Fig 1: Tinostamustine increases the effects of radiotherapy in glioma models in vitro (part I). a Clonogenic assay performed on U87MG, U251, A172, and T98G cells showing radio-sensitizing effects of tinostamustine (TINO). b Comparison between dose enhancement factor (DRE) calculated in U87MG, U251, A172, and T98G in cultures treated with Rt combined to SAHA, BDM, SAHA + BDM, and TINO and showing higher DRE values in the combination of TINO and RT. c Western blots for autophagy and necrosis marker expression in A172 cells used as representative cell line for this analyses. d ELISA determination of beclin 1 and p62 proteins in U87MG, U251, A172, and T98G cells. ELISA determinations performed in quintuplicate. Mean values (mean values ± standard errors, SD) were transformed as % versus relative controls. e Representative comet assay obtained in A172 cells treated with different doses of RT with or without 3.5 μM TINO. f Quantification of comet assays as percentage of comets (with intermediated/high comet tails) in U87MG, U251, A172, and T98G; Single results are representative of three different experiments performed in triplicate. Statistical analyses: *p < 0.005
Fig 2: Lysosomal accumulation of rLGALS9 results in halted autophagy. (A) LC3B-II levels after 24 h treatment of the indicated cell lines with rLGALS9 (300 nM), autophagy inhibitor chloroquine (100 μM) or autophagy inducer rapamycin (100 nM). (B) Analysis of SQSTM1 levels using ELISA after treatment of DLD-1 as in (A) (factor = rLGALS9/medium). (C) Time-course analysis of SQSTM1 levels in DLD-1 cells treated with rLGALS9 (24 h, 300 nM). (D) SQSTM1 formation induced by rLGALS9 treatment (24 h, 300 nM) in a panel of KRASmut cell lines (n = 5) and BRAFmut (n = 5). (E) Confocal microscopy pictures of DLD-1 cells treated for 24 h with DyLight-594-labeled rLGALS9 and costained with anti-LC3B-488. (F) LC3B/rLGALS9 colocalization was analyzed by Pearson's correlation. (G) Confocal microscopy pictures of DLD-1 cells treated with rLGALS9 (24 h, 300 nM) or chloroquine (24 h, 100 μM) and subsequently stained with anti-LAMP2 and anti-LC3B. LAMP2/LC3B colocalization was analyzed by Pearson's correlation. (H) Confocal microscopy pictures of DLD-1 cells transfected with a mCherry-GFP-LC3 construct treated with rLGALS9 (24 h, 300 nM) or chloroquine (24 h, 100 µM). mCherry/GFP ratio was analyzed using ImageJ software.
Fig 3: The intracellular level of p62 protein and the percentage of spontaneous apoptosis in PBMC of PD patients and HDs and their correlation. (a) Differences in the intracellular level of p62 protein (ng/mL) between PD patients and the HD group. (b) The percentages of cells undergoing spontaneous apoptosis among PBMC from PD patients and HD. (c) Correlation analysis between apoptotic cell percentage and p62 level (ng/mL) in PBMC from PD patients (d) from HDs. Solid lines, mean ± SEM. Pearson’s correlation coefficient was used; dotted line, 95% confidence interval.
Fig 4: ROC curve analysis showing HSPA6 gene expression levels and p62 protein levels as potential biomarkers of PD. (a) The HSPA6 mRNA levels in PBMC showed 75.0% sensitivity and 52.63% specificity in distinguishing PD. (b) The p62 protein level in PBMC showed 69.23% sensitivity and 62.86% specificity in distinguishing PD. (c) Combination of HSPA6 mRNA and p62 protein showed 77% sensitivity and 83% specificity in distinguishing PD. ROC, receiver operating characteristic; AUC, the area under the ROC curve; Sen, sensitivity; Spe, specificity.
Supplier Page from Enzo Life Sciences, Inc. for p62 ELISA kit