. ( A-E ) The cellular Glu ( A ), α-KG ( B ), ATP ( C ), GSH ( D ), and ROS levels (E, Scale bar: 100 µm) of A2780 cells stably transfected as indicated (n = 3). (F) Western blot indicating the protein levels of AAK1, Notch3, and GLS in A2780 and OVCAR3 cells stably transfected as indicated (n = 3). ( G ) Schematic diagram of AAK1 promots glutamine metabolism and malignant progression through the Notch3 pathway in ovarian cancer. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Western blotting assay was executed as previously reported , with rabbit anti-human antibodies for AAK1 (1:2000, PA5408, Abmart), Notch3 (1:1000, PS08936, Abmart), GLS (1:2000, T55719, Abmart), MMP-2 (1:1000, PA1748, Abmart), MMP-9 (1:2000, TA5228, Abmart), and β-actin (1:2000, GB11001, Servicebio).

Techniques: Stable Transfection, Transfection, Western Blot

Journal: Journal of Cancer

Article Title: Comprehensive analysis based on glycolytic and glutaminolytic pathways signature for predicting prognosis and immunotherapy in ovarian cancer

doi: 10.7150/jca.88359

Figure Lengend Snippet: Therapeutic knockdown of AAK1 suppresses OV progression and metastasis in vivo . ( A ) The representative images, ( B ) tumor growth, ( C ) tumor weight, ( D ) the representative IHC images and statistical analysis results of AAK1, Notch3, GLS, MMP-2, MMP-9, and Ki-67 of xenograft tumors established by subcutaneous injection of OVCAR3 cells that were stably transfected as indicated, respectively, respectively (n = 5 per group). Scale bar: 50 μm. ( E ) The lung metastasis images (left panel) and metastatic counts (right panel) of tumor lung metastasis assay formed by tail vein injection of OVCAR3 cells that were stably transfected as indicated, respectively (n = 5 per group). Scale bar: 100 μm. ( G ) K-M curves were employed to display the survival of nude mice with distinct treatments in the tumor lung metastasis assay. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Western blotting assay was executed as previously reported , with rabbit anti-human antibodies for AAK1 (1:2000, PA5408, Abmart), Notch3 (1:1000, PS08936, Abmart), GLS (1:2000, T55719, Abmart), MMP-2 (1:1000, PA1748, Abmart), MMP-9 (1:2000, TA5228, Abmart), and β-actin (1:2000, GB11001, Servicebio).

Techniques: Knockdown, In Vivo, Injection, Stable Transfection, Transfection

Journal: Journal of Cancer

Article Title: Correlation of ALDH1 and Notch3 Expression: Clinical implication in Ovarian Carcinomas

doi: 10.7150/jca.18955

Figure Lengend Snippet: Aldefluor assay and the expression of ALDH1 and Notch signaling genes in ovarian cancer stem cells. A. ALDH1 population in primarily-cultured human ovarian carcinoma cells was analyzed by Aldefluor assay. DEAB is a specific inhibitor of ALDH and was used to confirm gating areas. ALDH1 (+) population (6.74%) was displayed in the absence of DEAB. B. Expression of ALDH1 mRNA in parental carcinoma cells and spheroid forming cells from 5 primarily-cultured ovarian carcinoma cells (SCN 1-5). C. The mRNA expression of Notch signaling genes in FACS-sorted ALDH1 (+) ovarian cancer cells. Notch3 (1.8-fold, p = 0.046), Notch1 (1.2-fold, p = 0.007), and Hes1 (1.2-fold, p = 0.033) were significantly overexpressed in ALDH1 (+) cells compared with ALDH1 (-) cells.

Article Snippet: Briefly, blocks were cut at five micromiter thick and the sections dried at 62°C for 30 min, and then, subjected to antigen retrieval using cell conditioning solution containing Tris/borate/EDTA in a Ventana BenchMark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) for 60 min. For double staining, the slides were incubated for 3 h with anti-ALDH1 (biorbyt, Canbrige, UK) and anti-Notch3 antibody (GeneTex, San Antonio, USA) at a concentration of 1:100.

