Structured Review

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Recovery of the lower rates of fork progression in the presence of CPT in PARP-1 mutant is not observed under the deficiency of NHEJ capacity. (A and B) Distribution of the ratio of the rate of fork progression during <t>IdU</t> and <t>CldU</t> pulse labeling in each indicated DT40 cell. The total number of the forks analyzed in each cell is also indicated. (C) Model illustrating how PARP-1 affects replication fork progression on damaged DNA. Data bars are the means of three independent experiments, and error bars represent SEM. WT, wild type.
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Images

1) Product Images from "PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA"

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200806068

Recovery of the lower rates of fork progression in the presence of CPT in PARP-1 mutant is not observed under the deficiency of NHEJ capacity. (A and B) Distribution of the ratio of the rate of fork progression during IdU and CldU pulse labeling in each indicated DT40 cell. The total number of the forks analyzed in each cell is also indicated. (C) Model illustrating how PARP-1 affects replication fork progression on damaged DNA. Data bars are the means of three independent experiments, and error bars represent SEM. WT, wild type.
Figure Legend Snippet: Recovery of the lower rates of fork progression in the presence of CPT in PARP-1 mutant is not observed under the deficiency of NHEJ capacity. (A and B) Distribution of the ratio of the rate of fork progression during IdU and CldU pulse labeling in each indicated DT40 cell. The total number of the forks analyzed in each cell is also indicated. (C) Model illustrating how PARP-1 affects replication fork progression on damaged DNA. Data bars are the means of three independent experiments, and error bars represent SEM. WT, wild type.

Techniques Used: Cycling Probe Technology, Mutagenesis, Non-Homologous End Joining, Labeling

Replication fork progression in PARP-1 −/− DT40 cells. (A) Distribution of the ratio of the rate of fork progression in wild-type (WT), PARP-1 −/− DT40, and PARP-1 −/− + human PARP-1 (hPARP-1) DT40 cells. (B) Distribution of the rate of fork progression during IdU and CldU pulse labeling in each cell. (C and D) Distribution of the ratio of the rate of fork progression and the rate of fork progression during IdU and CldU pulse labeling in wild-type DT40 cells pretreated with NU1025. Data bars are the means of three independent experiments, and error bars represent SEM.
Figure Legend Snippet: Replication fork progression in PARP-1 −/− DT40 cells. (A) Distribution of the ratio of the rate of fork progression in wild-type (WT), PARP-1 −/− DT40, and PARP-1 −/− + human PARP-1 (hPARP-1) DT40 cells. (B) Distribution of the rate of fork progression during IdU and CldU pulse labeling in each cell. (C and D) Distribution of the ratio of the rate of fork progression and the rate of fork progression during IdU and CldU pulse labeling in wild-type DT40 cells pretreated with NU1025. Data bars are the means of three independent experiments, and error bars represent SEM.

Techniques Used: Labeling

Effects of PARP-1 knockdown on kinetics of replication forks. (A and B) Evaluation of PARP-1 knockdown in HeLa cells. The amount of PARP-1 in cells transfected with two different siRNA duplexes against PARP-1 and negative control siRNA (siNC) was evaluated by Western blotting (A) and immunofluorescence (B). β-Actin was used as a loading control. (C) Poly-ADP ribosylated proteins were detected as shown in Fig. 1 B . The arrow indicates the molecular mass of PARP-1. (D) Distribution of the ratio of the rate of fork progression in cells transfected with each siRNA. (E) Distribution of the rate of fork progression during IdU and CldU pulse labeling in cells transfected with each siRNA. (F) The tabular data are mean fork rates for each cell treated as indicated. The mean rates were calculated from the data described in E. Bar, 10 μm.
Figure Legend Snippet: Effects of PARP-1 knockdown on kinetics of replication forks. (A and B) Evaluation of PARP-1 knockdown in HeLa cells. The amount of PARP-1 in cells transfected with two different siRNA duplexes against PARP-1 and negative control siRNA (siNC) was evaluated by Western blotting (A) and immunofluorescence (B). β-Actin was used as a loading control. (C) Poly-ADP ribosylated proteins were detected as shown in Fig. 1 B . The arrow indicates the molecular mass of PARP-1. (D) Distribution of the ratio of the rate of fork progression in cells transfected with each siRNA. (E) Distribution of the rate of fork progression during IdU and CldU pulse labeling in cells transfected with each siRNA. (F) The tabular data are mean fork rates for each cell treated as indicated. The mean rates were calculated from the data described in E. Bar, 10 μm.

Techniques Used: Transfection, Negative Control, Western Blot, Immunofluorescence, Labeling

A PARP inhibitor abrogates CPT-induced replication fork slowing. (A) m5S cells were labeled with digoxigenin–deoxy-UTP and release cultured in the presence of CPT. Merged images show that γ-H2AX are detected only in S-phase nuclei. (B) Cells were pretreated with NU1025 before CPT treatment and incubated with FITC-NAD + and CPT for 15 min as described in Materials and methods. β-Actin was used as a loading control. (C) Fluorescent microscope images of poly-ADP ribose in cells were treated as indicated. Fluorescence intensities of each nucleus ( > 50 cells) were quantified. (D) Sensitivity of NU1025-treated cells to CPT. The mean of three independent experiments and standard deviation are shown. (E) A typical global image of DNA fibers. HeLa cells were pulse labeled with IdU (red) and sequentially with CldU (green). After molecular combing, replicated DNA was immunodetected as described in Materials and methods. (F) The images show the typical DNA fibers in cells treated with the indicated inhibitors. Cells were pretreated with NU1025 and pulse labeled with IdU for 20 min. After washing in PBS, cells were treated with CldU in the absence or presence of CPT for 20 min. (G) Distribution of the ratio of the rate of fork progression in cells treated as indicated. The p-value of the Kolmogorov-Smirnov test for the ratio distribution of NU1025-treated cells for CPT treatment compared with NU1025-untreated cells is shown. (H) Distribution of the rate of fork progression during IdU and CldU pulse labeling in each cell. The p-value of the Wilcoxon signed-rank test for the distribution of fork rate in each cell is shown. (I) The tabular data are mean fork rates for each cell treated as indicated. The mean rates were calculated from the data described in H. Bars, 10 kb.
Figure Legend Snippet: A PARP inhibitor abrogates CPT-induced replication fork slowing. (A) m5S cells were labeled with digoxigenin–deoxy-UTP and release cultured in the presence of CPT. Merged images show that γ-H2AX are detected only in S-phase nuclei. (B) Cells were pretreated with NU1025 before CPT treatment and incubated with FITC-NAD + and CPT for 15 min as described in Materials and methods. β-Actin was used as a loading control. (C) Fluorescent microscope images of poly-ADP ribose in cells were treated as indicated. Fluorescence intensities of each nucleus ( > 50 cells) were quantified. (D) Sensitivity of NU1025-treated cells to CPT. The mean of three independent experiments and standard deviation are shown. (E) A typical global image of DNA fibers. HeLa cells were pulse labeled with IdU (red) and sequentially with CldU (green). After molecular combing, replicated DNA was immunodetected as described in Materials and methods. (F) The images show the typical DNA fibers in cells treated with the indicated inhibitors. Cells were pretreated with NU1025 and pulse labeled with IdU for 20 min. After washing in PBS, cells were treated with CldU in the absence or presence of CPT for 20 min. (G) Distribution of the ratio of the rate of fork progression in cells treated as indicated. The p-value of the Kolmogorov-Smirnov test for the ratio distribution of NU1025-treated cells for CPT treatment compared with NU1025-untreated cells is shown. (H) Distribution of the rate of fork progression during IdU and CldU pulse labeling in each cell. The p-value of the Wilcoxon signed-rank test for the distribution of fork rate in each cell is shown. (I) The tabular data are mean fork rates for each cell treated as indicated. The mean rates were calculated from the data described in H. Bars, 10 kb.

Techniques Used: Cycling Probe Technology, Labeling, Cell Culture, Incubation, Microscopy, Fluorescence, Standard Deviation

2) Product Images from "Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis"

Article Title: Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis

Journal: Nature Communications

doi: 10.1038/s41467-017-02720-9

Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or Map2 (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and Nestin (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P
Figure Legend Snippet: Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or Map2 (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and Nestin (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P

Techniques Used: In Vivo, In Vitro, Staining, Knock-Out, Immunostaining, Serial Dilution, Software, Real-time Polymerase Chain Reaction

Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower right) Quantification of the proportion of Tuj1 + or MAP2 + cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67 + ( n = 2 or 3) or Nestin + ( n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF ( n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP + cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d ( n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f . DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (* P
Figure Legend Snippet: Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower right) Quantification of the proportion of Tuj1 + or MAP2 + cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67 + ( n = 2 or 3) or Nestin + ( n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF ( n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP + cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d ( n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f . DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (* P

Techniques Used: Immunostaining, Infection, Expressing, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation

Stau1 inhibits neurogenesis in a Klf4-dependent manner. a (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 (#1 and #3) lentivirus. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1, Stau2, and α-tubulin in samples analyzed in a were were detected by immunoblotting ( n = 2). See also Supplementary Fig. 8a . (Lower right) Corresponding quantification of number of Tuj1 + and Map2 + cells. b (Upper) Immunostaining with Nestin or Ki67 (both red) antibodies of samples analyzed in a . DAPI (blue). Scale bars, 50 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + ( n = 2 or 3) cells. c (Upper) Immunostaining of NPCs infected with pCDH-control or pCDH-Stau1 lentivirus with Tuj1 (green) or MAP2 (red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1 and α-tubulin in samples analyzed in c were detected by immunoblotting ( n = 2). (Lower) Corresponding quantification of data shown above. d (Upper) Immunostaining of samples equivalent to those shown in c with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + (Ctrl, n = 3; Stau1, n = 2) cells. e RT-qPCR of indicated transcripts in control- or Klf4-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). f RT-qPCR of indicated transcripts in control- or Stau1-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). Data are shown as mean ± SD. ANOVA tests were performed to calculate statistical significance (* P
Figure Legend Snippet: Stau1 inhibits neurogenesis in a Klf4-dependent manner. a (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 (#1 and #3) lentivirus. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1, Stau2, and α-tubulin in samples analyzed in a were were detected by immunoblotting ( n = 2). See also Supplementary Fig. 8a . (Lower right) Corresponding quantification of number of Tuj1 + and Map2 + cells. b (Upper) Immunostaining with Nestin or Ki67 (both red) antibodies of samples analyzed in a . DAPI (blue). Scale bars, 50 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + ( n = 2 or 3) cells. c (Upper) Immunostaining of NPCs infected with pCDH-control or pCDH-Stau1 lentivirus with Tuj1 (green) or MAP2 (red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1 and α-tubulin in samples analyzed in c were detected by immunoblotting ( n = 2). (Lower) Corresponding quantification of data shown above. d (Upper) Immunostaining of samples equivalent to those shown in c with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + (Ctrl, n = 3; Stau1, n = 2) cells. e RT-qPCR of indicated transcripts in control- or Klf4-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). f RT-qPCR of indicated transcripts in control- or Stau1-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). Data are shown as mean ± SD. ANOVA tests were performed to calculate statistical significance (* P

Techniques Used: Immunostaining, Infection, Quantitative RT-PCR, Cell Culture

3) Product Images from "Astrocyte activation and wound healing in intact-skull mouse after focal brain injury"

Article Title: Astrocyte activation and wound healing in intact-skull mouse after focal brain injury

Journal: The European journal of neuroscience

doi: 10.1111/j.1460-9568.2012.08280.x

Tissue sections showing the lesion core (LC) and perilesional reactive astrocytes. Neuronal nuclei and astrocytes were stained with anti-NeuN (green) and anti-GFAP (red) antibodies, respectively. (A) Low-magnification images of a sham-treated (no light
Figure Legend Snippet: Tissue sections showing the lesion core (LC) and perilesional reactive astrocytes. Neuronal nuclei and astrocytes were stained with anti-NeuN (green) and anti-GFAP (red) antibodies, respectively. (A) Low-magnification images of a sham-treated (no light

Techniques Used: Staining

4) Product Images from "Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation"

Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1400502

In the large intestine of CX3CR1 gfp/+ mice, five populations of myeloid cells can be defined (P1–P5). M2s emerge postinfection in populations P3 and P4 (both of which are subpopulations of Mφs). Following a high-level infection, the accumulation of M2s in the large intestine reaches a peak after the worms have been expelled. In contrast, following a low-level infection (where the worms are not expelled), the accumulation of M2s is less marked. CX3CR1 gfp/+ mice were infected with either a low or high level of T. muris ova. Another group of CX3CR1 gfp/+ mice was left uninfected. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live leukocytes were analyzed by gating on viability stain–negative CD45 + cells ( A ). Three populations of CD11b + leukocytes were identified by their differential expression of eGFP (CX3CR1) (A). The CD11b + CX3CR1 − cells were analyzed, and representative plots are shown in ( B ). The relative abundance of the CD11b + CX3CR1 int and CD11b + CX3CR1 hi populations over the time course of a high-level infection is shown in ( C ). CD11b + CX3CR1 + cells could be subdivided into five populations (P1 to P5) based on their differential expression of CX3CR1 and the presence or absence of Ly6C, I-A/I-E, F4/80, and CD11c. Representative plots illustrate how these different populations of cells were defined (A). Representative histogram plots of RELMα staining in populations P1 to P4 are shown for uninfected mice and for infected mice at selected time points ( D ). The data are shown at all time points in ( E ), where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p
Figure Legend Snippet: In the large intestine of CX3CR1 gfp/+ mice, five populations of myeloid cells can be defined (P1–P5). M2s emerge postinfection in populations P3 and P4 (both of which are subpopulations of Mφs). Following a high-level infection, the accumulation of M2s in the large intestine reaches a peak after the worms have been expelled. In contrast, following a low-level infection (where the worms are not expelled), the accumulation of M2s is less marked. CX3CR1 gfp/+ mice were infected with either a low or high level of T. muris ova. Another group of CX3CR1 gfp/+ mice was left uninfected. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live leukocytes were analyzed by gating on viability stain–negative CD45 + cells ( A ). Three populations of CD11b + leukocytes were identified by their differential expression of eGFP (CX3CR1) (A). The CD11b + CX3CR1 − cells were analyzed, and representative plots are shown in ( B ). The relative abundance of the CD11b + CX3CR1 int and CD11b + CX3CR1 hi populations over the time course of a high-level infection is shown in ( C ). CD11b + CX3CR1 + cells could be subdivided into five populations (P1 to P5) based on their differential expression of CX3CR1 and the presence or absence of Ly6C, I-A/I-E, F4/80, and CD11c. Representative plots illustrate how these different populations of cells were defined (A). Representative histogram plots of RELMα staining in populations P1 to P4 are shown for uninfected mice and for infected mice at selected time points ( D ). The data are shown at all time points in ( E ), where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p

Techniques Used: Mouse Assay, Infection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, Expressing

In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p
Figure Legend Snippet: In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p

Techniques Used: Mouse Assay, Infection, Immunohistochemistry, Staining, Isolation, Labeling, Flow Cytometry, Cytometry

The proliferation of Mφs following infection with T. muris . AKR, C57BL/6, BALB/c, and CX3CR1 gfp/+ mice were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. In AKR, C57BL/6, and BALB/c mice, live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative plots of Ki-67 and BrdU staining are shown at selected time points postinfection ( A ). The data are shown at all time points in ( B ), where the values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. Ki-67 and BrdU staining in CX3CR1 gfp/+ mice was analyzed by gating on each of the four populations of monocytes and Mϕs (P1–P4, as defined in Fig. 5A ). Representative plots at selected time points postinfection are shown in ( C ). The gates were defined by staining with fluorochrome-labeled isotype control Abs in parallel (shown in Supplemental Fig. 3 ). The data are shown at all time points in ( D ) where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p
Figure Legend Snippet: The proliferation of Mφs following infection with T. muris . AKR, C57BL/6, BALB/c, and CX3CR1 gfp/+ mice were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. In AKR, C57BL/6, and BALB/c mice, live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative plots of Ki-67 and BrdU staining are shown at selected time points postinfection ( A ). The data are shown at all time points in ( B ), where the values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. Ki-67 and BrdU staining in CX3CR1 gfp/+ mice was analyzed by gating on each of the four populations of monocytes and Mϕs (P1–P4, as defined in Fig. 5A ). Representative plots at selected time points postinfection are shown in ( C ). The gates were defined by staining with fluorochrome-labeled isotype control Abs in parallel (shown in Supplemental Fig. 3 ). The data are shown at all time points in ( D ) where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p

Techniques Used: Infection, Mouse Assay, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, BrdU Staining

Flow cytometric analysis of lamina propria Mφs confirms the kinetics of M2 accumulation in the large intestine postinfection. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were either left uninfected or infected with a high level of T. muris ova. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells as shown in ( A ). Representative histogram plots of RELMα staining are shown in ( B ). Quantitative analysis of the staining is shown in ( C ). The values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. * p
Figure Legend Snippet: Flow cytometric analysis of lamina propria Mφs confirms the kinetics of M2 accumulation in the large intestine postinfection. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were either left uninfected or infected with a high level of T. muris ova. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells as shown in ( A ). Representative histogram plots of RELMα staining are shown in ( B ). Quantitative analysis of the staining is shown in ( C ). The values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. * p

Techniques Used: Flow Cytometry, Infection, Isolation, Staining, Labeling, Cytometry, Mouse Assay

Mφs are the predominant type of leukocyte in the large intestine both before and postinfection with T. muris . C57BL/6 mice were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of leukocytes (CD45 + ), Mφs (F4/80 + ), or Th cells (CD4 + ) was conducted on sections of the proximal colon. ( A ) Representative photographs are shown of serial sections from one mouse, 21 d postinfection. Scale bars, 200 μm. Quantitative analysis of the staining in both uninfected (0 d postinfection) and infected (21 d postinfection) mice is shown in ( B ). The values represent the means + SEM of between five and seven mice in each group, and the results are representative of three separate experiments. ** p
Figure Legend Snippet: Mφs are the predominant type of leukocyte in the large intestine both before and postinfection with T. muris . C57BL/6 mice were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of leukocytes (CD45 + ), Mφs (F4/80 + ), or Th cells (CD4 + ) was conducted on sections of the proximal colon. ( A ) Representative photographs are shown of serial sections from one mouse, 21 d postinfection. Scale bars, 200 μm. Quantitative analysis of the staining in both uninfected (0 d postinfection) and infected (21 d postinfection) mice is shown in ( B ). The values represent the means + SEM of between five and seven mice in each group, and the results are representative of three separate experiments. ** p

Techniques Used: Mouse Assay, Infection, Immunohistochemistry, Staining

The vast majority of M2s do not proliferate. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative histogram plots of RELMα and BrdU staining are shown at selected time points postinfection ( A ). The RELMα + cells (M2s) were then analyzed for their BrdU content: the data are shown as the relative percentage of the BrdU + and BrdU − populations at all time points postinfection ( B ). The values are the means of five mice in each group, and the results are representative of two separate experiments.
Figure Legend Snippet: The vast majority of M2s do not proliferate. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative histogram plots of RELMα and BrdU staining are shown at selected time points postinfection ( A ). The RELMα + cells (M2s) were then analyzed for their BrdU content: the data are shown as the relative percentage of the BrdU + and BrdU − populations at all time points postinfection ( B ). The values are the means of five mice in each group, and the results are representative of two separate experiments.

Techniques Used: Infection, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, BrdU Staining, Mouse Assay

5) Product Images from "Three-dimensional culture and clinical drug responses of a highly metastatic human ovarian cancer HO-8910PM cells in nanofibrous microenvironments of three hydrogel biomaterials"

Article Title: Three-dimensional culture and clinical drug responses of a highly metastatic human ovarian cancer HO-8910PM cells in nanofibrous microenvironments of three hydrogel biomaterials

Journal: Journal of Nanobiotechnology

doi: 10.1186/s12951-020-00646-x

The cell distribution and molecular expression of integrin β1 ( a ), E-cadherin ( b ) and N-cadherin ( c ) in HO-8910PM cells cultured in RADA16-I hydrogel, Matrigel and collagen I for 7 days. The brown color indicated the viable proliferation cells, which revealed a solid tumor-like tissue architecture and MCTS formation that was stained by hematoxylin in sections. d Relative quantification of protein expression levels in RADA16-I hydrogel, Matrigel and collagen I. Immuno-expression of integrin β1, E-cadherin and N-cadherin was quantified as an index of positively staining area over total hematoxylin-staining section area in gel-cell clumps. **P
Figure Legend Snippet: The cell distribution and molecular expression of integrin β1 ( a ), E-cadherin ( b ) and N-cadherin ( c ) in HO-8910PM cells cultured in RADA16-I hydrogel, Matrigel and collagen I for 7 days. The brown color indicated the viable proliferation cells, which revealed a solid tumor-like tissue architecture and MCTS formation that was stained by hematoxylin in sections. d Relative quantification of protein expression levels in RADA16-I hydrogel, Matrigel and collagen I. Immuno-expression of integrin β1, E-cadherin and N-cadherin was quantified as an index of positively staining area over total hematoxylin-staining section area in gel-cell clumps. **P

Techniques Used: Expressing, Cell Culture, Staining

Western blot analysis of key cell adhesion proteins in HO-8910PM cells cultured in RADA16-I hydrogel, Matrigel and collagen I for 7 days. a Immunoblot showed qualitative molecular expression of integrin β1, E-cadherin and N-cadherin in different hydrogel biomaterials. b The curve graph indicated the densitometry quantitation of integrin β1, E-cadherin and N-cadherin protein expression levels normalized to GAPDH. Data represented the mean ± SD in three independent assays and showed statistical difference. *P
Figure Legend Snippet: Western blot analysis of key cell adhesion proteins in HO-8910PM cells cultured in RADA16-I hydrogel, Matrigel and collagen I for 7 days. a Immunoblot showed qualitative molecular expression of integrin β1, E-cadherin and N-cadherin in different hydrogel biomaterials. b The curve graph indicated the densitometry quantitation of integrin β1, E-cadherin and N-cadherin protein expression levels normalized to GAPDH. Data represented the mean ± SD in three independent assays and showed statistical difference. *P

Techniques Used: Western Blot, Cell Culture, Expressing, Quantitation Assay

Negatively staining TEM images showed the nanofiber morphology of RADA16-I ( a , d ), Matrigel ( b , e ), and collagen I ( c , f ). RADA16-I, Matrigel, and collagen I were dissolved in 0.1 × PBS solution and self-assembled spontaneously, respectively. Three types of hydrogels all presented a collection of interwoven and disorganized nanofiber networks ultra-microarchitectures in saline solution. Scale bar represented different resolution in TEM images (top panels ( a – c ): ×8000; bottom panels ( d – f ): ×15,000)
Figure Legend Snippet: Negatively staining TEM images showed the nanofiber morphology of RADA16-I ( a , d ), Matrigel ( b , e ), and collagen I ( c , f ). RADA16-I, Matrigel, and collagen I were dissolved in 0.1 × PBS solution and self-assembled spontaneously, respectively. Three types of hydrogels all presented a collection of interwoven and disorganized nanofiber networks ultra-microarchitectures in saline solution. Scale bar represented different resolution in TEM images (top panels ( a – c ): ×8000; bottom panels ( d – f ): ×15,000)

Techniques Used: Staining, Transmission Electron Microscopy

HO-8910PM cell viability and cell proliferation in 3D cell cultures. HO-8910PM cells were encapsulated in RADA16-I hydrogel, Matrigel and collagen I and cultivated for 1 day ( a ), 3 days ( b ), and 6 days ( c ), respectively. Gel-cell clumps were successfully harvested when 3D cell cultures were over at the desired time points. The photomicrographs were taken by phase contrast microscopy (top panels in a – c ). The green fluorescent calcein-AM staining for the living cells was performed to indicate cell viability and robust cell proliferation with distinct cell shapes in all hydrogel volume (bottom panels in a – c )
Figure Legend Snippet: HO-8910PM cell viability and cell proliferation in 3D cell cultures. HO-8910PM cells were encapsulated in RADA16-I hydrogel, Matrigel and collagen I and cultivated for 1 day ( a ), 3 days ( b ), and 6 days ( c ), respectively. Gel-cell clumps were successfully harvested when 3D cell cultures were over at the desired time points. The photomicrographs were taken by phase contrast microscopy (top panels in a – c ). The green fluorescent calcein-AM staining for the living cells was performed to indicate cell viability and robust cell proliferation with distinct cell shapes in all hydrogel volume (bottom panels in a – c )

Techniques Used: Microscopy, Staining

Cell-to-cell interactions and geometry arrangements of HO-8910PM cells in gel-cell clumps. HO-8910PM cells formed MCTS in three types of hydrogel matrices on days 6 ( a ) and days 12 ( b ). Gel-cell clumps of HO-8910PM cell line in RADA16-I, Matrigel and collagen I were harvested on day 6 and day 12. Immunofluorescence assay was performed to indicate cell-to-cell interactions and geometry arrangements in 3D cell culture. Red indicated F-actin and blue indicated DAPI-stained cell nuclear. Immunofluorescence images were obtained by an inverted Olympus IX71 microscope. The blue nuclear staining showed HO-8910PM cell arrangement in MCTS and the red staining surrounding blue nuclear envisioned the cell-to-cell adhesion or intercellular junction in MCTS
Figure Legend Snippet: Cell-to-cell interactions and geometry arrangements of HO-8910PM cells in gel-cell clumps. HO-8910PM cells formed MCTS in three types of hydrogel matrices on days 6 ( a ) and days 12 ( b ). Gel-cell clumps of HO-8910PM cell line in RADA16-I, Matrigel and collagen I were harvested on day 6 and day 12. Immunofluorescence assay was performed to indicate cell-to-cell interactions and geometry arrangements in 3D cell culture. Red indicated F-actin and blue indicated DAPI-stained cell nuclear. Immunofluorescence images were obtained by an inverted Olympus IX71 microscope. The blue nuclear staining showed HO-8910PM cell arrangement in MCTS and the red staining surrounding blue nuclear envisioned the cell-to-cell adhesion or intercellular junction in MCTS

Techniques Used: Immunofluorescence, Cell Culture, Staining, Microscopy

The viable cell proliferation curves of HO-8910PM cells cultured in RADA16-I hydrogel, Matrigel and collagen I on days 1, 3, 6 and 9. a HO-8910PM cell proliferation in different hydrogels was calculated from DNA content fold changes at the desired time points. b BrdU labelling assay was performed to evaluate the viable cell percentage of HO-8910PM cells seeded in different nanofiber scaffolds, expressed as the cell proliferation rate (%) in three assays (about 200 cells per microscopical view field) on cell culture days 1, 3, 6, and 9. Data showed statistically significant differences (*p
Figure Legend Snippet: The viable cell proliferation curves of HO-8910PM cells cultured in RADA16-I hydrogel, Matrigel and collagen I on days 1, 3, 6 and 9. a HO-8910PM cell proliferation in different hydrogels was calculated from DNA content fold changes at the desired time points. b BrdU labelling assay was performed to evaluate the viable cell percentage of HO-8910PM cells seeded in different nanofiber scaffolds, expressed as the cell proliferation rate (%) in three assays (about 200 cells per microscopical view field) on cell culture days 1, 3, 6, and 9. Data showed statistically significant differences (*p

Techniques Used: Cell Culture

6) Product Images from "Defining retinal progenitor cell competence in Xenopus laevis by clonal analysis"

Article Title: Defining retinal progenitor cell competence in Xenopus laevis by clonal analysis

Journal: Development (Cambridge, England)

doi: 10.1242/dev.027607

Mitotic landmarks of retinal development in Xenopus laevis and the pattern of genesis of the seven major cell types at the population level. ( A ) The timing of gfp transfection and BrdU injection according to three different scales: (1) hours post-fertilization
Figure Legend Snippet: Mitotic landmarks of retinal development in Xenopus laevis and the pattern of genesis of the seven major cell types at the population level. ( A ) The timing of gfp transfection and BrdU injection according to three different scales: (1) hours post-fertilization

Techniques Used: Transfection, Injection

Retinal sections of stage 41 Xenopus containing clusters of GFP-labeled cells. Sections were immunolabeled as in . (A,B,C,E,G,I) Monochrome images showing GFP labeling only. ( A ) BrdU injected at stage 30. This cluster has at least one member in
Figure Legend Snippet: Retinal sections of stage 41 Xenopus containing clusters of GFP-labeled cells. Sections were immunolabeled as in . (A,B,C,E,G,I) Monochrome images showing GFP labeling only. ( A ) BrdU injected at stage 30. This cluster has at least one member in

Techniques Used: Labeling, Immunolabeling, Injection

GFP + retinal sections of stage 41 Xenopus tadpoles. Immunolabeled for GFP (red), BrdU (green) and rhodopsin (blue). Sclerad is up, vitread is down. Each cell type has a readily recognized morphology. Am, amacrine cell; BP, bipolar cell; CPr, cone cell;
Figure Legend Snippet: GFP + retinal sections of stage 41 Xenopus tadpoles. Immunolabeled for GFP (red), BrdU (green) and rhodopsin (blue). Sclerad is up, vitread is down. Each cell type has a readily recognized morphology. Am, amacrine cell; BP, bipolar cell; CPr, cone cell;

Techniques Used: Immunolabeling

7) Product Images from "Increasing Proliferation of Intrinsic Tubular Cells after Renal Ischemia-reperfusion Injury in Adult Rat"

Article Title: Increasing Proliferation of Intrinsic Tubular Cells after Renal Ischemia-reperfusion Injury in Adult Rat

Journal: Aging and Disease

doi: 10.14336/AD.2014.0917

BrdU-positive cells expressed nestin and vimentin in adult kidney in rat. A. BrdU-positive cells (red) in the tubule (top panel) and the glomerulus (bottom panel) expressed nestin (green). B. BrdU-positive cells (red) in the tubule (top panel) and the glomerulus (bottom panel) expressed vimentin (green). Representative microphotographs of a kidney section were from 3 days after ischemia.
Figure Legend Snippet: BrdU-positive cells expressed nestin and vimentin in adult kidney in rat. A. BrdU-positive cells (red) in the tubule (top panel) and the glomerulus (bottom panel) expressed nestin (green). B. BrdU-positive cells (red) in the tubule (top panel) and the glomerulus (bottom panel) expressed vimentin (green). Representative microphotographs of a kidney section were from 3 days after ischemia.

Techniques Used:

8) Product Images from "MAINTAINING EPITHELIOPOIETIC POTENCY WHEN CULTURING OLFACTORY PROGENITORS"

Article Title: MAINTAINING EPITHELIOPOIETIC POTENCY WHEN CULTURING OLFACTORY PROGENITORS

Journal: Experimental neurology

doi: 10.1016/j.expneurol.2008.07.012

Epithelial islands contain cells expressing mixed phenotypic markers, i.e., CK14, a maker of HBCs in vivo , and CK18, a marker of sustentacular cells in vivo . (A, B) CK14(+) cells are prominent in cultures derived from MeBr-lesioned OE grown on laminin
Figure Legend Snippet: Epithelial islands contain cells expressing mixed phenotypic markers, i.e., CK14, a maker of HBCs in vivo , and CK18, a marker of sustentacular cells in vivo . (A, B) CK14(+) cells are prominent in cultures derived from MeBr-lesioned OE grown on laminin

Techniques Used: Expressing, In Vivo, Marker, Derivative Assay

Cells in spheres derived from MeBr-lesioned rat OE express a variety of differentiated cell markers when maintained in air-liquid interface culture as in the regenerating OE, including CK14 (A, B), CK18 (C, D), TuJ-1 (E, F). (G–H) Some of the
Figure Legend Snippet: Cells in spheres derived from MeBr-lesioned rat OE express a variety of differentiated cell markers when maintained in air-liquid interface culture as in the regenerating OE, including CK14 (A, B), CK18 (C, D), TuJ-1 (E, F). (G–H) Some of the

Techniques Used: Derivative Assay

9) Product Images from "Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis"

Article Title: Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20062299

Proliferative defect of Fbxw7-deficient mature T cells. (A) Representative flow cytometric analysis of surface expression of TCRβ and B220 (top) or of CD4 and CD8 (bottom) on spleen cells from Fbxw7 F/F (control) or Lck-Cre/ Fbxw7 F/F mice at 8 wk of age. The respective percentages are indicated. (B) Absolute cell numbers of splenocyte subsets determined as in A. Data are means ± SD of values from 8 Fbxw7 F/F (control) and 14 Lck-Cre/ Fbxw7 F/F mice. ***, P
Figure Legend Snippet: Proliferative defect of Fbxw7-deficient mature T cells. (A) Representative flow cytometric analysis of surface expression of TCRβ and B220 (top) or of CD4 and CD8 (bottom) on spleen cells from Fbxw7 F/F (control) or Lck-Cre/ Fbxw7 F/F mice at 8 wk of age. The respective percentages are indicated. (B) Absolute cell numbers of splenocyte subsets determined as in A. Data are means ± SD of values from 8 Fbxw7 F/F (control) and 14 Lck-Cre/ Fbxw7 F/F mice. ***, P

Techniques Used: Flow Cytometry, Expressing, Mouse Assay

10) Product Images from "Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis"

Article Title: Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20062299

Proliferative defect of Fbxw7-deficient mature T cells. (A) Representative flow cytometric analysis of surface expression of TCRβ and B220 (top) or of CD4 and CD8 (bottom) on spleen cells from Fbxw7 F/F (control) or Lck-Cre/ Fbxw7 F/F mice at 8 wk of age. The respective percentages are indicated. (B) Absolute cell numbers of splenocyte subsets determined as in A. Data are means ± SD of values from 8 Fbxw7 F/F (control) and 14 Lck-Cre/ Fbxw7 F/F mice. ***, P
Figure Legend Snippet: Proliferative defect of Fbxw7-deficient mature T cells. (A) Representative flow cytometric analysis of surface expression of TCRβ and B220 (top) or of CD4 and CD8 (bottom) on spleen cells from Fbxw7 F/F (control) or Lck-Cre/ Fbxw7 F/F mice at 8 wk of age. The respective percentages are indicated. (B) Absolute cell numbers of splenocyte subsets determined as in A. Data are means ± SD of values from 8 Fbxw7 F/F (control) and 14 Lck-Cre/ Fbxw7 F/F mice. ***, P

Techniques Used: Flow Cytometry, Expressing, Mouse Assay

11) Product Images from "Interactions between Sox9 and ?-catenin control chondrocyte differentiation"

Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

Journal: Genes & Development

doi: 10.1101/gad.1171104

Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) Schematic representation of the β -catenin deletion mutants. ( B ) In vitro binding of β -catenin deletion mutants to 6xHis-tagged Sox9 bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged Sox9. ( C ) Activation of TOPFLASH in C3H10T1/2 cells by Tcf3 is inhibited by Sox9 in a dose-dependent manner. ( D ) Sox9 inhibits binding of Tcf3 to β-catenin in vitro. Sox9 and 35 S-labeled Tcf3 proteins are incubated with 200 ng of 6xHis-tagged β-catenin immobilized to a nickel-resin. Of the input Tcf3 protein (lane 1 ) and bound Tcf3 proteins (lanes 2 - 4 ), 10% are resolved on SDS-PAGE. ( E ) Binding between Sox9 and β-catenin in Cos-7 cells. Cos-7 cells are cotransfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 for 24 h. Cell lysates are incubated with anti-β-catenin antibody and protein G-agarose beads under gentle agitation for 3 h at 4°C. The beads are washed three times with buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100), resuspended in 10 μL of SDS-polyacrylamide gel electrophoresis sample loading buffer, and boiled for 2 min. Western immunoblotting is then performed. Anti-HA:HRP monoclonal antibody (Roche) and anti-myc:HRP antibody (Invitrogen) are diluted 1:1000 and 1:5000, respectively.
Figure Legend Snippet: Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) Schematic representation of the β -catenin deletion mutants. ( B ) In vitro binding of β -catenin deletion mutants to 6xHis-tagged Sox9 bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged Sox9. ( C ) Activation of TOPFLASH in C3H10T1/2 cells by Tcf3 is inhibited by Sox9 in a dose-dependent manner. ( D ) Sox9 inhibits binding of Tcf3 to β-catenin in vitro. Sox9 and 35 S-labeled Tcf3 proteins are incubated with 200 ng of 6xHis-tagged β-catenin immobilized to a nickel-resin. Of the input Tcf3 protein (lane 1 ) and bound Tcf3 proteins (lanes 2 - 4 ), 10% are resolved on SDS-PAGE. ( E ) Binding between Sox9 and β-catenin in Cos-7 cells. Cos-7 cells are cotransfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 for 24 h. Cell lysates are incubated with anti-β-catenin antibody and protein G-agarose beads under gentle agitation for 3 h at 4°C. The beads are washed three times with buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100), resuspended in 10 μL of SDS-polyacrylamide gel electrophoresis sample loading buffer, and boiled for 2 min. Western immunoblotting is then performed. Anti-HA:HRP monoclonal antibody (Roche) and anti-myc:HRP antibody (Invitrogen) are diluted 1:1000 and 1:5000, respectively.

Techniques Used: Functional Assay, In Vitro, Binding Assay, Activation Assay, Labeling, Incubation, SDS Page, Polyacrylamide Gel Electrophoresis, Western Blot

( A,B ) Sox9 inhibits β-catenin-mediated secondary-axis formation in Xenopus embryos. ( A ) Representative tail bud stage Xenopus embryos injected with the indicated RNAs into a single ventral-vegetal blastomere at the four-cell stage. ( B ) Summary of second axis assays from two separate experiments. Injection of mRNA encoding wild-type β-catenin (40 pg) results in a high frequency (77.8%) of embryos with secondary axes. Coinjection of increasing levels of Sox9 mRNA (0.5, 1.0, and 2.0 ng) with β -catenin (40 pg) results in a dose-dependent inhibition of β-catenin-induced secondary-axis formation. Sox9 (1-304) mRNA (2.0 ng) does not inhibit β-catenin-induced secondary-axis formation. Semiquantitative analysis of each embryo positive for a secondary axis (1 = weak, 2 = moderate, 3 = strong) further indicates that β-catenin-mediated secondary-axis formation is inhibited by increasing levels of Sox9, but not by Sox9 (1-304). ( C ) Proteasome inhibitor, MG132, restores the levels of β-catenin and Sox9 in 293 cells transfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9. Mutant Sox9 (1-304) does not decrease the levels of β-catenin, and MG132 has no effect on the levels of either β-catenin or Sox9 in these experiments.
Figure Legend Snippet: ( A,B ) Sox9 inhibits β-catenin-mediated secondary-axis formation in Xenopus embryos. ( A ) Representative tail bud stage Xenopus embryos injected with the indicated RNAs into a single ventral-vegetal blastomere at the four-cell stage. ( B ) Summary of second axis assays from two separate experiments. Injection of mRNA encoding wild-type β-catenin (40 pg) results in a high frequency (77.8%) of embryos with secondary axes. Coinjection of increasing levels of Sox9 mRNA (0.5, 1.0, and 2.0 ng) with β -catenin (40 pg) results in a dose-dependent inhibition of β-catenin-induced secondary-axis formation. Sox9 (1-304) mRNA (2.0 ng) does not inhibit β-catenin-induced secondary-axis formation. Semiquantitative analysis of each embryo positive for a secondary axis (1 = weak, 2 = moderate, 3 = strong) further indicates that β-catenin-mediated secondary-axis formation is inhibited by increasing levels of Sox9, but not by Sox9 (1-304). ( C ) Proteasome inhibitor, MG132, restores the levels of β-catenin and Sox9 in 293 cells transfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9. Mutant Sox9 (1-304) does not decrease the levels of β-catenin, and MG132 has no effect on the levels of either β-catenin or Sox9 in these experiments.

Techniques Used: Injection, Inhibition, Transfection, Mutagenesis

Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) EMSAs performed using in vitro translated 3xHA-tagged Sox9 and 3xHA-tagged xTcf-3 proteins show that Sox9 does not bind to Tcf/Lef DNA-binding sites and that Tcf does not bind to Sox9 consensus DNA-binding sites. ( B ) Schematic representation of the Sox9 deletion mutants. ( C ) Activation of TOPFLASH by stβ-catenin is inhibited by Sox9 mutants containing the C-terminal transactivation domain. Cotransfection of 6x myc-tagged stβ-catenin and Sox9 mutants containing the C-terminal transactivation domain results in a reduction of the levels of these proteins. ( D ) In vitro binding of Sox9 deletion mutants to 6xHis-tagged β-catenin bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged β-catenin.
Figure Legend Snippet: Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) EMSAs performed using in vitro translated 3xHA-tagged Sox9 and 3xHA-tagged xTcf-3 proteins show that Sox9 does not bind to Tcf/Lef DNA-binding sites and that Tcf does not bind to Sox9 consensus DNA-binding sites. ( B ) Schematic representation of the Sox9 deletion mutants. ( C ) Activation of TOPFLASH by stβ-catenin is inhibited by Sox9 mutants containing the C-terminal transactivation domain. Cotransfection of 6x myc-tagged stβ-catenin and Sox9 mutants containing the C-terminal transactivation domain results in a reduction of the levels of these proteins. ( D ) In vitro binding of Sox9 deletion mutants to 6xHis-tagged β-catenin bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged β-catenin.

Techniques Used: Functional Assay, In Vitro, Binding Assay, Activation Assay, Cotransfection

Model describing functional and physical interactions between Sox9 and β-catenin in chondrocytes. β-Catenin activates the Cyclin D1 gene and regulates chondrocyte proliferation. Sox9 activates ECM genes including Col2a1 and regulates chondrocyte differentiation. Sox9 inhibits β-catenin/Tcf-Lef activity by competing with the binding sites of Tcf/Lef within β-catenin. Sox9 also promotes degradation of β-catenin by the ubiquitination/26S proteasome pathway through formation of a Sox9:β-catenin complex. This results in inhibition of proliferation, and in delayed hypertrophic chondrocyte differentiation. In addition, formation of a Sox9:β-catenin complex also causes degradation of Sox9, inhibiting overt chondrocyte differentiation and accelerating hypertrophic chondrocyte differentiation. The model predicts that the relative levels of Sox9 and β-catenin control chondrocyte differentiation.
Figure Legend Snippet: Model describing functional and physical interactions between Sox9 and β-catenin in chondrocytes. β-Catenin activates the Cyclin D1 gene and regulates chondrocyte proliferation. Sox9 activates ECM genes including Col2a1 and regulates chondrocyte differentiation. Sox9 inhibits β-catenin/Tcf-Lef activity by competing with the binding sites of Tcf/Lef within β-catenin. Sox9 also promotes degradation of β-catenin by the ubiquitination/26S proteasome pathway through formation of a Sox9:β-catenin complex. This results in inhibition of proliferation, and in delayed hypertrophic chondrocyte differentiation. In addition, formation of a Sox9:β-catenin complex also causes degradation of Sox9, inhibiting overt chondrocyte differentiation and accelerating hypertrophic chondrocyte differentiation. The model predicts that the relative levels of Sox9 and β-catenin control chondrocyte differentiation.

Techniques Used: Functional Assay, Activity Assay, Binding Assay, Inhibition

Severe, generalized chondrodysplasia in β -catenin +/ flox(ex3) ; Col2a1-Cre embryos. ( A ) Gross appearance of E16.5 embryos. ( B ) Skeletons of E18.0 embryos stained by alcian blue followed by alizarin red. Mutant embryos are characterized by a very severe and generalized chondrodysplasia. ( C ) Gross appearance of E12.5 mutant embryos is comparable to that of wild-type littermates. Histological analysis of limb buds stained by hematoxylin and Treosin at 12.5 dpc. Both wild-type and mutant limb buds have discernible chondrogenic mesenchymal condensations. Immunohistochemistry shows comparable expression of Sox9 protein in condensed mesenchymal cells in E12.5 wild-type and mutant embryos. ( D ) Alcian blue and nuclear fast-red staining, PCNA staining, and immunohistochemistry of Sox9 protein in ulna of E16.5 wild-type and mutant embryos, respectively. Only cells surrounded by alcian blue-stainable matrix are PCNA-positive and express Sox9. Small round cells (arrows) lack expression of Sox9.
Figure Legend Snippet: Severe, generalized chondrodysplasia in β -catenin +/ flox(ex3) ; Col2a1-Cre embryos. ( A ) Gross appearance of E16.5 embryos. ( B ) Skeletons of E18.0 embryos stained by alcian blue followed by alizarin red. Mutant embryos are characterized by a very severe and generalized chondrodysplasia. ( C ) Gross appearance of E12.5 mutant embryos is comparable to that of wild-type littermates. Histological analysis of limb buds stained by hematoxylin and Treosin at 12.5 dpc. Both wild-type and mutant limb buds have discernible chondrogenic mesenchymal condensations. Immunohistochemistry shows comparable expression of Sox9 protein in condensed mesenchymal cells in E12.5 wild-type and mutant embryos. ( D ) Alcian blue and nuclear fast-red staining, PCNA staining, and immunohistochemistry of Sox9 protein in ulna of E16.5 wild-type and mutant embryos, respectively. Only cells surrounded by alcian blue-stainable matrix are PCNA-positive and express Sox9. Small round cells (arrows) lack expression of Sox9.

Techniques Used: Staining, Mutagenesis, Immunohistochemistry, Expressing

Analysis of skeletal phenotypes in conditional β -catenin -null mutants with the Col2a1-Cre transgene. ( A ) Gross appearance of newborn mice. ( B ) A cleft secondary palate in mutant newborn mice (the arrow). ( C,D ) Skeletons of newborn mice stained by alcian blue followed by alizarin red. ( E ) Alcian blue staining of the radius in E16.5 mice shows delay of endochondral bone formation in the mutant embryos. Staining by von Kossa's method shows no mineral deposition of the radius in E16.5 mutant embryos. ( F ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) of the radius in E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius in E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p
Figure Legend Snippet: Analysis of skeletal phenotypes in conditional β -catenin -null mutants with the Col2a1-Cre transgene. ( A ) Gross appearance of newborn mice. ( B ) A cleft secondary palate in mutant newborn mice (the arrow). ( C,D ) Skeletons of newborn mice stained by alcian blue followed by alizarin red. ( E ) Alcian blue staining of the radius in E16.5 mice shows delay of endochondral bone formation in the mutant embryos. Staining by von Kossa's method shows no mineral deposition of the radius in E16.5 mutant embryos. ( F ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) of the radius in E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius in E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p

Techniques Used: Mouse Assay, Mutagenesis, Staining, BrdU Incorporation Assay

Inhibition of cell proliferation and Cyclin D1 expression in Col2a1/Sox9 knock-in mutant embryos, and negative functional interactions between Sox9 and β-catenin in vitro. ( A ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) in the radius of E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius of E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p
Figure Legend Snippet: Inhibition of cell proliferation and Cyclin D1 expression in Col2a1/Sox9 knock-in mutant embryos, and negative functional interactions between Sox9 and β-catenin in vitro. ( A ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) in the radius of E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius of E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p

Techniques Used: Inhibition, Expressing, Knock-In, Mutagenesis, Functional Assay, In Vitro, Staining, BrdU Incorporation Assay

12) Product Images from "Cellular prion protein promotes post-ischemic neuronal survival, angioneurogenesis and enhances neural progenitor cell homing via proteasome inhibition"

Article Title: Cellular prion protein promotes post-ischemic neuronal survival, angioneurogenesis and enhances neural progenitor cell homing via proteasome inhibition

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.365

Enhanced post-ischemic neurogenesis and angiogenesis in PrP−/− mice is a consequence of increased infarct size. ( a ) Infarct volume determined by TTC staining, ( b ) neuronal density in the striatum determined by NeuN immunohistochemistry, ( c ) cell proliferation measured by BrdU immunolabeling, ( d ) neurogenesis evaluated by co-labeling of the immature neuronal marker Dcx and BrdU, ( e ) neurogenesis assessed by co-labeling of the mature neuronal marker NeuN and BrdU, and ( f ) angiogenesis examined by co-labeling of the endothelial marker CD31 and BrdU in WT mice exposed to 45 min MCA occlusion and PrP−/− mice exposed to 30 min MCA occlusion followed by 24 h reperfusion ( a ) or 28 days reperfusion ( b – f ). Note that in the presence of very similar brain injury ( a and b ), post-ischemic cell proliferation ( c ), neurogenesis ( d – e ) and angiogenesis ( f ) do not differ between WT and PrP−/− mice
Figure Legend Snippet: Enhanced post-ischemic neurogenesis and angiogenesis in PrP−/− mice is a consequence of increased infarct size. ( a ) Infarct volume determined by TTC staining, ( b ) neuronal density in the striatum determined by NeuN immunohistochemistry, ( c ) cell proliferation measured by BrdU immunolabeling, ( d ) neurogenesis evaluated by co-labeling of the immature neuronal marker Dcx and BrdU, ( e ) neurogenesis assessed by co-labeling of the mature neuronal marker NeuN and BrdU, and ( f ) angiogenesis examined by co-labeling of the endothelial marker CD31 and BrdU in WT mice exposed to 45 min MCA occlusion and PrP−/− mice exposed to 30 min MCA occlusion followed by 24 h reperfusion ( a ) or 28 days reperfusion ( b – f ). Note that in the presence of very similar brain injury ( a and b ), post-ischemic cell proliferation ( c ), neurogenesis ( d – e ) and angiogenesis ( f ) do not differ between WT and PrP−/− mice

Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Immunolabeling, Labeling, Marker

Post-ischemic neurogenesis and angiogenesis are increased in PrP−/− and PrP+/+ mice. ( a ) Cell proliferation assessed by bromodeoxyuridine (BrdU) immunolabeling, ( b ) neurogenesis evaluated by co-labeling of the immature neuronal marker Dcx and BrdU, ( c ) neurogenesis determined by co-labeling of the mature neuronal marker NeuN and BrdU and ( d ) angiogenesis examined by co-labeling of the endothelial marker CD31 and BrdU in the striatum of WT, PrP−/− and PrP+/+ mice exposed to 45 min MCA occlusion followed by 28 days reperfusion. Representative photographs are presented from the core of the ischemic striatum from PrP+/+ mice. Scale bars: 50 μ m. *Significantly different from WT mice, P
Figure Legend Snippet: Post-ischemic neurogenesis and angiogenesis are increased in PrP−/− and PrP+/+ mice. ( a ) Cell proliferation assessed by bromodeoxyuridine (BrdU) immunolabeling, ( b ) neurogenesis evaluated by co-labeling of the immature neuronal marker Dcx and BrdU, ( c ) neurogenesis determined by co-labeling of the mature neuronal marker NeuN and BrdU and ( d ) angiogenesis examined by co-labeling of the endothelial marker CD31 and BrdU in the striatum of WT, PrP−/− and PrP+/+ mice exposed to 45 min MCA occlusion followed by 28 days reperfusion. Representative photographs are presented from the core of the ischemic striatum from PrP+/+ mice. Scale bars: 50 μ m. *Significantly different from WT mice, P

Techniques Used: Mouse Assay, Immunolabeling, Labeling, Marker

13) Product Images from "Propionyl-L-Carnitine is Efficacious in Ulcerative Colitis Through its Action on the Immune Function and Microvasculature"

Article Title: Propionyl-L-Carnitine is Efficacious in Ulcerative Colitis Through its Action on the Immune Function and Microvasculature

Journal: Clinical and Translational Gastroenterology

doi: 10.1038/ctg.2014.4

PLC treatment reduces inflammation and endothelial dysfunction in rat TNBS-induced acute colitis. Representative microphotographs and morphometric evaluation of ICAM-1, VCAM-1, iNOS, CD31, PlGF, and BrDU staining of rat colon tissue from the different experimental groups: vehicle (50% ethanol, v/v), PLC (25 mg/kg intrarectal, twice daily for 1 week), TNBS and TNBS plus PLC (25 mg/kg intrarectal, twice daily for 1 week). Student's t -test: *, **, and *** P
Figure Legend Snippet: PLC treatment reduces inflammation and endothelial dysfunction in rat TNBS-induced acute colitis. Representative microphotographs and morphometric evaluation of ICAM-1, VCAM-1, iNOS, CD31, PlGF, and BrDU staining of rat colon tissue from the different experimental groups: vehicle (50% ethanol, v/v), PLC (25 mg/kg intrarectal, twice daily for 1 week), TNBS and TNBS plus PLC (25 mg/kg intrarectal, twice daily for 1 week). Student's t -test: *, **, and *** P

Techniques Used: Planar Chromatography, BrdU Staining

14) Product Images from "Integrin-linked kinase is required for epidermal and hair follicle morphogenesis"

Article Title: Integrin-linked kinase is required for epidermal and hair follicle morphogenesis

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200608125

ILK-K5 HFs show normal β-catenin stability and hair-specific differentiation. (A) Control and mutant 2-wk skin sections stained for Ki67 and Lef1 show an increased number of Ki67+ cells in the ORS (arrowheads), yet retained Lef1 expression in the HM and DP of ILK-K5 HFs. (B) Control and mutant 2-wk skin sections stained for β-catenin revealed nuclear β-catenin (arrowheads) in precortical HM and proximal HS cortex in both control and long (▴) and short (▪) ILK-K5 HFs. (C) BatGal reporter mice were intercrossed with ILK-K5 and control animals. LacZ activity is present in precortical HM and HS cortex of both control and ILK-K5 HFs. (D) Immunostaining of K14 for the ORS, of keratin K6irs1 for the IRS, and of keratin hHa1 for HS cortex and α6 integrin. ILK-K5 HFs revealed the presence of a multilayered K14+ ORS. K6irs1 and hHa1 were expressed but mislocalized in short ILK-K5 HFs (arrowheads). Bars, 50 μm.
Figure Legend Snippet: ILK-K5 HFs show normal β-catenin stability and hair-specific differentiation. (A) Control and mutant 2-wk skin sections stained for Ki67 and Lef1 show an increased number of Ki67+ cells in the ORS (arrowheads), yet retained Lef1 expression in the HM and DP of ILK-K5 HFs. (B) Control and mutant 2-wk skin sections stained for β-catenin revealed nuclear β-catenin (arrowheads) in precortical HM and proximal HS cortex in both control and long (▴) and short (▪) ILK-K5 HFs. (C) BatGal reporter mice were intercrossed with ILK-K5 and control animals. LacZ activity is present in precortical HM and HS cortex of both control and ILK-K5 HFs. (D) Immunostaining of K14 for the ORS, of keratin K6irs1 for the IRS, and of keratin hHa1 for HS cortex and α6 integrin. ILK-K5 HFs revealed the presence of a multilayered K14+ ORS. K6irs1 and hHa1 were expressed but mislocalized in short ILK-K5 HFs (arrowheads). Bars, 50 μm.

Techniques Used: Mutagenesis, Staining, Expressing, Mouse Assay, Activity Assay, Immunostaining

Loss of ILK retards differentiation and disturbs polarity of epidermal keratinocytes. (A) Double immunostaining for K14, K10, or loricrin (Lor) and α6 integrin on back skin of 2-wk-old control and ILK-K5 animals. ILK-K5 epidermis shows several cell layers expressing K14 and loricrin, respectively. Integrin α6 expression is discontinuous in ILK-K5 skin and present on suprabasal cells (asterisks). Bar 25 μm. (B) Immunostaining for F-actin, β-catenin, and plakoglobin in 2-wk-old mouse skin. In control epidermis, F-actin and β-catenin are absent from the basal side of basal keratinocytes. In ILK-K5 epidermis, F-actin and β-catenin are found basally adjacent to nidogen (arrowheads). Plakoglobin localizes to the lateral–apical sides of basal keratinocytes of both control and ILK-K5 mice. Bar, 25 μm. (C) F-actin overlaps with α6 integrin in the mutant HF (arrowheads). Bars, 50 μm. (D) Western blot analysis reveals similar expression levels of E-cadherin and β-catenin in control and ILK-K5 epidermal lysates.
Figure Legend Snippet: Loss of ILK retards differentiation and disturbs polarity of epidermal keratinocytes. (A) Double immunostaining for K14, K10, or loricrin (Lor) and α6 integrin on back skin of 2-wk-old control and ILK-K5 animals. ILK-K5 epidermis shows several cell layers expressing K14 and loricrin, respectively. Integrin α6 expression is discontinuous in ILK-K5 skin and present on suprabasal cells (asterisks). Bar 25 μm. (B) Immunostaining for F-actin, β-catenin, and plakoglobin in 2-wk-old mouse skin. In control epidermis, F-actin and β-catenin are absent from the basal side of basal keratinocytes. In ILK-K5 epidermis, F-actin and β-catenin are found basally adjacent to nidogen (arrowheads). Plakoglobin localizes to the lateral–apical sides of basal keratinocytes of both control and ILK-K5 mice. Bar, 25 μm. (C) F-actin overlaps with α6 integrin in the mutant HF (arrowheads). Bars, 50 μm. (D) Western blot analysis reveals similar expression levels of E-cadherin and β-catenin in control and ILK-K5 epidermal lysates.

Techniques Used: Double Immunostaining, Expressing, Immunostaining, Mouse Assay, Mutagenesis, Western Blot

Keratinocyte-restricted deletion of ILK causes progressive hair loss. (A) ILK protein level in epidermal lysates of ILK Co and ILK-K5 mice. (B) Back skin of 2-wk-old ILK Co and ILK-K5 animals stained for ILK and α6 integrin. ILK is expressed in basal keratinocytes of the epidermis (E), ORS, HM, DP, arrector pili muscle (AP), and dermis (D). ILK-K5 skin retains ILK expression in DP and dermis but lacks ILK expression in epidermis, HM, and ORS. Bar, 25 μm. (C) Control and ILK-K5 animals at 8 wk of age.
Figure Legend Snippet: Keratinocyte-restricted deletion of ILK causes progressive hair loss. (A) ILK protein level in epidermal lysates of ILK Co and ILK-K5 mice. (B) Back skin of 2-wk-old ILK Co and ILK-K5 animals stained for ILK and α6 integrin. ILK is expressed in basal keratinocytes of the epidermis (E), ORS, HM, DP, arrector pili muscle (AP), and dermis (D). ILK-K5 skin retains ILK expression in DP and dermis but lacks ILK expression in epidermis, HM, and ORS. Bar, 25 μm. (C) Control and ILK-K5 animals at 8 wk of age.

Techniques Used: Mouse Assay, Staining, Expressing

15) Product Images from "Activation of the human FP prostanoid receptor disrupts mitosis progression and generates aneuploidy and polyploidy"

Article Title: Activation of the human FP prostanoid receptor disrupts mitosis progression and generates aneuploidy and polyploidy

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-005-5303-0

PGF 2 α treatment of HEK cells stably expressing the human FP prostanoid receptor increases expression of cyclin B1 ( A ) and increases Cdc2-mediated phosphorylation of histone H1 ( B ). ( A ) Upper panel: immunoblot of lysates prepared from hFP-HEK and pCEP4-HEK cells that had been treated with either vehicle (V) or 1 µM PGF 2 α for 24 h. Blots were probed with anti-cyclin B1 and anti-actin antibodies as described in Materials and methods. Lower panel: quantitative analysis of three independent experiments by densitometry of cyclin B1 immunoreactivity normalized to actin immunoreactivity (means ± SEs). ( B ) Upper panel: autoradiograph of Cdc2 kinase activity (phosphorylation of histone H1) and immunoblot of Cdc2 kinase expression in lysates prepared from hFP-HEK and pCEP4-HEK cells that had been treated with either vehicle (V) or 1 µM PGF 2 α for 24 h. Cdc2 kinase assays and Cdc2 immunoblotting were done as described in Materials and methods. Lower panel: quantitative analysis of three independent experiments by densitometry of Cdc2 kinase activity (histone H1 phosphorylation) normalized to Cdc2 immunoreactivity (means ± SEs).
Figure Legend Snippet: PGF 2 α treatment of HEK cells stably expressing the human FP prostanoid receptor increases expression of cyclin B1 ( A ) and increases Cdc2-mediated phosphorylation of histone H1 ( B ). ( A ) Upper panel: immunoblot of lysates prepared from hFP-HEK and pCEP4-HEK cells that had been treated with either vehicle (V) or 1 µM PGF 2 α for 24 h. Blots were probed with anti-cyclin B1 and anti-actin antibodies as described in Materials and methods. Lower panel: quantitative analysis of three independent experiments by densitometry of cyclin B1 immunoreactivity normalized to actin immunoreactivity (means ± SEs). ( B ) Upper panel: autoradiograph of Cdc2 kinase activity (phosphorylation of histone H1) and immunoblot of Cdc2 kinase expression in lysates prepared from hFP-HEK and pCEP4-HEK cells that had been treated with either vehicle (V) or 1 µM PGF 2 α for 24 h. Cdc2 kinase assays and Cdc2 immunoblotting were done as described in Materials and methods. Lower panel: quantitative analysis of three independent experiments by densitometry of Cdc2 kinase activity (histone H1 phosphorylation) normalized to Cdc2 immunoreactivity (means ± SEs).

Techniques Used: Stable Transfection, Expressing, Autoradiography, Activity Assay

16) Product Images from "Proliferative Defects and Formation of a Double Cortex in Mice Lacking Mltt4 and Cdh2 in the Dorsal Telencephalon"

Article Title: Proliferative Defects and Formation of a Double Cortex in Mice Lacking Mltt4 and Cdh2 in the Dorsal Telencephalon

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1793-14.2014

Afadin-cKO mice present disruption of the cortical adherens junctions. A , Coronal section of the brain of control mice at E13.5 immunostained for afadin protein. Note the accumulation of afadin at the ventricular surface of the pallium and subpallium. Scale bar, 100 μm. B , Coronal section of the E13.5 developing cortex of control mice showing the VZ immunostained for the junctional proteins CDH2 (green) and afadin (red). Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. C , Coronal section of the E13.5 developing cortex of control mice showing the VZ immunostained for the junctional proteins αE-catenin (green) and β-catenin (red). Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. D , Coronal section of the brain of Afadin-cKO mice at E13.5 immunostained for afadin protein. Note the disappearance of afadin immunostaining in the dorsal telencephalon, whereas afadin expression is maintained in the subpallium. Arrows point toward blood vessels in the dorsal pallium, which also continue to express afadin. Scale bar, 100 μm. E , Coronal section of the E13.5 developing cortex of Afadin-cKO mice showing the VZ immunostained with the junctional proteins CDH2 (green) and afadin (red). Note the absence of afadin expression and the altered localization of CDH2 at the apical–junctional complex. Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. F , Coronal section of the E13.5 developing cortex of Afadin-cKO mice showing the VZ immunostained with the junctional proteins αE-catenin (green) and β-catenin (red). Note the altered localization of both proteins at the apical–junctional complex. Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. PSB, Pallial–subpallial boundary.
Figure Legend Snippet: Afadin-cKO mice present disruption of the cortical adherens junctions. A , Coronal section of the brain of control mice at E13.5 immunostained for afadin protein. Note the accumulation of afadin at the ventricular surface of the pallium and subpallium. Scale bar, 100 μm. B , Coronal section of the E13.5 developing cortex of control mice showing the VZ immunostained for the junctional proteins CDH2 (green) and afadin (red). Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. C , Coronal section of the E13.5 developing cortex of control mice showing the VZ immunostained for the junctional proteins αE-catenin (green) and β-catenin (red). Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. D , Coronal section of the brain of Afadin-cKO mice at E13.5 immunostained for afadin protein. Note the disappearance of afadin immunostaining in the dorsal telencephalon, whereas afadin expression is maintained in the subpallium. Arrows point toward blood vessels in the dorsal pallium, which also continue to express afadin. Scale bar, 100 μm. E , Coronal section of the E13.5 developing cortex of Afadin-cKO mice showing the VZ immunostained with the junctional proteins CDH2 (green) and afadin (red). Note the absence of afadin expression and the altered localization of CDH2 at the apical–junctional complex. Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. F , Coronal section of the E13.5 developing cortex of Afadin-cKO mice showing the VZ immunostained with the junctional proteins αE-catenin (green) and β-catenin (red). Note the altered localization of both proteins at the apical–junctional complex. Nuclei stained with DAPI are shown in blue. Scale bars, 5 μm. PSB, Pallial–subpallial boundary.

Techniques Used: Mouse Assay, Staining, Immunostaining, Expressing

Disruption of adherens junctions and massively increased proliferation in the neocortex of Cdh2-cKO mice. A , B , Disruption of the apical–junctional complex in Cdh2-cKO mice as visualized by the immunostaining for β-catenin (red), αE-catenin (green; A ), as well as afadin (red) and CDH2 (green; B ). Scale bars, 5 μm. C , Overview of the developing neocortex from control and Cdh2-cKO mice at E13.5 immunostained with the RGC marker Pax6 (red) and the intermediate progenitor marker Tbr2 (green). Note the cortical disorganization and severe hyperplasia in the mutant mice. Note that despite of the disorganization each marker is primarily expressed for different cell populations. Scale bar, 100 μm. D , Overview of the developing neocortex from control and Cdh2-cKO mice at E13.5 immunostained with the neuronal marker Tuj1. Note the presence of multiple rosette-like structures in the Cdh2-cKO mice. Scale bar, 100 μm. E , Overview of the developing neocortex from control and Cdh2-cKO mice at E13.5 immunostained with the Ki67 (red) and pH3 (green). Note the dramatic increase in the number of proliferative cells present in the enlarged cortex of the Cdh2 mutant mice. Scale bar, 100 μm. F , Analysis of the number of Pax6 + cells, Tbr2 + cells, and Ki67 + cells in controls and Cdh2-cKO mice at E13.5 reveals massive increases in the number of progenitor cells in the developing cortex of the mutant mice (mean ± SEM). * p
Figure Legend Snippet: Disruption of adherens junctions and massively increased proliferation in the neocortex of Cdh2-cKO mice. A , B , Disruption of the apical–junctional complex in Cdh2-cKO mice as visualized by the immunostaining for β-catenin (red), αE-catenin (green; A ), as well as afadin (red) and CDH2 (green; B ). Scale bars, 5 μm. C , Overview of the developing neocortex from control and Cdh2-cKO mice at E13.5 immunostained with the RGC marker Pax6 (red) and the intermediate progenitor marker Tbr2 (green). Note the cortical disorganization and severe hyperplasia in the mutant mice. Note that despite of the disorganization each marker is primarily expressed for different cell populations. Scale bar, 100 μm. D , Overview of the developing neocortex from control and Cdh2-cKO mice at E13.5 immunostained with the neuronal marker Tuj1. Note the presence of multiple rosette-like structures in the Cdh2-cKO mice. Scale bar, 100 μm. E , Overview of the developing neocortex from control and Cdh2-cKO mice at E13.5 immunostained with the Ki67 (red) and pH3 (green). Note the dramatic increase in the number of proliferative cells present in the enlarged cortex of the Cdh2 mutant mice. Scale bar, 100 μm. F , Analysis of the number of Pax6 + cells, Tbr2 + cells, and Ki67 + cells in controls and Cdh2-cKO mice at E13.5 reveals massive increases in the number of progenitor cells in the developing cortex of the mutant mice (mean ± SEM). * p

Techniques Used: Mouse Assay, Immunostaining, Marker, Mutagenesis

17) Product Images from "Heparin-Binding Epidermal Growth Factor-Like Growth Factor: Hypoxia-Inducible Expression In Vitro and Stimulation of Neurogenesis In Vitro and In Vivo"

Article Title: Heparin-Binding Epidermal Growth Factor-Like Growth Factor: Hypoxia-Inducible Expression In Vitro and Stimulation of Neurogenesis In Vitro and In Vivo

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.22-13-05365.2002

Hypoxia-inducible expression of HB-EGF is associated with both mature and immature neurons. A , Cultures were exposed to hypoxia for 8 hr and labeled with an antibody against a marker of mature neurons ( NeuN , MAP2 ), immature neurons ( βIII-tubulin ), or neuroepithelial precursor cells ( Nestin ) ( a ); an antibody against HB-EGF ( b ); and the nuclear stain DAPI ( c ). Merged images are shown in d . NeuN-, MAP2-, βIII-tubulin-, and nestin-immunopositive cells all coexpressed HB-EGF. B , Cultures were treated with BrdU, exposed to hypoxia for 8 hr, and labeled with an antibody against BrdU ( a ); an antibody against the mature neuronal marker NeuN, the late immature to mature neuronal marker Hu, the neuroepithelial precursor cell marker nestin, or the astroglial marker GFAP ( b ); and the nuclear stain DAPI ( c ). Merged images are shown in d . Only nestin-immunopositive cells were labeled with BrdU. Data in A and B are representative fields from at least three independent experiments per row. Original magnification, 40×.
Figure Legend Snippet: Hypoxia-inducible expression of HB-EGF is associated with both mature and immature neurons. A , Cultures were exposed to hypoxia for 8 hr and labeled with an antibody against a marker of mature neurons ( NeuN , MAP2 ), immature neurons ( βIII-tubulin ), or neuroepithelial precursor cells ( Nestin ) ( a ); an antibody against HB-EGF ( b ); and the nuclear stain DAPI ( c ). Merged images are shown in d . NeuN-, MAP2-, βIII-tubulin-, and nestin-immunopositive cells all coexpressed HB-EGF. B , Cultures were treated with BrdU, exposed to hypoxia for 8 hr, and labeled with an antibody against BrdU ( a ); an antibody against the mature neuronal marker NeuN, the late immature to mature neuronal marker Hu, the neuroepithelial precursor cell marker nestin, or the astroglial marker GFAP ( b ); and the nuclear stain DAPI ( c ). Merged images are shown in d . Only nestin-immunopositive cells were labeled with BrdU. Data in A and B are representative fields from at least three independent experiments per row. Original magnification, 40×.

Techniques Used: Expressing, Labeling, Marker, Staining

18) Product Images from "Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis"

Article Title: Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis

Journal: Nature Communications

doi: 10.1038/s41467-017-02720-9

Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or Map2 (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and Nestin (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P
Figure Legend Snippet: Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or Map2 (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and Nestin (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P

Techniques Used: In Vivo, In Vitro, Staining, Knock-Out, Immunostaining, Serial Dilution, Software, Real-time Polymerase Chain Reaction

Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower right) Quantification of the proportion of Tuj1 + or MAP2 + cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67 + ( n = 2 or 3) or Nestin + ( n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF ( n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP + cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d ( n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f . DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (* P
Figure Legend Snippet: Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower right) Quantification of the proportion of Tuj1 + or MAP2 + cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67 + ( n = 2 or 3) or Nestin + ( n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF ( n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP + cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d ( n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f . DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (* P

Techniques Used: Immunostaining, Infection, Expressing, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation

Stau1 inhibits neurogenesis in a Klf4-dependent manner. a (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 (#1 and #3) lentivirus. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1, Stau2, and α-tubulin in samples analyzed in a were were detected by immunoblotting ( n . (Lower right) Corresponding quantification of number of Tuj1 + and Map2 + cells. b (Upper) Immunostaining with Nestin or Ki67 (both red) antibodies of samples analyzed in a . DAPI (blue). Scale bars, 50 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + ( n = 2 or 3) cells. c (Upper) Immunostaining of NPCs infected with pCDH-control or pCDH-Stau1 lentivirus with Tuj1 (green) or MAP2 (red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1 and α-tubulin in samples analyzed in c were detected by immunoblotting ( n = 2). (Lower) Corresponding quantification of data shown above. d (Upper) Immunostaining of samples equivalent to those shown in c with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + (Ctrl, n = 3; Stau1, n = 2) cells. e RT-qPCR of indicated transcripts in control- or Klf4-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). f RT-qPCR of indicated transcripts in control- or Stau1-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). Data are shown as mean ± SD. ANOVA tests were performed to calculate statistical significance (* P
Figure Legend Snippet: Stau1 inhibits neurogenesis in a Klf4-dependent manner. a (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 (#1 and #3) lentivirus. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1, Stau2, and α-tubulin in samples analyzed in a were were detected by immunoblotting ( n . (Lower right) Corresponding quantification of number of Tuj1 + and Map2 + cells. b (Upper) Immunostaining with Nestin or Ki67 (both red) antibodies of samples analyzed in a . DAPI (blue). Scale bars, 50 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + ( n = 2 or 3) cells. c (Upper) Immunostaining of NPCs infected with pCDH-control or pCDH-Stau1 lentivirus with Tuj1 (green) or MAP2 (red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1 and α-tubulin in samples analyzed in c were detected by immunoblotting ( n = 2). (Lower) Corresponding quantification of data shown above. d (Upper) Immunostaining of samples equivalent to those shown in c with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + (Ctrl, n = 3; Stau1, n = 2) cells. e RT-qPCR of indicated transcripts in control- or Klf4-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). f RT-qPCR of indicated transcripts in control- or Stau1-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). Data are shown as mean ± SD. ANOVA tests were performed to calculate statistical significance (* P

Techniques Used: Immunostaining, Infection, Quantitative RT-PCR, Cell Culture

19) Product Images from "A20 deficiency in multipotent progenitors perturbs quiescence of hematopoietic stem cells"

Article Title: A20 deficiency in multipotent progenitors perturbs quiescence of hematopoietic stem cells

Journal: Stem cell research

doi: 10.1016/j.scr.2018.10.020

A20 deficiency in MPPs leads to modest changes in hematopoiesis. (A) Cellularity of BM (two femurs and two tibias), thymus and spleen of 8 weeks old A20 F/F Flt3 cre/+ and control mice (n = 4–6). (B) Genotyping PCR for Tnfaip3 gene from BM of A20F/FFlt3+/+, A20F/+Flt3cre/+ and A20F/FFlt3cre/+ mice. (C) Frequencies of RFP+ cells in the indicated hematopoietic organs from A20F/FFlt3cre/+ RosaRFP mice. (D) Representative histograms indicating frequencies of RFP+ cells in the specified hematopoietic subsets from A20F/FFlt3cre/+ RosaRFP mice. (E) Cumulative data indicating frequencies of RFP+ cells in hematopoietic subsets of A20F/FFlt3cre/+ RosaRFP mice (n = 15–16). (F) Frequencies of CD11b+, Ter119+, CD19+ and CD3e+, cells in the BM of A20F/FFlt3cre/+ and control mice (n = 13–18). (G) Absolute number of myeloid cells (CD11b+, 1st panel), erythroid cells (Ter119+, 2nd panel), B cells (CD19+, 3rd panel) and T cells (CD3+, 4th panel) from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 9–10). (H) Frequencies of CD11b+, Ter119+, CD19+ and CD3e+, cells in the Spleen of A20F/FFlt3cre/+ and control mice (n = 12–17). (I) Absolute number of myeloid cells (CD11b+, 1st panel), erythroid cells (Ter119+, 2nd panel), B cells (CD19+, 3rd panel) and T cells (CD3+, 4th panel) from the spleen of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 9–10). (J) Frequencies of CD4+CD8+, CD4+CD8−, CD4−CD8+ and CD4−CD8− in the thymus of A20F/FFlt3cre/+ and control mice (n = 11–15). (K) Frequencies of myeloid cells (CD11b+, 1st panel), B cells (CD19+, 2nd panel) and T cells (CD3+, 3rd panel) from the peripheral blood of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 9–10). All data represent mean ± SEM. Two-tailed student’s t -tests were used to assess statistical significance (*P
Figure Legend Snippet: A20 deficiency in MPPs leads to modest changes in hematopoiesis. (A) Cellularity of BM (two femurs and two tibias), thymus and spleen of 8 weeks old A20 F/F Flt3 cre/+ and control mice (n = 4–6). (B) Genotyping PCR for Tnfaip3 gene from BM of A20F/FFlt3+/+, A20F/+Flt3cre/+ and A20F/FFlt3cre/+ mice. (C) Frequencies of RFP+ cells in the indicated hematopoietic organs from A20F/FFlt3cre/+ RosaRFP mice. (D) Representative histograms indicating frequencies of RFP+ cells in the specified hematopoietic subsets from A20F/FFlt3cre/+ RosaRFP mice. (E) Cumulative data indicating frequencies of RFP+ cells in hematopoietic subsets of A20F/FFlt3cre/+ RosaRFP mice (n = 15–16). (F) Frequencies of CD11b+, Ter119+, CD19+ and CD3e+, cells in the BM of A20F/FFlt3cre/+ and control mice (n = 13–18). (G) Absolute number of myeloid cells (CD11b+, 1st panel), erythroid cells (Ter119+, 2nd panel), B cells (CD19+, 3rd panel) and T cells (CD3+, 4th panel) from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 9–10). (H) Frequencies of CD11b+, Ter119+, CD19+ and CD3e+, cells in the Spleen of A20F/FFlt3cre/+ and control mice (n = 12–17). (I) Absolute number of myeloid cells (CD11b+, 1st panel), erythroid cells (Ter119+, 2nd panel), B cells (CD19+, 3rd panel) and T cells (CD3+, 4th panel) from the spleen of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 9–10). (J) Frequencies of CD4+CD8+, CD4+CD8−, CD4−CD8+ and CD4−CD8− in the thymus of A20F/FFlt3cre/+ and control mice (n = 11–15). (K) Frequencies of myeloid cells (CD11b+, 1st panel), B cells (CD19+, 2nd panel) and T cells (CD3+, 3rd panel) from the peripheral blood of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 9–10). All data represent mean ± SEM. Two-tailed student’s t -tests were used to assess statistical significance (*P

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Two Tailed Test

Loss of A20 in MPPs leads to loss of quiescence of HSPCs. (A) Schematic of serial transplantation experiments. (B) Survival curve of lethally-irradiated WT congenic (CD45.1+) secondary recipients (n = 9). Total BM cells from A20F/FFlt3cre/+ and control groups were injected into lethally irradiated WT congenic primary recipients (n = 10). After 12 weeks of transplantation primary recipients were sacrificed and their BM cells were injected into lethally irradiated WT congenic secondary recipients (n = 9). (C) Frequencies of donor (CD45.2+)-derived cells in the peripheral blood of secondary recipients after 12 weeks of BMT (n = 7). (D) Frequencies of donor (CD45.2+) and recipient (CD45.1+) derived CD11b+ , CD19+ and CD3e+ cells in the peripheral blood of secondary recipients at 12 weeks of transplantation (n = 7). (E) Apoptosis assay of LSK cells from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 5). Representative FACS plots (left) and dot plots (right). (F) Cell cycle analysis (Hoechst and Pyronin Y) of LSK cells from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice. (G–J) Cell cycle analysis (Hoechst and Ki67) of LSK cells (G and H) and CD150+ CD48− LSK cells (I and J) from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice. Representative FACS plots (G and I) and dot plots (H and J) (n = 5). (K and L) BrdU assay of HSPCs from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 3). Representative FACS plots (K) and dot plots (L). All data represent mean ± SEM. Two-tailed student’s t -tests were used to assess statistical significance (*P
Figure Legend Snippet: Loss of A20 in MPPs leads to loss of quiescence of HSPCs. (A) Schematic of serial transplantation experiments. (B) Survival curve of lethally-irradiated WT congenic (CD45.1+) secondary recipients (n = 9). Total BM cells from A20F/FFlt3cre/+ and control groups were injected into lethally irradiated WT congenic primary recipients (n = 10). After 12 weeks of transplantation primary recipients were sacrificed and their BM cells were injected into lethally irradiated WT congenic secondary recipients (n = 9). (C) Frequencies of donor (CD45.2+)-derived cells in the peripheral blood of secondary recipients after 12 weeks of BMT (n = 7). (D) Frequencies of donor (CD45.2+) and recipient (CD45.1+) derived CD11b+ , CD19+ and CD3e+ cells in the peripheral blood of secondary recipients at 12 weeks of transplantation (n = 7). (E) Apoptosis assay of LSK cells from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 5). Representative FACS plots (left) and dot plots (right). (F) Cell cycle analysis (Hoechst and Pyronin Y) of LSK cells from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice. (G–J) Cell cycle analysis (Hoechst and Ki67) of LSK cells (G and H) and CD150+ CD48− LSK cells (I and J) from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice. Representative FACS plots (G and I) and dot plots (H and J) (n = 5). (K and L) BrdU assay of HSPCs from the BM of 8 weeks old A20F/FFlt3cre/+ and control mice (n = 3). Representative FACS plots (K) and dot plots (L). All data represent mean ± SEM. Two-tailed student’s t -tests were used to assess statistical significance (*P

Techniques Used: Transplantation Assay, Irradiation, Injection, Derivative Assay, Apoptosis Assay, Mouse Assay, FACS, Cell Cycle Assay, BrdU Staining, Two Tailed Test

20) Product Images from "Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys"

Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2012121236

Increase in cell proliferation, apoptosis, and fibrosis in kidneys of Cdc42 conditional knockout mice. (A and B) BrdU incorporation, a marker of active cell division, is significantly increased in sections of kidneys from the Cdc42 kidney-specific knockout
Figure Legend Snippet: Increase in cell proliferation, apoptosis, and fibrosis in kidneys of Cdc42 conditional knockout mice. (A and B) BrdU incorporation, a marker of active cell division, is significantly increased in sections of kidneys from the Cdc42 kidney-specific knockout

Techniques Used: Knock-Out, Mouse Assay, BrdU Incorporation Assay, Marker

Active pERK is increased in the kidneys of Cdc42 kidney-specific knockout mice. (A) Immunoblot analysis of proteins extracted from the whole kidneys of P6 control and Cdc42 kidney-specific knockout mice, showed increased pERK levels in Cdc42 knockout
Figure Legend Snippet: Active pERK is increased in the kidneys of Cdc42 kidney-specific knockout mice. (A) Immunoblot analysis of proteins extracted from the whole kidneys of P6 control and Cdc42 kidney-specific knockout mice, showed increased pERK levels in Cdc42 knockout

Techniques Used: Knock-Out, Mouse Assay

Primary ciliogenesis is inhibited in the cysts of Cdc42 kidney-specific knockout mice. (A and B) Immunostaining of acetylated α-tubulin (red) in kidneys from control (A) and Cdc42 kidney-specific knockout mice (B) at P4, shows lack of cilia in
Figure Legend Snippet: Primary ciliogenesis is inhibited in the cysts of Cdc42 kidney-specific knockout mice. (A and B) Immunostaining of acetylated α-tubulin (red) in kidneys from control (A) and Cdc42 kidney-specific knockout mice (B) at P4, shows lack of cilia in

Techniques Used: Knock-Out, Mouse Assay, Immunostaining

Lack of Cdc42 in kidney tubule cells leads to an early postnatal death. KspCre;Cdc42 fl/+ mice were mated to Cdc42 fl/fl mice as illustrated in . Of 65 pups tested from E16.5 to P10, there were 15 Cdc42 fl/fl , 25 Cdc42 fl/+ , 12 KspCre;Cdc42 fl/+ ,
Figure Legend Snippet: Lack of Cdc42 in kidney tubule cells leads to an early postnatal death. KspCre;Cdc42 fl/+ mice were mated to Cdc42 fl/fl mice as illustrated in . Of 65 pups tested from E16.5 to P10, there were 15 Cdc42 fl/fl , 25 Cdc42 fl/+ , 12 KspCre;Cdc42 fl/+ ,

Techniques Used: Mouse Assay

cdc42 and sec10 genetically interact. A synergistic interaction resulting in hydrocephalus (arrowhead), small eyes (arrow), pericardial edema (*), and tail defects was observed upon co-injection of suboptimal doses of 2 ng cdc42MO plus 7.5 ng sec10MO
Figure Legend Snippet: cdc42 and sec10 genetically interact. A synergistic interaction resulting in hydrocephalus (arrowhead), small eyes (arrow), pericardial edema (*), and tail defects was observed upon co-injection of suboptimal doses of 2 ng cdc42MO plus 7.5 ng sec10MO

Techniques Used: Injection

Kidney tubule cell-specific Cdc42 knockout mice were successfully generated. (A) Targeting scheme showing the Cdc42 gene (wild type), the targeting construct, and the conditional allele after homologous recombination of the targeting construct and removal
Figure Legend Snippet: Kidney tubule cell-specific Cdc42 knockout mice were successfully generated. (A) Targeting scheme showing the Cdc42 gene (wild type), the targeting construct, and the conditional allele after homologous recombination of the targeting construct and removal

Techniques Used: Knock-Out, Mouse Assay, Generated, Construct, Homologous Recombination

cdc42 expression occurs in the zebrafish kidney, eye, and brain, and cdc42 knockdown by antisense MOs results in abnormal phenotypes. (A) Lateral views of whole mount in situ hybridization of zebrafish embryos at 3 dpf with antisense (upper part) and
Figure Legend Snippet: cdc42 expression occurs in the zebrafish kidney, eye, and brain, and cdc42 knockdown by antisense MOs results in abnormal phenotypes. (A) Lateral views of whole mount in situ hybridization of zebrafish embryos at 3 dpf with antisense (upper part) and

Techniques Used: Expressing, In Situ Hybridization

Model for the role of Cdc42 in delivery of ciliary proteins. (A) Our data support a model in which the exocyst complex is localized to the primary cilium by Cdc42, which is located all along the apical surface but is activated at the primary cilium by
Figure Legend Snippet: Model for the role of Cdc42 in delivery of ciliary proteins. (A) Our data support a model in which the exocyst complex is localized to the primary cilium by Cdc42, which is located all along the apical surface but is activated at the primary cilium by

Techniques Used:

KspCre;Cdc42 fl/fl mice develop renal cysts in the distal and collecting tubules. (A–D) Hematoxylin and eosin–stained sections of kidneys from control (A and C) and Cdc42 kidney-specific knockout mice (B and D) at P4 (A and B) and P6 (C
Figure Legend Snippet: KspCre;Cdc42 fl/fl mice develop renal cysts in the distal and collecting tubules. (A–D) Hematoxylin and eosin–stained sections of kidneys from control (A and C) and Cdc42 kidney-specific knockout mice (B and D) at P4 (A and B) and P6 (C

Techniques Used: Mouse Assay, Staining, Knock-Out

21) Product Images from "Interleukin 7 Regulates the Survival and Generation of Memory CD4 Cells"

Article Title: Interleukin 7 Regulates the Survival and Generation of Memory CD4 Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20030735

In vivo–primed TCR transgenic memory CD4 cells survive in response to rIL-7 in vitro. Purified naive OT-II Thy 1.1 CD4 cells were transferred into C57BL/6 Rag2 − mice (5 × 10 6 cells/recipient) and immunized with OVA protein and adjuvant as indicated in Materials and Methods. 1 mo later, resting memory OT-II cells were isolated and compared with freshly isolated naive OT-II cells. (A) Phenotype of memory OT-II cells. Naive and memory OT-II cells were stained for expression of CD62L, CD44, and IL-7Rα and analyzed by flow cytometry (shaded histograms; unshaded histograms denote background staining). (B) Frequencies of effector cytokine producers among memory OT-II cells. Memory OT-II Thy 1.1 cells were restimulated with OVA peptide in the presence of splenic APC and tested for secretion of IFN-γ at 12 h by ICS, and after enrichment of Thy 1.1 cells, for production of IL-4 or IL-13 at 24 h by ELISPOT analysis. (C) IL-7 promotes survival of OT-II cells. Naive and memory OT-II Thy 1.1 cells were cultured at 10 6 /ml for the indicated number of days without or with rIL-7 at 10 ng/ml (left and right, respectively). (D) Blocking IL-7 prevents up-regulation of Bcl-2. Naive (top left) and memory (top right) OT-II Thy 1.1 CD4 cells were stained for expression of Bcl-2 (light gray histograms). The cells were cultured as in C for 6 d with 10 ng/ml rIL-7 and stained for BcL-2 (dark gray histograms). Memory cell cultures were also treated with either 10 μg/ml each anti–IL-7 and anti–IL-7R mAb or with an equivalent amount of rat and mouse IgG (bottom, rIgG and mIgG, respectively). The cells were stained for Bcl-2 on day 6 after culture with blocking mAb (shaded histogram) or with control mAb (unshaded histogram).
Figure Legend Snippet: In vivo–primed TCR transgenic memory CD4 cells survive in response to rIL-7 in vitro. Purified naive OT-II Thy 1.1 CD4 cells were transferred into C57BL/6 Rag2 − mice (5 × 10 6 cells/recipient) and immunized with OVA protein and adjuvant as indicated in Materials and Methods. 1 mo later, resting memory OT-II cells were isolated and compared with freshly isolated naive OT-II cells. (A) Phenotype of memory OT-II cells. Naive and memory OT-II cells were stained for expression of CD62L, CD44, and IL-7Rα and analyzed by flow cytometry (shaded histograms; unshaded histograms denote background staining). (B) Frequencies of effector cytokine producers among memory OT-II cells. Memory OT-II Thy 1.1 cells were restimulated with OVA peptide in the presence of splenic APC and tested for secretion of IFN-γ at 12 h by ICS, and after enrichment of Thy 1.1 cells, for production of IL-4 or IL-13 at 24 h by ELISPOT analysis. (C) IL-7 promotes survival of OT-II cells. Naive and memory OT-II Thy 1.1 cells were cultured at 10 6 /ml for the indicated number of days without or with rIL-7 at 10 ng/ml (left and right, respectively). (D) Blocking IL-7 prevents up-regulation of Bcl-2. Naive (top left) and memory (top right) OT-II Thy 1.1 CD4 cells were stained for expression of Bcl-2 (light gray histograms). The cells were cultured as in C for 6 d with 10 ng/ml rIL-7 and stained for BcL-2 (dark gray histograms). Memory cell cultures were also treated with either 10 μg/ml each anti–IL-7 and anti–IL-7R mAb or with an equivalent amount of rat and mouse IgG (bottom, rIgG and mIgG, respectively). The cells were stained for Bcl-2 on day 6 after culture with blocking mAb (shaded histogram) or with control mAb (unshaded histogram).

Techniques Used: In Vivo, Transgenic Assay, In Vitro, Purification, Mouse Assay, Isolation, Staining, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Cell Culture, Blocking Assay

22) Product Images from "FGF19 functions as autocrine growth factor for hepatoblastoma"

Article Title: FGF19 functions as autocrine growth factor for hepatoblastoma

Journal: Genes & Cancer

doi: 10.18632/genesandcancer.101

FGF19 downregulation results in inhibition of hepatoblastoma proliferation A. shRNA-mediated silencing of FGF19 mRNA expression in Hep293TT cells. Hep293TT cells were infected with lentiviruses expressing shRNAs against FGF19 or control shRNA and were selected with 2 μg/ml puromycin. Four days after infection, total RNA was isolated and the expression of FGF19 was analyzed by RT-PCR. RNA polymerase II (Pol II) serves as a loading control. B. Suppression of secreted FGF19 protein levels by shRNAs. Hep293TT cells were infected with lentiviruses expressing shRNAs against FGF19 or control shRNA and were selected with 2 μg/ml puromycin. Four days after infection, the levels of secreted FGF19 protein were assessed by ELISA. C. - E. FGF19 silencing inhibits Hep293TT cell proliferation. Hep293TT cells were infected with lentiviruses expressing shRNAs against FGF19 or control shRNA. Four days after infection, cells were labeled with BrdU for 6 hours and immunofluorescence microscopy was used to score the percentage of BrdU-positive cells (C); cells were stained for Ki-67 (D); and the dephosphorylation of Rb was analyzed by immunoblotting (E; nucleolin serves as a loading control). Asterisks denote p
Figure Legend Snippet: FGF19 downregulation results in inhibition of hepatoblastoma proliferation A. shRNA-mediated silencing of FGF19 mRNA expression in Hep293TT cells. Hep293TT cells were infected with lentiviruses expressing shRNAs against FGF19 or control shRNA and were selected with 2 μg/ml puromycin. Four days after infection, total RNA was isolated and the expression of FGF19 was analyzed by RT-PCR. RNA polymerase II (Pol II) serves as a loading control. B. Suppression of secreted FGF19 protein levels by shRNAs. Hep293TT cells were infected with lentiviruses expressing shRNAs against FGF19 or control shRNA and were selected with 2 μg/ml puromycin. Four days after infection, the levels of secreted FGF19 protein were assessed by ELISA. C. - E. FGF19 silencing inhibits Hep293TT cell proliferation. Hep293TT cells were infected with lentiviruses expressing shRNAs against FGF19 or control shRNA. Four days after infection, cells were labeled with BrdU for 6 hours and immunofluorescence microscopy was used to score the percentage of BrdU-positive cells (C); cells were stained for Ki-67 (D); and the dephosphorylation of Rb was analyzed by immunoblotting (E; nucleolin serves as a loading control). Asterisks denote p

Techniques Used: Inhibition, shRNA, Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Labeling, Immunofluorescence, Microscopy, Staining, De-Phosphorylation Assay

23) Product Images from "Origin of Climbing Fiber Neurons and Their Developmental Dependence on Ptf1a"

Article Title: Origin of Climbing Fiber Neurons and Their Developmental Dependence on Ptf1a

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1423-07.2007

Expression of Ptf1a in the embryonic caudal hindbrain. A , B , Serial transverse frozen sections of r7 hindbrain at E11.5. Localization of Math1 transcripts (visualized by in situ hybridization) and Ptf1a and Ngn1 proteins (visualized by immunohistochemistry) are shown. C , Schematic diagram of expression of bHLH transcription factors/genes in the caudal hindbrain at E11.5. Lmx1a ). D–F , Double immunostaining with anti-Ptf1a and BrdU antibodies within the Ptf1a domain of the E11.5 caudal hindbrain. Pregnant mice were given BrdU injections 1 h before embryo harvest and fixation. Many Ptf1a-positive cells incorporate BrdU (arrowheads in F ), indicating that they are mitotic. G–I , Double immunostaining with anti-Ptf1a and HuC/D antibodies around the Ptf1a domain of the caudal hindbrain at E11.5. Ptf1a-positive cells do not express HuC/D, suggesting that they do not include postmitotic neurons. Scale bars: A , B , 100 μm; D–I , 20 μm.
Figure Legend Snippet: Expression of Ptf1a in the embryonic caudal hindbrain. A , B , Serial transverse frozen sections of r7 hindbrain at E11.5. Localization of Math1 transcripts (visualized by in situ hybridization) and Ptf1a and Ngn1 proteins (visualized by immunohistochemistry) are shown. C , Schematic diagram of expression of bHLH transcription factors/genes in the caudal hindbrain at E11.5. Lmx1a ). D–F , Double immunostaining with anti-Ptf1a and BrdU antibodies within the Ptf1a domain of the E11.5 caudal hindbrain. Pregnant mice were given BrdU injections 1 h before embryo harvest and fixation. Many Ptf1a-positive cells incorporate BrdU (arrowheads in F ), indicating that they are mitotic. G–I , Double immunostaining with anti-Ptf1a and HuC/D antibodies around the Ptf1a domain of the caudal hindbrain at E11.5. Ptf1a-positive cells do not express HuC/D, suggesting that they do not include postmitotic neurons. Scale bars: A , B , 100 μm; D–I , 20 μm.

Techniques Used: Expressing, In Situ Hybridization, Immunohistochemistry, Double Immunostaining, Mouse Assay

24) Product Images from "Deletion of lysophosphatidic acid receptor LPA1 reduces neurogenesis in the mouse dentate gyrus"

Article Title: Deletion of lysophosphatidic acid receptor LPA1 reduces neurogenesis in the mouse dentate gyrus

Journal: Molecular and cellular neurosciences

doi: 10.1016/j.mcn.2008.07.014

Reduced PSA-NCAM and doublecortin expression and altered expression pattern in absence of LPA 1 receptor
Figure Legend Snippet: Reduced PSA-NCAM and doublecortin expression and altered expression pattern in absence of LPA 1 receptor

Techniques Used: Expressing

25) Product Images from "Nondividing, Postpubertal Rat Sertoli Cells Resumed Proliferation after Transplantation 1"

Article Title: Nondividing, Postpubertal Rat Sertoli Cells Resumed Proliferation after Transplantation 1

Journal: Biology of Reproduction

doi: 10.1095/biolreprod.113.110197

Transplanted SC were positive for the proliferation markers PCNA and MKI67. Double immunofluorescence staining for GATA4 (green, A and D ), PCNA (red, B ) or MKI67 (red, E ) was performed with tissue sections obtained from graft-bearing kidneys collected
Figure Legend Snippet: Transplanted SC were positive for the proliferation markers PCNA and MKI67. Double immunofluorescence staining for GATA4 (green, A and D ), PCNA (red, B ) or MKI67 (red, E ) was performed with tissue sections obtained from graft-bearing kidneys collected

Techniques Used: Double Immunofluorescence Staining

26) Product Images from "Reduced Cell Turnover in Bovine Leukemia Virus-Infected, Persistently Lymphocytotic Cattle"

Article Title: Reduced Cell Turnover in Bovine Leukemia Virus-Infected, Persistently Lymphocytotic Cattle

Journal: Journal of Virology

doi: 10.1128/JVI.77.24.13073-13083.2003

Cell proliferation and viral expression appear mutually exclusive. Three days post-BrdU injection, PBMCs from noninfected (PBK, 109322), aleukemic (BKL, Wysoka), and PL (BKL-2, Stara) cows were isolated and cultivated for 18 h. The cells were then fixed and incubated with anti-p24 antibody 4′G9, which recognizes the viral capsid protein, and with a phycoerythrin-conjugated secondary antibody. Finally, cells were stained with anti-BrdU fluorescein isothiocyanate conjugate containing DNase and analyzed by flow cytometry. A representative experiment (out of three) is represented as dot plots (10,000 gated events). Numbers represent the percentages of positively stained cells in each quadrant.
Figure Legend Snippet: Cell proliferation and viral expression appear mutually exclusive. Three days post-BrdU injection, PBMCs from noninfected (PBK, 109322), aleukemic (BKL, Wysoka), and PL (BKL-2, Stara) cows were isolated and cultivated for 18 h. The cells were then fixed and incubated with anti-p24 antibody 4′G9, which recognizes the viral capsid protein, and with a phycoerythrin-conjugated secondary antibody. Finally, cells were stained with anti-BrdU fluorescein isothiocyanate conjugate containing DNase and analyzed by flow cytometry. A representative experiment (out of three) is represented as dot plots (10,000 gated events). Numbers represent the percentages of positively stained cells in each quadrant.

Techniques Used: Expressing, Injection, Isolation, Incubation, Staining, Flow Cytometry, Cytometry

Bromodeoxyuridine incorporates into B lymphocytes in vivo. Two PL (BKL-2, Stara) and aleukemic (BKL, Wysoka) BLV-infected cattle and three controls (PBK, BK, 109322) (PBK and 109322 are represented) were injected intravenously with 3 g of BrdU, and an aliquot of blood (1 ml) was collected 6 days later. After lysis of the red blood cells, B cells were labeled with biotinylated 1H4 monoclonal antibody and streptavidin-phycoerythrin (PE) conjugate. Then, the cells were stained with anti-BrdU fluorescein isothiocyanate antibody in the presence of DNase and analyzed by two-color flow cytometry ( x axis = BrdU; y axis = B lymphocytes). Ten thousand cells (lymphocytes, monocytes, and granulocytes) were acquired and PBMCs were selected by the forward/side scatter gating method. The total numbers of B cells are indicated in the upper quadrants.
Figure Legend Snippet: Bromodeoxyuridine incorporates into B lymphocytes in vivo. Two PL (BKL-2, Stara) and aleukemic (BKL, Wysoka) BLV-infected cattle and three controls (PBK, BK, 109322) (PBK and 109322 are represented) were injected intravenously with 3 g of BrdU, and an aliquot of blood (1 ml) was collected 6 days later. After lysis of the red blood cells, B cells were labeled with biotinylated 1H4 monoclonal antibody and streptavidin-phycoerythrin (PE) conjugate. Then, the cells were stained with anti-BrdU fluorescein isothiocyanate antibody in the presence of DNase and analyzed by two-color flow cytometry ( x axis = BrdU; y axis = B lymphocytes). Ten thousand cells (lymphocytes, monocytes, and granulocytes) were acquired and PBMCs were selected by the forward/side scatter gating method. The total numbers of B cells are indicated in the upper quadrants.

Techniques Used: In Vivo, Infection, Injection, Lysis, Labeling, Staining, Flow Cytometry, Cytometry

27) Product Images from "Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity"

Article Title: Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200805155

Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of BrdU incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, CRALBP, Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P
Figure Legend Snippet: Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of BrdU incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, CRALBP, Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P

Techniques Used: In Vivo, Staining, Mouse Assay, BrdU Incorporation Assay, Marker, Transfection

Gli2 is required for the Shh effects on proliferation and cell fate. Retinal explants were cultured from wild-type (Wt; n = 3) and Gli2 −/− ( n = 3) mice at E18 for 3 d in culture with or without a Smo agonist. IHC was performed on dissociated cells using anti-BrdU, anti-CRALBP, anti-rhodopsin, and anti-recoverin antibodies. Values represent the fold induction of positive cells in Wt + Ag or Gli2 −/− + Ag cultures compared with nontreated explants. Error bars represent SEM. *, P
Figure Legend Snippet: Gli2 is required for the Shh effects on proliferation and cell fate. Retinal explants were cultured from wild-type (Wt; n = 3) and Gli2 −/− ( n = 3) mice at E18 for 3 d in culture with or without a Smo agonist. IHC was performed on dissociated cells using anti-BrdU, anti-CRALBP, anti-rhodopsin, and anti-recoverin antibodies. Values represent the fold induction of positive cells in Wt + Ag or Gli2 −/− + Ag cultures compared with nontreated explants. Error bars represent SEM. *, P

Techniques Used: Cell Culture, Mouse Assay, Immunohistochemistry

28) Product Images from "Steroid Receptor RNA Activator Stimulates Proliferation as Well as Apoptosis In Vivo"

Article Title: Steroid Receptor RNA Activator Stimulates Proliferation as Well as Apoptosis In Vivo

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.20.7163-7176.2003

Histopathology of SRA expression in the male accessory sex glands. Tissues from the male urogenital tract are shown by gross anatomy (a and b), H E-stained histology (c to h), and Mac-3-immunohistochemistry (d). Compared to the wild type (a), SV from SRA-transgenic mice were grossly malformed (b), showed inflammation in the lumen (c) and muscular surrounding tissue (d), and displayed hyperplastic luminal epithelia with marked apoptotic activity (e). The inset in panel c shows brown hemosiderin, granulocytes, and dense-staining lymphocytes in the luminal portion of the SV; the inset in panel d shows H E staining of the inflammation in the muscular wall of the SV, and the inset in panel e is a higher magnification of a section of the coagulating-gland epithelia showing multiple apoptotic bodies. The luminal epithelial cells of the different lobes of SRA-transgenic prostate (coagulating gland [f], dorsal prostate [g], and ventral prostate [h]) displayed various levels of hyperplasia. Bl indicates the urinary bladder.
Figure Legend Snippet: Histopathology of SRA expression in the male accessory sex glands. Tissues from the male urogenital tract are shown by gross anatomy (a and b), H E-stained histology (c to h), and Mac-3-immunohistochemistry (d). Compared to the wild type (a), SV from SRA-transgenic mice were grossly malformed (b), showed inflammation in the lumen (c) and muscular surrounding tissue (d), and displayed hyperplastic luminal epithelia with marked apoptotic activity (e). The inset in panel c shows brown hemosiderin, granulocytes, and dense-staining lymphocytes in the luminal portion of the SV; the inset in panel d shows H E staining of the inflammation in the muscular wall of the SV, and the inset in panel e is a higher magnification of a section of the coagulating-gland epithelia showing multiple apoptotic bodies. The luminal epithelial cells of the different lobes of SRA-transgenic prostate (coagulating gland [f], dorsal prostate [g], and ventral prostate [h]) displayed various levels of hyperplasia. Bl indicates the urinary bladder.

Techniques Used: Histopathology, Expressing, Staining, Immunohistochemistry, Transgenic Assay, Mouse Assay, Activity Assay

Germinal aplasia and elevated testosterone levels in SRA mice. (A) SRA-transgenic testes displayed dysgenesis of the seminifeous tubules. H E histology (micrographs a to d), PAS staining (inset in micrograph d), and Mac-3 immunohistochemistry (micrograph d) of wild-type (micrograph a) and SRA-transgenic (micrographs b to d) testes. The seminiferous tubules appear vacuolated (micrograph b), show sloughing of germ cell elements into the lumen (micrograph c), and have greatly disrupted luminal seminiferous epithelia (inset in micrograph d). Intraluminal eosinophilic secretions, hemosiderin, and infiltrates of lymphocytes and monocytes (micrograph d) indicate a breakdown of the blood-testis barrier formed by the Sertoli-Sertoli junctional complex. (B) Leydig cell hyperplasia and elevated serum testosterone levels. Histology of periodic acid-Schiff-stained sections from an SRA-transgenic testis illustrate numerous intrastitial Leydig cells with copious and vacuolated cytoplasm (inset, H E staining). Critically elevated serum testosterone levels in adolescent and mature SRA-transgenic males were detected by radioimmunoassays (chart). **, Significant differences between transgenic (Tg) and wild-type (WT) mice; 6 weeks: n WT = 14, n Tg = 12 ( t test, P = 0.00035); 6 months: n WT = 3, n Tg = 6 ( t test, P = 0.00009).
Figure Legend Snippet: Germinal aplasia and elevated testosterone levels in SRA mice. (A) SRA-transgenic testes displayed dysgenesis of the seminifeous tubules. H E histology (micrographs a to d), PAS staining (inset in micrograph d), and Mac-3 immunohistochemistry (micrograph d) of wild-type (micrograph a) and SRA-transgenic (micrographs b to d) testes. The seminiferous tubules appear vacuolated (micrograph b), show sloughing of germ cell elements into the lumen (micrograph c), and have greatly disrupted luminal seminiferous epithelia (inset in micrograph d). Intraluminal eosinophilic secretions, hemosiderin, and infiltrates of lymphocytes and monocytes (micrograph d) indicate a breakdown of the blood-testis barrier formed by the Sertoli-Sertoli junctional complex. (B) Leydig cell hyperplasia and elevated serum testosterone levels. Histology of periodic acid-Schiff-stained sections from an SRA-transgenic testis illustrate numerous intrastitial Leydig cells with copious and vacuolated cytoplasm (inset, H E staining). Critically elevated serum testosterone levels in adolescent and mature SRA-transgenic males were detected by radioimmunoassays (chart). **, Significant differences between transgenic (Tg) and wild-type (WT) mice; 6 weeks: n WT = 14, n Tg = 12 ( t test, P = 0.00035); 6 months: n WT = 3, n Tg = 6 ( t test, P = 0.00009).

Techniques Used: Mouse Assay, Transgenic Assay, Staining, Immunohistochemistry

29) Product Images from "Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice"

Article Title: Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice

Journal: Scientific Reports

doi: 10.1038/s41598-018-35716-6

Multiple types of analyses show that GDF11 does not cross the BBB. ( a ) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. 7a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.
Figure Legend Snippet: Multiple types of analyses show that GDF11 does not cross the BBB. ( a ) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. 7a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay, Clone Assay, Differentiation Assay, Recombinant, Staining

30) Product Images from "Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development"

Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0047719

Cross-reactivity and validation of antibody staining. Section of an embryo exposed to IdU shows specific staining using an antibody against IdU (Panel A). When an antibody against CldU is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.
Figure Legend Snippet: Cross-reactivity and validation of antibody staining. Section of an embryo exposed to IdU shows specific staining using an antibody against IdU (Panel A). When an antibody against CldU is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.

Techniques Used: Staining

Image analysis and visualisation. Panel A. Using a Sytox green staining, all nuclei are first detected based on a local-maxima threshold. All detected objects that were at least twice as large as the median object size were processed to separate these fused nuclei (inserts). Panel B shows a schematic overview of the image processing steps involved in the recognition of IdU- and CldU-positive nuclei. After the detection of the Sytox green stained nuclei, each nucleus is individually processed. A zone is selected around all nuclei which will be excluded in the following measurement (gray zone). For each nucleus within the region of interest (myocardium), the algorithm measures the signal in (red area) and around (green area) the nucleus in the IdU and CldU channels. The measurement of the local background excludes the locations at which other nuclei were detected (gray zone). When the signal in the nucleus is at least a standard deviation above the background, the nucleus is classified as positively labelled. The program generates control images both for the nuclei detection as well as for which nuclei are positive for the proliferation markers. The difference between the two proliferation markers is used to determine ΔF (number of green nuclei divided by total number of nuclei). Panel C shows how the quantitative information can be projected onto a reconstruction or onto the original section. Each unit in the boxel representation has a volume of approximately 21 3 µm 3 , and is the central boxel of the sampling volume of approximately 105 3 µm 3 that is required for reliable measurement of the labelling indices [15] .
Figure Legend Snippet: Image analysis and visualisation. Panel A. Using a Sytox green staining, all nuclei are first detected based on a local-maxima threshold. All detected objects that were at least twice as large as the median object size were processed to separate these fused nuclei (inserts). Panel B shows a schematic overview of the image processing steps involved in the recognition of IdU- and CldU-positive nuclei. After the detection of the Sytox green stained nuclei, each nucleus is individually processed. A zone is selected around all nuclei which will be excluded in the following measurement (gray zone). For each nucleus within the region of interest (myocardium), the algorithm measures the signal in (red area) and around (green area) the nucleus in the IdU and CldU channels. The measurement of the local background excludes the locations at which other nuclei were detected (gray zone). When the signal in the nucleus is at least a standard deviation above the background, the nucleus is classified as positively labelled. The program generates control images both for the nuclei detection as well as for which nuclei are positive for the proliferation markers. The difference between the two proliferation markers is used to determine ΔF (number of green nuclei divided by total number of nuclei). Panel C shows how the quantitative information can be projected onto a reconstruction or onto the original section. Each unit in the boxel representation has a volume of approximately 21 3 µm 3 , and is the central boxel of the sampling volume of approximately 105 3 µm 3 that is required for reliable measurement of the labelling indices [15] .

Techniques Used: Staining, Standard Deviation, Sampling

Application in heart development. 3D visualisation of cell cycle length in the heart at stages HH9 (Panel A), HH12 (Panel B) and HH16 (Panel C) of chicken embryonic development. The pointer in panel B indicates the region with a high proliferation rate at the site of early ventricle formation. Panel C shows the quantitative reconstructions of the individual labelling indices for CldU and IdU, on which the cycle lengths are based. The pointers in the CldU reconstruction indicate areas in which a low fraction of cells is positive in both CldU and IdU reconstructions, resulting in a low labelling difference and thus a long cell cycle length. The pointers in the IdU reconstruction show large differences in IdU and CldU labelling indices, indicating short cell cycle lengths. Note the heterogeneity in cell cycle lengths in different parts of the heart at every stage. Interactive versions of the 3D-reconstructions can be found in Interactive 3D-pdf S1 .
Figure Legend Snippet: Application in heart development. 3D visualisation of cell cycle length in the heart at stages HH9 (Panel A), HH12 (Panel B) and HH16 (Panel C) of chicken embryonic development. The pointer in panel B indicates the region with a high proliferation rate at the site of early ventricle formation. Panel C shows the quantitative reconstructions of the individual labelling indices for CldU and IdU, on which the cycle lengths are based. The pointers in the CldU reconstruction indicate areas in which a low fraction of cells is positive in both CldU and IdU reconstructions, resulting in a low labelling difference and thus a long cell cycle length. The pointers in the IdU reconstruction show large differences in IdU and CldU labelling indices, indicating short cell cycle lengths. Note the heterogeneity in cell cycle lengths in different parts of the heart at every stage. Interactive versions of the 3D-reconstructions can be found in Interactive 3D-pdf S1 .

Techniques Used:

31) Product Images from "Defective neurogenesis and schizophrenia-like behavior in PARP-1-deficient mice"

Article Title: Defective neurogenesis and schizophrenia-like behavior in PARP-1-deficient mice

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-2174-0

PARP-1-deficient embryonic NSCs exhibited defects in proliferation. a NSCs were cultured from E13.5 PARP-1 littermate embryos and sphere formation assay was performed. Representative photographs are shown in the top panels (scale bar = 100 μm). The number (bottom left panel; n = 4 independent cultures) and diameter (bottom right; n = 32; pooled from four independent cultures) of the primary neurospheres were determined at days in vitro 3. b Embryonic NSCs were transfected with PARP-1 siRNA and then assayed for neurosphere formation at day 6 (left panel, n = 4 independent cultures; right panel, n = 20; pooled from four independent cultures). c NSCs prepared from WT or PARP-1 KOs were infected with retroviruses carrying GFP or GFP-PARP-1 (PARP-1) and then assayed for neurosphere formation at day 4 (WT GFP, n = 289; KO GFP, n = 19; KO PARP-1, n = 66; data pooled from four independent cultures). d Neurospheres were formed in the presence of a PARP-1 inhibitor DPQ (50 μM) for 5 days (DMSO, n = 40; DPQ, n = 39, data pooled from four independent cultures). e – f PARP-1 littermate embryonic NSCs ( e ) or NSCs infected with retroviruses as indicated ( f ) were labeled with BrdU and then quantified ( n = 6 for each group). g PARP-1 siRNA-transfected NSCs were labeled with BrdU and then counted ( n = 4 for each group). h PARP-1 NSCs were incubated with DPQ and then BrdU-labeled ( n = 44 for each group, pooled from four independent cultures). i – j PARP-1 NSCs were immunostained for p27 (I; n = 8 for each group) and p21 (J; WT, n = 4; KO, n = 5) and then the immunoreactive cells were counted. Representative images are shown in the upper panels (scale bar = 20 μm). k WT NSCs were incubated with increasing concentrations of DPQ (8, 20, 50 μM) and then immunoblotted to monitor the changes in the protein amounts of p21 and p27. Molecular weights on the left in kDa. Bar graphs show means ± SD and box plots show median, boxed 25 and 75% percentiles and whiskers 10 and 90% percentiles ( a b , d , e , g , i , j : two-tailed unpaired t -test; c , f : one-way ANOVA with Bonferroni post hoc comparisons; h : two-way ANOVA with Bonferroni post hoc comparisons; * p
Figure Legend Snippet: PARP-1-deficient embryonic NSCs exhibited defects in proliferation. a NSCs were cultured from E13.5 PARP-1 littermate embryos and sphere formation assay was performed. Representative photographs are shown in the top panels (scale bar = 100 μm). The number (bottom left panel; n = 4 independent cultures) and diameter (bottom right; n = 32; pooled from four independent cultures) of the primary neurospheres were determined at days in vitro 3. b Embryonic NSCs were transfected with PARP-1 siRNA and then assayed for neurosphere formation at day 6 (left panel, n = 4 independent cultures; right panel, n = 20; pooled from four independent cultures). c NSCs prepared from WT or PARP-1 KOs were infected with retroviruses carrying GFP or GFP-PARP-1 (PARP-1) and then assayed for neurosphere formation at day 4 (WT GFP, n = 289; KO GFP, n = 19; KO PARP-1, n = 66; data pooled from four independent cultures). d Neurospheres were formed in the presence of a PARP-1 inhibitor DPQ (50 μM) for 5 days (DMSO, n = 40; DPQ, n = 39, data pooled from four independent cultures). e – f PARP-1 littermate embryonic NSCs ( e ) or NSCs infected with retroviruses as indicated ( f ) were labeled with BrdU and then quantified ( n = 6 for each group). g PARP-1 siRNA-transfected NSCs were labeled with BrdU and then counted ( n = 4 for each group). h PARP-1 NSCs were incubated with DPQ and then BrdU-labeled ( n = 44 for each group, pooled from four independent cultures). i – j PARP-1 NSCs were immunostained for p27 (I; n = 8 for each group) and p21 (J; WT, n = 4; KO, n = 5) and then the immunoreactive cells were counted. Representative images are shown in the upper panels (scale bar = 20 μm). k WT NSCs were incubated with increasing concentrations of DPQ (8, 20, 50 μM) and then immunoblotted to monitor the changes in the protein amounts of p21 and p27. Molecular weights on the left in kDa. Bar graphs show means ± SD and box plots show median, boxed 25 and 75% percentiles and whiskers 10 and 90% percentiles ( a b , d , e , g , i , j : two-tailed unpaired t -test; c , f : one-way ANOVA with Bonferroni post hoc comparisons; h : two-way ANOVA with Bonferroni post hoc comparisons; * p

Techniques Used: Cell Culture, Tube Formation Assay, In Vitro, Transfection, Infection, Labeling, Incubation, Two Tailed Test

PARP-1 negatively regulated the expression of ESP. a mRNA level of ESP was examined in the PARP-1 WT and KO NSCs by RT-PCR. β-Actin served as an internal control. b – d ESP promoter-luciferase activity was determined in the PARP-1 NSCs (B; n = 5); in the NSCs following overexpression of PARP-1 ( c : n = 5); in the NSCs incubated with DPQ ( d: n = 4). e mRNA level of ESP after DPQ treatment was determined in the NSCs by RT-PCR. f–h PARP-1 NSCs were incubated to form neurospheres following transfection of siRNA for ESP. Knockdown of ESP was confirmed by RT-PCR ( f ). g Representative images of ESP-knocked down NSCs are shown (scale bar = 200 μm) and the quantification of the neurosphere diameter is shown in the right panel (WT, n = 26 each; KO, n = 27 each; data pooled from four independent cultures). h PARP-1 NSCs were transfected with ESP siRNA and then the rate of proliferation was monitored by BrdU labeling (WT CTL, n = 7; WT siESP, n = 6; KO CTL, n = 15; KO siESP, n = 7; data pooled from two independent cultures). i – j Activation of PI3K, Akt, and ERK was examined by immunoblotting following knockdown of ESP in the PARP-1 KO NSCs. The membranes were reprobed to examine the changes in the level of p21, p27 and phospho-FOXO1. A blot with anti-tubulin served as a loading control. Molecular weights on the left in kDa ( i ). ESP knockdown was confirmed by RT-PCR ( j ). Bar graphs show means ± SD and box plots show median, boxed 25 and 75% percentiles and whiskers 10 and 90% percentiles ( b : two-tailed unpaired t -test; c , g : one-way ANOVA with Bonferroni post hoc comparisons; d : Kruskal–Wallis test with Dunn’s post hoc comparisons; h : two-way ANOVA with Bonferroni post hoc comparisons; ** p
Figure Legend Snippet: PARP-1 negatively regulated the expression of ESP. a mRNA level of ESP was examined in the PARP-1 WT and KO NSCs by RT-PCR. β-Actin served as an internal control. b – d ESP promoter-luciferase activity was determined in the PARP-1 NSCs (B; n = 5); in the NSCs following overexpression of PARP-1 ( c : n = 5); in the NSCs incubated with DPQ ( d: n = 4). e mRNA level of ESP after DPQ treatment was determined in the NSCs by RT-PCR. f–h PARP-1 NSCs were incubated to form neurospheres following transfection of siRNA for ESP. Knockdown of ESP was confirmed by RT-PCR ( f ). g Representative images of ESP-knocked down NSCs are shown (scale bar = 200 μm) and the quantification of the neurosphere diameter is shown in the right panel (WT, n = 26 each; KO, n = 27 each; data pooled from four independent cultures). h PARP-1 NSCs were transfected with ESP siRNA and then the rate of proliferation was monitored by BrdU labeling (WT CTL, n = 7; WT siESP, n = 6; KO CTL, n = 15; KO siESP, n = 7; data pooled from two independent cultures). i – j Activation of PI3K, Akt, and ERK was examined by immunoblotting following knockdown of ESP in the PARP-1 KO NSCs. The membranes were reprobed to examine the changes in the level of p21, p27 and phospho-FOXO1. A blot with anti-tubulin served as a loading control. Molecular weights on the left in kDa ( i ). ESP knockdown was confirmed by RT-PCR ( j ). Bar graphs show means ± SD and box plots show median, boxed 25 and 75% percentiles and whiskers 10 and 90% percentiles ( b : two-tailed unpaired t -test; c , g : one-way ANOVA with Bonferroni post hoc comparisons; d : Kruskal–Wallis test with Dunn’s post hoc comparisons; h : two-way ANOVA with Bonferroni post hoc comparisons; ** p

Techniques Used: Expressing, End-sequence Profiling, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Over Expression, Incubation, Transfection, Labeling, CTL Assay, Activation Assay, Two Tailed Test

32) Product Images from "Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory"

Article Title: Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3100-11.2011

Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and CldU (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with BrdU at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
Figure Legend Snippet: Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and CldU (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with BrdU at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p

Techniques Used: Mouse Assay, Injection

Functional integration of stimulation-induced neurons into hippocampal memory networks. a , After unilateral stimulation, mice were injected with IdU during the period of increased proliferation and CldU once proliferation returned to baseline ( n = 17). Mice were trained 6 weeks after CldU treatment, and spatial memory was assessed 4 weeks after the completion of training. b , Mice searched selectively in the probe test. A density plot for grouped data (left), with accompanying color scale, represents the number of visits per mouse per 5 × 5 cm area. Mice spent more time (right graph) searching the target zone (T) compared with other (O) zones. c , Representative confocal images of tissue after water-maze testing showing IdU + , CldU + , and Fos + DGCs and a DAPI counterstained merge image with a representative IdU + /Fos + colabeled cell (scale bar, 20 μm). d , Numbers of IdU + and CldU + cells ipsilateral (I) versus contralateral (C) to stimulation site. e , Fos expression in the DG after probe testing was similar on both sides. f , After the probe test, the probability of Fos + cells being XdU + ipsilateral (I) and contralateral (C) to stimulation ( y -axis notation denotes the conditional probability of a cell being XdU + given it is Fos + , or P(XdU + |Fos + )). g , As the number of IdU + cells increases, so does their contribution to the population of activated neurons (i.e., P(IdU + |Fos + )). h , A separate group of mice underwent identical treatment except were trained 1 week after CldU treatment and had spatial memory assessed 9 weeks after the completion of training ( n = 15). i , Mice searched selectively in the probe test (left density plot), spending more time (right graph) searching the target zone compared with other zones. j , Numbers of IdU + and CldU + cells ipsilateral (I) versus contralateral (C) to stimulation site. k , Fos expression in the DG after probe testing was similar on both sides. l , The probability of Fos + cells being XdU + (or adult generated neurons) was equivalently low ipsilateral and contralateral to stimulation for IdU + and CldU + cells. m , The stimulation-induced increase in the availability of adult-generated neurons did not produce a proportional increase in their contribution to the population of activated neurons, suggesting that neurons 1 week old at the time of training are not functionally integrated. ** p
Figure Legend Snippet: Functional integration of stimulation-induced neurons into hippocampal memory networks. a , After unilateral stimulation, mice were injected with IdU during the period of increased proliferation and CldU once proliferation returned to baseline ( n = 17). Mice were trained 6 weeks after CldU treatment, and spatial memory was assessed 4 weeks after the completion of training. b , Mice searched selectively in the probe test. A density plot for grouped data (left), with accompanying color scale, represents the number of visits per mouse per 5 × 5 cm area. Mice spent more time (right graph) searching the target zone (T) compared with other (O) zones. c , Representative confocal images of tissue after water-maze testing showing IdU + , CldU + , and Fos + DGCs and a DAPI counterstained merge image with a representative IdU + /Fos + colabeled cell (scale bar, 20 μm). d , Numbers of IdU + and CldU + cells ipsilateral (I) versus contralateral (C) to stimulation site. e , Fos expression in the DG after probe testing was similar on both sides. f , After the probe test, the probability of Fos + cells being XdU + ipsilateral (I) and contralateral (C) to stimulation ( y -axis notation denotes the conditional probability of a cell being XdU + given it is Fos + , or P(XdU + |Fos + )). g , As the number of IdU + cells increases, so does their contribution to the population of activated neurons (i.e., P(IdU + |Fos + )). h , A separate group of mice underwent identical treatment except were trained 1 week after CldU treatment and had spatial memory assessed 9 weeks after the completion of training ( n = 15). i , Mice searched selectively in the probe test (left density plot), spending more time (right graph) searching the target zone compared with other zones. j , Numbers of IdU + and CldU + cells ipsilateral (I) versus contralateral (C) to stimulation site. k , Fos expression in the DG after probe testing was similar on both sides. l , The probability of Fos + cells being XdU + (or adult generated neurons) was equivalently low ipsilateral and contralateral to stimulation for IdU + and CldU + cells. m , The stimulation-induced increase in the availability of adult-generated neurons did not produce a proportional increase in their contribution to the population of activated neurons, suggesting that neurons 1 week old at the time of training are not functionally integrated. ** p

Techniques Used: Functional Assay, Mouse Assay, Injection, Expressing, Generated

33) Product Images from "Robo1 Modulates Proliferation and Neurogenesis in the Developing Neocortex"

Article Title: Robo1 Modulates Proliferation and Neurogenesis in the Developing Neocortex

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4256-13.2014

Robo1 regulates activity of genes involved in proliferation, apoptosis, and tumorogenesis during development
Figure Legend Snippet: Robo1 regulates activity of genes involved in proliferation, apoptosis, and tumorogenesis during development

Techniques Used: Activity Assay

Increase in proliferative activity in the absence of Robo1 is cell autonomous. ( A – B′ ) Immunohistochemistry in the dorsal cortex of C57BL/6J mice at E14.5, 48 h after in utero electroporation of either control shRNA ( A , A′ ) or
Figure Legend Snippet: Increase in proliferative activity in the absence of Robo1 is cell autonomous. ( A – B′ ) Immunohistochemistry in the dorsal cortex of C57BL/6J mice at E14.5, 48 h after in utero electroporation of either control shRNA ( A , A′ ) or

Techniques Used: Activity Assay, Immunohistochemistry, Mouse Assay, In Utero, Electroporation, shRNA

Increased PH-3-labeling in the cortices of Robo1 −/− / Robo2 −/− and Slit1 −/− mice at E14.5. Histograms show number of PH-3 + apical and basal progenitor cells in Robo1 −/− / Robo2 −/−
Figure Legend Snippet: Increased PH-3-labeling in the cortices of Robo1 −/− / Robo2 −/− and Slit1 −/− mice at E14.5. Histograms show number of PH-3 + apical and basal progenitor cells in Robo1 −/− / Robo2 −/−

Techniques Used: Labeling, Mouse Assay

Reduced proliferation in Robo1 +/+ , but not in Robo1 −/− dissociated cortical cell cultures following Slit1/Slit2 treatment. A , B , Histograms show percentage of BrdU + E15.5 rat apical (Pax6 + ; A ) and basal (Tbr2 + ; B ) progenitor cells following
Figure Legend Snippet: Reduced proliferation in Robo1 +/+ , but not in Robo1 −/− dissociated cortical cell cultures following Slit1/Slit2 treatment. A , B , Histograms show percentage of BrdU + E15.5 rat apical (Pax6 + ; A ) and basal (Tbr2 + ; B ) progenitor cells following

Techniques Used:

Increased PH-3-labeling in the telencephalon in Robo1 −/− , but not Robo2 −/− or Robo3 −/− mice during corticogenesis. ( A – F′ ) Images of coronal sections through the cortex ( A , A′ , D ,
Figure Legend Snippet: Increased PH-3-labeling in the telencephalon in Robo1 −/− , but not Robo2 −/− or Robo3 −/− mice during corticogenesis. ( A – F′ ) Images of coronal sections through the cortex ( A , A′ , D ,

Techniques Used: Labeling, Mouse Assay

Coexpression of Robo1 protein with the proliferation marker, PH-3. Immunohistochemical localization of Robo1 (green) and PH-3 (red) in coronal sections through the telencephalon ( A , B ), and through the VZ of the MGE ( C , D ) and cortex of C57BL/6J mice
Figure Legend Snippet: Coexpression of Robo1 protein with the proliferation marker, PH-3. Immunohistochemical localization of Robo1 (green) and PH-3 (red) in coronal sections through the telencephalon ( A , B ), and through the VZ of the MGE ( C , D ) and cortex of C57BL/6J mice

Techniques Used: Marker, Immunohistochemistry, Mouse Assay

Increased expression of proliferation markers in the dorsal cortices of Robo1 −/− mice at E14.5. ( A – B‴ ) Immunohistochemistry in the cortices of Robo1 +/+ ( A – A‴ ) and Robo1 −/− ( B – B‴
Figure Legend Snippet: Increased expression of proliferation markers in the dorsal cortices of Robo1 −/− mice at E14.5. ( A – B‴ ) Immunohistochemistry in the cortices of Robo1 +/+ ( A – A‴ ) and Robo1 −/− ( B – B‴

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

Reduced number of microglia and apoptotic cells in early and middle stages of corticogenesis in Robo1 −/− mice. A–F , Immunohistochemistry in the cortices of Robo1 +/+ ( A , C , E ) and Robo1 −/− ( B , D , F ) mice for Iba1
Figure Legend Snippet: Reduced number of microglia and apoptotic cells in early and middle stages of corticogenesis in Robo1 −/− mice. A–F , Immunohistochemistry in the cortices of Robo1 +/+ ( A , C , E ) and Robo1 −/− ( B , D , F ) mice for Iba1

Techniques Used: Mouse Assay, Immunohistochemistry

Increase in upper and lower layer pyramidal neurons in the cortices of Robo1 −/− mice at E18.5. ( A – B″ ) Immunohistochemistry in the cortex of Robo1 +/+ ( A – A″ ) and Robo1 −/− ( B – B″
Figure Legend Snippet: Increase in upper and lower layer pyramidal neurons in the cortices of Robo1 −/− mice at E18.5. ( A – B″ ) Immunohistochemistry in the cortex of Robo1 +/+ ( A – A″ ) and Robo1 −/− ( B – B″

Techniques Used: Mouse Assay, Immunohistochemistry

34) Product Images from "Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling"

Article Title: Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

Journal: Scientific Reports

doi: 10.1038/s41598-018-21073-x

Effects of HIF inhibitors on HPF cell proliferation. HPFs were treated with the HIF-1α inhibitor KC7F2 or the HIF-2α inhibitor TC-S 7009 and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. ( A , B ) Cell proliferation as determined by BrdU assay. Cells were incubated with BrdU for 12 hrs. ( C , D ) Cell viability as determined by LDH assay. Data were expressed as a percentage of control (0 µM inhibitor) for each oxygen condition. The absorbance of control at normoxia for proliferation was 0.39 ± 0.06 (n = 3) and for hypoxia was 0.66 ± 0.16 (n = 3). The viability (%) was calculated as 100% -cytotoxicity %. Cytotoxicity % = [(LDH activity of sample -Spontaneous LDH activity)/(Maximum LDH activity -Spontaneous LDH activity)] × 100. The absorbance of control at normoxia for LDH activity was 0.17 (n = 2) and for hypoxia was 0.16 (n = 2). Maximum and spontaneous LDH activity values for normoxia were 0.26 and 0.17 (n = 2), respectively. Maximum and spontaneous LDH activity values for hypoxia were 0.27 and 0.17 (n = 2), respectively. Values represent means ± SE for proliferation and means for viability.
Figure Legend Snippet: Effects of HIF inhibitors on HPF cell proliferation. HPFs were treated with the HIF-1α inhibitor KC7F2 or the HIF-2α inhibitor TC-S 7009 and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. ( A , B ) Cell proliferation as determined by BrdU assay. Cells were incubated with BrdU for 12 hrs. ( C , D ) Cell viability as determined by LDH assay. Data were expressed as a percentage of control (0 µM inhibitor) for each oxygen condition. The absorbance of control at normoxia for proliferation was 0.39 ± 0.06 (n = 3) and for hypoxia was 0.66 ± 0.16 (n = 3). The viability (%) was calculated as 100% -cytotoxicity %. Cytotoxicity % = [(LDH activity of sample -Spontaneous LDH activity)/(Maximum LDH activity -Spontaneous LDH activity)] × 100. The absorbance of control at normoxia for LDH activity was 0.17 (n = 2) and for hypoxia was 0.16 (n = 2). Maximum and spontaneous LDH activity values for normoxia were 0.26 and 0.17 (n = 2), respectively. Maximum and spontaneous LDH activity values for hypoxia were 0.27 and 0.17 (n = 2), respectively. Values represent means ± SE for proliferation and means for viability.

Techniques Used: BrdU Staining, Incubation, Lactate Dehydrogenase Assay, Activity Assay

Inhibition and silencing of HIF-2α reduces hypoxia-mediated NFATc2 nuclear translocation. ( A ) Immunofluorescence staining of NFATc2 in HPFs treated with HIF-1α and HIF-2α inhibitors (KC7F2, 10 µM and TC-S 7009, 50 µM, respectively) and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( B ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( C ) Lentiviral infection efficiency. BC = Blank control, VC = Vector control. ( D ) Western blot showing HIF-1α and HIF-2α silencing efficiency. ( E ) Quantitative representation of protein expression of HIF-1α and HIF-2α protein expression with HIF silencing. ( F ) Immunofluorescence staining of NFATc2 in HPFs treated with shRNA lentiviral constructs (MOI 100) of HIF-1α and HIF-2α and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( G ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( H ) HEK 293Ts were co-transfected with an NFAT reporter plasmid and HIF-1α or HIF-2α expression vector for 24 hrs. The reporter activities were measured as the ratio of Firefly/Renilla luciferase activities. Data represent means ± SE. *p
Figure Legend Snippet: Inhibition and silencing of HIF-2α reduces hypoxia-mediated NFATc2 nuclear translocation. ( A ) Immunofluorescence staining of NFATc2 in HPFs treated with HIF-1α and HIF-2α inhibitors (KC7F2, 10 µM and TC-S 7009, 50 µM, respectively) and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( B ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( C ) Lentiviral infection efficiency. BC = Blank control, VC = Vector control. ( D ) Western blot showing HIF-1α and HIF-2α silencing efficiency. ( E ) Quantitative representation of protein expression of HIF-1α and HIF-2α protein expression with HIF silencing. ( F ) Immunofluorescence staining of NFATc2 in HPFs treated with shRNA lentiviral constructs (MOI 100) of HIF-1α and HIF-2α and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( G ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( H ) HEK 293Ts were co-transfected with an NFAT reporter plasmid and HIF-1α or HIF-2α expression vector for 24 hrs. The reporter activities were measured as the ratio of Firefly/Renilla luciferase activities. Data represent means ± SE. *p

Techniques Used: Inhibition, Translocation Assay, Immunofluorescence, Staining, Infection, Plasmid Preparation, Western Blot, Expressing, shRNA, Construct, Transfection, Luciferase

35) Product Images from "Muscle regeneration is undisturbed by repeated polytraumatic injury"

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury

Journal: European Journal of Trauma and Emergency Surgery

doi: 10.1007/s00068-010-0034-9

Skeletal muscle morphology 1 week after trauma. Red color represents the nuclear Hoechst staining. Green color refers to BrdU staining. Co-localization of the signals ( yellow color ) represents the newly divided cells. Blue color refers to laminin-staining. Three different regions can be identified: intact muscle, a necrotic core, and a penumbra which is characterized by disturbed myofibers and a high percentage of proliferating cells
Figure Legend Snippet: Skeletal muscle morphology 1 week after trauma. Red color represents the nuclear Hoechst staining. Green color refers to BrdU staining. Co-localization of the signals ( yellow color ) represents the newly divided cells. Blue color refers to laminin-staining. Three different regions can be identified: intact muscle, a necrotic core, and a penumbra which is characterized by disturbed myofibers and a high percentage of proliferating cells

Techniques Used: Staining, BrdU Staining

Newly formed muscle fiber with BrDU positive nuclei. Five weeks after the trauma, clear laminin staining and completely regenerated muscular structure can be investigated. Arrows show BrDU positive nuclei incorporated into the myofiber
Figure Legend Snippet: Newly formed muscle fiber with BrDU positive nuclei. Five weeks after the trauma, clear laminin staining and completely regenerated muscular structure can be investigated. Arrows show BrDU positive nuclei incorporated into the myofiber

Techniques Used: Staining

36) Product Images from "Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis"

Article Title: Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis

Journal: Journal of Virology

doi: 10.1128/JVI.78.5.2336-2347.2004

Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with BrdU (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with anti-BrdU-R-PE and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.
Figure Legend Snippet: Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with BrdU (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with anti-BrdU-R-PE and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.

Techniques Used: Cell Culture, Infection, Immunofluorescence, Staining, Flow Cytometry, Cytometry

37) Product Images from "Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells"

Article Title: Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00848

CD1d trafficking through early endosomes, late endosomes, and lysosomes. (A–C) In double-positive (DP) thymocytes, super-resolution microscopy was used to determine intracellular localization of CD1d molecules in early endosomes, late endosomes, and lysosomes visualized in the red channel by early endosome antigen 1 (EEA1), Rab7, and lysosome-associated membrane protein 1 (LAMP1), respectively. Co-localization areas were presented in white (right panels). DAPI was applied to visualize the nucleus (bar = 5 µm). (D) DP thymocytes were analyzed for co-localization between green and red signals using the ImageJ’s co-localization plugin, and the ratio of co-localized and total green area was plotted and statistically analyzed using the unpaired t-test. Although a significant shift from late to toward early endosomes could be observed in Vav Cre GCS f/f DP thymocytes, the amount of CD1d in lysosomes was equal. Shown are means ± SEM, N = 20 cells per group. (E) A tendency toward less but larger LAMP1 + lysosomes could be seen in DP thymocytes of Vav Cre GCS f/f mice, however, the difference was not statistically significant. The bars show means ± SEM, N = 7 cells per group.
Figure Legend Snippet: CD1d trafficking through early endosomes, late endosomes, and lysosomes. (A–C) In double-positive (DP) thymocytes, super-resolution microscopy was used to determine intracellular localization of CD1d molecules in early endosomes, late endosomes, and lysosomes visualized in the red channel by early endosome antigen 1 (EEA1), Rab7, and lysosome-associated membrane protein 1 (LAMP1), respectively. Co-localization areas were presented in white (right panels). DAPI was applied to visualize the nucleus (bar = 5 µm). (D) DP thymocytes were analyzed for co-localization between green and red signals using the ImageJ’s co-localization plugin, and the ratio of co-localized and total green area was plotted and statistically analyzed using the unpaired t-test. Although a significant shift from late to toward early endosomes could be observed in Vav Cre GCS f/f DP thymocytes, the amount of CD1d in lysosomes was equal. Shown are means ± SEM, N = 20 cells per group. (E) A tendency toward less but larger LAMP1 + lysosomes could be seen in DP thymocytes of Vav Cre GCS f/f mice, however, the difference was not statistically significant. The bars show means ± SEM, N = 7 cells per group.

Techniques Used: Microscopy, Mouse Assay

Antigen presentation and recognition in Vav Cre GCS f/f mice. (A,B) Double-positive (DP) thymocytes were tested for their antigen presentation capacity toward invariant natural killer T (iNKT) cells in vitro . To this end, iNKT-depleted wild-type (WT), Vav Cre GCS f/f , and CD1d –/– DP thymocytes were exposed to increasing concentrations of αGalCer and co-incubated with responder WT iNKT cells enriched from livers of TCRVα14-Jα281 transgenic mice. The activation measured as secretion of IFNγ (A) and IL4 (B) did not differ between WT and Vav Cre GCS f/f DP thymocytes. CD1d –/– DP thymocytes served as negative controls, and the corresponding bars cannot be discriminated from the zero line in all but one concentration. Shown are means ± SEM, N = 6–9 per group. (C) Activation of iNKT cells was tested in vivo . WT and Vav Cre GCS f/f mice were i.p. injected with either 0.2 or 3 µg αGalCer. Eight hours later, splenic iNKT cells were analyzed for surface CD69 expression by flow cytometry by gating on CD19 − /PBS57-CD1d + /CD44 + lymphocytes. Expression of CD69 did not differ between WT and Vav Cre GCS f/f DP thymocytes. In parallel, serum was analyzed for IFNγ and IL4 levels. In Vav Cre GCS f/f mice injected with 3 µg αGalCer, IFNγ levels were significantly lower than in the WT controls. All other measurements did not show a statistically significant difference. Shown are means ± SEM, N = 3 per group. (D) Activation of iNKT cells was tested in vitro . iNKT cells from livers and spleens of WT and Vav Cre GCS f/f mice were exposed to αGalCer-loaded WT DP thymocytes. The activation measured as IFNγ secretion did not differ between WT and Vav Cre GCS f/f iNKT cells. Shown are means ± SEM, n = 3–6 per group. (E) Splenic conventional T cells were tested for their T cell receptor (TCR)-independent and TCR-dependent activation in vitro . WT and Vav Cre GCS f/f splenic T cells were activated by PMA/calcium ionophore A23187 or by plate-bound anti-CD3/anti-CD28 antibodies. Vehicle (media)-treated cells served as controls. No statistically significant differences could be found in the IFNγ secretion between WT and Vav Cre GCS f/f T cells. Shown are means ± SEM, n = 6 per group.
Figure Legend Snippet: Antigen presentation and recognition in Vav Cre GCS f/f mice. (A,B) Double-positive (DP) thymocytes were tested for their antigen presentation capacity toward invariant natural killer T (iNKT) cells in vitro . To this end, iNKT-depleted wild-type (WT), Vav Cre GCS f/f , and CD1d –/– DP thymocytes were exposed to increasing concentrations of αGalCer and co-incubated with responder WT iNKT cells enriched from livers of TCRVα14-Jα281 transgenic mice. The activation measured as secretion of IFNγ (A) and IL4 (B) did not differ between WT and Vav Cre GCS f/f DP thymocytes. CD1d –/– DP thymocytes served as negative controls, and the corresponding bars cannot be discriminated from the zero line in all but one concentration. Shown are means ± SEM, N = 6–9 per group. (C) Activation of iNKT cells was tested in vivo . WT and Vav Cre GCS f/f mice were i.p. injected with either 0.2 or 3 µg αGalCer. Eight hours later, splenic iNKT cells were analyzed for surface CD69 expression by flow cytometry by gating on CD19 − /PBS57-CD1d + /CD44 + lymphocytes. Expression of CD69 did not differ between WT and Vav Cre GCS f/f DP thymocytes. In parallel, serum was analyzed for IFNγ and IL4 levels. In Vav Cre GCS f/f mice injected with 3 µg αGalCer, IFNγ levels were significantly lower than in the WT controls. All other measurements did not show a statistically significant difference. Shown are means ± SEM, N = 3 per group. (D) Activation of iNKT cells was tested in vitro . iNKT cells from livers and spleens of WT and Vav Cre GCS f/f mice were exposed to αGalCer-loaded WT DP thymocytes. The activation measured as IFNγ secretion did not differ between WT and Vav Cre GCS f/f iNKT cells. Shown are means ± SEM, n = 3–6 per group. (E) Splenic conventional T cells were tested for their T cell receptor (TCR)-independent and TCR-dependent activation in vitro . WT and Vav Cre GCS f/f splenic T cells were activated by PMA/calcium ionophore A23187 or by plate-bound anti-CD3/anti-CD28 antibodies. Vehicle (media)-treated cells served as controls. No statistically significant differences could be found in the IFNγ secretion between WT and Vav Cre GCS f/f T cells. Shown are means ± SEM, n = 6 per group.

Techniques Used: Mouse Assay, In Vitro, Incubation, Transgenic Assay, Activation Assay, Concentration Assay, In Vivo, Injection, Expressing, Flow Cytometry, Cytometry

Vav Cre GCS f/f mice showed a typical thymocyte development and an unaltered expression of CD1d, SLAM and Ly108 molecules. (A) No statistically significant differences were observed between 8 week old WT and Vav Cre GCS f/f mice in terms of body weight and the weight and cellularity of spleen and thymus. Bars show means ± SEM, N = 6 per group. (B) Thymocyte development was investigated in 8 week old mice by flow cytometry using antibodies against CD4 and CD8. The double-negative (DN) stage was further subdivided into DN1 (CD25 – /CD44 + ), DN2 (CD25 + /CD44 + ), DN3 (CD25 + /CD44 – ), and DN4 (CD25 – /CD44 – ). Representative dot plots (upper panels) as well as relative and absolute numbers (lower panels) are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (C) Frequencies of B (CD3 – /CD19 + ) and T (CD3 + /CD19 – ) cells were measured by flow cytometry in spleens of 8 week old mice. Representative dot-plots and quantifications are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (D,E) Expression of CD1d was measured on DP (CD4 + /CD8 + ) thymocytes (D) and splenic DC (CD11c + /MHCII + ) (E) and expressed as mean fluorescence intensity (MFI). No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 6 per group in panel (D) and 4 per group in panel (E) . (F,G) Expression of SLAM (CD150) and Ly108, respectively, was measured on DP (CD4 + /CD8 + ) thymocytes and expressed as MFI. No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 4 per group.
Figure Legend Snippet: Vav Cre GCS f/f mice showed a typical thymocyte development and an unaltered expression of CD1d, SLAM and Ly108 molecules. (A) No statistically significant differences were observed between 8 week old WT and Vav Cre GCS f/f mice in terms of body weight and the weight and cellularity of spleen and thymus. Bars show means ± SEM, N = 6 per group. (B) Thymocyte development was investigated in 8 week old mice by flow cytometry using antibodies against CD4 and CD8. The double-negative (DN) stage was further subdivided into DN1 (CD25 – /CD44 + ), DN2 (CD25 + /CD44 + ), DN3 (CD25 + /CD44 – ), and DN4 (CD25 – /CD44 – ). Representative dot plots (upper panels) as well as relative and absolute numbers (lower panels) are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (C) Frequencies of B (CD3 – /CD19 + ) and T (CD3 + /CD19 – ) cells were measured by flow cytometry in spleens of 8 week old mice. Representative dot-plots and quantifications are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (D,E) Expression of CD1d was measured on DP (CD4 + /CD8 + ) thymocytes (D) and splenic DC (CD11c + /MHCII + ) (E) and expressed as mean fluorescence intensity (MFI). No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 6 per group in panel (D) and 4 per group in panel (E) . (F,G) Expression of SLAM (CD150) and Ly108, respectively, was measured on DP (CD4 + /CD8 + ) thymocytes and expressed as MFI. No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 4 per group.

Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence

Vav Cre GCS f/f showed a significant reduction of the invariant natural killer T (iNKT) cell population. (A) In thymi, spleens, and livers of 8-week-old wild-type (WT), Vav Cre GCS f/f and CD1d –/– mice, frequencies and absolute numbers of iNKT cells were measured by flow cytometry using PBS57-loaded CD1d tetramers and anti-CD44 antibodies. In spleens and livers, CD19 + cells were gated out. In Vav Cre GCS f/f mice, iNKT cells frequencies and numbers were significantly reduced in all three organs. CD1d-deficient mice served as negative controls. N = 10–13/group. (B) Thymic development of iNKT cells was investigated in 8-week-old mice. Antibodies against NK1.1 and CD44 were used to subdivide the developmental stages in immature (CD44 − /NK1.1 − ), semi-mature (CD44 + /NK1.1 − ), and mature (CD44 + /NK1.1 + ). Analyses were gated on iNKT cells defined as CD3 + /PBS57-CD1d + thymocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells with the corresponding phenotype. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three stages). The significant reduction in absolute numbers reflected the overall diminished iNKT cell population in Vav Cre GCS f/f mice. N = 16/group in the left panel and 10/group in the right panel, respectively. (C) Usage of TCRVβ-chains by splenic iNKT cells was investigated in 8-week-old mice. Analyses were gated on CD19 − /PBS57-CD1d + /CD44 + splenocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells expressing the corresponding TCRVβ-chain. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three TCRVβ-chains). The reduction in the absolute numbers reflected the diminished iNKT cell population in Vav Cre GCS f/f mice. N = 9/group in the left panel and 6/group in the right panel, respectively. (D,E) Proliferation and apoptosis of thymic iNKT cells were measured in 8-week-old mice using BrdU incorporation and Annexin V staining, respectively. In Vav Cre GCS f/f mice, iNKT cells (CD3 + /PBS57-CD1d + thymocytes) showed a significantly reduced proliferation and an increased apoptosis as compared to WT controls. By contrast, conventional thymocytes were unaffected. N = 5/group. Bars represent means ± SEM; * p
Figure Legend Snippet: Vav Cre GCS f/f showed a significant reduction of the invariant natural killer T (iNKT) cell population. (A) In thymi, spleens, and livers of 8-week-old wild-type (WT), Vav Cre GCS f/f and CD1d –/– mice, frequencies and absolute numbers of iNKT cells were measured by flow cytometry using PBS57-loaded CD1d tetramers and anti-CD44 antibodies. In spleens and livers, CD19 + cells were gated out. In Vav Cre GCS f/f mice, iNKT cells frequencies and numbers were significantly reduced in all three organs. CD1d-deficient mice served as negative controls. N = 10–13/group. (B) Thymic development of iNKT cells was investigated in 8-week-old mice. Antibodies against NK1.1 and CD44 were used to subdivide the developmental stages in immature (CD44 − /NK1.1 − ), semi-mature (CD44 + /NK1.1 − ), and mature (CD44 + /NK1.1 + ). Analyses were gated on iNKT cells defined as CD3 + /PBS57-CD1d + thymocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells with the corresponding phenotype. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three stages). The significant reduction in absolute numbers reflected the overall diminished iNKT cell population in Vav Cre GCS f/f mice. N = 16/group in the left panel and 10/group in the right panel, respectively. (C) Usage of TCRVβ-chains by splenic iNKT cells was investigated in 8-week-old mice. Analyses were gated on CD19 − /PBS57-CD1d + /CD44 + splenocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells expressing the corresponding TCRVβ-chain. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three TCRVβ-chains). The reduction in the absolute numbers reflected the diminished iNKT cell population in Vav Cre GCS f/f mice. N = 9/group in the left panel and 6/group in the right panel, respectively. (D,E) Proliferation and apoptosis of thymic iNKT cells were measured in 8-week-old mice using BrdU incorporation and Annexin V staining, respectively. In Vav Cre GCS f/f mice, iNKT cells (CD3 + /PBS57-CD1d + thymocytes) showed a significantly reduced proliferation and an increased apoptosis as compared to WT controls. By contrast, conventional thymocytes were unaffected. N = 5/group. Bars represent means ± SEM; * p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, BrdU Incorporation Assay, Staining

38) Product Images from "A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase"

Article Title: A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201007118

Relationship between the Nup133–CENP-F–NudE/EL and RanBP2–BICD2 pathways in centrosome tethering to the NE in U2OS cells. (A) Distances between centrosomes and nuclear periphery, measured in phospho-H3–positive U2OS cells treated for 3 d with scramble, CENP-F, BICD2, a combination of CENP-F and BICD2, or NudE/EL siRNA duplexes. Distances are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated ( n ). ***, P
Figure Legend Snippet: Relationship between the Nup133–CENP-F–NudE/EL and RanBP2–BICD2 pathways in centrosome tethering to the NE in U2OS cells. (A) Distances between centrosomes and nuclear periphery, measured in phospho-H3–positive U2OS cells treated for 3 d with scramble, CENP-F, BICD2, a combination of CENP-F and BICD2, or NudE/EL siRNA duplexes. Distances are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated ( n ). ***, P

Techniques Used:

The N-terminal domain of hNup133 tethers CENP-F at the NE in prophase. (Aa) Schematic representation of hNup133 constructs used in this study outlining its previously described β-propeller (N-terminal domain, NTD, blue) and α-solenoid (C-terminal domain, CTD, red) domains (adapted from Berke et al., 2004 ). (Ab) Yeast two-hybrid interactions between hNup133 (aa 12–1156), hNup133 CTD (aa 466–1156) or hNup133 NTD (aa 1–500), and hNup107 (aa 784–924), or CENP-F (aa 2644–3065) were analyzed as described in Materials and methods. Empty bait and prey vectors were used as negative controls (−). (B and C) Control HeLa cells (wild-type) or cell lines stably expressing GFP-hNup133 CTD or GFP 3x -mNup133 were transfected with a scramble siRNA (siScr) or with an siRNA duplex targeting the N-terminal domain of human Nup133. Cells were fixed after 3 d and processed for immunofluorescence using anti-RanGAP1, anti-CENP-F, and anti–phospho-H3 antibodies. Typical G2/M phospho-H3–positive cells are presented. Bars, 10 µm. In C, the anti-RanGAP1 signal is shown for cells on the bottom row. Line scans (with distances in pixels) measuring the intensity of CENP-F (red lines) and RanGAP1 (blue lines) reveal the peak of CENP-F that colocalizes with RanGAP1 at the NE in GFP-hNup133 CTD treated with the scramble siRNA duplexes (left panels) or in GFP 3x -mNup133 cells depleted for endogenous Nup133 (right panels), but not in Nup133-depleted GFP-hNup133 CTD cells (middle panels). In some prophase cells, the bright intranuclear foci reflect the early recruitment of CENP-F at kinetochores.
Figure Legend Snippet: The N-terminal domain of hNup133 tethers CENP-F at the NE in prophase. (Aa) Schematic representation of hNup133 constructs used in this study outlining its previously described β-propeller (N-terminal domain, NTD, blue) and α-solenoid (C-terminal domain, CTD, red) domains (adapted from Berke et al., 2004 ). (Ab) Yeast two-hybrid interactions between hNup133 (aa 12–1156), hNup133 CTD (aa 466–1156) or hNup133 NTD (aa 1–500), and hNup107 (aa 784–924), or CENP-F (aa 2644–3065) were analyzed as described in Materials and methods. Empty bait and prey vectors were used as negative controls (−). (B and C) Control HeLa cells (wild-type) or cell lines stably expressing GFP-hNup133 CTD or GFP 3x -mNup133 were transfected with a scramble siRNA (siScr) or with an siRNA duplex targeting the N-terminal domain of human Nup133. Cells were fixed after 3 d and processed for immunofluorescence using anti-RanGAP1, anti-CENP-F, and anti–phospho-H3 antibodies. Typical G2/M phospho-H3–positive cells are presented. Bars, 10 µm. In C, the anti-RanGAP1 signal is shown for cells on the bottom row. Line scans (with distances in pixels) measuring the intensity of CENP-F (red lines) and RanGAP1 (blue lines) reveal the peak of CENP-F that colocalizes with RanGAP1 at the NE in GFP-hNup133 CTD treated with the scramble siRNA duplexes (left panels) or in GFP 3x -mNup133 cells depleted for endogenous Nup133 (right panels), but not in Nup133-depleted GFP-hNup133 CTD cells (middle panels). In some prophase cells, the bright intranuclear foci reflect the early recruitment of CENP-F at kinetochores.

Techniques Used: Construct, Stable Transfection, Expressing, Transfection, Immunofluorescence

Centrosome movement away from the nuclear periphery requires microtubules and Eg5 activity. (A) HeLa cells transfected with scramble, CENP-F, or NudE/EL siRNA duplexes were either fixed (top row) or incubated with 20 µM nocodazole for 30 min or with 100 µM monastrol for 1 h before fixation. They were then stained with anti-pericentrin and anti–Phospho-H3 antibodies. Note that under those conditions, all phospho-H3–positive cells had entered prophase in the absence of microtubules or before Eg5 activation. All images arise from a single experimental dataset, although they were captured at different times using slightly different acquisition settings. Either a unique plane or maximum intensity projections of stacks are presented, as needed, depending on the locations of the centrosomes relative to the focal plane. Bar, 10 µm. (B) Distances between centrosomes and the NE, measured in phospho-H3–positive cells processed as above, are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated ( n ). ***, P
Figure Legend Snippet: Centrosome movement away from the nuclear periphery requires microtubules and Eg5 activity. (A) HeLa cells transfected with scramble, CENP-F, or NudE/EL siRNA duplexes were either fixed (top row) or incubated with 20 µM nocodazole for 30 min or with 100 µM monastrol for 1 h before fixation. They were then stained with anti-pericentrin and anti–Phospho-H3 antibodies. Note that under those conditions, all phospho-H3–positive cells had entered prophase in the absence of microtubules or before Eg5 activation. All images arise from a single experimental dataset, although they were captured at different times using slightly different acquisition settings. Either a unique plane or maximum intensity projections of stacks are presented, as needed, depending on the locations of the centrosomes relative to the focal plane. Bar, 10 µm. (B) Distances between centrosomes and the NE, measured in phospho-H3–positive cells processed as above, are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated ( n ). ***, P

Techniques Used: Activity Assay, Transfection, Incubation, Staining, Activation Assay

Interfering with Nup133-anchored dynein/dynactin impairs the tethering of centrosomes to the NE. (A) Time-lapse imaging of HeLa cells expressing EB3-GFP (green) and H2B-mCherry (red) and transfected with CENP-F (c and c′), NudE/EL siRNA duplexes (d and d′), or with a CFP-p50/dynamitin construct (e), or of cells stably expressing GFP-hNup133 CTD treated with hNup133 siRNAs and subsequently transfected with plasmids encoding EB3-GFP and H2B-mCherry (b). Time (in min:sec) was set at 0:00 when centrosome splitting just became detectable. Bars, 10 µm. See also Videos 1–6 . (B) Tracks representing centrosome (CTR) movements in a control and a CENP-F–depleted cell (see Aa, Ac′, and Videos 1 and 4 ). Trajectories before centrosome separation (red) and tracks of the separated centrosomes (blue and green) were superimposed on a schematic representation of the cell border and nuclear position (gray shading) at the beginning of the video. White and black dots indicate the positions of centrosomes at the beginning and at the end of the videos, respectively. (C) Analysis of the centrosome–NE distance over time in HeLa cells expressing EB3-GFP and H2B-mCherry and treated with scramble or CENP-F siRNA duplexes. For each cell entering mitosis, the maximum distance between the centrosomes and the NE reached during the G2/M transition was plotted over the time centrosomes spent > 3 µm away from the NE. Each dot represents a single cell. The number of cell quantified is indicated.
Figure Legend Snippet: Interfering with Nup133-anchored dynein/dynactin impairs the tethering of centrosomes to the NE. (A) Time-lapse imaging of HeLa cells expressing EB3-GFP (green) and H2B-mCherry (red) and transfected with CENP-F (c and c′), NudE/EL siRNA duplexes (d and d′), or with a CFP-p50/dynamitin construct (e), or of cells stably expressing GFP-hNup133 CTD treated with hNup133 siRNAs and subsequently transfected with plasmids encoding EB3-GFP and H2B-mCherry (b). Time (in min:sec) was set at 0:00 when centrosome splitting just became detectable. Bars, 10 µm. See also Videos 1–6 . (B) Tracks representing centrosome (CTR) movements in a control and a CENP-F–depleted cell (see Aa, Ac′, and Videos 1 and 4 ). Trajectories before centrosome separation (red) and tracks of the separated centrosomes (blue and green) were superimposed on a schematic representation of the cell border and nuclear position (gray shading) at the beginning of the video. White and black dots indicate the positions of centrosomes at the beginning and at the end of the videos, respectively. (C) Analysis of the centrosome–NE distance over time in HeLa cells expressing EB3-GFP and H2B-mCherry and treated with scramble or CENP-F siRNA duplexes. For each cell entering mitosis, the maximum distance between the centrosomes and the NE reached during the G2/M transition was plotted over the time centrosomes spent > 3 µm away from the NE. Each dot represents a single cell. The number of cell quantified is indicated.

Techniques Used: Imaging, Expressing, Transfection, Construct, Stable Transfection, Size-exclusion Chromatography

CENP-F depletion impairs the NE localization of NudE/EL at the G2/M transition in HeLa cells. HeLa cells transfected with scramble or CENP-F siRNA duplexes were preextracted, fixed, and stained with anti-NudE/EL, anti-RanGAP1, and anti–phospho-H3 antibodies. Bars, 10 µm. See also Fig. S2 .
Figure Legend Snippet: CENP-F depletion impairs the NE localization of NudE/EL at the G2/M transition in HeLa cells. HeLa cells transfected with scramble or CENP-F siRNA duplexes were preextracted, fixed, and stained with anti-NudE/EL, anti-RanGAP1, and anti–phospho-H3 antibodies. Bars, 10 µm. See also Fig. S2 .

Techniques Used: Transfection, Staining

hNup133 contributes to dynactin anchoring at the NE at the G2/M transition via CENP-F and NudE/EL. (A) GFP-hNup133 CTD or GFP 3x -mNup133 cells (a) or wild-type HeLa cells (b) transfected with the indicated siRNA duplexes were processed for immunofluorescence using anti-p150 Glued and anti–phospho-H3 antibodies. Bars, 10 µm. See also Fig. S3 . (B) Schematic representation of the interaction networks connecting Nup133 to dynein/dynactin. Proteins are represented on approximate scale except for CENP-F. Boxes indicate the minimal domains involved in the interactions between Nup133 and CENP-F (black boxes; this paper and Zuccolo et al., 2007 ), CENP-F and NudE/EL (gray boxes), and between NudE/EL and dynein (dashed area overlapping with the CENP-F interaction domain; Liang et al., 2007 ; Stehman et al., 2007 ; Vergnolle and Taylor, 2007 ). Although not represented on this scheme, association of CENP-F with the pool of Nup133 localized on the nuclear side of NPCs cannot be excluded.
Figure Legend Snippet: hNup133 contributes to dynactin anchoring at the NE at the G2/M transition via CENP-F and NudE/EL. (A) GFP-hNup133 CTD or GFP 3x -mNup133 cells (a) or wild-type HeLa cells (b) transfected with the indicated siRNA duplexes were processed for immunofluorescence using anti-p150 Glued and anti–phospho-H3 antibodies. Bars, 10 µm. See also Fig. S3 . (B) Schematic representation of the interaction networks connecting Nup133 to dynein/dynactin. Proteins are represented on approximate scale except for CENP-F. Boxes indicate the minimal domains involved in the interactions between Nup133 and CENP-F (black boxes; this paper and Zuccolo et al., 2007 ), CENP-F and NudE/EL (gray boxes), and between NudE/EL and dynein (dashed area overlapping with the CENP-F interaction domain; Liang et al., 2007 ; Stehman et al., 2007 ; Vergnolle and Taylor, 2007 ). Although not represented on this scheme, association of CENP-F with the pool of Nup133 localized on the nuclear side of NPCs cannot be excluded.

Techniques Used: Transfection, Immunofluorescence

The Nup133-anchored network tethers centrosomes to the NPCs specifically in prophase. (A) GFP 3x -mNup133 or GFP-hNup133 CTD cells treated for 3 d with scramble or hNup133 siRNAs (a), HeLa cells treated for 2 d with scramble, CENP-F, or NudE/EL siRNA duplexes (b), or HeLa cells transfected with a GFP-p50/dynamitin or a DsRec-p150cc1 construct (c) were processed for immunofluorescence using anti-pericentrin and anti–phospho-H3 antibodies. In b, cells were incubated before fixation with 40 µM BrdU for 3 h and anti-BrdU antibodies were further used. Bars, 10 µm. (B) Distances between centrosomes and the NE were measured on cells processed as in A. Prophase cells were identified by phospho-H3 staining and S/early G2 cells as BrdU-positive cells after a 3-h pulse with BrdU (G1 cells were not analyzed because centrioles were reported to be very mobile at that stage of the cell cycle; Piel et al., 2000 ). Distances are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated ( n ). ***, P
Figure Legend Snippet: The Nup133-anchored network tethers centrosomes to the NPCs specifically in prophase. (A) GFP 3x -mNup133 or GFP-hNup133 CTD cells treated for 3 d with scramble or hNup133 siRNAs (a), HeLa cells treated for 2 d with scramble, CENP-F, or NudE/EL siRNA duplexes (b), or HeLa cells transfected with a GFP-p50/dynamitin or a DsRec-p150cc1 construct (c) were processed for immunofluorescence using anti-pericentrin and anti–phospho-H3 antibodies. In b, cells were incubated before fixation with 40 µM BrdU for 3 h and anti-BrdU antibodies were further used. Bars, 10 µm. (B) Distances between centrosomes and the NE were measured on cells processed as in A. Prophase cells were identified by phospho-H3 staining and S/early G2 cells as BrdU-positive cells after a 3-h pulse with BrdU (G1 cells were not analyzed because centrioles were reported to be very mobile at that stage of the cell cycle; Piel et al., 2000 ). Distances are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated ( n ). ***, P

Techniques Used: Transfection, Construct, Immunofluorescence, Incubation, Staining

39) Product Images from "YAP/TAZ direct commitment and maturation of lymph node fibroblastic reticular cells"

Article Title: YAP/TAZ direct commitment and maturation of lymph node fibroblastic reticular cells

Journal: Nature Communications

doi: 10.1038/s41467-020-14293-1

YAP/TAZ support growth and structural organization of LNs by FRCs. a Diagram for generation of indicated mice and their analyses at 8 weeks after birth. b , c Representative images of YAP or TAZ in PDGFRβ + or CCL19 + FRCs in WT and Yap / Taz ∆FRC mice. FRCs around high endothelial venule (HEV) within the white dashed-line box are magnified in the lower panels with single-channel YAP or TAZ image. Scale bars, 250 µm. d Comparisons of body weight, inguinal LN weight and total number of cells within the inguinal LN in WT ( n = 11; body weight) and Yap / Taz ∆FRC mice ( n = 9; body weight). e Representative flow cytometric analysis and comparison of proportion of PDPN + CD31 − FRCs (red box) gated from CD45 − stromal cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. f Representative images and comparison of Ki-67 + FRCs (white arrows) in WT and Yap / Taz ∆FRC mice. Scale bars, 50 µm. g Comparison of indicated stromal cell counts gated from CD45 − cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. BECs ( n = 5), blood endothelial cells; LECs ( n = 6), lymphatic endothelial cells. h Representative images of distinction between B and T cells (white dashed line) beneath the LN capsule (white line) in WT and Yap / Taz ∆FRC mice. Scale bars, 200 µm. i Comparison of indicated mRNA expression in FRCs sorted from WT and Yap / Taz ∆FRC mice (quintuplicate values using n = 10–15 mice/group). j , k Representative images and comparison of DsRed + B cells and GFP + T cells within the inguinal LN at 24 h after the adoptive transfer in WT and Yap / Taz ∆FRC mice. Scale bars, 500 µm. l Changes in body weight after 1 × 10 3 pfu of A/PR/8 influenza viral infection ( n = 13). m Flow cytometric analyses and comparisons of IFN-γ+CD8 + T cells in gated CD3ε + T cells. n = 5 (CO) or 7 (IM) mice. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 4 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two‐tailed Mann–Whitney U test. NS, not significant.
Figure Legend Snippet: YAP/TAZ support growth and structural organization of LNs by FRCs. a Diagram for generation of indicated mice and their analyses at 8 weeks after birth. b , c Representative images of YAP or TAZ in PDGFRβ + or CCL19 + FRCs in WT and Yap / Taz ∆FRC mice. FRCs around high endothelial venule (HEV) within the white dashed-line box are magnified in the lower panels with single-channel YAP or TAZ image. Scale bars, 250 µm. d Comparisons of body weight, inguinal LN weight and total number of cells within the inguinal LN in WT ( n = 11; body weight) and Yap / Taz ∆FRC mice ( n = 9; body weight). e Representative flow cytometric analysis and comparison of proportion of PDPN + CD31 − FRCs (red box) gated from CD45 − stromal cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. f Representative images and comparison of Ki-67 + FRCs (white arrows) in WT and Yap / Taz ∆FRC mice. Scale bars, 50 µm. g Comparison of indicated stromal cell counts gated from CD45 − cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. BECs ( n = 5), blood endothelial cells; LECs ( n = 6), lymphatic endothelial cells. h Representative images of distinction between B and T cells (white dashed line) beneath the LN capsule (white line) in WT and Yap / Taz ∆FRC mice. Scale bars, 200 µm. i Comparison of indicated mRNA expression in FRCs sorted from WT and Yap / Taz ∆FRC mice (quintuplicate values using n = 10–15 mice/group). j , k Representative images and comparison of DsRed + B cells and GFP + T cells within the inguinal LN at 24 h after the adoptive transfer in WT and Yap / Taz ∆FRC mice. Scale bars, 500 µm. l Changes in body weight after 1 × 10 3 pfu of A/PR/8 influenza viral infection ( n = 13). m Flow cytometric analyses and comparisons of IFN-γ+CD8 + T cells in gated CD3ε + T cells. n = 5 (CO) or 7 (IM) mice. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 4 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two‐tailed Mann–Whitney U test. NS, not significant.

Techniques Used: Mouse Assay, Flow Cytometry, Expressing, Adoptive Transfer Assay, Infection, Two Tailed Test, MANN-WHITNEY

YAP/TAZ hyperactivation impairs differentiation and maturation of FRCs. a Diagram for analyses of indicated mice at P14. b Representative images of PDGFRβ + FRCs and CD31 + vessels in WT and Lats1 / 2 ∆FRC mice ( n = 5). Scale bars, 500 µm. c Comparisons of LN weight ( n = 4–7) and total number of cells ( n = 6–10) in WT and Lats1 / 2 ∆FRC mice. d Diagram for analyses of indicated mice at E18.5 or P14. e Representative images of LN anlagen (dashed line) at E18.5 showing CD4 + LTi cells in WT and Lats1 / 2 ΔFRC mice ( n = 6). Scale bars, 200 μm. f , Representative images of indicated markers (dashed box) within the inguinal LN (dotted-line) in WT and Lats1 / 2 ΔFRC mice at P14 ( n = 6). Scale bars, 500 µm. g , h Diagram and representative images for analyses of WT ΔFRC-TR mice ( n = 6) that were injected with anti-CD3ε for 5 days to induce T cell depletion. Scale bars, 100 μm. i Representative flow cytometric plots and comparison of proportion of PDPN + CD31 − FRCs (red box) and PDPN − CD31 − double-negative (DN) cells of skin-draining LNs in WT and Lats1 / 2 ∆FRC ( n = 5–6) mice. j Representative images and comparison of YAP expression and nuclear localization (green-arrowheads) in LN of WT and Lats1 / 2 ∆FRC mice ( n = 5). Scale bars, 20 µm. k Comparison of indicated mRNA expression in FRCs sorted from WT ΔFRC-TR and Lats1 / 2 iΔFRC-TR mice ( n = 4). l Representative images and comparisons of indicated marker expressions in LNs of WT and Lats1 / 2 ∆FRC mice ( n = 4–5). Scale bars, 20 µm. m Comparison of indicated mRNA expression in FRCs sorted from WT ΔFRC-TR and Lats1 / 2 iΔFRC-TR mice. n Diagram for analyses of indicated mice at P14. o Representative images of YAP expression in LNs of WT and L1 / 2-Y / T ∆FRC mice. Scale bars, 100 µm. p , q Representative images of indicated markers in LNs of WT and L1 / 2-Y / T ∆FRC mice. Scale bars, 500 µm. Unless otherwise denoted, each dot indicates a value obtained from inguinal LN and n = 4 mice. Horizontal bars indicate mean ± SD and P values versus WT or WT ΔFRC-TR by two‐tailed Mann‐Whitney U test except for ( k ) and ( m ) (two-tailed Student’s t -test). NS, not significant.
Figure Legend Snippet: YAP/TAZ hyperactivation impairs differentiation and maturation of FRCs. a Diagram for analyses of indicated mice at P14. b Representative images of PDGFRβ + FRCs and CD31 + vessels in WT and Lats1 / 2 ∆FRC mice ( n = 5). Scale bars, 500 µm. c Comparisons of LN weight ( n = 4–7) and total number of cells ( n = 6–10) in WT and Lats1 / 2 ∆FRC mice. d Diagram for analyses of indicated mice at E18.5 or P14. e Representative images of LN anlagen (dashed line) at E18.5 showing CD4 + LTi cells in WT and Lats1 / 2 ΔFRC mice ( n = 6). Scale bars, 200 μm. f , Representative images of indicated markers (dashed box) within the inguinal LN (dotted-line) in WT and Lats1 / 2 ΔFRC mice at P14 ( n = 6). Scale bars, 500 µm. g , h Diagram and representative images for analyses of WT ΔFRC-TR mice ( n = 6) that were injected with anti-CD3ε for 5 days to induce T cell depletion. Scale bars, 100 μm. i Representative flow cytometric plots and comparison of proportion of PDPN + CD31 − FRCs (red box) and PDPN − CD31 − double-negative (DN) cells of skin-draining LNs in WT and Lats1 / 2 ∆FRC ( n = 5–6) mice. j Representative images and comparison of YAP expression and nuclear localization (green-arrowheads) in LN of WT and Lats1 / 2 ∆FRC mice ( n = 5). Scale bars, 20 µm. k Comparison of indicated mRNA expression in FRCs sorted from WT ΔFRC-TR and Lats1 / 2 iΔFRC-TR mice ( n = 4). l Representative images and comparisons of indicated marker expressions in LNs of WT and Lats1 / 2 ∆FRC mice ( n = 4–5). Scale bars, 20 µm. m Comparison of indicated mRNA expression in FRCs sorted from WT ΔFRC-TR and Lats1 / 2 iΔFRC-TR mice. n Diagram for analyses of indicated mice at P14. o Representative images of YAP expression in LNs of WT and L1 / 2-Y / T ∆FRC mice. Scale bars, 100 µm. p , q Representative images of indicated markers in LNs of WT and L1 / 2-Y / T ∆FRC mice. Scale bars, 500 µm. Unless otherwise denoted, each dot indicates a value obtained from inguinal LN and n = 4 mice. Horizontal bars indicate mean ± SD and P values versus WT or WT ΔFRC-TR by two‐tailed Mann‐Whitney U test except for ( k ) and ( m ) (two-tailed Student’s t -test). NS, not significant.

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Expressing, Marker, Two Tailed Test, MANN-WHITNEY

Canonical Hippo pathway LATS1/2-YAP/TAZ governs FRCs. a Diagram for generation of indicated mice and their analyses at 8-weeks old after the tamoxifen injection from 4-weeks old. b Comparisons of the inguinal LN weight and cellularity within the inguinal LN in i-WT ΔFRC-TR and i- Yap / Taz ΔFRC-TR mice. c Representative images of intact border between B and T cell zones (white dashed line) beneath the LN capsule (white line) in i-WT ΔFRC-TR and i- Yap / Taz ΔFRC-TR mice ( n = 4). Scale bars, 200 μm. d Representative images of preserved LYVE-1 + lymphatic vessels and CD31 + blood vessels within the inguinal LN in i-WT ΔFRC-TR and i- Yap / Taz ΔFRC-TR mice ( n = 4). The regions within the white dashed-line box around subcapsular sinuses (SCS), medullary sinus (MS) and HEVs are magnified as indicated. Scale bars, 500 μm. e Diagram for generation of indicated mice for their analyses at 8-weeks old after the tamoxifen delivery from 6-weeks old. f Comparisons of the inguinal LN weight and total number of cells within the inguinal LN in WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. g Representative images of inguinal LN in WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. Scale bars, 500 μm. h , i Representative images and comparison of YAP nuclear localization (white arrowheads) in inguinal LN of WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. Scale bars, 40 µm. j , k Representative images and comparisons of indicated marker expressions in FRCs around T cell zone of inguinal LN in WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. Scale bars, 60 µm. l Heatmap and hierarchical clustering of differentially expressed genes of RNA-Seq data in isolated FRCs from WT and i- Lats1 / 2 ∆FRC mice and list of selected downregulated genes (green) encoding cytokines and chemokines and upregulated genes (red) involved in TGF-β signaling. m Canonical IPA-annotated pathways listed in absolute IPA activation Z-score ( P
Figure Legend Snippet: Canonical Hippo pathway LATS1/2-YAP/TAZ governs FRCs. a Diagram for generation of indicated mice and their analyses at 8-weeks old after the tamoxifen injection from 4-weeks old. b Comparisons of the inguinal LN weight and cellularity within the inguinal LN in i-WT ΔFRC-TR and i- Yap / Taz ΔFRC-TR mice. c Representative images of intact border between B and T cell zones (white dashed line) beneath the LN capsule (white line) in i-WT ΔFRC-TR and i- Yap / Taz ΔFRC-TR mice ( n = 4). Scale bars, 200 μm. d Representative images of preserved LYVE-1 + lymphatic vessels and CD31 + blood vessels within the inguinal LN in i-WT ΔFRC-TR and i- Yap / Taz ΔFRC-TR mice ( n = 4). The regions within the white dashed-line box around subcapsular sinuses (SCS), medullary sinus (MS) and HEVs are magnified as indicated. Scale bars, 500 μm. e Diagram for generation of indicated mice for their analyses at 8-weeks old after the tamoxifen delivery from 6-weeks old. f Comparisons of the inguinal LN weight and total number of cells within the inguinal LN in WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. g Representative images of inguinal LN in WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. Scale bars, 500 μm. h , i Representative images and comparison of YAP nuclear localization (white arrowheads) in inguinal LN of WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. Scale bars, 40 µm. j , k Representative images and comparisons of indicated marker expressions in FRCs around T cell zone of inguinal LN in WT, i- Lats1 / 2 ∆FRC or i- L1 / 2-Y / T ∆FRC mice. Scale bars, 60 µm. l Heatmap and hierarchical clustering of differentially expressed genes of RNA-Seq data in isolated FRCs from WT and i- Lats1 / 2 ∆FRC mice and list of selected downregulated genes (green) encoding cytokines and chemokines and upregulated genes (red) involved in TGF-β signaling. m Canonical IPA-annotated pathways listed in absolute IPA activation Z-score ( P

Techniques Used: Mouse Assay, Injection, Mass Spectrometry, Marker, RNA Sequencing Assay, Isolation, Indirect Immunoperoxidase Assay, Activation Assay

40) Product Images from "Loss of T cell receptor-induced Bmi-1 in the KLRG1+ senescent CD8+ T lymphocyte"

Article Title: Loss of T cell receptor-induced Bmi-1 in the KLRG1+ senescent CD8+ T lymphocyte

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0706040104

Enhanced homeostatic expansion of CD8 + T cells expressing ectopic Bmi-1. Rat Thy1 + OT-I cells (4 × 10 5 ) that had been transduced with pMig/Thy1 or pMig/Thy1/Bmi-1 during proliferation in the presence of IL-2 and IL-7 were adoptively transferred to Rag2 −/− recipients. After 215 d, the number of rat Thy1 + CD8 + T cells in the blood, spleen, femur, and liver was measured, with recovery from the latter two sites being normalized by counting Gr-1 + cells. The means and SE are shown for each determination. The experiment has been repeated with similar results. *, P = 0.043 (paired Student's t test).
Figure Legend Snippet: Enhanced homeostatic expansion of CD8 + T cells expressing ectopic Bmi-1. Rat Thy1 + OT-I cells (4 × 10 5 ) that had been transduced with pMig/Thy1 or pMig/Thy1/Bmi-1 during proliferation in the presence of IL-2 and IL-7 were adoptively transferred to Rag2 −/− recipients. After 215 d, the number of rat Thy1 + CD8 + T cells in the blood, spleen, femur, and liver was measured, with recovery from the latter two sites being normalized by counting Gr-1 + cells. The means and SE are shown for each determination. The experiment has been repeated with similar results. *, P = 0.043 (paired Student's t test).

Techniques Used: Expressing, Transduction

Maintenance of Bmi-1 expression by IL-2. Naïve OT-I cells were cultured for 24 h in medium with 2.5 nM SIINFEKL peptide. The cells were then transferred to fresh medium alone or medium containing incremental concentrations of IL-2 and cultured for an additional 20 h, after which Bmi-1 expression was assessed by staining with isotype control (black line) or anti-Bmi-1 antibody (red line). The mean fluorescent intensity for specific Bmi-1 staining is represented by the number in each histogram. The experiments shown were performed multiple times with comparable results.
Figure Legend Snippet: Maintenance of Bmi-1 expression by IL-2. Naïve OT-I cells were cultured for 24 h in medium with 2.5 nM SIINFEKL peptide. The cells were then transferred to fresh medium alone or medium containing incremental concentrations of IL-2 and cultured for an additional 20 h, after which Bmi-1 expression was assessed by staining with isotype control (black line) or anti-Bmi-1 antibody (red line). The mean fluorescent intensity for specific Bmi-1 staining is represented by the number in each histogram. The experiments shown were performed multiple times with comparable results.

Techniques Used: Expressing, Cell Culture, Staining

TCR signaling and induction of Bmi-1 expression in CD8 + T cells. ( a ) Naïve OT-I T cells were cultured for 24 h in medium alone or in medium containing incremental concentrations of SIINFEKL peptide and assessed by flow cytometry for Bmi-1 expression after staining with isotype control (black line) or anti-Bmi-1 antibody (red line). ( b ) Whole-cell lysates of naïve OT-I cells were generated 0 and 20 h after culturing cells in medium containing 2.5 nM SIINFEKL peptide and were assessed by immunoblot for Bmi-1 and β-actin. ( c and d ) Naïve OT-I cells were cultured in the presence of 2.5 nM SIINFEKL peptide for various lengths of time and then assessed for Bmi-1 expression by flow cytometry after staining with isotype control (black line) or anti-Bmi-1 antibody (red line) ( c ) and assessed for Bmi-1 mRNA by quantitative RT-PCR ( d ). The mean fluorescent intensity for specific Bmi-1 staining is represented by the number in each histogram. These experiments were performed multiple times with comparable results.
Figure Legend Snippet: TCR signaling and induction of Bmi-1 expression in CD8 + T cells. ( a ) Naïve OT-I T cells were cultured for 24 h in medium alone or in medium containing incremental concentrations of SIINFEKL peptide and assessed by flow cytometry for Bmi-1 expression after staining with isotype control (black line) or anti-Bmi-1 antibody (red line). ( b ) Whole-cell lysates of naïve OT-I cells were generated 0 and 20 h after culturing cells in medium containing 2.5 nM SIINFEKL peptide and were assessed by immunoblot for Bmi-1 and β-actin. ( c and d ) Naïve OT-I cells were cultured in the presence of 2.5 nM SIINFEKL peptide for various lengths of time and then assessed for Bmi-1 expression by flow cytometry after staining with isotype control (black line) or anti-Bmi-1 antibody (red line) ( c ) and assessed for Bmi-1 mRNA by quantitative RT-PCR ( d ). The mean fluorescent intensity for specific Bmi-1 staining is represented by the number in each histogram. These experiments were performed multiple times with comparable results.

Techniques Used: Expressing, Cell Culture, Flow Cytometry, Cytometry, Staining, Generated, Quantitative RT-PCR

Inhibition of proliferation of CD8 + T cells by a lentivirus expressing an shRNA specific for Bmi-1. CTLL-2 cells maintained with IL-2 were infected at an moi of 5 with lentiviral vectors expressing EGFP alone (pll3.7) or together with a Bmi-1-specific shRNA (pll3.7-shBmi-1). ( a ) Whole-cell lysates of CTLL-2 cells (lane 1), cells infected with pll3.7 (lane 2), or cells infected with pll3.7-shBmi-1 (lane 3) were subjected to immunoblot analysis with antibodies against Bmi-1 and β-actin. ( b ) CTLL-2 cells infected with either the pll3.7 or pll3.7-shBmi-1 lentiviral vector were assessed for Bmi-1 and Mel-18 mRNA by using quantitative RT-PCR. ( c ) The number of CTLL-2 cells infected with pll3.7 (open squares) or pll3.7-shBmi-1 (filled squares) was measured during continuous culture in the presence of IL-2. ( d ) CTLL-2 cells were transduced with a retroviral vector expressing human CD2 alone (circles) or together with Bmi-1 containing silent mutations at the shRNA binding site (triangles). Transduced cells were superinfected with either pll3.7 (open triangles) or pll3.7-shBmi-1 (filled triangles and filled circles), and the number of EGFP + , lentivirally transduced cells was assayed periodically during growth in the presence of IL-2. ( e ) The number of EGFP + OT-I cells infected with pll3.7 (open squares) or pll3.7-shBmi-1 (filled squares) was measured during continuous culture in the presence of IL-2 and IL-7. The experiments shown were performed multiple times with comparable results.
Figure Legend Snippet: Inhibition of proliferation of CD8 + T cells by a lentivirus expressing an shRNA specific for Bmi-1. CTLL-2 cells maintained with IL-2 were infected at an moi of 5 with lentiviral vectors expressing EGFP alone (pll3.7) or together with a Bmi-1-specific shRNA (pll3.7-shBmi-1). ( a ) Whole-cell lysates of CTLL-2 cells (lane 1), cells infected with pll3.7 (lane 2), or cells infected with pll3.7-shBmi-1 (lane 3) were subjected to immunoblot analysis with antibodies against Bmi-1 and β-actin. ( b ) CTLL-2 cells infected with either the pll3.7 or pll3.7-shBmi-1 lentiviral vector were assessed for Bmi-1 and Mel-18 mRNA by using quantitative RT-PCR. ( c ) The number of CTLL-2 cells infected with pll3.7 (open squares) or pll3.7-shBmi-1 (filled squares) was measured during continuous culture in the presence of IL-2. ( d ) CTLL-2 cells were transduced with a retroviral vector expressing human CD2 alone (circles) or together with Bmi-1 containing silent mutations at the shRNA binding site (triangles). Transduced cells were superinfected with either pll3.7 (open triangles) or pll3.7-shBmi-1 (filled triangles and filled circles), and the number of EGFP + , lentivirally transduced cells was assayed periodically during growth in the presence of IL-2. ( e ) The number of EGFP + OT-I cells infected with pll3.7 (open squares) or pll3.7-shBmi-1 (filled squares) was measured during continuous culture in the presence of IL-2 and IL-7. The experiments shown were performed multiple times with comparable results.

Techniques Used: Inhibition, Expressing, shRNA, Infection, Plasmid Preparation, Quantitative RT-PCR, Transduction, Binding Assay

Enhanced proliferation in vitro of CD8 + T cells expressing ectopic Bmi-1. ( a ) OT-I cells that had been activated with anti-CD3ε in the presence of IL-2 and IL-7 were transduced with pMig/Thy1 or with pMig/Thy1/Bmi-1 and maintained for 6 d in the presence of IL-2 and IL-7. On day 4 after transduction, cells expressing rat Thy1 were purified, and lysates were subjected to analysis by Western blot with antibodies specific for Bmi-1, HA, and β-actin. ( b ) In a replicate experiment, OT-I cells were transduced with pMig/Thy1 (open squares) or pMig/Thy1/Bmi-1 (filled squares) and assessed for growth in the presence of IL-2 and IL-7. The experiments shown were performed multiple times with comparable results, except for the Western blot, which was performed once.
Figure Legend Snippet: Enhanced proliferation in vitro of CD8 + T cells expressing ectopic Bmi-1. ( a ) OT-I cells that had been activated with anti-CD3ε in the presence of IL-2 and IL-7 were transduced with pMig/Thy1 or with pMig/Thy1/Bmi-1 and maintained for 6 d in the presence of IL-2 and IL-7. On day 4 after transduction, cells expressing rat Thy1 were purified, and lysates were subjected to analysis by Western blot with antibodies specific for Bmi-1, HA, and β-actin. ( b ) In a replicate experiment, OT-I cells were transduced with pMig/Thy1 (open squares) or pMig/Thy1/Bmi-1 (filled squares) and assessed for growth in the presence of IL-2 and IL-7. The experiments shown were performed multiple times with comparable results, except for the Western blot, which was performed once.

Techniques Used: In Vitro, Expressing, Transduction, Purification, Western Blot

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Article Title: REG? is a strong candidate for the regulation of cell cycle, proliferation and the invasion by poorly differentiated thyroid carcinoma cells
Article Snippet: Mouse monoclonal anti-p21 was purchased from BD (USA).

Staining:

Article Title: Physiological pathway of differentiation of hematopoietic stem cell population into mural cells
Article Snippet: .. The mAbs used in immunofluorescence staining were anti-CD45, anti–c-kit, and anti-lineage (a mixture of ter119, Gr-1, Mac-1, B220, CD4, and CD8) antibodies (all purchased from BD Biosciences). .. All mAbs were purified and conjugated with either FITC, PE, or biotin.

Immunofluorescence:

Article Title: Physiological pathway of differentiation of hematopoietic stem cell population into mural cells
Article Snippet: .. The mAbs used in immunofluorescence staining were anti-CD45, anti–c-kit, and anti-lineage (a mixture of ter119, Gr-1, Mac-1, B220, CD4, and CD8) antibodies (all purchased from BD Biosciences). .. All mAbs were purified and conjugated with either FITC, PE, or biotin.

Software:

Article Title: IL-21 Regulates the Differentiation of a Human ?? T Cell Subset Equipped with B Cell Helper Activity
Article Snippet: .. Vγ9Vδ2 cells were incubated in U-bottom 96-well plates with labelled mAbs in PBS containing 1% FCS, for 30 min at 4°C according to manufacturers’ recommendations, washed, and analyzed by flow cytometry on an FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). ..

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    Becton Dickinson anti bromodeoxyuridine brdu
    Expression of Ptf1a in the embryonic caudal hindbrain. A , B , Serial transverse frozen sections of r7 hindbrain at E11.5. Localization of Math1 transcripts (visualized by in situ hybridization) and Ptf1a and <t>Ngn1</t> proteins (visualized by immunohistochemistry) are shown. C , Schematic diagram of expression of bHLH transcription factors/genes in the caudal hindbrain at E11.5. Lmx1a ). D–F , Double immunostaining with anti-Ptf1a and <t>BrdU</t> antibodies within the Ptf1a domain of the E11.5 caudal hindbrain. Pregnant mice were given BrdU injections 1 h before embryo harvest and fixation. Many Ptf1a-positive cells incorporate BrdU (arrowheads in F ), indicating that they are mitotic. G–I , Double immunostaining with anti-Ptf1a and HuC/D antibodies around the Ptf1a domain of the caudal hindbrain at E11.5. Ptf1a-positive cells do not express HuC/D, suggesting that they do not include postmitotic neurons. Scale bars: A , B , 100 μm; D–I , 20 μm.
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    Becton Dickinson mouse monoclonal anti brdu
    Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of <t>BrdU</t> incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, <t>CRALBP,</t> Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P
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    Becton Dickinson anti idu
    Cross-reactivity and validation of antibody staining. Section of an embryo exposed to <t>IdU</t> shows specific staining using an antibody against IdU (Panel A). When an antibody against <t>CldU</t> is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.
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    Becton Dickinson anti brdu r pe conjugated mouse mab
    Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with <t>BrdU</t> (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with <t>anti-BrdU-R-PE</t> and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.
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    Image Search Results


    Expression of Ptf1a in the embryonic caudal hindbrain. A , B , Serial transverse frozen sections of r7 hindbrain at E11.5. Localization of Math1 transcripts (visualized by in situ hybridization) and Ptf1a and Ngn1 proteins (visualized by immunohistochemistry) are shown. C , Schematic diagram of expression of bHLH transcription factors/genes in the caudal hindbrain at E11.5. Lmx1a ). D–F , Double immunostaining with anti-Ptf1a and BrdU antibodies within the Ptf1a domain of the E11.5 caudal hindbrain. Pregnant mice were given BrdU injections 1 h before embryo harvest and fixation. Many Ptf1a-positive cells incorporate BrdU (arrowheads in F ), indicating that they are mitotic. G–I , Double immunostaining with anti-Ptf1a and HuC/D antibodies around the Ptf1a domain of the caudal hindbrain at E11.5. Ptf1a-positive cells do not express HuC/D, suggesting that they do not include postmitotic neurons. Scale bars: A , B , 100 μm; D–I , 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Origin of Climbing Fiber Neurons and Their Developmental Dependence on Ptf1a

    doi: 10.1523/JNEUROSCI.1423-07.2007

    Figure Lengend Snippet: Expression of Ptf1a in the embryonic caudal hindbrain. A , B , Serial transverse frozen sections of r7 hindbrain at E11.5. Localization of Math1 transcripts (visualized by in situ hybridization) and Ptf1a and Ngn1 proteins (visualized by immunohistochemistry) are shown. C , Schematic diagram of expression of bHLH transcription factors/genes in the caudal hindbrain at E11.5. Lmx1a ). D–F , Double immunostaining with anti-Ptf1a and BrdU antibodies within the Ptf1a domain of the E11.5 caudal hindbrain. Pregnant mice were given BrdU injections 1 h before embryo harvest and fixation. Many Ptf1a-positive cells incorporate BrdU (arrowheads in F ), indicating that they are mitotic. G–I , Double immunostaining with anti-Ptf1a and HuC/D antibodies around the Ptf1a domain of the caudal hindbrain at E11.5. Ptf1a-positive cells do not express HuC/D, suggesting that they do not include postmitotic neurons. Scale bars: A , B , 100 μm; D–I , 20 μm.

    Article Snippet: Primary antibodies used in this study were anti-Brn3a (brain-specific homeobox/POU domain protein 3A) (1:10, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Brn3a (1:10, rabbit polyclonal; Abcam, Cambridge, UK), anti-Brn3b (1:25, goat; Santa Cruz Biotechnology), anti-GABA (1:500, rabbit; Sigma, St. Louis, MO), anti-HuC/D (1:500, mouse monoclonal; Invitrogen, Carlsbad, CA), anti-β-galactosidase (β-gal) (1:400, goat; Biogenesis, Poole, UK), anti-β-galactosidase (1:1600, rabbit; Cappel, Aurora, OH), anti-glutaminase (1:600, rabbit) , anti-Ngn1 (Neurogenin1) (1:100, goat; Santa Cruz Biotechnology), anti-bromodeoxyuridine (BrdU) (1:75, mouse monoclonal; BD, Franklin Lakes, NJ), anti-Fluorogold (1:2000, rabbit; Chemicon, Temecula, CA), anti-cleaved Caspase-3 (1:100, rabbit; Cell Signaling Technology, Beverly, MA), and anti-Ptf1a (1:3000).

    Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Double Immunostaining, Mouse Assay

    Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of BrdU incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, CRALBP, Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

    doi: 10.1083/jcb.200805155

    Figure Lengend Snippet: Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of BrdU incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, CRALBP, Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P

    Article Snippet: Antibodies used in this study include rabbit polyclonal anti-CRALBP (a kind gift from J. Saari, University of Washington, Seattle, WA), mouse monoclonal anti-BrdU (BD), mouse monoclonal anti-rhodopsin , rabbit polyclonal anti-recoverin (Millipore), sheep polyclonal anti-Chx10 (a gift from R. Bremner, Toronto Western Research Institute, Toronto, Ontario, Canada), rabbit polyclonal phosphohistone H3 (Millipore), and rabbit polyclonal anti-GFP (Invitrogen).

    Techniques: In Vivo, Staining, Mouse Assay, BrdU Incorporation Assay, Marker, Transfection

    Gli2 is required for the Shh effects on proliferation and cell fate. Retinal explants were cultured from wild-type (Wt; n = 3) and Gli2 −/− ( n = 3) mice at E18 for 3 d in culture with or without a Smo agonist. IHC was performed on dissociated cells using anti-BrdU, anti-CRALBP, anti-rhodopsin, and anti-recoverin antibodies. Values represent the fold induction of positive cells in Wt + Ag or Gli2 −/− + Ag cultures compared with nontreated explants. Error bars represent SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

    doi: 10.1083/jcb.200805155

    Figure Lengend Snippet: Gli2 is required for the Shh effects on proliferation and cell fate. Retinal explants were cultured from wild-type (Wt; n = 3) and Gli2 −/− ( n = 3) mice at E18 for 3 d in culture with or without a Smo agonist. IHC was performed on dissociated cells using anti-BrdU, anti-CRALBP, anti-rhodopsin, and anti-recoverin antibodies. Values represent the fold induction of positive cells in Wt + Ag or Gli2 −/− + Ag cultures compared with nontreated explants. Error bars represent SEM. *, P

    Article Snippet: Antibodies used in this study include rabbit polyclonal anti-CRALBP (a kind gift from J. Saari, University of Washington, Seattle, WA), mouse monoclonal anti-BrdU (BD), mouse monoclonal anti-rhodopsin , rabbit polyclonal anti-recoverin (Millipore), sheep polyclonal anti-Chx10 (a gift from R. Bremner, Toronto Western Research Institute, Toronto, Ontario, Canada), rabbit polyclonal phosphohistone H3 (Millipore), and rabbit polyclonal anti-GFP (Invitrogen).

    Techniques: Cell Culture, Mouse Assay, Immunohistochemistry

    Cross-reactivity and validation of antibody staining. Section of an embryo exposed to IdU shows specific staining using an antibody against IdU (Panel A). When an antibody against CldU is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.

    Journal: PLoS ONE

    Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

    doi: 10.1371/journal.pone.0047719

    Figure Lengend Snippet: Cross-reactivity and validation of antibody staining. Section of an embryo exposed to IdU shows specific staining using an antibody against IdU (Panel A). When an antibody against CldU is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.

    Article Snippet: Each section was exposed overnight to a mixture of anti-IdU (mouse-monoclonal anti-BrdU; BD, 347580), anti-CldU (rat-monoclonal anti-BrdU; Serotec, OBT0030CX) and anti-cTnI (rabbit polyclonal; HyTest, 4T21/2) followed by incubation for at least 2 hrs with a mixture of the fluorescent antibodies, goat-anti-mouse-Alexa 680, goat-anti-rat-Alexa 568, goat-anti-rabbit-Alexa 405 (Invitrogen), and Sytox green 488 (Invitrogen).

    Techniques: Staining

    Image analysis and visualisation. Panel A. Using a Sytox green staining, all nuclei are first detected based on a local-maxima threshold. All detected objects that were at least twice as large as the median object size were processed to separate these fused nuclei (inserts). Panel B shows a schematic overview of the image processing steps involved in the recognition of IdU- and CldU-positive nuclei. After the detection of the Sytox green stained nuclei, each nucleus is individually processed. A zone is selected around all nuclei which will be excluded in the following measurement (gray zone). For each nucleus within the region of interest (myocardium), the algorithm measures the signal in (red area) and around (green area) the nucleus in the IdU and CldU channels. The measurement of the local background excludes the locations at which other nuclei were detected (gray zone). When the signal in the nucleus is at least a standard deviation above the background, the nucleus is classified as positively labelled. The program generates control images both for the nuclei detection as well as for which nuclei are positive for the proliferation markers. The difference between the two proliferation markers is used to determine ΔF (number of green nuclei divided by total number of nuclei). Panel C shows how the quantitative information can be projected onto a reconstruction or onto the original section. Each unit in the boxel representation has a volume of approximately 21 3 µm 3 , and is the central boxel of the sampling volume of approximately 105 3 µm 3 that is required for reliable measurement of the labelling indices [15] .

    Journal: PLoS ONE

    Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

    doi: 10.1371/journal.pone.0047719

    Figure Lengend Snippet: Image analysis and visualisation. Panel A. Using a Sytox green staining, all nuclei are first detected based on a local-maxima threshold. All detected objects that were at least twice as large as the median object size were processed to separate these fused nuclei (inserts). Panel B shows a schematic overview of the image processing steps involved in the recognition of IdU- and CldU-positive nuclei. After the detection of the Sytox green stained nuclei, each nucleus is individually processed. A zone is selected around all nuclei which will be excluded in the following measurement (gray zone). For each nucleus within the region of interest (myocardium), the algorithm measures the signal in (red area) and around (green area) the nucleus in the IdU and CldU channels. The measurement of the local background excludes the locations at which other nuclei were detected (gray zone). When the signal in the nucleus is at least a standard deviation above the background, the nucleus is classified as positively labelled. The program generates control images both for the nuclei detection as well as for which nuclei are positive for the proliferation markers. The difference between the two proliferation markers is used to determine ΔF (number of green nuclei divided by total number of nuclei). Panel C shows how the quantitative information can be projected onto a reconstruction or onto the original section. Each unit in the boxel representation has a volume of approximately 21 3 µm 3 , and is the central boxel of the sampling volume of approximately 105 3 µm 3 that is required for reliable measurement of the labelling indices [15] .

    Article Snippet: Each section was exposed overnight to a mixture of anti-IdU (mouse-monoclonal anti-BrdU; BD, 347580), anti-CldU (rat-monoclonal anti-BrdU; Serotec, OBT0030CX) and anti-cTnI (rabbit polyclonal; HyTest, 4T21/2) followed by incubation for at least 2 hrs with a mixture of the fluorescent antibodies, goat-anti-mouse-Alexa 680, goat-anti-rat-Alexa 568, goat-anti-rabbit-Alexa 405 (Invitrogen), and Sytox green 488 (Invitrogen).

    Techniques: Staining, Standard Deviation, Sampling

    Application in heart development. 3D visualisation of cell cycle length in the heart at stages HH9 (Panel A), HH12 (Panel B) and HH16 (Panel C) of chicken embryonic development. The pointer in panel B indicates the region with a high proliferation rate at the site of early ventricle formation. Panel C shows the quantitative reconstructions of the individual labelling indices for CldU and IdU, on which the cycle lengths are based. The pointers in the CldU reconstruction indicate areas in which a low fraction of cells is positive in both CldU and IdU reconstructions, resulting in a low labelling difference and thus a long cell cycle length. The pointers in the IdU reconstruction show large differences in IdU and CldU labelling indices, indicating short cell cycle lengths. Note the heterogeneity in cell cycle lengths in different parts of the heart at every stage. Interactive versions of the 3D-reconstructions can be found in Interactive 3D-pdf S1 .

    Journal: PLoS ONE

    Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

    doi: 10.1371/journal.pone.0047719

    Figure Lengend Snippet: Application in heart development. 3D visualisation of cell cycle length in the heart at stages HH9 (Panel A), HH12 (Panel B) and HH16 (Panel C) of chicken embryonic development. The pointer in panel B indicates the region with a high proliferation rate at the site of early ventricle formation. Panel C shows the quantitative reconstructions of the individual labelling indices for CldU and IdU, on which the cycle lengths are based. The pointers in the CldU reconstruction indicate areas in which a low fraction of cells is positive in both CldU and IdU reconstructions, resulting in a low labelling difference and thus a long cell cycle length. The pointers in the IdU reconstruction show large differences in IdU and CldU labelling indices, indicating short cell cycle lengths. Note the heterogeneity in cell cycle lengths in different parts of the heart at every stage. Interactive versions of the 3D-reconstructions can be found in Interactive 3D-pdf S1 .

    Article Snippet: Each section was exposed overnight to a mixture of anti-IdU (mouse-monoclonal anti-BrdU; BD, 347580), anti-CldU (rat-monoclonal anti-BrdU; Serotec, OBT0030CX) and anti-cTnI (rabbit polyclonal; HyTest, 4T21/2) followed by incubation for at least 2 hrs with a mixture of the fluorescent antibodies, goat-anti-mouse-Alexa 680, goat-anti-rat-Alexa 568, goat-anti-rabbit-Alexa 405 (Invitrogen), and Sytox green 488 (Invitrogen).

    Techniques:

    Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with BrdU (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with anti-BrdU-R-PE and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.

    Journal: Journal of Virology

    Article Title: Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/JVI.78.5.2336-2347.2004

    Figure Lengend Snippet: Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with BrdU (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with anti-BrdU-R-PE and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.

    Article Snippet: BrdU staining was then performed by adding of 50 μl of anti-BrdU-R-PE-conjugated mouse MAb (Becton Dickinson) for 45 min at room temperature.

    Techniques: Cell Culture, Infection, Immunofluorescence, Staining, Flow Cytometry, Cytometry