To the manufacturer’s instructions. Fluorescence was correlated to protein content.Statistical analysisAll experiments were PD-1/PD-L1 inhibitor 1 repeated at least three times and the results are presented as the means and standard deviations of independent samples. Data were statistically evaluated using a nonparametric Kruskal-Wallis test, followed by Mann-Whitney U test for comparison of two groups. P values #0.05 were considered to be significant and marked with an asterisk in figures.Lipid measurementsUnesterified cholesterol content was measured in cell lysates using the Amplex Red Cholesterol Assay Kit (Invitrogen, Paisley, UK), as described by the manufacturer. Cholesterol amount was correlated to protein content. Sphingomyelin content was analyzed according to a previously described method [28].Supporting InformationFigure S1 Viability of human fibroblasts after MSDH exposureImmunocytochemistryCells were prepared for immuno-cytochemistry as described elsewhere [20]. Antibodies against LAMP-2 (Southern Biotech, Birmingham, AL, USA), followed by antibodies conjugated to Alexa Fluor (Molecular Probes), were used. To visualize unesterified cholesterol, cells were stained with filipin (125 mg/ml; SigmaAldrich) for 1 h at room temperature. Cover slips were washed and mounted using Prolong gold (Invitrogen). Cells were examined using a Nikon Eclipse E600 laser scanning confocal microscope (Nikon, Tokyo, Japan) together with the EZC1 3.7 software (Nikon Instruments, Melville, NY, USA) or a Nikon Eclipse TE2000U microscope (Nikon) with a Bio-Rad Radiance 2100 MP confocal system (Carl Zeiss, Jena, Germany).as assessed by crystal violet staining. Human wt fibroblasts were treated with U18666A or quinacrine to induce cholesterol accumulation, and NPC1-mutant fibroblasts were treated with methyl-b-cyclodextrin (MbCD) or 25-hydroxy cholesterol (25-HC) to revert cholesterol storage. Viability of cultures assessed by crystal violet staining (n = 4). Viability is expressed as percentage of untreated cultures. Data are presented as the mean 6 SD, * p#0.05. (TIF)Figure SMeasurement of cholesterol content in AKT inhibitor 2 primary neuronal cultures. Cultures of rat neurons were treated with U18666A (0.5? mg/ml, 48 h) and the unesterified cholesterolLysosomal Stability Is Regulated by Cholesterolcontent was measured 1317923 (n = 3). Data are presented as the mean 6 SD, ns; non-significant. (TIF)Author Contributions?Conceived and designed the experiments: HA LS KB PS BG KO KK. ?Performed the experiments: HA LS. Analyzed the data: HA LS BG KO ?KK. Wrote the paper: HA LS KB PS BG KO KK.AcknowledgmentsWe thank Ida Eriksson for technical assistance, Sangeeta Nath for help with confocal microscopy and Ann-Charlotte Johansson and Alex Schneede for scientific input.
Pancreatic b cell transplantation, either in the form of harvested pancreatic islets, or as cells derived from embryonic precursors or following trans-differentiation in vitro, has the potential to restore the recipients’ ability to respond to blood glucose levels and secrete insulin in a physiological manner [1]. However major problems in achieving this ideal include lack of donor islets available for transplantation; loss of the valuable resource of islets during the harvesting procedure; and loss of islets following transplantation due to immune mediated allo-rejection plus lack of trophic support [2]. Although future advances in regenerative medicine may alleviate the problem of availability, all these issues arecompounded by continuin.To the manufacturer’s instructions. Fluorescence was correlated to protein content.Statistical analysisAll experiments were repeated at least three times and the results are presented as the means and standard deviations of independent samples. Data were statistically evaluated using a nonparametric Kruskal-Wallis test, followed by Mann-Whitney U test for comparison of two groups. P values #0.05 were considered to be significant and marked with an asterisk in figures.Lipid measurementsUnesterified cholesterol content was measured in cell lysates using the Amplex Red Cholesterol Assay Kit (Invitrogen, Paisley, UK), as described by the manufacturer. Cholesterol amount was correlated to protein content. Sphingomyelin content was analyzed according to a previously described method [28].Supporting InformationFigure S1 Viability of human fibroblasts after MSDH exposureImmunocytochemistryCells were prepared for immuno-cytochemistry as described elsewhere [20]. Antibodies against LAMP-2 (Southern Biotech, Birmingham, AL, USA), followed by antibodies conjugated to Alexa Fluor (Molecular Probes), were used. To visualize unesterified cholesterol, cells were stained with filipin (125 mg/ml; SigmaAldrich) for 1 h at room temperature. Cover slips were washed and mounted using Prolong gold (Invitrogen). Cells were examined using a Nikon Eclipse E600 laser scanning confocal microscope (Nikon, Tokyo, Japan) together with the EZC1 3.7 software (Nikon Instruments, Melville, NY, USA) or a Nikon Eclipse TE2000U microscope (Nikon) with a Bio-Rad Radiance 2100 MP confocal system (Carl Zeiss, Jena, Germany).as assessed by crystal violet staining. Human wt fibroblasts were treated with U18666A or quinacrine to induce cholesterol accumulation, and NPC1-mutant fibroblasts were treated with methyl-b-cyclodextrin (MbCD) or 25-hydroxy cholesterol (25-HC) to revert cholesterol storage. Viability of cultures assessed by crystal violet staining (n = 4). Viability is expressed as percentage of untreated cultures. Data are presented as the mean 6 SD, * p#0.05. (TIF)Figure SMeasurement of cholesterol content in primary neuronal cultures. Cultures of rat neurons were treated with U18666A (0.5? mg/ml, 48 h) and the unesterified cholesterolLysosomal Stability Is Regulated by Cholesterolcontent was measured 1317923 (n = 3). Data are presented as the mean 6 SD, ns; non-significant. (TIF)Author Contributions?Conceived and designed the experiments: HA LS KB PS BG KO KK. ?Performed the experiments: HA LS. Analyzed the data: HA LS BG KO ?KK. Wrote the paper: HA LS KB PS BG KO KK.AcknowledgmentsWe thank Ida Eriksson for technical assistance, Sangeeta Nath for help with confocal microscopy and Ann-Charlotte Johansson and Alex Schneede for scientific input.
Pancreatic b cell transplantation, either in the form of harvested pancreatic islets, or as cells derived from embryonic precursors or following trans-differentiation in vitro, has the potential to restore the recipients’ ability to respond to blood glucose levels and secrete insulin in a physiological manner [1]. However major problems in achieving this ideal include lack of donor islets available for transplantation; loss of the valuable resource of islets during the harvesting procedure; and loss of islets following transplantation due to immune mediated allo-rejection plus lack of trophic support [2]. Although future advances in regenerative medicine may alleviate the problem of availability, all these issues arecompounded by continuin.