Techniques: Expressing, Cell Culture

Journal: Journal of Cancer

Article Title: Correlation of ALDH1 and Notch3 Expression: Clinical implication in Ovarian Carcinomas

doi: 10.7150/jca.18955

Figure Lengend Snippet: The mRNA expression of ALDH1 and Notch signaling genes in various subtypes of ovarian carcinomas and ovarian cancer cell lines. A . The mRNA expression of ALDH1 (2.6-fold, p = 0.024) and Notch3 (13.7-fold, p = 0.000) was significantly upregulated in ovarian carcinomas (OC) compared with normal epithelium (OE). Each experiment was done in triplicate (* p < 0.05). B. The mRNA expression of ALDH1 was significantly elevated in mucinous (3.8-fold, p = 0.014) and endometrioid (2.5-fold, p = 0.029) types of OC compared with OE. Serous carcinoma (2.6-fold, p = 0.24) also showed higher ALDH1 expression. Each experiment was done in triplicate (* p < 0.05). C. The mRNA expression of Notch1 (3.0-fold, p < 0.001), Notch3 (3.4-fold, p < 0.001), Jagged2 (2.6-fold, p = 0.014), Hes1 (2.7-fold, p = 0.003), and ALDH1 (2.8-fold, p < 0.001) was significantly upregulated in the chemoresistant group compared with the chemosensitive group of OSCs. D. The correlation between ALDH1 and Notch3 expression. Notch3 expression was positively correlated with ALDH1 expression (R = 0.429, p = 0.007, Spearman's correlation test). E. Dual immunohistochemical stain for ALDH1 and Notch 3 proteins in chemoresistant OSCs. The carcinoma cells showing diffuse cytoplasmic and nuclear staining with Notch 3 (red) were also stained with ALDH1 in the cytoplasm (blue-green). F. The mRNA expression of ALDH1 and Notch signaling genes in PTX-resistant SKpac cells and PTX-sensitive parent SKOV3 cells. ALDH1 and Notch 3 mRNA levels (>25-fold than parent cells) were significantly elevated in SKpac cells compared with SKOV3 cells. Each experiment was done in triplicate (* p < 0.05). G. The expression of ALDH1 and Notch signaling genes in cisplatin-resistant A2780cis cells and cisplatin-sensitive A2780 cells. ALDH1 (4.2-fold, p=0.016), Notch1 (2.5-fold,p=0.034), Notch3 (1.3-fold,p=0.312), and Jagged2 (3.6-fold,p=0.001) mRNA levels were elevated in A2780cis cells compared with A2780. Each experiment was done in triplicate. (* p < 0.05)

Article Snippet: Briefly, blocks were cut at five micromiter thick and the sections dried at 62°C for 30 min, and then, subjected to antigen retrieval using cell conditioning solution containing Tris/borate/EDTA in a Ventana BenchMark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) for 60 min. For double staining, the slides were incubated for 3 h with anti-ALDH1 (biorbyt, Canbrige, UK) and anti-Notch3 antibody (GeneTex, San Antonio, USA) at a concentration of 1:100.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: Correlation of ALDH1 and Notch3 Expression: Clinical implication in Ovarian Carcinomas

doi: 10.7150/jca.18955

Figure Lengend Snippet: Clinicopathological correlation of Notch signaling genes and ALDH1

Article Snippet: Briefly, blocks were cut at five micromiter thick and the sections dried at 62°C for 30 min, and then, subjected to antigen retrieval using cell conditioning solution containing Tris/borate/EDTA in a Ventana BenchMark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) for 60 min. For double staining, the slides were incubated for 3 h with anti-ALDH1 (biorbyt, Canbrige, UK) and anti-Notch3 antibody (GeneTex, San Antonio, USA) at a concentration of 1:100.

Techniques:

Journal: Journal of Cancer

Article Title: Correlation of ALDH1 and Notch3 Expression: Clinical implication in Ovarian Carcinomas

doi: 10.7150/jca.18955

Figure Lengend Snippet: Multivariate survival analysis (Cox regression) for the patients of ovarian serous carcinomas.

Article Snippet: Briefly, blocks were cut at five micromiter thick and the sections dried at 62°C for 30 min, and then, subjected to antigen retrieval using cell conditioning solution containing Tris/borate/EDTA in a Ventana BenchMark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) for 60 min. For double staining, the slides were incubated for 3 h with anti-ALDH1 (biorbyt, Canbrige, UK) and anti-Notch3 antibody (GeneTex, San Antonio, USA) at a concentration of 1:100.

Techniques: