Journal: eLife

Article Title: Single-cell transcriptome analysis of cavernous tissues reveals the key roles of pericytes in diabetic erectile dysfunction

doi: 10.7554/eLife.88942

Figure Lengend Snippet: ( A and B ) Representative images of immunofluorescence staining of LBH (green)/CD140b (red) in cavernosum tissues and LBH (red) in MCPs under normal and diabetic conditions (in vivo and in vitro). Nuclei were labeled with DAPI (blue). Scale bar, 100 µm. ( C and D ) Quantification of LBH or CD140b expression in in vivo and in vitro by using ImageJ, and results are presented as means ± SEM (n=4). ( E ) Representative western blots for LBH of MCPs under NG and HG conditions, and mouse penis tissues from age-matched control and diabetic mice. ( F ) Normalized band intensity ratio of LBH to β-actin was quantified using ImageJ, and results are presented as means ± SEM (n=4). ( G ) Representative intracavernous pressure (ICP) responses for the age-matched control and diabetic mice stimulated at 2 weeks after intracavernous injections with PBS, lentiviruses ORF control particles (NC), and ORF clone of mouse Lbh (LBH O/E) (20 µL for PBS, 5×10 4 IFU/mouse for lentiviral particles). The stimulus interval is indicated by a solid bar. ( H ) Ratios of mean maximal ICP and total ICP (area under the curve) versus MSBP were calculated for each group, and the results are presented as means ± SEM (n=5). ( I and J ) Cavernous CD31 (endothelial cell, red), NG2 (pericyte, green), neurofilament (red), and neuronal nitric oxide synthase (nNOS) (green) staining in cavernous tissues from age-matched control ( C ) and diabetic mice stimulated at 2 weeks after intracavernous injections with lentiviruses ORF control particles (NC) and ORF clone of mouse Lbh (LBH O/E). Scale bars, 100 µm (left), 25 µm (right). ( K–N ) Quantitative analysis of cavernous endothelial cell, pericyte, and neuronal cell content were quantified by ImageJ, and results are presented as means ± SEM (n=4). The relative ratio in the NG or control group was defined as 1. **p<0.01; ***p<0.001. DM, diabetes mellitus; PBS, phosphate-buffered saline; MCPs, mouse cavernous pericytes; NG, normal glucose; HG, high glucose; DAPI, 4,6-diamidino-2-phenylindole; MSBP, mean systolic blood pressure; ns, not significant.

Article Snippet: In order to examine the effect of LBH overexpression under NG (5 mM glucose, Sigma-Aldrich) or high glucose (HG, 30 mM glucose, Sigma-Aldrich) conditions, MCPs were infected with lentiviruses ORF control particles (NC, 5×10 5 infection units per milliliter cultured medium; Origene Technology) or ORF clone of mouse Lbh (LBH O/E, 5×10 5 infection units per milliliter cultured medium; Origene Technology) under HG conditions for at least 3 days.

Techniques: Immunofluorescence, Staining, In Vivo, In Vitro, Labeling, Expressing, Western Blot, Control, Saline

Journal: eLife

Article Title: Single-cell transcriptome analysis of cavernous tissues reveals the key roles of pericytes in diabetic erectile dysfunction

doi: 10.7554/eLife.88942

Figure Lengend Snippet: ( A ) Immunofluorescence staining of CD31 (green) and LBH (red) in corpus cavernosum tissues treated with lentiviruses ORF control particles (NC) and ORF clone of mouse Lbh (LBH O/E) in diabetic mice. Nuclei were labeled with DAPI (blue). Scale bars, 50 µm. ( B ) Quantification of LBH-positive area by ImageJ, and results are presented as means ± SEM (n=4). The relative ratio in the control group was defined as 1. *p<0.05; ***p<0.001. DM, diabetes mellitus; DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: In order to examine the effect of LBH overexpression under NG (5 mM glucose, Sigma-Aldrich) or high glucose (HG, 30 mM glucose, Sigma-Aldrich) conditions, MCPs were infected with lentiviruses ORF control particles (NC, 5×10 5 infection units per milliliter cultured medium; Origene Technology) or ORF clone of mouse Lbh (LBH O/E, 5×10 5 infection units per milliliter cultured medium; Origene Technology) under HG conditions for at least 3 days.

Techniques: Immunofluorescence, Staining, Control, Labeling

Journal: eLife

Article Title: Single-cell transcriptome analysis of cavernous tissues reveals the key roles of pericytes in diabetic erectile dysfunction

doi: 10.7554/eLife.88942

Figure Lengend Snippet: (A, B, C, G, and I ) TUNEL assay ( A , green), immunofluorescence staining of PH3 ( B , red), migration ( C ), tube formation ( G ) in MCPs, and immunofluorescence staining with neurofilament in MPG tissues ( I , green) treated with lentiviruses ORF control particles (NC) and ORF clone of mouse Lbh (LBH O/E) under HG conditions. Nuclei were labeled with DAPI (blue). Scale bars, 50 µm. ( D, E, F, H , and J ) Quantification of number of TUNEL-positive cells (arrow indicated, D ), PH3-positive cells (arrow indicated, E ), ratio of migrated cells (cells in red frame dot line, F ), master junctions ( H ), and fold change of MPG sprouting ( J ) by ImageJ, and results are presented as means ± SEM (n=4). The relative ratio in the NG group was defined as 1. *p<0.05; **p<0.01; ***p<0.001. TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling; NG, normal glucose; HG, high glucose; DM, diabetes mellitus; DAPI, 4,6-diamidino-2-phenylindole; MPG, major pelvic ganglion.

Article Snippet: In order to examine the effect of LBH overexpression under NG (5 mM glucose, Sigma-Aldrich) or high glucose (HG, 30 mM glucose, Sigma-Aldrich) conditions, MCPs were infected with lentiviruses ORF control particles (NC, 5×10 5 infection units per milliliter cultured medium; Origene Technology) or ORF clone of mouse Lbh (LBH O/E, 5×10 5 infection units per milliliter cultured medium; Origene Technology) under HG conditions for at least 3 days.

Techniques: TUNEL Assay, Immunofluorescence, Staining, Migration, Control, Labeling, End Labeling

Journal: Scientific Reports

Article Title: Rigid Cooperation of Per1 and Per2 proteins

doi: 10.1038/srep32769

Figure Lengend Snippet: Under conditions of gradually changing light, both Per1 (−/−) and Per2 (−/−) mice were entrained to a broader range of period length than WT mice. ( A ) Number and genotype of mice used in this experiment. ( B ) Components of 12-LED–based lighting system: (a) main unit, (b) infrared sensor and LED, (c) interface. ( C ) Block diagram of LED control device. ( D ) Light intensity (lux) measured by luminance meter on the floor of the rack just below the LED at each light level (0–255) with (a) normal and (b) 1/100 electric current. We used 1/100 current so that mice would not sense the light as constant. ( E ) Pattern of gradually changing light level programmed in this experiment. ( F ) Behavioural period (the circadian period of locomotor activity) determined by chi-square periodogram and environmental period (the period of the light cycle) under gradually changing light with period of 22 h to 27 h for (a) 2 weeks (b) final 1 week. We performed both chi-square periodograms for a duration of 2 weeks and one with a final week for each environmental period. The red line shows the concordance of environmental and behavioural periods. The light intensity was approximately 0.01 Lux at the trough and approximately 5.4 Lux at the peak at the centre bottom of the cage. P-values were calculated using one-way ANOVA and Tukey’s post hoc tests. The F and P-values from the Tukey’s test are shown in .

Article Snippet: Per1 (−/−) Per2 (−/−) males (kindly provided by Genshiro A. Sunagawa of RIKEN) were mated with WT Per1 (+ / +) Per2 (+ / +) C57B6/J females (Japan SLC) to generate F1 Per1 (+ / −) Per2 (+ / −) mice for balancing genetic backgrounds.

Techniques: Blocking Assay, Control, Activity Assay

Journal: Scientific Reports

Article Title: Rigid Cooperation of Per1 and Per2 proteins

doi: 10.1038/srep32769

Figure Lengend Snippet: ( A ) Schematic diagram of ES cell differentiation system. ( B ) Real-time monitoring of luminescence of Per2::Luc KI/KI ES cells on day 20 with photomultiplier tube; cpm, counts per min. ( C ) Histogram and ( D ) dot plot of results of autocorrelation analysis of period length using Per2::Luc KI/KI ES cells. Values in ( C ) are mean % of total samples of six independent experiments. Values in ( D ) represent individual samples in six independent experiments. GMEM, Glasgow Minimum Essential Medium; KSR, KnockOut Serum Replacement; FBS, foetal bovine serum; 2i, 2 inhibitors (CHIR99021 and PD0325901); LIF, leukaemia inhibitory factor; DMEM, Dulbecco’s Modified Eagle Medium; NEAA, Non-Essential Amino Acids; L-Glu: L-glutamine. β-ME: β-mercaptoethanol; RA, retinoic acid. Each line and dot indicates each 35-mm dish sample.

Article Snippet: Per1 (−/−) Per2 (−/−) males (kindly provided by Genshiro A. Sunagawa of RIKEN) were mated with WT Per1 (+ / +) Per2 (+ / +) C57B6/J females (Japan SLC) to generate F1 Per1 (+ / −) Per2 (+ / −) mice for balancing genetic backgrounds.

Techniques: Cell Differentiation, Knock-Out, Modification

Journal: Scientific Reports

Article Title: Rigid Cooperation of Per1 and Per2 proteins

doi: 10.1038/srep32769

Figure Lengend Snippet: ( A ) Schematic diagram of Per2 knockout-rescue targeting. We introduced Per2 into the Rosa26 locus of Per2 (−/−) ES cells using TALEN, picked colonies, and checked genome structure with Arm PCR and quantitative PCR. ES clones containing only one copy of the rescue construct were differentiated. ( B ) Schematic diagram of homologous recombination between targeting vector and Rosa26 locus. ( C ) Detrend/baseline oscillation of two independent lines each of WT and FASPS mutant ES cells. ( D ) Period length calculated by autocorrelation analysis in individual (a) lines and (b) genotypes. FASPS, familial advanced sleep phase syndrome; FRT, flippase recognition target; P(PGK), mouse phosphoglycerate kinase 1 promoter; DT-A, diphtheria toxin fragment A.

Article Snippet: Per1 (−/−) Per2 (−/−) males (kindly provided by Genshiro A. Sunagawa of RIKEN) were mated with WT Per1 (+ / +) Per2 (+ / +) C57B6/J females (Japan SLC) to generate F1 Per1 (+ / −) Per2 (+ / −) mice for balancing genetic backgrounds.

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Clone Assay, Construct, Homologous Recombination, Plasmid Preparation, Mutagenesis

Journal: Scientific Reports

Article Title: Rigid Cooperation of Per1 and Per2 proteins

doi: 10.1038/srep32769

Figure Lengend Snippet: The presence of the coding regions, not the promoters, seems to be important in the difference between Per1 and Per2 . ( A–C ) Comparison of Per2 knockout cells rescued with and without Per2 coding region. ( A ) Rescue constructs with or without Per2 coding region. ( B ) Detrend/baseline oscillation of ES cells rescued with or without Per2 coding region. Results of analysis of two independent experiments are shown. Data with Per2 coding region were generated using one cell line (n = 2, twice). Data without coding region were generated using two lines (n = 2, 2 lines * 2 conditions, twice). ( C ) Rate of oscillation period identification (a) and comparison of period length (b). ( D–F ) Comparison of rescue with Per2 and Per1 coding regions. ( D ) Rescue constructs containing Per2 or Per1 coding region. ( E ) Detrend/baseline oscillation of ES cells rescued with Per2 or Per1 coding region. ( F ) Comparison of the period length calculated by autocorrelation analysis in individual (a) lines and (b) genotypes. The P-value was calculated using the Mann Whitney U test.

Article Snippet: Per1 (−/−) Per2 (−/−) males (kindly provided by Genshiro A. Sunagawa of RIKEN) were mated with WT Per1 (+ / +) Per2 (+ / +) C57B6/J females (Japan SLC) to generate F1 Per1 (+ / −) Per2 (+ / −) mice for balancing genetic backgrounds.

Techniques: Comparison, Knock-Out, Construct, Generated, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Rigid Cooperation of Per1 and Per2 proteins

doi: 10.1038/srep32769

Figure Lengend Snippet: ( A ) Data from all rescued Per2 knockout ES cells are shown. Each point represents one 35-mm dish. These data include outliers, but not dishes for which oscillation was not determined. P-values were calculated using one-way ANOVA and Tukey’s post hoc tests. The F and P-values from the Tukey’s test are shown in . ( B ) Degradation curve (a) and half-lives (b) of Per2(WT), Per2(FASPS), and Per1 are plotted against time. ( C ) Model of short period-associated Per1 and long period-associated Per2 proteins. ( D ) Our hypothesis on the factors that determine endogenous period length. Period length at a greater concentration of Per2 (e.g. Per1 knockout) is longer than the period length observed at a greater concentration of Per1 (e.g. Per2 knockout) because the degradation rate of Per2 is higher and its rate of accumulation is slower than Per1. The study by Gerard et al . that modelled the negative autoregulation of gene expression showed that the period increases with an increased degradation rate when degradation is described by a Michaelis-Menten function. This was in accord with the results of the current study that indicated a difference in the period length and half-lives of Per1 and Per2.

Article Snippet: Per1 (−/−) Per2 (−/−) males (kindly provided by Genshiro A. Sunagawa of RIKEN) were mated with WT Per1 (+ / +) Per2 (+ / +) C57B6/J females (Japan SLC) to generate F1 Per1 (+ / −) Per2 (+ / −) mice for balancing genetic backgrounds.

Techniques: Knock-Out, Concentration Assay, Gene Expression