Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: In vitro analysis also shows a decreased response to hedgehog protein in mouse embryonic fibroblasts (MEFs). Wild-type, but not mutant, MEFs show increased cell growth upon treatment with SHH (A; *p = 0.001 for wt; n = 4). Quantitative RT-PCR analysis also shows a reduced response of Gli1 (B; *p = 0.002 for wt, p = 0.01 for rud ; n = 3) and Patched (C; *p = 0.001 for wt; n = 3) upon SHH treatment in mutant MEFs. β-galactosidase accumulates in SHH-treated wild-type; Ptc-lacZ MEFs, but not rud;Ptc-lacZ MEFs (D; *p = 0.005 for wt; n = 4). (E) PzP53Med ( Ptc-lacZ;p53 medulloblastoma) cells infected and clonally selected with control plKO.1 lentivirus respond to SHH treatment with accumulation of β-galactosidase (* p = 0.07 for wt; n = 4). Two independent clonal lines infected with Hsd17b7 RNAi constructs do not respond as robustly to SHH treatment.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: In Vitro, Mutagenesis, Quantitative RT-PCR, Infection, Control, Construct

Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: Wild-type (A–I) and rudolph (J–R) mutant MEFs were transfected with a SMO-GFP plasmid and acetylated α-tubulin immunocytochemistry was used to identify the primary cilium. SMO-GFP in untreated cells is seen outside the cilium (A–C, J–L), accumulating at the base of the axoneme (not shown), or throughout the cilium (D–F, M–O). Treatment with SHH protein localizes SMO-GFP throughout the cilium in the majority of cells observed (G–I, P–R). All images were taken at the same magnification and the scale bar in C represent 10 µm.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Immunocytochemistry

Journal: Stem cell research

Article Title: Dynamic blebbing: A bottleneck to human embryonic stem cell culture that can be overcome by Laminin-Integrin signaling

doi: 10.1016/j.scr.2018.10.022

Figure Lengend Snippet: Comparison of dynamic blebbing in four cell types. All cell types produced dynamic blebs: (A) human prostate cancer cell (HU145), (B) mouse embryonic fibroblast (mEF), (C) mouse embryonic stem cell (mESC), and (D) human embryonic stem cell (hESC). (E) Number of blebs/cell in different cell types in each frame before attachment. One of three independent experiments is shown. The insert shows the number of blebs/cell averaged over all frames in all three experiments. The number of blebs/cell was significantly higher in hESC than in the other cell types by one-way ANOVA. (F, G) The percentage of blebbing cells (F) and attached cells (G) for the four cell types over 100 min. Videos were collected using a BioStation IM with 62 s intervals for hESC, 60 s intervals for prostate cancer cells and mEF, and 120 s intervals for mESC. The percentage of blebbing cells was significantly higher at the initial time point for hESC than for the other cell types (F). The percentage of attached ESC was significantly lower than the other cells at the final time point (G). F and G were analyzed by two-way ANOVA. *** = p < .001. (H, I) Morphological and temporal comparisons of four patterns of blebbing behavior in hESC. (H) Phase contrast images of hESC at various times in culture showing the four patterns of behavior (DB = dynamic blebbing; A = attached; AB = apoptotic blebbing; R = rounding). Dynamic blebs appear dense, while apoptotic blebs are bright. (I) Percentage of cells in each of the behavior groups shown in H over 225 min of incubation. Each group is based on a count of 25 cells. The average time to attachment, rounding, or apoptotic blebbing ± the standard deviation is shown. The percentage of cells in each group is given on the Y axis. (J) Gray means of dynamic blebs and apoptotic blebs were analyzed using ImageJ followed by a t -test. *** p < .001.

Article Snippet: Mouse embryonic fibroblasts (mEF) were derived from 12.5-day to 13.5-day pregnant mouse using the ATCC protocol and then frozen in liquid nitrogen ( ; ; ). mEF were expanded by plating on 0.1% gelatin (Sigma 128-K-0066, St Louis MO.) in T-25 flasks.

Techniques: Comparison, Produced, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

doi: 10.1038/s41598-019-43115-8

Figure Lengend Snippet: Viability values (%) of 3T3 cells exposed to different concentrations of E. faecalis phage Max and E. faecium phage Zip for 24 h ( A ) Phage lysate; ( B ) Phages purified by PEG/NaCl precipitation. Viability was calculated as percentage of negative control (3T3 cells without phages). Error bars represent standard deviations from three independent experiments performed in duplicate.

Article Snippet: Mouse embryonic fibroblast 3T3 cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Purification, Negative Control

Journal: Scientific Reports

Article Title: Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

doi: 10.1038/s41598-019-43115-8

Figure Lengend Snippet: Efficacy of phages against bacteria colonizing 3T3 cells. ( A ) Concentration of viable bacterial cells; and ( B ) number of 3T3 cells in control (without treatment) and after 6 and 24 h of phage treatment. Error bars represent standard deviations from three independent experiments performed in duplicate. *Statistically significant (p < 0.05) conditions between control and test assays.

Article Snippet: Mouse embryonic fibroblast 3T3 cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Bacteria, Concentration Assay, Control

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 4. Purification of cardiomyocytes from differentiating mouse embryonic stem cells (mESCs) with cardiomyocyte-specific molecular beacons (MBs). A, Schematic of the protocol used for differentiating mESCs to the cardiac lineage. EB indicates embryoid body; and ES, embryonic stem cell. B, Percent expression of Tnnt2 at days 4 and 9 during mESC differentiation into cardiomyocytes; n=3. C, Immunocytochemistry results of mESC-derived cardiomyocytes at day 9 for Tnnt2, Tnnt3, and Actn2. Scale bars, 20 µm. D, Flow cytometry analyses of MHC1-MB (an MB that targeted myosin heavy chain 6/7 mRNA) signals in mESC differentiation culture at day 9; n=6. FSC indicates forward scatter. E, Flow cytometry analysis of Tnnt2 expression in mESCs sorted with MHC1-MB and fluorescence- activated cell sorting (FACS) at differentiation day 9; n=3. Numbers represent percentages of MB-positive cells (D, E). F, FACS-sorted MHC1-MB–positive cells exhibited Tnnt2 and Actn2 in immunocytochemistry. Scale bars, 20 µm. G, Quantitative reverse-transcriptase polymerase chain reaction analyses showing difference in gene expression levels between mouse tail tip fibroblasts (MTTF) and presorted (PRE) and postsorted (POST) mESCs. The cardiomyocyte genes Tnnt2, Myh6, Myh7, and Myl2 were significantly enriched in postsorted cells with MHC1-MB, and noncardiac lineage genes (Acta2, Ddr2, Gata1, and Sox17) were substantially reduced compared with presorted cells. The y axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *P<0.05 compared with presorted group; n=3.

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Purification, Expressing, Immunocytochemistry, Derivative Assay, Flow Cytometry, Fluorescence, FACS, Reverse Transcription, Polymerase Chain Reaction, Gene Expression

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 5. Purification of cardiomyocytes from differentiating human embryonic stem cells (hESCs) by cell sorting via molecular beacon (MB) and fluorescence-activated cell sorting (FACS). A, Schematic of the protocol to differentiate human pluripotent stem cells (hPSCs) to the cardiac lineage. BMP4 indicates bone morphogenetic protein 4; END-2, mouse endoderm-like cells; ES, embryonic stem cell; and FGF2, basic fibroblast growth factor. B, Percent expression of TNNI3 at days 9 and 13 determined by flow cytometry during hESC differentiation into cardiomyocytes; n=3. C, Flow cytometry results of MHC1-MB–positive cells in hESC differentiation culture at day 13; n=6. FSC indicates forward scatter; and MHC1-MB, an MB that targeted myosin heavy chain 6/7 mRNA. D, Flow cytometry results showing TNNI3 expression of FACS-sorted hESCs at day 13 of differentiation after applying MHC1-MB. Numbers represent percentages of MB-positive cells (C, D). n=3.

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Purification, FACS, Fluorescence, Expressing, Flow Cytometry

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 5. (continued) E, Immunocytochemistry for TNNI3 and TNNT2 on MHC1-MB–positive cells sorted from hESC cultures. Scale bars, 20 µm. F, mRNA expression levels of cardiac and noncardiac genes measured by quantitative reverse-transcriptase polymerase chain reaction. Comparisons were made for human dermal fibroblast (HDF), presorted hESCs at day 13 (PRE), and post-FACS–sorted cells at day 13 (POST) using MHC1-MB. Cardiomyocyte genes (TNNT2, MYH6, MYH7, and MYL2) were significantly higher in sorted hESCs than in presorted hESCs and HDF. Expression of the noncardiac lineage genes (PECAM1, CALPONIN, THY1, MYOD, and NEUROD) was significantly lower in sorted cells than in others. The y axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *P<0.05 compared with presorted group; n=3. G, mRNA expression levels of cardiac genes from both MHC1-MB–positive and –negative cells measured by quantitative reverse-transcriptase polymerase chain reaction. H, Action potentials of MHC1-MB–positive cardiomyocytes. Shown are representative configurations of nodal (top), atrial (middle), and ventricular (bottom) action potentials.

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Immunocytochemistry, Expressing, Reverse Transcription, Polymerase Chain Reaction

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 6. Purification of cardiomyocytes from differentiating human induced pluripotent stem cells (hiPSCs) by cell sorting via molecular beacon (MB) and fluorescence-activated cell sorting (FACS). A, Schematic of the protocol to differentiate hiPSCs to the cardiac lineage. BMP4 indicates bone morphogenetic protein 4; END-2, mouse endoderm-like cells; ES, embryonic stem cells; and FGF2, basic fibroblast growth factor. B, Percent expression of TNNI3 at days 9 and 13 during hiPSC differentiation into cardiomyocytes determined by flow cytometry; n=3. C, Flow cytometric analysis of MHC1-MB signals in hiPSC differentiation culture at day 13; n=6. MHC1-MB indicates an MB that targeted myosin heavy chain 6/7 mRNA. D, Flow cytometric results showing TNNI3 expression of FACS-sorted hiPSCs at day 13 of differentiation after applying MHC1-MB. Numbers represent percentages of MB-positive cells (C, D). n=3. E, Immunocytochemistry for TNNI3 and TNNT2 on MHC1-MB–positive cells sorted from hiPSC cultures. Scale bars, 20 µm. F, mRNA expression levels of cardiac and noncardiac genes measured by quantitative reverse-transcriptase polymerase chain reaction. Comparisons were made among human dermal fibroblast (HDF), presorted hiPSCs at day 13 (PRE), and MB-based FACS-sorted hiPSCs at day 13 (POST). The y axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *P<0.05 compared to presorted group; n=3. G, Calcium imaging of MHC1-MB–positive cardiomyocytes sorted from hiPSCs. Left, Confocal scan of representative cells loaded with Fluo-4 AM, with magnification of line-scanned region indicated with red dashed line (white scale bars, 20 µm; legend shows increasing calcium levels, with blue being low calcium). Right, Time course of [Ca]2+I, measured at line-scan region in cell pictured and paced at 0.5 Hz by field stimulation. [Ca]2+ is plotted as fluorescence intensity normalized to baseline (F/F0).

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Purification, FACS, Fluorescence, Expressing, Flow Cytometry, Immunocytochemistry, Reverse Transcription, Polymerase Chain Reaction, Imaging