Protective effects of maternal immunization with naturally oxidized LDL (nLDL) on male offspring fed 0.5% cholesterol diet.

<p>(A) Glucose response in an OGTT in offspring after 29 weeks on diet. (B) Insulin responses in the same OGTT. AUCs for glucose and insulin are shown as insets; statistical significances for individual time points are not indicated. n = 26.</p

Overview of the experimental design and aims.

<p>Overview of the experimental design and aims.</p

Protective effects of nLDL immunization in euglycemic female mice fed regular chow.

<p>To assess direct effects of immunization on glucose responses, OGTTs were performed on separate groups of female mice 40 and 250 days after the primary immunization. Controls were non-immunized. (<b>A</b>) Body weights at both time points. (<b>B</b>) Glucose responses after 40 days. (<b>C,D</b>) Glucose and insulin responses after 250 days. AUC, Area under the curve. n = 24 (40 days) and 25 (250 days).</p

Characterization of experimental diets.

<p>(<b>A</b>) Effects of regular chow, 0.5% cholesterol diet and 60% sucrose diet on (<b>A</b>) body weight, (<b>B</b>) lipoprotein profiles and (<b>C,D</b>) glucose response in male mice after 78 days on diet (n = 31). FPLC analysis was performed on pooled plasma samples from 2–3 mice per group. Glucose responses were assessed by OGTT) on 31 mice (C) and differences in the area under the curve (AUC) determined by unpaired T-test (D). (<b>E–I</b>) Plasma concentrations of major free fatty acids (FFAs) in male offspring of OxLDL-immunized (OxLDL) and non-immunized mothers (controls) after 24 weeks on 60% sucrose or regular diet (n = 32). (<b>J</b>) Total FFAs.</p

Effects of maternal OxLDL immunization on the activity of antioxidant enzymes and glutathion concentration in offspring.

<p>Hepatic activity of (A) cytosolic superoxide dismutase (SOD), (B) total SOD, (C) glutathione peroxidase (GPx), (D) catalase, and (E) mitochondrial SOD. (F) Hepatic glutathion concentration. Ventricular activities of mitochondrial SOD (G) and (H) GPx.</p

Protective effects of maternal OxLDL immunization in male offspring.

<p>(<b>A,B</b>) Glucose and insulin responses during an OGTT in male offspring of mothers immunized with OxLDL or PBS and non-immunized control mothers, after 28 weeks on 60% sucrose diet (n = 26). AUCs are shown as insets. (<b>C–H</b>) Pancreas immunohistochemistry of the same animals. Alpha cells identified by glucagon-staining in offspring of OxLDL-immunized (<b>C</b>) and control mothers (<b>D</b>). No-primary-antibody control (<b>E</b>). Beta cells staining for insulin in offspring of (<b>F</b>) OxLDL-immunized, (<b>G</b>) PBS-immunized and (<b>H</b>) control mothers (<b>I–K</b>)<b>.</b> An additional group of male offspring of OxLDL-immunized and control mothers was fed sucrose for 38 weeks and then subjected to hyperinsulinemic euglycemic clamp (n = 16). Body weights (I) and fasting plasma glucose levels (J) were not significantly different, whereas the amounts of glucose infused during the euglycemic phase was markedly greater in offspring of OxLDL-immunized mothers (K).</p

Effect of maternal OxLDL immunization on hepatic expression of genes of relevance for diabetes.

<p>mRNA expression of genes known to be regulated in diabetic conditions was assessed by commercial gene array in the liver of male offspring of OxLDL-immunized and control mice after 28 weeks on 60% sucrose diet (experiment #4). Only genes significantly regulated more than 1.5 fold are shown. IL-10 is included even though it failed to reach significance.</p

Protective effects of maternal OxLDL immunization in female offspring.

<p>(<b>A–C</b>) Female offspring of mothers immunized with OxLDL or PBS and non-immunized controls fed 60% sucrose diet for 24 weeks (n = 27). Body weights, glucose responses and insulin responses during an OGTT at this time are shown, together with respective AUCs (insets). (<b>D–F</b>) A second group of female offspring was reverted to regular chow for 19 weeks after insulin resistance had been induced by 36 weeks on the sucrose diet. Body weights, plasma glucose and insulin levels (after 6 hr. fasting) were determined at 60 weeks of age. Numbers of mice with glucose levels ≥230 mg/dl and total number in each group are indicated in the bars of panel E.</p

Protective effects of OxLDL immunization in male mice fed 60% sucrose.

<p>To assess direct effects of immunization with extensively oxidized LDL in immunized mice exposed to more obesogenic conditions, 4 month old male LDLR^-/- mice immunized with OxLDL or PBS and controls were fed 60% sucrose for 38 weeks. Age-matched non-immunized mice fed regular chow served as additional control. (<b>A</b>) Body weights. (<b>B,C</b>) OGTT glucose response curves and AUCs. (<b>D,E</b>) Corresponding OGTT insulin curves and AUCs. n = 22.</p

Additional file 1 of Therapeutic targeting of P2X4 receptor and mitochondrial metabolism in clear cell renal carcinoma models

Additional file 1: Suppl. Figure 1. P2X4 in clear cell renal carcinoma correlates with mitochondrial antioxidant proteins by mRNA seq data bases. Suppl. Figure 2. (A) Representative MRI image of a patient with kidney carcinoma. (B) Organoid diameters after different time of culture. Data represent 3 different measurements of 3-5 biological replicates. Scale bars = 50 μm (C) Representative hematoxylin-eosin (H&E) staining of different clear cell carcinomas biopsies together with the bright-field microscope images of corresponding H&E stained ccRCC organoids (inset). Scale bars = 50 μm. Suppl. Figure 3. (A) Representative image of organoids treated with DMSO vehicle or 5-BDBD for different times and then stained with Calcein-AM (green) indicating vital cells or PI (red) indicating necrotic ones. Scale bar = 100 µm. (B) Concentration response to 5-BDBD determined in 3D culture assay measured at day 10 yielding an IC50 value of 7.556 µM (C) Schematic representation of plate view software dosage of 5-BDBD tested on 20 replicates for individual dose in single patient analyzed by CELIGO software. (D) Comparison of organoids growth for 3 days than treated with DMSO or 5-BDBD (5µM) for additional 7 days. Scale bar 100µm (E) Colony formation assay. The number of colony formed by A-498 cells 105 per well in presence of Vehicle (DMSO) or 5-BDBD at 5μM were cultured for 14 days colonies were stained with crystal violet and then photographed. Suppl. Figure 4. (A) Phase contrast image of representative organoids from different patients (PDO#1-5) treated with different doses of Everolimus for 7 days. (B,C) Quantification by MTT assay of the vitality reported as ratio to non treated organoids x100 from different patients (PDO#1-5) treated for 7 days with increasing doses of Everolimus or EZD8055, as indicated. Data are means of 20 replicate organoids from the same patient for each dose. Error bars are SD of 29 biological replicated for each dose. Table 1. Clinical features of the Clear Cell Renal Carcinomas. Table 2. Imaging characteristics of renal lesions