Additional file 1 of Evaluation of the effect of tofogliflozin on the tissue characteristics of the carotid wall—a sub-analysis of the UTOPIA trial

Additional file 1: Tables S1. Between-group comparison of changes in clinical parameters during the treatment period. Table S2. Changes in concomitantly used anti-diabetic agents. Table S3. Changes in concomitantly used cardiovascular medications. Table S4. The changes of GSM-CCA on the basis of tertiles of changes in mean-IMT during observation perio

Expression levels of genes associated with viral infection increased following PIC transfection.

<p>Quantitative RT-PCR analysis was performed with RNA extracted from MIN6 cells with or without PIC transfection. IFNα, IFNβ, CXCL10, Fas (a), TLR3, RIG-I, MDA5, LGP2 (b), ISG15, Mx1, OAS1, and PKR (c) gene expressions were significantly increased compared with those of the control (Lipofectamine only) cells (n = 3). ELISA was also performed with cell lysates of MIN6 cells with or without PIC transfection. The levels of IFNα and IFNβ were significantly increased in PIC-transfected MIN6 cells compared with those in the control cells (Fig 1d, n = 3). The data were normalized to β-actin gene expression, with the relative gene expressions of the PIC-transfected cells arbitrarily set to 100. The error bars represent SE. The asterisk indicates significant difference (p<0.05).</p

Examination of a Viral Infection Mimetic Model in Human iPS Cell-Derived Insulin-Producing Cells and the Anti-Apoptotic Effect of GLP-1 Analogue

<div><p>Aims</p><p>Viral infection is associated with pancreatic beta cell destruction in fulminant type 1 diabetes mellitus. The aim of this study was to investigate the acceleration and protective mechanisms of beta cell destruction by establishing a model of viral infection in pancreatic beta cells.</p><p>Methods</p><p>Polyinosinic:polycytidylic acid was transfected into MIN6 cells and insulin-producing cells differentiated from human induced pluripotent stem cells via small molecule applications. Gene expression was analyzed by real-time PCR, and apoptosis was evaluated by caspase-3 activity and TUNEL staining. The anti-apoptotic effect of Exendin-4 was also evaluated.</p><p>Results</p><p>Polyinosinic:polycytidylic acid transfection led to elevated expression of the genes encoding IFNα, IFNβ, CXCL10, Fas, viral receptors, and IFN-inducible antiviral effectors in MIN6 cells. Exendin-4 treatment suppressed the elevated gene expression levels and reduced polyinosinic:polycytidylic acid-induced apoptosis both in MIN6 cells and in insulin-producing cells from human induced pluripotent stem cells. Glucagon-like peptide-1 receptor, protein kinase A, and phosphatidylinositol-3 kinase inhibitors counteracted the anti-apoptotic effect of Exendin-4.</p><p>Conclusions</p><p>Polyinosinic:polycytidylic acid transfection can mimic viral infection, and Exendin-4 exerted an anti-apoptotic effect both in MIN6 and insulin-producing cells from human induced pluripotent stem cells.</p></div

PIC transfection increased IFNβ in insulin-producing cells differentiated from human iPS cells.

<p>(a) Immunostaining of insulin-producing cells differentiated from human iPS cells. Insulin-positive cells were detected in purple, transfected cells in red with TI, and IFNβ-positive cells in green. Scale bars = 100 μm. (b) IFNβ staining of control cells and PIC-transfected cells. Scale bars = 200 μm. (c) IFNβ-positive ratio in TI-positive insulin-producing cells. The error bars represent SE. The asterisk indicates significant difference (p<0.05).</p

Effects of Src inhibitor on adiponectin-induced changes in peripheral blood monocyte-derived macrophages (PBDMs).

<p>PBDMs were preincubated with 10 µM PP1 for 30 min and then incubated with adiponectin for 6 hours (A–D). The mRNA expression levels of COX-2 (A), TIMP-1 (B), IL-6 (C), and VEGF-C (D) were quantified by real-time PCR. Values are normalized to the level of GAPDH mRNA and expressed as mean ± SEM (n = 3). ***P<0.001. N.S not significant.</p

PIC transfection stimulated apoptosis while Ex4 treatment reduced PIC-induced apoptosis in MIN6 cells.

<p>(a) Caspase-3 activities of PIC-transfected cells with or without Ex4 (10 nM and 100 nM). The data were expressed as the caspase-3-to-protein content ratio, with that of the control cells arbitrarily set to 1. The error bars represent SE. The asterisk indicates significant difference (p<0.05). (b) TUNEL staining of MIN6 cells. Nuclei were detected in blue with Hoechst 33342, transfected cells in red with TI, and TUNEL-positive cells in green. Scale bars = 100 μm. (c) TUNEL staining of control MIN6 cells (Lipofectamine only) with or without 100nM Ex4 and PIC-transfected cells with or without 100 nM Ex4. Scale bars = 200 μm. (d) TUNEL-positive cells in TI-positive MIN6 cells. The error bars represent SE. The asterisk indicates significant difference (p<0.05). NS represents no significant difference.</p

PIC stimulated apoptosis while Ex4 reduced PIC-induced apoptosis in insulin-producing cells from human iPS cells.

<p>(a) Immunostaining of insulin-producing cells differentiated from human iPS cells. Insulin-positive cells were detected in purple, transfected cells in red with TI, and TUNEL-positive cells in green. Scale bars = 200 μm. (b) TUNEL staining of control cells with or without 100nM Ex4 and PIC-transfected cells with or without 100 nM Ex4. (c) TUNEL-positive cells in TI-positive insulin-producing cells. The error bars represent SE. The asterisk indicates significant difference (p<0.05). NS represents no significant difference.</p

Ex4 inhibits the PIC-induced increase in cytokine and chemokine gene expression.

<p>Quantitative RT-PCR analysis of IFNα, IFNβ, CXCL10, and Fas was performed in PIC-transfected MIN6 cells with or without Ex4 (10 nM and 100 nM, n = 3). The data were normalized to β-actin gene expression, with the relative gene expressions of the PIC-transfected cells arbitrarily set to 100. The error bars represent SE. The asterisk indicates significant difference (p<0.05).</p

Ex4 mitigated PIC-induced apoptosis via the GLP-1 receptor and both the PKA and PI-3K pathways.

<p>(a) Quantitative RT-PCR analysis of the GLP-1 receptor was performed in PIC-transfected MIN6 cells with or without Ex4 (10 nM and 100 nM, n = 3). The data were normalized to β-actin gene expression, with the relative gene expressions of the PIC-transfected cells arbitrarily set to 100. The error bars represent SE. (b–d) Caspase-3 activities of PIC-transfected MIN6 cells treated with 100 nM Ex4 and Ex9 (b), H89 (c), or LY294002 (d). The data are expressed as the caspase-3-to-protein content ratio, with that of the PIC-transfected cells without Ex4/Ex9, H89, or LY294002 arbitrarily set to 100. The error bars represent SE. The asterisk indicates significant difference (p<0.05). NS represents no significant difference.</p

Adiponectin-induced phosphorylation of ERK in peripheral blood monocyte-derived macrophages (PBDMs).

<p>PBDMs were incubated for 20 min with 10 µg/ml of adiponectin protein. Cell lysates were subjected to SDS-PAGE followed by western blotting with anti-phosphorylated ERK1/2, and anti-ERK1/2 antibodies (A). PBDMs were preincubated with 10 µM BAY 61-3606 (BAY) for 30 min and then treated for 20 min with 10 µg/ml of adiponectin protein. Cell lysates were subjected to SDS-PAGE followed by western blotting with anti-phosphorylated ERK1/2, and anti-ERK1/2 antibodies (B). Equal protein loading and transfer were confirmed with Ponceau staining.</p