Journal: Gene therapy

Article Title: Tumor cells expressing a fusion protein of MULT1 and Fas are rejected in vivo by apoptosis and NK cell activation.

doi: 10.1038/gt.2008.77

Figure Lengend Snippet: Figure 3 Mouse UL16-binding protein-like transcript 1 E (MULT1E)/FasTI induces apoptosis. A total of 1 106 cells of TC-1 and clones L-5, L-7 and L-10 were treated with 1 mg/ml NKG2D/Fc for 16 h. The cells were then analyzed for apoptosis and necrosis using Annexin V assay (a–c) or caspase-3 assay (d) according to the manufacturers’ protocols. (a) An example of the fluorescence-activated cell sorting (FACS) data. (b, c) Summaries of data from three separate experiments. The statistical analyses were conducted between the controls (open bars) and NKG2D/Fc-treated cells (solid bars) using two-way analysis of variance (ANOVA). The difference between NKG2D/Fc-treated L-5 cells and NKG2D/Fc-treated L-10 cells was also compared using Student’s t-test. *Po0.05; **Po0.01 and ***Po0.001.

Article Snippet: Induction of apoptosis in cells expressing the fusion protein To determine if cells expressing the fusion protein can be induced to undergo apoptosis, one million cells of TC-1 and clones L-5, L-7 and L-10 were treated with 1 mg/ml of NKG2D/Fc for 16 h. Apoptosis of the cells was measured using two systems: a TACS Annexin V-FITC Apoptosis Kit (R&D Systems) and a caspase-3 fluorometric assay (R&D Systems).

Techniques: Binding Assay, Clone Assay, Annexin V Assay, Caspase-3 Assay, FACS

Journal: Frontiers in Oncology

Article Title: AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma

doi: 10.3389/fonc.2022.903874

Figure Lengend Snippet: Downregulation of AXL expression attenuates metformin-induced apoptotic cell death in EAC cells. SK-GT-4-shCtrl and SK-GT-4-shAXL cells (A) or FLO-1-shCtrl and FLO-1-shAXL cells (B) were subjected to CCK-8 cell viability assay following treatment with the indicated metformin concentrations for 72 h. (C) SK-GT-4-shCtrl and SK-GT-4-shAXL cells were treated with vehicle or metformin (15 mM) for 96 h. Apoptosis was determined by Annexin-V/FITC staining and FACS analysis. (D) Immunoblot analysis of AXL, caspase-3, and PARP proteins in cell lysates from SK-GT-4-shCtrl and SK-GT-4-shAXL cells treated with vehicle or 15 mM metformin for 96 h. (E) Apoptosis in FLO-1-shCtrl and FLO-1-shAXL cells after treatment with vehicle or 10 mM metformin for 72 h was assessed as in panel (C) . (F) Western blot analysis of AXL, caspase-3, and PARP proteins in FLO-1-shCtrl and FLO-1-shAXL cells treated with vehicle or 10 mM metformin for 72 h. Gel loading was normalized for equal β-actin. Data are representative of three independent experiments and statistical analysis was evaluated by one-way ANOVA followed by the Newman-Keuls post hoc test.

Article Snippet: Briefly, cells were harvested and stained with Annexin-V fluorescein isothiocyanate (FITC) using TACS Annexin V-FITC Apoptosis Detection Kit (4830-250-K, R&D Systems) and propidium iodide (PI) (P4170, Sigma-Aldrich).

Techniques: Expressing, CCK-8 Assay, Viability Assay, Staining, Western Blot

Journal: Frontiers in Oncology

Article Title: AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma

doi: 10.3389/fonc.2022.903874

Figure Lengend Snippet: Metformin-induced apoptosis requires autophagy in EAC cells. (A) FLO-1 cells stably expressing control shRNA (shCtrl) or Beclin 1 shRNA (shBeclin1) were treated with increasing concentrations of metformin for 72 h and then subjected to CCK-8 cell viability assay. (B) Western blot analysis of Beclin 1, caspase-3, and PARP proteins in FLO-1-shCtrl and FLO-1-shBeclin1 cells treated with vehicle or 10 mM metformin for 72 h. (C) Immunoblotting of ATG7, Caspase-3, and PARP proteins in FLO-1-shCtrl and FLO-1-shATG7 cells treated with vehicle or 10 mM metformin for 72 h. (D) Western blot analysis of ATG7, caspase-3, and PARP proteins in SK-GT-4-shCtrl and SK-GT-4-shATG7 cells treated with 15 mM metformin for 96 h. (E) Western blot analysis of Beclin 1, p-AMPK (T172), AMPK, and LC3B proteins in FLO-1-shCtrl and FLO-1-shBeclin1 cells treated with vehicle or 10 mM metformin for 2 h. (F) Western blot analysis of ATG7, p-AMPK (T172), AMPK, and LC3B proteins in FLO-1-shCtrl and FLO-1-shATG7 cells treated with vehicle or 10 mM metformin for 2 h. (G) Immunoblotting of ATG7, p-AMPK (T172), AMPK, and LC3B proteins in SK-GT-4-shCtrl and SK-GT-4-shATG7 cells treated with 15 mM metformin for 2 h. Gel loading was normalized for equal β-actin.

Article Snippet: Briefly, cells were harvested and stained with Annexin-V fluorescein isothiocyanate (FITC) using TACS Annexin V-FITC Apoptosis Detection Kit (4830-250-K, R&D Systems) and propidium iodide (PI) (P4170, Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Control, shRNA, CCK-8 Assay, Viability Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma

doi: 10.3389/fonc.2022.903874

Figure Lengend Snippet: Expression of AXL sensitizes tumors to metformin in a xenograft mouse model. Four groups (10 mice per group) of Rag2 mice were injected on both hip flanks with FLO-1-shCtrl (two groups, no treatment and treatment) or FLO-1-shAXL cells (two groups, no treatment and treatment). When tumors reached a size of approximately 250 mm 3 , the mice were randomized and not treated or treated by oral gavage with metformin (250 mg/kg) daily for 12 days. The metformin dose was then increased to 500 mg/kg daily until the end of the experiment (day 28). (A) Treatment with metformin significantly reduced tumor size in shCtrl cells ( P = 0.004) relative to nontreated tumors. Knockdown of AXL expression (shAXL cells) completely abrogated the suppressive effect of metformin on tumor growth. (B) Representative images of endpoint xenografts depicting the effect of metformin on tumor size. Images (200x magnification) of H&E staining (C) and IHC analysis for expression for Ki-67, a marker of proliferation (C , D) ; cleaved caspase-3, a marker of apoptosis (C , E) ; and p-AMPK (T172), a marker of autophagy (C , F) in representative endpoint tumors of all metformin-treated and non-treated animal groups. (G) Real-time PCR data depicting relative AXL mRNA expression in representative endpoint tumors of all the animal groups. n.s., not significant.

Article Snippet: Briefly, cells were harvested and stained with Annexin-V fluorescein isothiocyanate (FITC) using TACS Annexin V-FITC Apoptosis Detection Kit (4830-250-K, R&D Systems) and propidium iodide (PI) (P4170, Sigma-Aldrich).

Techniques: Expressing, Injection, Knockdown, Staining, Marker, Real-time Polymerase Chain Reaction