Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Osimertinib drug‐tolerant persister (DTP) cells show resistance to osimertinib and the Notch pathway was activated in DTP cells. (A) PC‐9 and H1975 cells were treated with osimertinib for 72 h. Cell viability was assessed using MTT assays ( n = 3; mean ± SD). (B) DTP cells were isolated from three cell lines after treatment with 3 μmol/L osimertinib for 9 days. (C) Parental cells and osimertinib DTP cells were treated with osimertinib for 72 h and cell viability was assessed using the MTT assay. Three independent experiments were performed ( n = 3; mean ± SD). * p < 0.05. (D) Differential gene expression between samples of parental PC‐9 cells or PC‐9 DTP cells were calculated. The results are shown as a volcano plot. Upregulated NOTCH1 ‐responsive genes (marked in orange) as observed in RNA‐seq analyses are shown.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Isolation, MTT Assay, Expressing, RNA Sequencing Assay

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Combined osimertinib and γ‐secretase inhibitor (GSI) suppresses the Notch pathway in drug‐tolerant persister (DTP) cells. (A) PC‐9, H1975, and HCC827 cells were incubated with vehicle, 3 μmol/L osimertinib, 1 μmol/L GSI, or a combination for 9 days. The medium was replenished every 72 h. The cells were lysed and the indicated proteins were detected by western blotting ( n = 3). (B) Relative mRNA expression of Notch1 , HES1 , and HEY1 was determined by qRT‐PCR ( n = 3; mean ± SD). * p < 0.05.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Incubation, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Notch1 knockdown inhibits the emergence of osimertinib drug‐tolerant persister (DTP) cells and suppresses phospho‐ERK in osimertinib DTP cells. (A) Stable PC‐9 and H1975 cells were generated by introducing short hairpin RNA (shRNA) inhibiting Notch1 expression (shNotch1) and control non‐targeting shRNA (shControl). Notch1 knockdown cells and shControl cells were treated with vehicle or 3 μmol/L osimertinib. The medium was replenished every 72 h for 28 days. Tumor cell counts were visually checked every 72 h and plotted over time ( n = 3; mean ± SD). (B, C) Notch1 knockdown cells and shControl cells were incubated with vehicle or 3 μmol/L osimertinib for 9 days. The medium was replenished every 72 h. The cells were lysed and the indicated proteins were detected by western blotting ( n = 3).

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Generated, shRNA, Expressing, Incubation, Western Blot

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: The combination of osimertinib and GSI suppresses phospho‐ERK and the Notch pathway in vivo. (A, B) After subcutaneous injection of PC‐9 or H1975 cells into nude mice, vehicle (control), 5 mg/kg osimertinib, 3.3 mg/kg γ‐secretase inhibitor (GSI), or osimertinib plus GSI was administered for 9 days. The cells were lysed and the indicated proteins were detected by western blotting ( n = 3). (C) Relative mRNA expression of Notch1 , HES1 , and HEY1 was determined by qRT‐PCR ( n = 3; mean ± SD). * p < 0.05.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: In Vivo, Injection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Notch1 or HES1 expression is enhanced after EGFR tyrosine kinase inhibitor (TKI) treatment in human tumor tissues from EGFR ‐mutated patients with non–small‐cell lung cancer (NSCLC). (A, B) Immunohistochemical staining of Notch1 and HES1 in NSCLC tissues. Notch1 expression was detected in the cytoplasm and/or nucleus (scale bars, 50 μm). HES1 expression was observed in the nucleus (scale bars, 50 μm). Notch1 and HES1 levels before and after EGFR‐TKI treatment were compared using paired t ‐tests in patients with lung adenocarcinoma. Line graphs show changes in Notch1 and HES1 protein expression after EGFR‐TKI treatment. Each line represents one patient. Red lines represent patients who showed increased expression levels after therapy, whereas black lines represent patients who did not show increased expression levels after therapy.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Notch1 Signaling Regulates the Proliferation and Self-Renewal of Human Dental Follicle Cells by Modulating the G1/S Phase Transition and Telomerase Activity

doi: 10.1371/journal.pone.0069967

Figure Lengend Snippet: HDFC-C (uninfected parental HDFCs), HDFC-GFP (HDFCs infected with GFP gene), HDFC-ICN (HDFCs infected with the ICN1 gene), HDFC-CS (HDFCs infected with control shRNA lentiviral particles) and HDFC-NS (HDFCs infected with Notch-1 shRNA lentiviral particles) cells were cultured in DMEM containing 10% FBS. At approximately 80% confluence, these cells were starved for an additional 24 h and harvested for qPCR and western blot analyses. ( A ) qPCR analysis of Notch1 transcript levels in different HDFC groups. The data are normalized to β-actin levels and presented as mean values ± SD of three independent experiments. # P <0.01. ( B ) Western blot analysis of cleaved Notch1 protein levels in different HDFC groups. The representative blots show the expression of cleaved Notch1 protein, whereas the bar graph below shows the photodensitometric analysis of the bands of cleaved Notch1 protein, using β-actin as an internal control. The data are presented as mean values ± SD of three independent experiments. # P <0.01.

Article Snippet: Notch1 shRNA lentiviral particles (sc-36095-V) and control lentiviral particles expressing a scrambled shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Control, shRNA, Cell Culture, Western Blot, Expressing