Journal: Oncotarget

Article Title: Autocrine IL-6 mediates pituitary tumor senescence

doi: 10.18632/oncotarget.13577

Figure Lengend Snippet: A . SA-β-gal activity was measured by flow cytometry. The bar graph represents the SA-β-gal activity measured by the mean fluorescence intensity (MFI). B . To assay pRb and p16 INK4a protein levels, cell extracts were subjected to western blot (WB) with the indicated antibodies. β-Actin was used as loading control. C . IL-6 secretion by MtT/S cells under basal and stimulated conditions with LPS was measured by ELISA. Values represent the average fold stimulation with respect to basal. Absolute values of basal (similar to vehicle) IL-6: 72.71±0.74 pg/ml (30,000 cells/well). D-E . Effect of LPS and rhIL-6 on MtT/S cell proliferation, measured with the WST-1 assay (A 450nm ). F-H . Effect of LPS, rhIL-6 and siRNA IL-6 on MtT/S SA-β-gal activity. The grey scale used in the histogram plot correspond to the grey scale used in the bar graph, which represent the SA-β-gal activity measured by the mean fluorescence intensity (MFI). One representative experiment from three independent experiments (N= 4 for each condition in each experiment) with similar results is shown. Results are expressed as mean ± SEM. *p<0.05 and **p<0.01 compared with cells treated with vehicle (C-G) or cells transfected with siRNA GL3 as control (H). ANOVA with Scheffé's test.

Article Snippet: Proteins were blotted onto nitrocellulose membranes using standard procedures, and incubated with p16 INK4a (1:1000), STAT3 (1:1000), phosphoSTAT3 (1:1000), β-Actin (1:3000) (Santa Cruz Biotechnologies, California, USA), pRb (1:1000, Cell Signalling Technology, Danvers, Massachusetts, USA), GH (1:1000) or GAPDH (1:10000) (Abcam, Cambridge, Massachusetts, USA) antibodies overnight, followed by corresponding secondary antibodies incubation.

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Western Blot, Control, Enzyme-linked Immunosorbent Assay, WST-1 Assay, Transfection

Journal: Oncotarget

Article Title: Autocrine IL-6 mediates pituitary tumor senescence

doi: 10.18632/oncotarget.13577

Figure Lengend Snippet: A . SA-β-gal activity was measured on MtT/S wild type (MtT/S), MtT/S control (MtT/S scr) and MtT/S IL-6 silenced (MtT/S shIL-6) clones, by flow cytometry. The bar graph represents the SA-β-gal activity measured by the mean fluorescence intensity (MFI). B . For pRb and p16 INK4a protein levels, cell extracts were subjected to western blot (WB) with the indicated antibodies. GAPDH was used as loading control. C-D . Cell proliferation, measured by WST-1 assay (A 450nm ), and cell invasion, measured using transwells, were performed on MtT/S, MtT/S scr and MtT/S shIL-6 clones. One representative experiment from three independent experiments (N= 3 for each condition in each experiment) with similar results is shown (A-D). Results are expressed as mean ± SEM. *p<0.05 compared with MtT/S and MtT/S scr, ANOVA with Scheffé's test (A, C and D). E . MtT/S scr and MtT/S shIL-6 clones were injected subcutaneously (5×10 5 cells for each clone). Photographs were taken at 70 days. One mouse with similar results to all the others of each group is shown (i.e. no tumor in all the animals injected with MtT/S scr cells and tumors similar to the one shown in the picture in all animals injected with MtT/S shIL-6). Volume of each of the tumors of one of three independent experiments with six mice in each group in each experiment with similar results is shown. The six MtT/S scr mice did not form tumors and overlap in one red line and the volume from each of the tumors from the MtT/S shIL-6 group is shown in green. F . MtT/S scr with TtT/GF and MtT/S shIL-6 cells were injected subcutaneously in 2 groups of five male nude mice (5×10 5 cells for each clone and 1,8×10 5 cell for TtT/GF). Photographs of mice were taken at 70 days. Tumor weights were obtained at the end of the experiment. One mouse and one tumor with similar results to the others of each group are shown. Three independent experiments with five mice in each group in each experiment were performed, showing similar results. Weights of one of these experiments are shown in the graph. G . 6μm tumor cryosections (upper panel, x20 magnification) and primary cell cultures derived from the same tumor (lower panel, x10 magnification) were stained for SA-β-gal. Arrows and dotted line arrows indicate examples of SA-β-gal positive and negative cells, respectively. Quantification of positive SA-β-gal cells is shown. Results obtained from 20 independent pictures for each condition are expressed as mean ± SEM. H-I . 6μm tumor sections were stained by double immunofluorescence with p16 INK4a (yellow; 1:50), pRb (red; 1:50) and 4′,6-diamidino-2-phenylindole (DAPI) for staining cell nuclei (blue). Tumors developed by the injected cells were verified by the detection of eGFP (green). Images were acquired at x63 magnification (scale bar: 20μm). p16 INK4a and pRb were quantified by the mean fluorescence intensity (MFI) and relativized to DAPI signal. Results obtained from six independent pictures for each condition are expressed as mean ± SEM. One representative picture for each condition is shown. *p<0.05 compared with tumors developed by the co-injection of MtT/S scr and TtT/GF, Student t test (H-I).

