Journal: bioRxiv

Article Title: Oncogenic mutations convert MET from a pro-apoptotic tumor suppressor to an oncogenic driver

doi: 10.1101/2025.11.10.687614

Figure Lengend Snippet: (A) Immunoblot analysis of MET-induced downstream signaling pathways in edited 16HBE cells after 30 ng/mL HGF treatment for the indicated time. Immunoblotting is representative of three biological replicates. (B-D) Quantitative analysis of phosphorylated MET (p-MET), AKT (p-AKT), and ERK (p-ERK) normalized to their respective total protein levels from western blot experiments. Data are presented as means ± SEM from three independent experiments. Statistical significance was determined by two-way ANOVA followed by appropriate post hoc tests. ns (not significant), * ( p < 0.05), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001). (E-F) Quantification of relative wound density after cell in the presence or absence of HGF in migration assays after 8 h post-HGF stimulation (E) and invasion assays with Matrigel 3 days post-HGF stimulation (F) following stimulation with 30 ng/mL HGF. Values represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA followed by appropriate post hoc tests. ns (not significant), * ( p < 0.05), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001). (G) Quantification of cell scattering based on cell density after 48 h stimulation with 30 ng/mL HGF. At least 15 cellular islets were analyzed. Data are presented as box-and-whiskers plots showing minimum to maximum values; in each box, the point represents the mean, and the horizontal line indicates the median. Statistical significance was determined using two-way ANOVA followed by appropriate post hoc tests. ns (not significant), ** ( p < 0.01), **** ( p < 0.0001). (H-K) Cell confluence (H), membrane permeability (I), Annexin V staining (J), and active caspase 3/7 staining (K) were measured five days after serum starvation. For (I–K), fluorescence signals were normalized to cell confluence. n=6; mean ± SD; representative of three independent experiments. Statistical significance was determined by one-way ANOVA. ns (not significant), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001). (L) Representative images showing nuclear staining with Hoechst (blue) and cleaved caspase 3 staining (green) after five hours of treatment with 1 µM staurosporine. Adjacent quantification shows the percentage of apoptotic cells, defined as cleaved caspase 3 positive cells with fragmented nuclei, from at least 200 counted cells per experiment. Data represent means ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA. * ( p < 0.05), *** ( p < 0.001), **** ( p < 0.0001).

Article Snippet: Three complementary fluorescent readouts were used: Annexin V staining (#4641 Annexin V Green Reagent for Apoptosis, Sartorius) to detect phosphatidylserine externalization, caspase 3/7 activity (#4440 Caspase 3/7 Green Apoptosis Assay Reagent, Sartorius), and membrane permeability (#4632 Cytotox Red Reagent, Sartorius).

Techniques: Western Blot, Protein-Protein interactions, Migration, Membrane, Permeability, Staining, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic mutations convert MET from a pro-apoptotic tumor suppressor to an oncogenic driver

doi: 10.1101/2025.11.10.687614

Figure Lengend Snippet: (A) Immunoblot analysis of MET-induced downstream signalling pathways in edited A549 cells after treatment with 30 ng/mL HGF for the indicated time. Immunoblotting is representative of three biological replicates. (B-D) Membrane permeability (B), Annexin V staining (C), and active caspase 3/7 staining (D) were measured after seven days of serum starvation. Fluorescence signals were normalized to cell confluence. n=5; mean ± SD; representative of three independent experiments. Statistical significance was determined by one-way ANOVA. **** ( p < 0.0001).

Article Snippet: Three complementary fluorescent readouts were used: Annexin V staining (#4641 Annexin V Green Reagent for Apoptosis, Sartorius) to detect phosphatidylserine externalization, caspase 3/7 activity (#4440 Caspase 3/7 Green Apoptosis Assay Reagent, Sartorius), and membrane permeability (#4632 Cytotox Red Reagent, Sartorius).

Techniques: Western Blot, Membrane, Permeability, Staining, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic mutations convert MET from a pro-apoptotic tumor suppressor to an oncogenic driver

doi: 10.1101/2025.11.10.687614

Figure Lengend Snippet: (A-B) Membrane permeability (A), and active caspase 3/7 staining (B) were measured in edited 16HBE cells after 8 h of staurosporine treatment at 1 µM. Fluorescence signals were normalized to cell confluence. n=5; mean ± SD; representative of three independent experiments. Statistical significance was determined by one-way ANOVA. * ( p < 0.05), ** ( p < 0.01), **** ( p < 0.0001). (C-E) Membrane permeability (C), Annexin V staining (D), and active caspase 3/7 staining (E) were measured 24 h after staurosporine treatment at 1 µM. Fluorescence signals were normalized to cell confluence. n=5; mean ± SD; representative of three independent experiments. Statistical significance was determined by one-way ANOVA. **** ( p < 0.0001).

Article Snippet: Three complementary fluorescent readouts were used: Annexin V staining (#4641 Annexin V Green Reagent for Apoptosis, Sartorius) to detect phosphatidylserine externalization, caspase 3/7 activity (#4440 Caspase 3/7 Green Apoptosis Assay Reagent, Sartorius), and membrane permeability (#4632 Cytotox Red Reagent, Sartorius).

Techniques: Membrane, Permeability, Staining, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic mutations convert MET from a pro-apoptotic tumor suppressor to an oncogenic driver

doi: 10.1101/2025.11.10.687614

Figure Lengend Snippet: (A) Immunoblot analysis showing generation of the p40MET fragment following in vitro cleavage of MET by recombinant caspase 3 in 16HBE cell lysates. MET-mutated proteins level was analyzed using two anti-MET antibodies: MET-KC, recognizing the kinase-proximal C-terminal region and detecting MET even after caspase-mediated C-terminal cleavage, and MET-EC, recognizing the extreme C-terminal tail, which does not detect the cleaved receptor. Immunoblots are representative of three independent biological experiments. (B-F) Cell confluence (B), viability (C), membrane permeability (D), Annexin V staining (E), and active caspase 3/7 staining (F) were measured five days after serum starvation. For (C), viability was measured five days after serum starvation in an AlamarBlue assay. For (D–F), fluorescence signals were normalized to cell confluence. n=6; mean ± SD; representative of three independent experiments. Statistical significance was determined by one-way ANOVA. ns (not significant), * ( p < 0.05), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001).

Article Snippet: Three complementary fluorescent readouts were used: Annexin V staining (#4641 Annexin V Green Reagent for Apoptosis, Sartorius) to detect phosphatidylserine externalization, caspase 3/7 activity (#4440 Caspase 3/7 Green Apoptosis Assay Reagent, Sartorius), and membrane permeability (#4632 Cytotox Red Reagent, Sartorius).

Techniques: Western Blot, In Vitro, Recombinant, Membrane, Permeability, Staining, Alamar Blue Assay, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic mutations convert MET from a pro-apoptotic tumor suppressor to an oncogenic driver

doi: 10.1101/2025.11.10.687614

Figure Lengend Snippet: Immunoblot analysis showing generation of the p40MET fragment following in vitro cleavage of MET by recombinant caspase 3 in A549 cell lysates. Immunoblots are representative of three independent biological experiments.

Article Snippet: Three complementary fluorescent readouts were used: Annexin V staining (#4641 Annexin V Green Reagent for Apoptosis, Sartorius) to detect phosphatidylserine externalization, caspase 3/7 activity (#4440 Caspase 3/7 Green Apoptosis Assay Reagent, Sartorius), and membrane permeability (#4632 Cytotox Red Reagent, Sartorius).

Techniques: Western Blot, In Vitro, Recombinant

Journal: Frontiers in Immunology

Article Title: Senescent human fibroblasts have increased FasL expression and impair the tumor immune response

doi: 10.3389/fimmu.2025.1685269

Figure Lengend Snippet: The SASP triggers apoptosis of immune cells and promotes tumor cell survival in vitro. (A) Concentration of soluble FasL (sFasL) as detected by ELISA in the conditioned media (CM) of non-senescent and senescent HDF populations. Each dot represents an independent experiment (n=3), and t-tests determined statistical significance between groups. (B) Graphs showing the absolute counts of CD45 + (left panel) and CD3 + (right panel) live cells as determined by flow cytometry after 48 hours of culture in CM collected from the indicated HDF populations. Shown is the mean ± SEM from three independent experiments. Multiple t-tests determined statistical significance between groups. *p < 0.05; **p < 0.01; ***P < 0.001. (C) Representative images of LEC-4T tumor cells (expressing mPlum in red) after 72 hours co-cultured with PBMCs in CM collected from the indicated HDF populations. The Caspase-3/7 Green reagent was added to detect cells undergoing apoptosis. Scale bars represent 400 μm. (D) Scatter dot plot showing the number of caspase 3/7 positive tumor cells (red and green overlap) after 72 hours of co-culture as determined using the IncuCyte ® live-cell analysis software. Shown is the mean ± SEM from four independent experiments. Multiple t-tests determined statistical significance between groups. *p < 0.05. (E) Scatter dot plot showing the number of LEC-4T tumor cells (in red) after 48 hours of co-culture as determined using the IncuCyte ® live-cell analysis software. Shown is the mean ± SEM from four independent experiments. Multiple t-tests determined statistical significance between groups. **p < 0.01; ***P < 0.001. (F) Absolute counts of live LEC-4T cells as measured by flow cytometry at the end of the 72-hours co-culture period. Each dot represents the average of five technical replicates. Shown is the mean ± SEM from four independent experiments. Multiple t-tests determined statistical significance between groups. **p < 0.01; ***P < 0.001.

Article Snippet: To detect apoptotic cells we used the IncuCyte S3 (Essen Bioscience) live cell imaging system in combination with propidium iodide (PI) or the caspase 3/7 specific activity dye.

Techniques: In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Cell Culture, Co-Culture Assay, Cell Analysis, Software