Fig 2: Drug sensibilization in senescent U87-MG glioblastoma cell line. (A) Representative FACS dot plots showing the percentages of multidrug resistance protein (MDR)-positive U87-MG cells in different conditions as indicated. After inducing senescence with etoposide, the percentage of MDR-positive cells rose to 6.36%, and the 48 h treatment with rhLAV-BPIFB4 reduced this value to 0.72%. (B) Bar graph reporting the mean percentage values ± SD of MDR+ viable cells from 3 independent experiments. (C) Cytofluorometric analysis of Spider-ß galactosidase in the U87-MG glioma cell line after 24 h treatment with TMZ (100 µM) and 5 days resting in the absence or presence with rhLAV-BPIFB4 for the last 48 h. The right panel reports the Western blot analysis of p16 and p21 senescence-related proteins in the same experimental condition. ß-Actin was used as a control for quantitation of the sample protein. (D) BrdU proliferation assay on the U87-MG cell line after treatment with temozolomide (96 h, 100 µM). The LAV-BPIFB4 co-treatment induced a higher sensibilization to the drug in a dose–response manner. (E) Cytofluorimetric analysis of the HLA-E expression. The panel on left side shows a representative, of three independent experiments, histogram profile of HLA-E staining on U87-MG cells’ surface in the control and TMZ-treated cells with or without rhLAV-BPIFB4 for the last 48 h of the 5 days resting. (F) The right panel is a bar graph of percentage positive cells. The results are representative of 3 independent experiments expressed as the mean ± SD. (G,H) Induction of apoptosis measured by Annexin V and propidium iodide (PI) double-staining through flow cytometry in the U87-MG glioma cell line after 24 h of treatment with TMZ (100 µM) and 5 days resting in the absence or presence of rhLAV-BPIFB4 for the last 48 h. The left panel is a representative density plot of cytofluorometric analysis. Histograms on the right indicate total percentage of early (Annexin V-positive cells/PI-negative cells) and late apoptotic events (Annexin V/PI-double positive cells) as well as necrotic cells (Annexin V-negative cells/PI-positive cells). The results are representative of 3 independent experiments performed in duplicate and expressed as mean ± SD. Statistical analysis by two-way ANOVA with Tukey’s test for multiple comparison was conducted (* p < 0.01, ** p <0.001, *** p <0.0001, **** p < 0.00001).