Journal: PLoS ONE

Article Title: Lack of Wdr13 Gene in Mice Leads to Enhanced Pancreatic Beta Cell Proliferation, Hyperinsulinemia and Mild Obesity

doi: 10.1371/journal.pone.0038685

Figure Lengend Snippet: A) Transfection with Ad Wdr 13 and AdGFP viruses shows overexpression of WDR13 protein in MIN6 cells as visualized by immunoblotting using anti WDR13 antibody. Lower panel shows beta actin as loading control. Overexpression of WDR13 protein results in retardation in cell proliferation after 48 h of transfection with 100 MOI. B) Overexpression of WDR13 protein results in accumulation of p21, whereas Cyclin D1, Cyclin D2, Cyclin E1 and p27 levels remain unaffected. C) siRNA knockdown of WDR13 in MIN6 cells. MIN6 cells were transfected with nonspecific scrambled (Scr) siRNAs and WDR13 specific siRNA. Immunoblot analysis shows Knockdown of WDR13 protein. Actin was used as loading control. Immunoblot, using p21 antibody shows reduction of p21 levels in WDR13 knockdown MIN6 cell. D) Cell cycle inhibitor p21 expression in purified pancreatic islets of Wdr 13 knockout mice and that of wild type littermates by western blot analysis. Beta actin was used as loading control. p21 expression is less in the islets of knockout mice. E) Occupancy by WDR13 at p21 promoter revealed by chromatin immunoprecipitation using primers specific for p21 and GAPDH.

Article Snippet: 10,000 MIN6 cells (obtained from National Centre for Cell Science, Pune, India) were seeded per well of 24 well plates in DMEM media containing 10% FBS and were transfected either with AdGFP or Ad Wdr13 using a titer of 100 MOI.

Techniques: Transfection, Over Expression, Western Blot, Control, Knockdown, Expressing, Purification, Knock-Out, Chromatin Immunoprecipitation

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Images of the spheroids from static culture for morphological analysis and the determination of properties such as spheroid size and circularity ( n = 12; data are means ± STDV). (A) Morphological differences can be seen during the aggregation of the different β‐cell lines within 7 days. The images of INS‐1, MIN6, and MSCs were reduced by 60%, whereas the 1.1B4 images were reduced by 73% ( * ). In both cases the scale bar represents 100 μm. (B) The growth kinetics of the spheroids reveal the differences between cell lines. The INS‐1 cells (squares) showed fast aggregation and a continuous increase in spheroid size, whereas the MIN6 cells (circles) needed 3 days to form stable spheroids before slow growth was observed. The 1.1B4 cells (triangles) aggregated slowly over 5–7 days, represented by a peak on day 2 followed by a decline in size until a stable spheroid was formed and a volume increase was observed. The hMSC‐TERTs (crosses) and ad‐MSCs (stars) showed no growth and the spheroid size declined over time. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The viability of spheroids from static cultures determined by staining with calcein AM and ethidium after 7 days. (A) INS‐1 spheroids featured a dead core and a viable mantle, whereas MIN6 spheroids featured a heterogenous distribution of dead cells. The loose structure of the 1.1B4 spheroids promoted sufficient mass transfer resulting in a high viability. (B) Insulin secretion profiles of INS‐1 cells cultured as monolayers and spheroids cultured under static (96‐well plate) and dynamic (shaking flask) conditions ( n = 3; data are means ± STDV; significance intervals are * p < 0.05, ** p < 0.01, and *** p < 0.001). 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Cell Culture, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The insulin profiles of β‐cell spheroids in static culture was measured by GSIS ( n = 3; error = STDV)

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques:

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Aggregation of INS‐1 (upper row), MIN6 (middle row) and 1.1B4 (lower row) cells with hMSC‐TERTs at different ratios after 24 h. MSCs were stained blue (VPD) and β‐cells were stained with the green dye CFSE. Starting with monospheroids in the first (MSCs, blue) and second (β‐cells, green) columns, the cell ratios increase from left to right. Due to different scaling of the images, the MSC spheroids seem to have a different size in each setup, but the seeding density was always 1000 cells per well. In all cases the scale bar represents 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; CFSE, 5‐(and 6)‐carboxyfluorescein diacetate, succinimidyl ester; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Bright‐field and viability images (at day 7) of monospheroids (0–1 = MSC only; 1–0 = β‐cell only) and heterospheroids INS‐1 (upper row), MIN6 (middle row), and 1.1B4 (lower row) cocultured with hMSC‐TERTs at different cell ratios. The stated viabilities of the spheroids were assessed by measuring the red (core) and green (whole spheroid) diameter and the resulting volume to describe the real “3D viability,” but the displayed images only represent two dimensions of the spheroids, which could provide a deceptive impression. Scale bar = 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A ) The images of MIN6 cells infected with HCV at 24, 48, 72 and 96 hpi. MIN6 cells were mock-infected or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1. Scale bar, 10 µm. ( B ) MIN6 cells were infected with HCV at different MOIs. At indicated time points post-infection, the percentage of viable cells was assessed by MTT assay and plotted versus time. The activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). * P <0.05, ** P <0.01, compared with respective controls.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, MTT Assay, Activity Assay

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A, B ) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) ( A ) were obtained and cell viability ( B ) of MIN6 cells was determined by MTT assay. ( C, D ) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV ( C ) or BILN 2061 ( D ) at 96 hpi, cell viability was assessed by MTT assay. ( E ) MIN6 cells were infected with HCV as in . At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. ( F ) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. ( A–F ) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). * P <0.05, ** P <0.01, compared with respective controls.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Purification, Cell Culture, Light Microscopy, MTT Assay, Staining, Activity Assay

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A–C ) Confocal image of MIN6 cells stained with Hoechst 33258. Scale bar, 10 µm. Cells were mock infected or infected with 1.0 MOI HCV for 96 h. Cells treated with CHX for 48 h served as an apoptosis positive control. White arrows indicate nuclei ( A ). MIN6 cells were treated as in ( B ). Cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of RBV (50 µM) or BILN 2061 (10 µM) at 96 hpi ( C ). ( D ) Electron microscopic analysis as in (A). Scale bar, 2 µm. All measurements were done in triplicates.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Staining, Infection, Positive Control

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. ( A ) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. ( B ) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. ( C ) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. ( D ) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). ( A, B, D ) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). ** P <0.01, compared with respective controls.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Staining, TUNEL Assay, Activity Assay, Western Blot, Positive Control, Flow Cytometry

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. ( A ) Mitochondrial transmembrane potential changes at 48 hpi. Representative images from JC-1 staining are shown. The aggregated form of JC-1, characteristic of mitochondrial integrity (red) and monomeric JC-1 (green) were examined under laser confocal scanning microscope. ( B ) Electron microscopy of mitochondrial morphologies at 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Black and white arrows indicate mitochondria and insulin granules, respectively. Scale bar, 1 µm.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Staining, Microscopy, Electron Microscopy, Positive Control

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A ) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). * P <0.05, compared with respective controls. ( B ) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A ) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. ( B ) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H 2 O). ( C–D ) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody ( C ) or HCV dsRNA labeled with J2 antibody (green) ( D ) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. ( E ) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Isolation, Amplification, Positive Control, Labeling, Immunofluorescence, Fluorescence, Staining, Immunoprecipitation, Purification, Lysis, Western Blot