Journal: Biology of reproduction

Article Title: The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

doi: 10.1095/biolreprod.106.055236

Figure Lengend Snippet: FIG. 2. Construction of the reporter genes for transgenic mice. Three kinds of DNA fragment were inserted bidirectionally in EGFP and DsRed reporter genes. Arrows indicate the directions of haspin (white) and integrin aE (black) transcription. The AseI-NheI DNA fragments that included reporter genes were used as transgenes.

Article Snippet: The filters were then reacted with anti-EGFP rat monoclonal antibody [3] or anti-DsRed rabbit serum (Clontech) in TBS for 1 h at 258C and washed in TBS, once for 3 min, then three times for 5 min each.

Techniques: Transgenic Assay

Journal: Biology of reproduction

Article Title: The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

doi: 10.1095/biolreprod.106.055236

Figure Lengend Snippet: FIG. 4. Developmental expression of EGFP and DsRed mRNA in prepubertal mice; 10 lg of total RNA was used for Northern blotting. Numbers associated with each lane indicate the ages of transgenic mouse.

Article Snippet: The filters were then reacted with anti-EGFP rat monoclonal antibody [3] or anti-DsRed rabbit serum (Clontech) in TBS for 1 h at 258C and washed in TBS, once for 3 min, then three times for 5 min each.

Techniques: Expressing, Northern Blot, Transgenic Assay

Journal: Biology of reproduction

Article Title: The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

doi: 10.1095/biolreprod.106.055236

Figure Lengend Snippet: FIG. 5. Histochemical detection of the reporter gene product, EGFP. Expression of EGFP was observed under UV light using a fluorescence microscope. Constructions for transgenic mice are presented schemati- cally. A and C) Whole testes. Green signals were detected in seminiferous tubules. B and D) Cross sections of transgenic mice. RS, Round spermatids; ES, elongated spermatids. White arrowheads indicate pachytene spermatocytes. Bar ¼ 100 lm.

Article Snippet: The filters were then reacted with anti-EGFP rat monoclonal antibody [3] or anti-DsRed rabbit serum (Clontech) in TBS for 1 h at 258C and washed in TBS, once for 3 min, then three times for 5 min each.

Techniques: Expressing, Fluorescence, Microscopy, Transgenic Assay

Journal: Biology of reproduction

Article Title: The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

doi: 10.1095/biolreprod.106.055236

Figure Lengend Snippet: FIG. 3. Tissue blot analysis of reporter gene products in transgenic mice. Tissue distributions of reporter genes (BiPRO-1) were examined using Northern and Western blotting; 10 lg of total RNA (A) and 50 lg of protein (B–E) from tissue were applied in each blot. Western blotting was performed using anti-EGFP (B, D, and E) and anti-DsRed (C) antibodies. The subcellular fractions of testicular cells were loaded (E). The monoclonal antibody, TRA54, was used as a marker of a germ cell fraction [34]. Molecular masses are shown in the left margin (Mr).

Article Snippet: The filters were then reacted with anti-EGFP rat monoclonal antibody [3] or anti-DsRed rabbit serum (Clontech) in TBS for 1 h at 258C and washed in TBS, once for 3 min, then three times for 5 min each.

Techniques: Transgenic Assay, Northern Blot, Western Blot, Marker

Journal: Biology of reproduction

Article Title: The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

doi: 10.1095/biolreprod.106.055236

Figure Lengend Snippet: FIG. 6. Synchronous expression from the Gsg2 bidirectional promoter in haploid germ cells. A) Phase contrast. The expres- sion of DsRed was observed in germ cells expressing EGFP. The expression of EGFP and DsRed was observed using a blue filter (B) and a red filter (C). Bar ¼ 100 lm.

Article Snippet: The filters were then reacted with anti-EGFP rat monoclonal antibody [3] or anti-DsRed rabbit serum (Clontech) in TBS for 1 h at 258C and washed in TBS, once for 3 min, then three times for 5 min each.

Techniques: Expressing

Journal: JCI Insight

Article Title: Brd4 modulates diet-induced obesity via PPAR γ -dependent Gdf3 expression in adipose tissue macrophages

doi: 10.1172/jci.insight.143379

Figure Lengend Snippet: ( A ) PCA of RNA-Seq data derived from CD11b + ATMs of WT and Brd4 -CKO mice fed a HFD for 20 weeks. ( B ) Volcano plot of mRNA-Seq analysis in ATMs as indicated in A . Brown dots represent genes increased in ATMs of Brd4 -CKO mice vs. WT mice (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value). Blue dots represent genes decreased in ATMs of Brd4 -CKO mice vs. WT mice (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value). Gray dots represent genes without significantly altered expression. Clusters of significantly altered genes (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value) were identified using gene ontology terms ( C ) and KEGG pathways ( D ). ( E ) Heat map of the relative expression levels (scaled Z -score) of cytokine-cytokine receptor interaction-related genes clustered in ( D ). ( F ) mRNA levels of Gdf3 in CD11b + ATMs isolated from WT or Brd4 -CKO mice fed a HFD for 20 weeks. ( G ) Left panel: Gdf3 or F4/80 IHC staining of eWAT of WT or Brd4-CKO mice fed a HFD for 20 weeks. Right panel: statistical analysis of Gdf3-positive or F4/80-positive area percentage, the ratio of Gdf3-positive cells in F4/80-positive cells in eWAT of WT and Brd4 -CKO mice fed a HFD. Data are mean and SD and are determined by an unpaired 2-tailed Student’s t test. n = 3 mice. ** P < 0.01. Brd4 -CKO, myeloid lineage-specific Brd4 knockout; HFD, high-fat diet–induced; eWAT, epididymal WAT; ATMs, adipose tissue macrophages; PCA, principal component analysis.

Article Snippet: Four-μm continuous sections were prepared for immunostaining with Gdf3 antibody (AF958, R&D Systems) or F4/80 antibody (BM4008, Origene).

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Isolation, Immunohistochemistry, Knock-Out

Journal: bioRxiv

Article Title: Disrupted Cerebral Peri-Microvascular Glycogen Promotes Capillary Constrictions and Aggravates Ischemia in Mice

doi: 10.1101/2022.08.24.505172

Figure Lengend Snippet: CD13 positive capillary pericyte mediated constrictions were evaluated in Swiss albino, wild-type (WT) and GYS1 Nestin-KO mice. (A) Experiments performed in adult Swiss albino male and female mice intracerebroventricularly (i.c.v) administered with saline (vehicle) or DAB (as indicated in figure) sacrificed after 30 mins, 1h, 3h, 6h, 9h and 24h (shown left to right respectively). Lycopersicon Esculentum Lectin (upper panel-green) and CD13 (middle panel-red) double labelling (merged as lower panel) reveals increased microvascular constrictions 30 minutes after DAB injections shown by arrows. Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (B) Microvascular constrictions in ipsilateral and contralateral hemispheres were quantified semi-stereologically. DAB (i.c.v) injections result in robust increase of constrictions after 30 minutes, persists for 6 hours. Although number of constrictions started to decline after 9 hours, impact of DAB on these constrictions are not fully reversible even after 24 hours. Quantification of CD13+ pericyte mediated microvascular constrictions in ten fields fields for each hemisphere spanning MCA territory (240 μm × 160 μm) per animal (n = 3 animals per group). Two-way analysis of Kruskal-Wallis and Mann-Whitney U, n = 3; *P < 0.05. Black: ipsilateral hemisphere, Orange: Contralateral hemisphere. (C) Adult naïve wild-type (WT) and GYS1 Nestin-KO mice are sacrificed, and brain sections are labeled with Lycopersicon Esculentum Lectin (upper panel-green) and CD13 (middle panel-red and merged as lower panel). GYS1 Nestin-KO mice demonstrate higher number of microvascular constrictions as shown by arrows. Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (D) CD13+ pericyte mediated microvascular constrictions in adult naïve wild-type (WT) and GYS1 Nestin-KO mice are quantified. Transgenic mice have higher number of constrictions. Quantification of CD13+ pericyte mediated microvascular constrictions in ten fields for each hemisphere spanning MCA territory (240 μm × 160 μm) per animal (n = 3 animals per group). Mann-Whitney U, n = 3; *P < 0.05.

Article Snippet: The sections were blocked in 1% bovine serum albumin containing 0.3% Triton-X, 10% normal goat serum (NGS) (0.3% TBS-T / 1% BSA, 10% NGS) solution for 1 hour at room temperature and then incubated with primary antibodies (anti-glycogen antibodies ESG1A9 and IV58B6 (courtesy of Dr. Hitoshi Ashida and Dr. Otto Baba), CD13 (Acris Antibodies GmBH, AM26636AF-N) for pericytes) in 0.3% TBS-T overnight at + 4°C.

Techniques: MANN-WHITNEY, Labeling, Transgenic Assay

Journal: bioRxiv

Article Title: Disrupted Cerebral Peri-Microvascular Glycogen Promotes Capillary Constrictions and Aggravates Ischemia in Mice

doi: 10.1101/2022.08.24.505172

Figure Lengend Snippet: Permanent MCAo experiments performed in Saline, DAB injected WT, naïve WT and GYS1 Nestin-KO mice (n = 3 animals per group). (A) Representative images taken from Cresyl-violet stained sections of ischemic mice via phase contrast microscopy (as indicated in figure) that underwent 2-hour MCAo. Red dots are placed over the border between core and peri-infarct areas. Scale bars, 500 μm. (B) Infarct volumes after 2-hour MCAo are measured then quantified with volume correction (Swanson, JCBFM 1990). Ischemic infarct volumes after MCAo were significantly higher in DAB treated mice than saline treated groups (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). Infarct volumes of GYS1 Nestin-KO group were also higher when compared to wild type (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). (C) Quantification of microvascular constrictions after MCAo in Saline, DAB injected WT, naïve WT and GYS1 Nestin-KO mice (n = 3 animals per group). Quantification of CD13+ pericyte mediated microvascular constrictions are held in ten fields fields for each hemisphere spanning MCA territory (240 μm × 160 μm) per animal. Ischemia further resulted in higher number of microvascular constrictions in peri-microvascular glycogen disrupted mice (DAB injected and GYS1 Nestin-KO ) compared to ischemic controls (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). Black: ipsilateral hemisphere, Orange: Contralateral hemisphere. (D) Lycopersicon Esculentum Lectin (upper panel-green) and CD13 (middle panel-red) double labelling (merged as lower panel) reveals increased microvascular constrictions (arrows) 2 hours after MCAo in Saline, DAB injected WT, naïve WT and GYS1 Nestin-KO mice (left to right, respectively). Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (E) Lycopersicon Esculentum Lectin (left panel-green) and CD13 (middle panel-red) double labelling (merged as right panel) in i.c.v DAB+ L/D-lactate injected mice. Double labelling shows that L-lactate (upper panel) reverses the DAB’s impact on CD13+ pericyte mediated constrictions contrary to its enantiomer D-Lactate (lower panel). Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (F) Quantification of CD13+ pericyte mediated microvascular constrictions in i.c.v DAB+ L/D-lactate injected mice compared to controls (n = 3 animals per group). L-lactate can reverse the DAB-induced constrictions; however, D-lactate demonstrates similar number of microvascular constrictions with DAB (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). Black: ipsilateral hemisphere, Brown: Contralateral hemisphere.

Article Snippet: The sections were blocked in 1% bovine serum albumin containing 0.3% Triton-X, 10% normal goat serum (NGS) (0.3% TBS-T / 1% BSA, 10% NGS) solution for 1 hour at room temperature and then incubated with primary antibodies (anti-glycogen antibodies ESG1A9 and IV58B6 (courtesy of Dr. Hitoshi Ashida and Dr. Otto Baba), CD13 (Acris Antibodies GmBH, AM26636AF-N) for pericytes) in 0.3% TBS-T overnight at + 4°C.

Techniques: Injection, Staining, Microscopy, MANN-WHITNEY

Journal: Journal of hepatology

Article Title: Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

doi: 10.1016/j.jhep.2018.10.040

Figure Lengend Snippet: (A) Male C57BL/6 mice were subcutaneously inoculated with 1 × 106 RIL-175 HCC cells. The size of the tumors in the HCC xenograft mouse models was blindly measured by a caliper. Then, 1 to 2 weeks later, 5 × 106 CIK cells were intravenously injected into the tumor-bearing mice. n=5 for control, n=5 for CIK, Student’s t-test, *P=0.0369. (B) CD11b expression was evaluated by quantitative real-time PCR. n=5 for control, n=5 for CIK, Student’s t-test, *P<0.05. The frequency of (C) intratumoral and (D) splenic MDSCs was determined by flow cytometry. Total MDSCs were defined as CD45+CD11b+Gr1+ cells, while PMN-MDSCs and M-MDSCs were gated as CD11b+Ly6G+Ly6Clow and CD11b+Ly6G−Ly6Chigh cells, respectively. The gating strategy from a representative sample is shown in Supplemental Fig. 2. n=5, Student’s t-test, *P<0.05. Mean ± SEM. (E) A cytokine array using conditioned media from cocultured RIL-175 and CIK cells versus splenocytes as a control. Three independent experiments are shown.

Article Snippet: Tissues were fixed and stained with anti-Ly6G (clone 6D17, Acris Antibodies GmbH, Germany).

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Journal of hepatology

Article Title: Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

doi: 10.1016/j.jhep.2018.10.040

Figure Lengend Snippet: Cells were generated or isolated from C57BL/6 mice. CIK cells were incubated with different ratios of (A) CD11b+Ly6G+Ly6Clow PMN-MDSCs or splenocytes, as indicated and (B) CD11b+Ly6G−Ly6Chigh M-MDSCs or splenocytes as indicated. After 24 hr, RIL-175 cells were added at a ratio of 10:1 (E:T), and lysis was determined by LDH cytotoxicity assay. Cumulative results from three independent experiments are shown. Mean ± SEM. *P=0.001.

Article Snippet: Tissues were fixed and stained with anti-Ly6G (clone 6D17, Acris Antibodies GmbH, Germany).

Techniques: Generated, Isolation, Incubation, Lysis, LDH Cytotoxicity Assay

Journal: Journal of hepatology

Article Title: Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

doi: 10.1016/j.jhep.2018.10.040

Figure Lengend Snippet: Cells were generated or isolated from C57BL/6 mice. CIK cells and (A) CD11b+Ly6G+Ly6Clow PMN-MDSCs or (B) CD11b+Ly6G−Ly6Chigh M-MDSCs were co-cultured in the presence or absence of N(G)-monomethyl-L-arginine (L-NMMA), N-omega-hydroxy-L-arginine (L-NOHA) (10 μmol/L each), tadalafil (100 μmol/L each), and/or media alone as indicated. After 36 hours, RIL-175 cells were added at a ratio of 10:1 (E:T) and lysis was determined by LDH cytotoxicity assay. CIK cells, purified MDSCs (defined as CD11b+Ly6G+Ly6Clow or CD11b+Ly6G−Ly6Chigh cells), and target cells (5,000 cells per well) were co-cultured at a ratio of 10:1:1 (CIK cells: MDSCs: tumor cells). Cumulative results from three independent experiments are shown. Mean ± SEM. *P=0.001.

Article Snippet: Tissues were fixed and stained with anti-Ly6G (clone 6D17, Acris Antibodies GmbH, Germany).

Techniques: Generated, Isolation, Cell Culture, Lysis, LDH Cytotoxicity Assay, Purification

Journal: Journal of hepatology

Article Title: Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

doi: 10.1016/j.jhep.2018.10.040

Figure Lengend Snippet: (A) Male C57BL/6 mice were subcutaneously inoculated with 1 × 106 RIL-175 HCC cells. An investigator blinded to experimental treatment group assignments used calipers to measure the tumor size. Then, 1 to 2 weeks later, tadalafil (2 mg/kg), a PDE5 inhibitor, was administered to tumor-bearing mice daily for 5 days by i.p. injection. n=5 for control, n=5 for tadalafil, Student’s t-test, *n.s. not significant. The frequency of (B) intratumoral and (C) splenic MDSCs was determined by flow cytometry. Total MDSCs were defined as CD45+CD11b+GR1+ cells, while PMN-MDSCs and M-MDSCs were gated as CD11b+Ly6G+Ly6Clow and CD11b+Ly6G−Ly6Chigh cells, respectively. The gating strategy from a representative sample is shown in Supplemental Fig. 2. n=5, Student’s t-test, *P<0.05. Mean ± SEM. (D) CD11b expression was evaluated by quantitative real-time PCR. n=5 for control, n=5 for tadalafil, Student’s t-test, *P<0.05. (E) A cytokine array compared sera from tadalafil-treated RIL-175 tumor-bearing mice with sera of control mice. Data were expressed relative to control mice. n=5 independent experiments. (F & G) CIK cells were incubated with different ratios of both subsets of MDSCs, which were isolated from tadalafil-treated RIL-175 tumor-bearing mice, as indicated. After 24 hr, RIL-175 cells were added at a ratio of 10:1 (E:T), and lysis was determined by LDH cytotoxicity assay. Cumulative results from three independent experiments are shown. Mean ± SEM.

Article Snippet: Tissues were fixed and stained with anti-Ly6G (clone 6D17, Acris Antibodies GmbH, Germany).

Techniques: Injection, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Incubation, Isolation, Lysis, LDH Cytotoxicity Assay

Journal: Journal of hepatology

Article Title: Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

doi: 10.1016/j.jhep.2018.10.040

Figure Lengend Snippet: (A) RIL-175 hepatoma cells (5 × 105 /20 μL) were orthotopically implanted into the livers of recipient male B6(Cg)-Tyrc-2J/J mice. The establishment and growth of tumors were blindly monitored by BLI with the Xenogen IVIS. BLI represents the proliferation rate through total flux signals of luciferase. The mice were followed-up for 21 days. Tumor-bearing mice were intravenously injected with 5 × 106 CIK cells on day 7. Tadalafil (2 mg/kg), a PDE5 inhibitor, was daily administered to tumor-bearing mice by i.p. injection. (B) Analysis of BLI images was performed by Living Image 2.50 software (PerkinElmer, Waltham, MA, USA). Calculated ROIs are graphed. n=5 for control, n=5 for CIK, n=5 for tadalafil, and n=5 for the combination, two-way ANOVA. *P=0.0028 for Control vs. Combination. (C) The liver/body weight ratio was significantly reduced in the combination treatment (CIK cells + tadalafil) group compared with the other groups. n=5 for control, n=5 for CIK, n=5 for tadalafil, and n=5 for the combination, Student’s t-test. *P=0.0022 for Control vs. Combination; **P=0.0011 for CIK vs. Combination; †=0.0465 for tadalafil vs. Combination. (D) The tumor/liver weight ratio was significantly reduced in the combination treatment group compared with the other groups. n=5 for control, n=5 for CIK, n=5 for tadalafil, and n=5 for the combination, Student’s t-test. *P = 0.0184 for Control vs. Combination; **P = 0.0158 for CIK vs. Combination; †P = 0.0473 for tadalafil vs. Combination. Accumulation of (E) intrahepatic tumoral and (F) splenic MDSCs was determined by flow cytometry. Total MDSCs were defined as CD45+CD11b+GR1+ cells, while PMN-MDSCs and M-MDSCs were gated as CD11b+Ly6G+Ly6Clow and CD11b+Ly6G−Ly6Chigh cells, respectively. Student’s t-test. P < 0.001 for Control vs. CIK. **P < 0.05 for CIK vs. Combination. Control (black dots; n=5), CIK-treated (purple reverse triangle; n=5), tadalafil-treated (red rectangles; n=5), and combination-treated (blue triangles; n=5) mice. Mean ± SEM. n.s., not significant.

Article Snippet: Tissues were fixed and stained with anti-Ly6G (clone 6D17, Acris Antibodies GmbH, Germany).

Techniques: Luciferase, Injection, Software, Flow Cytometry