Article Snippet: Proteins were blotted onto nitrocellulose membranes using standard procedures, and incubated with p16 INK4a (1:1000), STAT3 (1:1000), phosphoSTAT3 (1:1000), β-Actin (1:3000) (Santa Cruz Biotechnologies, California, USA), pRb (1:1000, Cell Signalling Technology, Danvers, Massachusetts, USA), GH (1:1000) or GAPDH (1:10000) (Abcam, Cambridge, Massachusetts, USA) antibodies overnight, followed by corresponding secondary antibodies incubation.

Techniques: Activity Assay, Control, Clone Assay, Flow Cytometry, Fluorescence, Western Blot, WST-1 Assay, Injection, Derivative Assay, Staining, Immunofluorescence

Journal: Oncotarget

Article Title: Autocrine IL-6 mediates pituitary tumor senescence

doi: 10.18632/oncotarget.13577

Figure Lengend Snippet: A . Primary human pituitary cell cultures prepared from 61 samples of human pituitary tumor samples were stained for SA-β-gal (6 plurihormonal, 6 PRL-secreting, 7 ACTH-secreting, 4 LH/FSH-secreting, 15 GH-secreting and 23 non-functioning tumors). Black zone of the bars represents positive samples, while grey zone represents negative samples. B . Photographs of positive SA-β-gal staining from a non-functioning and a GH-secreting tumor are shown. C . Photographs of positive p16 INK4a (yellow; 1:50) and pRb (red, 1:50) staining from a non-functioning and a GH-secreting tumor are shown. 4′,6-diamidino-2-phenylindole (DAPI) were used for staining cell nuclei (blue). Images were acquired at x63 magnification (scale bar: 20μm). D . By Pearson correlation analysis it was determined that senescence correlates negatively with the cellular marker of proliferation Ki-67 in human pituitary adenomas (Pearson coefficient=-0.3245, p<0.05). E . Positive samples of human pituitary tumors (N=34) were electroporeted with 10mM hsiRNA IL-6 or 10mM siRNA GL3 and then stained for SA-β-gal. Black zone of the bars represents samples in which SA-β-gal staining were significantly diminished (Student t test, p<0.05), while grey zone represents samples in which SA-β-gal staining did not vary. F . Photographs of a representative non-functioning tumor with significant decrease in SA-β-gal with hsiRNA IL-6 are shown. Images were acquired at x40 magnification. G . Photographs of a representative non-functioning tumor with significant increase in c-myc (red, 1:50) with hsiRNA IL-6 are shown. 4′,6-diamidino-2-phenylindole (DAPI) were used for staining cell nuclei (blue). Images were acquired at x40 magnification (scale bar: 20μm).

Article Snippet: Proteins were blotted onto nitrocellulose membranes using standard procedures, and incubated with p16 INK4a (1:1000), STAT3 (1:1000), phosphoSTAT3 (1:1000), β-Actin (1:3000) (Santa Cruz Biotechnologies, California, USA), pRb (1:1000, Cell Signalling Technology, Danvers, Massachusetts, USA), GH (1:1000) or GAPDH (1:10000) (Abcam, Cambridge, Massachusetts, USA) antibodies overnight, followed by corresponding secondary antibodies incubation.

Techniques: Staining, Marker

Journal: Cost Effectiveness and Resource Allocation : C/E

Article Title: Early budget impact analysis of magnetic balloon technology for facilitating the completion of difficult colonoscopies

doi: 10.1186/s12962-025-00676-y

Figure Lengend Snippet: Summary of the budget impact analysis considering 1,313,100 colonoscopies/year (hospital and NHS perspectives)

Article Snippet: The Lombardy Region reported 108,502 colonoscopies in 2018 [ ] for a regional population in the same year of 10,010,833 individuals (ISTAT), i.e., approximately 11 procedures per 1,000 inhabitants.

Techniques:

Journal: Cost Effectiveness and Resource Allocation : C/E

Article Title: Early budget impact analysis of magnetic balloon technology for facilitating the completion of difficult colonoscopies

doi: 10.1186/s12962-025-00676-y

Figure Lengend Snippet: Summary of the budget impact analysis considering the 11% of incomplete colonoscopies (hospital and NHS perspectives)

Article Snippet: The Lombardy Region reported 108,502 colonoscopies in 2018 [ ] for a regional population in the same year of 10,010,833 individuals (ISTAT), i.e., approximately 11 procedures per 1,000 inhabitants.

Techniques:

Journal: Cost Effectiveness and Resource Allocation : C/E

Article Title: Early budget impact analysis of magnetic balloon technology for facilitating the completion of difficult colonoscopies

doi: 10.1186/s12962-025-00676-y

Figure Lengend Snippet: Summary of the budget impact analysis considering 2 additional hospital days in case of incomplete colonoscopy in hospitalised patients (hospital perspective)

Article Snippet: The Lombardy Region reported 108,502 colonoscopies in 2018 [ ] for a regional population in the same year of 10,010,833 individuals (ISTAT), i.e., approximately 11 procedures per 1,000 inhabitants.

Techniques: