Journal: International neurourology journal

Article Title: Striatal ZBTB16 Is Associated With Cognitive Deficits in Alzheimer Disease Mice.

doi: 10.5213/inj.2244254.127

Figure Lengend Snippet: Fig. 1. Quantitative gene expression profiling analysis of the stri atal transcriptome of the amyloid precursor protein/presenilin 1 (APP/PS1) mice revealed striatum-specific differentially ex pressed genes (DEGs) including ZBTB16. (A) Summarized flow chart of the 3’ mRNA-sequencing analysis showing the criteria of DEG selection using the fold change (FC) values from the quan titative RNA sequencing data. (B) Heat-map presentation of the gene expression FC values of 33 DEGs from the mRNA-se quencing results from individual samples of wildtype (WT) and APP/PS1 mice. (C, D) Additional validation of gene expression of ZBTB16 in the mRNA level (C, quantitative polymerase chain reaction [qPCR]) and in the protein level (D, western blot analy sis). AD, Alzheimer disease. *P<0.05. **P<0.01.

Article Snippet: Knockdown shRNA against ZBTB16 (TL507125, Origene, Rockville, MD, USA), shRNA scrambled control was packaged into pseudo-lentivirus particles following the manufacturer instructions (EndoFectin, EFL1001-01, Genecopoeia, Rockville, MD, USA).

Techniques: Gene Expression, Sequencing, Selection, RNA Sequencing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Western Blot

Journal: International neurourology journal

Article Title: Striatal ZBTB16 Is Associated With Cognitive Deficits in Alzheimer Disease Mice.

doi: 10.5213/inj.2244254.127

Figure Lengend Snippet: Fig. 2. DNA methylation analysis with the resolution of single-cell via Brain Cell Methylation Viewer showed specific mCH (methyl ated non-CpG cytosine, H denotes A, C, or T) patterns of the ZBTB16 gene body. Single-cell methylome analysis via Brain Cell Meth ylation Viewer was used to compare the level of the mCH and the methylated CpG levels of 33 differentially expressed genes (DEGs). When confirming the gene body methylation level of the 12 increased DEGs of the early Alzheimer disease (bottom panels), the mCH level for the gene body of ZBTB16 was specifically increased (upper box). And this increase in mCH methylation was specific to D1 MSN and D2 MSN, which are the main neuron types of striatum.

Article Snippet: Knockdown shRNA against ZBTB16 (TL507125, Origene, Rockville, MD, USA), shRNA scrambled control was packaged into pseudo-lentivirus particles following the manufacturer instructions (EndoFectin, EFL1001-01, Genecopoeia, Rockville, MD, USA).

Techniques: DNA Methylation Assay, Methylation

Journal: International neurourology journal

Article Title: Striatal ZBTB16 Is Associated With Cognitive Deficits in Alzheimer Disease Mice.

doi: 10.5213/inj.2244254.127

Figure Lengend Snippet: Fig. 3. Transcriptomic alteration of the striatal region of amyloid precursor protein/presenilin 1 (APP/PS1) was associated with the function of zinc finger and BTB domain-containing protein 16 (ZBTB16) as a transcription factor. (A) Brief schematic flowchart of the chromatin immunoprecipitation-X enrichment analysis 3 (ChEA3) transcription factor enrichment analysis of the gene sets se lected from the APP/PS1 striatal transcriptome changes in chapter 1. (B) Detail information of ZBTB16 from the ChEA3 transcrip tion factor enrichment analysis for the altered transcriptome of the early Alzheimer disease (AD) and the late AD. Abbreviated 5 la bels in the last column matched with the information of each library listed in panel A. (C) Functional annotation categories for the downstream overlapping genes of ZBTB16. WT, wildtype; DEG, differentially expressed genes; FC, fold change.

Article Snippet: Knockdown shRNA against ZBTB16 (TL507125, Origene, Rockville, MD, USA), shRNA scrambled control was packaged into pseudo-lentivirus particles following the manufacturer instructions (EndoFectin, EFL1001-01, Genecopoeia, Rockville, MD, USA).

Techniques: Chromatin Immunoprecipitation, Functional Assay

Journal: International neurourology journal

Article Title: Striatal ZBTB16 Is Associated With Cognitive Deficits in Alzheimer Disease Mice.

doi: 10.5213/inj.2244254.127

Figure Lengend Snippet: Fig. 4. Behavioral defects were rescued by knockdown of striatal ZBTB16 of 6-month-old amyloid precursor protein/presenilin 1 (APP/PS1). (A) Cognitive function of wildtype (WT) and APP/PS1 mice of 6 months of age to validate the zinc finger and BTB do main-containing protein 16 (ZBTB16) knockdown effect. Memory function of WT+GFP (green fluorescent protein), APP/PS1+GFP, and APP/PS1+shZBTB16. Alterations in Y-maze test (YMT) and recognition index of noble object recognition (NOR) test are indi cated by percentage. N and F indicate Novel and Familiar objects, respectively. (B) Time spent in chambers shown as a measure of so ciability in the first session of the 3-chamber test (3CT) of WT+GFP, APP/PS1+GFP, and APP/PS1+shZBTB16 groups. And Time spent in chambers as a measure of novelty-seeking in the second session of 3CT. The early stage of APP/PS1 showed deficit in social novelty phenotype. And these deficits were rescued by ZBTB16 knockdown. S1 indicates stranger 1, S2 indicates stranger 2, and O in dicates object. (C) In elevated plus maze (EPM) and light-dark box (LDB) test, time spent on the open arm and light box were shown in seconds. ns, not significant. *P<0.05. **P<0.01. ***P<0.001.

Article Snippet: Knockdown shRNA against ZBTB16 (TL507125, Origene, Rockville, MD, USA), shRNA scrambled control was packaged into pseudo-lentivirus particles following the manufacturer instructions (EndoFectin, EFL1001-01, Genecopoeia, Rockville, MD, USA).

Techniques: Knockdown

Journal: International neurourology journal

Article Title: Striatal ZBTB16 Is Associated With Cognitive Deficits in Alzheimer Disease Mice.

doi: 10.5213/inj.2244254.127

Figure Lengend Snippet: Fig. 5. Knockdown of zinc finger and BTB domain-containing protein 16 (ZBTB16) at 5 months rescued behavioral defects in 12-month-old amyloid precursor protein/presenilin 1 (APP/PS1). (A) Similar rescue patterns in 3-chamber test (3CT) were also ob served in the 12-month-old APP/PS1+shZBTB16 group (social novelty). (B) In Y-maze test (YMT) and noble object recognition (NOR) test, cognitive impairments in APP/PS1 (12-month-old) were also rescued by ZBTB16 knockdown. (C) Deficits in anxiety- like phenotypes of APP/PS1+GFP (green fluorescent protein) were not rescued by ZBTB16 knockdown in the elevated plus maze (EPM) and light-dark box (LDB) tests. (D) Bar graphs showing the cumulative number of firings from all stimulation currents (from 125 nA to 1,000 nA currents). (E, F) Representative heat-map image and quantification (histogram densitometry) bar graph of neuro nal activity on the multielectrode array probes. APP/PS1+ZBTB16 knockdown group showed rescued patterns. ns, not significant. *P<0.05. **P<0.01. ***P<0.001. ****P<0.0001.

Article Snippet: Knockdown shRNA against ZBTB16 (TL507125, Origene, Rockville, MD, USA), shRNA scrambled control was packaged into pseudo-lentivirus particles following the manufacturer instructions (EndoFectin, EFL1001-01, Genecopoeia, Rockville, MD, USA).

Techniques: Knockdown, Activity Assay

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 1. Endometrial stromal and glandular cells display enhanced ZBTB16 immunoreactivity in women administered depo-medroxyprogesterone acetate (DMPA). (A) Representative ZBTB16 im- munostaining (brown) in paraffin sections from paired endometria obtained from pre- and 3 months post-DMPA administration. Original magnification ×40. Enhanced ZBTB16 immunoreactivity was observed in post-DMPA endometrial compared to pre-DMPA. A strong ZBTB16 immunoreactivity is seen in post-DPMA vascular endothelium (arrows) in the inset micrographs. Original magnification ×100. (B) HSCOREs for ZBTB16 immunoreactivity confirmed significantly higher ZBTB16 immunore- activity in both endometrial stromal (185.4 ± 9.2 vs. 92.1 ± 13.8) and glandular cells (181.3 ± 20.4 vs. 47.7 ± 14.7), as well as endothelial cells (185.3 ± 19.9 vs. 45.4 ± 16.3; p < 0.001) in post- vs. pre-DMPA endometria. Bars represent the mean ± SEM; n = 7/each; *** p < 0.001 vs. pre-DMPA analyzed by the t-test.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Activity Assay

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 2. MPA administration induces endometrial ZBTB16 expression in guinea pigs. (A) Repre- sentative images of ZBTB16 immunostaining in endometria obtained from ovariectomized guinea pigs treated 21 days with placebo as the control (Cont; n = 5), or estradiol (E2; n = 4), or medrox- yprogesterone acetate (MPA; n = 5), or E2 + MPA (n = 6). Original magnification: ×20. The insert represents negative control staining. (B) HSCORE analysis of ZBTB16 immunoreactivity in stromal and glandular cells of endometria. Bars represent the mean ± SEM; * p < 0.05 vs. the control or E2 treatment in stromal cells, and * p < 0.05 vs. the control or E2 treatment in glandular epithelial cells analyzed by one-way ANOVA followed by the Holm–Sidak method.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Expressing, Immunostaining, Control, Negative Control, Staining

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 3. Increased ZBTB16 expression during decidualization of cultured HESCs. (A) ZBTB16 mRNA levels in HESCs treated with 10−8 M E2 + 10−7 MPA + 5 × 10−5 cyclic AMP (EMC) for 0, 3, or 6 days by qPCR. The data represent the fold change as the mean ± SEM; n = 5/each; *** p < 0.001 vs. Day 0 analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. (B) MPA mediated upregulation of ZBTB16 mRNA levels in HESCs treated with vehicle (control; Cont) or E2 or MPA or cAMP for 6 days. Bars represent mean ± SEM; n = 4/each; * p < 0.05 vs. Cont or E2 or cAMP analyzed by one way ANOVA followed by Student-Newman-Keuls method.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Expressing, Cell Culture, Control

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 4. pLARCs induce ZBTB16 mRNA and protein levels in cultured HESCs. ZBTB16 mRNA (A) and protein (B) levels were analyzed by qPCR and immunoblotting, respectively, in HESCs treated with 10−8 M E2 ± 10−7 M ORG, ETO, LNG, MPA, or DEX for 7 days. Bars represent the mean ± SEM; (A) n = 4 for mRNA fold change; * p < 0.05 vs. E2 alone; and (B) n = 3 for protein levels after normalization to β-actin; * p < 0.05 vs. E2 alone analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. E2: estradiol, ORG: Organon 2058, ETO: etonogestrel, LNG: levonorgestrel, MPA: medroxyprogesterone acetate, DEX: dexamethasone. (C) NR3C1, ZBTB16, and FKBP5 mRNA levels in HESCs transfected with either the nonspecific control (Cont) or NR3C1- specific siRNA and treated with EMC for 3 days. Bars represent the mean ± SEM; n = 3; *** p < 0.001 vs. control siRNA by the t-test.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Cell Culture, Western Blot, Transfection, Control

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 5. ZBTB16 overexpression induces decidualization markers, as well as F3, IL-8, and PTGS2 levels in HESCs. (A) Decidualization markers’, PRL and IGFBP1, mRNA levels in ZBTB16-vector (ZBTB16-v) or control-vector (Cont-v)-transfected HESCs treated with or without 10−8 M estra- diol + 10−7 M medroxyprogesterone acetate + 5 × 10−5 M cAMP (EMC) for 3 days. Bars represent the mean ± SEM; n = 4; *** p < 0.05 vs. control-v-EMC; + p < 0.05 vs. ZBTB16-v + EMC. (B) Tissue factor (F3) mRNA levels in control- (Cont-v) or ZBTB16-vector (ZBTB16-v)-transfected HESCs treated with or without EMC for 3 days ± 1 U/mL of thrombin (THR) for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; + p < 0.05 vs. Cont-v; # p < 0.05 vs. Cont-v + THR. (C) Interleukin 8 (IL-8) and prostaglandin endoperoxide synthase 2 (PTGS2 aka cyclooxygenase 2; COX2) mRNA levels in the control- or ZBTB16-vector-transfected HESCs treated with EMC for 3 days ± 1 U/mL THR for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; + p < 0.05 vs. Cont-v; # p < 0.05 vs. Cont-v + THR. The data were analyzed by one-way ANOVA followed by the Student–Newman–Keuls method.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Control, Transfection

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 6. Schematic demonstration of the role of elevated ZBTB16 expression in the pathogenesis of pLARC-induced AUB. Administration of progestin-only, long-acting, reversible contraception (pLARCs) reduce uterine blood flow, which causes local hypoxia [8,9] and induces HESC decidu- alization, which increases tissue factor [15,23] and ZBTB16 levels. Increased ZBTB16 levels induce excess tissue factor expression, which generates thrombin. The resulting excess thrombin binds to protease-activated receptors (PARs), which increase the expression of several inflammatory factors and angiogenic factors, such as VEGF or IL-8 [19,20,48]. These factors promote excess angiogenesis, which results in abnormal uterine bleeding. pLARC-induced excess ZBTB16 exacerbates thrombin- induced endometrial angiogenesis and inflammation by increasing IL8 and PTGS2 levels in HESCs.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

doi: 10.18632/oncotarget.4045

Figure Lengend Snippet: Figure 1: Plzf expression is elevated in aged mouse testes. A, qPCR analysis of Plzf mRNA in young and aged testes normalized to Ddx4. Graphs represent mean value ± s.e.m. n = 9 mice per group. *P < 0.05. B, representative images of IHC for Plzf in young and aged testes. Images are representative of testes from 3 mice examined. Arrow, low Plzf. Arrowhead, high Plzf. *, degenerative seminiferous tubule. C, representative flow profile of detecting Plzf-expressing cells by intracellular staining and FACS by using anti-Plzf antibody (PE- conjugated). D, quantification of the percentage of Plzf-positive cells analyzed by FACS in panel C. Graphs represent mean value ± s.e.m. n = 6 mice per group. *P < 0.05. E, Plzf intensity in Plzf-expressing cells from young versus aged mice. F, Quantification of Plzf expression levels in young and aged testes. Graphs represent mean value ± s.e.m. n = 4 mice per group. *P < 0.05.

Article Snippet: Plzf expression plasmid was obtained from Origene (RC206745).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

doi: 10.18632/oncotarget.4045

Figure Lengend Snippet: Figure 5: Plzf expression inhibits RA-induced Stra8 transcription in F9 cells and correlates with a lack of Stra8- expressing cells in aged testes. A, qPCR analysis for Stra8 mRNA in F9 cells normalized to β-actin. Graphs represent mean value ± s.e.m. n = 3 independent cultures per group. *P < 0.05. B, western blotting analysis of Stra8 in F9 cells. Data are representative of 2 independent sets of experiments. C, dual-IF staining for Plzf and Stra8 in young (left) and aged (right) testes. Arrow, Plzf-expressing cells. Arrowhead, Stra8-expressing cells. D, IHC for Stra8 in aged testes. *, degenerative seminiferous tubule.

Article Snippet: Plzf expression plasmid was obtained from Origene (RC206745).

Techniques: Expressing, Western Blot, Staining

Journal: Oncotarget

Article Title: Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

doi: 10.18632/oncotarget.4045

Figure Lengend Snippet: Figure 6: Schematics of Plzf overexpression and spermatogonia aging. Loss of Plzf exhausts spermatogonia pool by promoting their differentiation at the expense of self-renewal. On the contrary, age-related increase in Plzf expression causes spermatogonia hypermaintenance and aging by preventing their differentiation.

Article Snippet: Plzf expression plasmid was obtained from Origene (RC206745).

Techniques: Over Expression, Expressing

Journal: The Journal of Experimental Medicine

Article Title: STAT-3–independent production of IL-17 by mouse innate-like αβ T cells controls ocular infection

doi: 10.1084/jem.20170369

Figure Lengend Snippet: IL-17–producing memory-like αβ T cells in TKO mice express the innate-associated transcription factor PLZF and do not phosphorylate STAT-3. (A) CD44 hi CD62L low CD4 + , CD8 + , DNT, and iNKT cells were sorted, stimulated with IL-1β and/or αCD3 for 72 h, fixed, and permeabilized. Cells were then stained for intracellular IL-17, PLZF, and lineage-specific markers. The representative histogram (from two independent experiments) depicts the PLZF expression in IL-17 + cells from the specified populations. (B) CD44 hi CD8 + and DNT cells from TKO mice were sorted and stimulated with CD3 and IL-1β in the presence of siRNA against PLZF for 72 h, and brefeldin A was added the last 6 h of culture. Cells were then stained for intracellular IL-17 and PLZF and analyzed by flow cytometry. Black bars represent IL-17 production, and white bars represent PLZF expression in the IL-17 + population. (C) CD4 + and CD8 + CD44 hi CD62L low αβ TCR + γδ TCR − cells from pooled ( n = 5) LNs of WT or β2m −/− mice. Data represent two independent experiments. (D) CD44 hi CD62L low CD4 + or CD8 + /DNT cells were sorted as stated previously from IL-6/21R/23R TKO and cultured with IL-1β and αCD3 for 72 h. After stimulation, cells were stained for IL-17 and Vβ6. Data represent two individual experiments. (E) CD44 hi CD62L low CD8 + and DNT cells were isolated, combined, and cultured with IL-1β and/or αCD3 for 30 min and 18 h. After stimulation, cells were harvested, fixed, permeabilized, and stained for phospho–STAT-3. Symbols represent individual mice from two independent experiments. Bars represent mean ± SEM. MFI of pSTAT-3 in live cells. One-way ANOVA determined significance (*, P = 0.0295; ***, P < 0.0036). (F–H) WT CD44 hi CD62L low CD8 + , DN, and iNKT cells were sorted as described previously. Each population was cultured with either IL-1β and αCD3 with or without αIL-6/αIL-6Rα/αIL-21/αIL-23p19/αIL-23R. After 72 h, cells were harvested, fixed, permeabilized, and stained for (F) intracellular IL-17 and (G and H) phospho–STAT-3. Symbols represent technical replicates from a single experiment that consisted of pooling cells from three mice. Bars represent the mean (E) IL-17% or (G) pSTAT-3 MFI ± SEM, and data are representative of two independent experiments. Student’s t test was used to determine significance (for F, *, P = 0.0274; ***, P = 0.0005; ****, P < 0.0001; for G, *, P = 0.0449; **, P < 0.0021).

Article Snippet: Three siRNA constructs targeting PLZF (Zbtb16 ID235320 Trilencer-27 Mouse siRNA, catalog SR41884) were purchased from OriGene.

Techniques: Staining, Expressing, Flow Cytometry, Cell Culture, Isolation

Journal: The Journal of Experimental Medicine

Article Title: STAT-3–independent production of IL-17 by mouse innate-like αβ T cells controls ocular infection

doi: 10.1084/jem.20170369

Figure Lengend Snippet: STAT-3–independent IL-17–producing innate-like T cells exist in the thymus. (A and B) Single-cell suspensions of thymuses from 6-wk-old WT, TKO, or STAT-3 KO (S3K) mice were either stained for PLZF expression (A) or stimulated with CD3 and IL-1β for 72 h (B). Gating strategies are depicted in Fig. S2 A. Histograms represent the PLZF staining. Flow plots represent staining pattern in the thymus after stimulation with CD3 plus IL-1β. Bars in graphs represent the MFI of PLZF staining ± SEM ( n = 3 for WT and TKO and n = 5 for S3K; *, P = 0.0130; ***, P = 0.0006). Data represent two independent experiments. (B) Flow plots represent the staining pattern after stimulation with either CD3 or CD3 and IL-1β (no stim samples yielded very few cells). Brefeldin A was added the last 6 h of culture, and IL-17 was assessed by flow cytometry. Bars represent the mean frequency ± SEM of cells that were IL-17 + ( n = 3 for WT and TKO and n = 5 for S3K; *, P < 0.05; **, P < 0.01; ****, P < 0.0001). Statistical significance was determined using ANOVA. (C) 2 × 10 4 CD44 hi CD8/DNT S3K thymocytes or 2 × 10 5 naive CD44 hi CD8/DNT S3K cells were transferred i.v. into Rag2/Il2rg DKO mice. After 4 wk, splenic cells were isolated and stimulated with CD3 and IL-1β and/or IL-23 for 72 h. Brefeldin A was added the last 6 h of culture, and cells were stained for intracellular IL-17. Bars represent the mean frequency ± SEM of IL-17 + cells after culture ( n = 3 for WT and S3K naive, n = 5 for S3K Thym.). Data represent two independent experiments. Statistical significance was determine by ANOVA (**, P = 0.002).

Article Snippet: Three siRNA constructs targeting PLZF (Zbtb16 ID235320 Trilencer-27 Mouse siRNA, catalog SR41884) were purchased from OriGene.

Techniques: Staining, Expressing, Flow Cytometry, Isolation

Journal: Methods in Molecular Biology (Clifton, N.j.)

Article Title: Isolation of undifferentiated and early differentiating Type A spermatogonia from Pou5f1-GFP reporter mice

doi: 10.1007/978-1-61779-436-0_3

Figure Lengend Snippet: Undifferentiated and early differentiating spermatogonia isolated through Fluorescence Activated Cell Sorting (FACS). (A) Flow-cytometric analysis and sorting of wild-type and Pou5f1-GFPMann cells stained with APC-Kit antibody. (B) Real-time-PCR analysis of Kit and canonical markers of undifferentiated spermatogonia in Pou5f1+/Kit- and Pou5f1+/Kit+ cells sorted from A. Cells were processed for RT-PCR using the TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems, Carlsbad, CA) according to manufacturer’s instructions. The expression value of each gene was normalized to the amount of an internal control gene (Eif3land Rps3) (29) and a relative quantitative fold change was determined using the ΔΔ Ct method. Experiments were performed in triplicate and data are represented as mean ± standard error. Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA) used for specific transcripts were: Mm00460859_m1 (Eif3l), Mm00833897_m1 (Gfra1), Mm00445212_m1 (Kit), Mm00437606_s1 (Neurog3), Mm00658129_gH (Pou5f1), Mm00656272_m1 (Rps3), Mm00493681_m1 (Thy1), and Mm01176868_m1 (Zbtb16). Normalization to Eif3l and Rps3 produced identical results.

Article Snippet: Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA) used for specific transcripts were: Mm00460859_m1 ( Eif3l ), Mm00833897_m1 ( Gfra1 ), Mm00445212_m1 ( Kit ), Mm00437606_s1 ( Neurog3 ), Mm00658129_gH ( Pou5f1 ), Mm00656272_m1 ( Rps3 ), Mm00493681_m1 ( Thy1 ), and Mm01176868_m1 ( Zbtb16 ).

Techniques: Isolation, Fluorescence, FACS, Staining, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Control, Produced