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

Journal: Journal of Cellular Biochemistry

Article Title: ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1002/jcb.25634

Figure Lengend Snippet: ZBTB16 expression depends on OIM during osteoblastic differentiation. (A) Three hMSCs (hMSCs #1, #2, and #3) were cultured with or without OIM (containing AA, β‐GP, and Dex) for 4, 7, and 14 d. The mRNA levels of ZBTB16 were evaluated by TaqMan assays. N.D.: not detectable. The bars represent the mean ± S.D. of triplicate wells. The results are representative of three independent experiments. β‐Actin was used as an internal control. (B) hBMMSCs were cultured with or without OIM for 4, 7, and 14 d. (C) Western blot analysis of ZBTB16 using cell lysates from hMSCs cultured with or without OIM for 4, 7, and 14 d. β‐Actin was used as an internal control. Representative data from three different samples are shown in (B) and (C); a similar tendency was observed in other samples. The experiments shown in Figure A and B were performed in triplicate.

Article Snippet: The primers used were as follows: ZBTB16 (Hs00957433_m1), Osx (Hs00541729_m1), RUNX2 (Hs00231692_m1), OCN (Hs01587814_g1), BSP (Hs00173720_m1), and β‐actin (43263251E; all of the probes were obtained from Applied Biosystems).

Techniques: Expressing, Cell Culture, Control, Western Blot

Journal: Journal of Cellular Biochemistry

Article Title: ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1002/jcb.25634

Figure Lengend Snippet: The inhibitory effect of siZBTB16 on ALP activity, mineralized nodule formation, and osteogenic gene expression in hMSCs. (A) The efficacy of the siRNA inhibiting ZBTB16 mRNA expression. (B) The effect of siRNA targeting ZBTB16 in hMSCs on the osteoblastic differentiation of hMSCs. The ALP activity of hMSCs after transfection and culture with or without OIM for 5 d. (C) The effect of siRNA targeting ZBTB16 on the osteoblastic differentiation of hBMMSCs. The ALP activity of hBMMSCs after they were transfected and cultured with or without OIM for 5 d. (D) The cell viability of hMSCs was measured by MTS assay after they were transfected and cultured with or without OIM for 5 d. * P < 0.05, compared with non‐siRNA and siControl. (E) Alizarin red S staining of hMSCs after the cells were transfected with siZBTB16 and cultured with or without OIM for 21 d. (F) Bound stain was eluted with a solution of 10% cetylpyridinium chloride and quantified by measuring the absorbance at a wavelength of 570 nm using a plate reader. (G) Effect of a siRNA targeting ZBTB16 in hMSCs on OCN and BSP expression after hMSCs were transfected and cultured with OIM for 5 d. * P < 0.05, compared with siControl. (−), non‐siRNA; siCont., siControl. These figures show representative data from three different samples; a similar tendency was observed in other hMSCs. The experiments shown in Figure A, B–D, F, and G were performed in triplicate.

Article Snippet: The primers used were as follows: ZBTB16 (Hs00957433_m1), Osx (Hs00541729_m1), RUNX2 (Hs00231692_m1), OCN (Hs01587814_g1), BSP (Hs00173720_m1), and β‐actin (43263251E; all of the probes were obtained from Applied Biosystems).

Techniques: Activity Assay, Gene Expression, Expressing, Transfection, Cell Culture, MTS Assay, Staining

Journal: Journal of Cellular Biochemistry

Article Title: ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1002/jcb.25634

Figure Lengend Snippet: The mRNA and protein levels of ZBTB16 were decreased by siOsx. (A) hMSCs were transfected with non (siCont.)‐ or Osx (siOsx1 and siOsx2)‐specific siRNA and cultured with OIM. RNA was extracted, and real‐time RT‐PCR was performed for Osx and ZBTB16. (B) hMSCs were transfected with non (siCont.)‐ or RUNX2 (siRUNX2)‐specific siRNA and cultured with OIM. RNA was extracted, and real‐time RT‐PCR was performed for RUNX2 and ZBTB16. (C) Western blot analysis of ZBTB16 using cell lysates from hMSCs after they were transfected and cultured with OIM. * P < 0.05, compared with siControl. These figures show representative data from three different samples; a similar tendency was observed in other hMSCs. The experiments shown in Figure A and B were performed in triplicate.

Article Snippet: The primers used were as follows: ZBTB16 (Hs00957433_m1), Osx (Hs00541729_m1), RUNX2 (Hs00231692_m1), OCN (Hs01587814_g1), BSP (Hs00173720_m1), and β‐actin (43263251E; all of the probes were obtained from Applied Biosystems).

Techniques: Transfection, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Journal of Cellular Biochemistry

Article Title: ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1002/jcb.25634

Figure Lengend Snippet: Osx binds to the Sp1 sequence of the ZBTB16 promoter region. (A) The diagram shows the location of the primers used to amplify the Sp1 or the non‐Sp1 site of the ZBTB16 promoter region in the ChIP assay. TSS, transcription start site of ZBTB16; P1, primer 1 set; P2, primer 2 set; P3 (negative control), primer 3 set. (B) hMSCs were cultured with OIM and rhBMP‐6 (200 ng/ml) for 7 d. ChIP assay was performed with antibodies against Osx and IgG (negative control). Ten percent of the input was loaded as a control. This figure shows representative data from three different samples; a similar tendency was observed in other hMSCs.

Article Snippet: The primers used were as follows: ZBTB16 (Hs00957433_m1), Osx (Hs00541729_m1), RUNX2 (Hs00231692_m1), OCN (Hs01587814_g1), BSP (Hs00173720_m1), and β‐actin (43263251E; all of the probes were obtained from Applied Biosystems).

Techniques: Sequencing, Negative Control, Cell Culture, Control

Journal: bioRxiv

Article Title: Differential retinoic acid responses across testicular development in vitro

doi: 10.64898/2025.12.30.696417

Figure Lengend Snippet: CIVMs derived from neonatal testis from PND 5 and PND 10 testis were exposure to isotretinoin for 24 hours. (A) Expression of Stra8 in PND 5 derived CIVMs determined by RT-qPCR. In the control, 0.3, 3, and 30 nM isotretinoin dose groups the expression of Stra8 contained samples below the threshold of quantification. The expression of Stra8 was significantly increased in the 300 nM isotretinoin dose group (p-value <0.0001). (B) Expression of Plzf in PND 5 derived CIVMs determined by RT-qPCR. All samples were above the threshold of quantification. The expression of Plzf in the 300 nM isotretinoin dose group was significantly lower than control (p-value of 0.026). (C) Expression of Stra8 in PND 10 derived CIVMs determined by RT-qPCR. Aside from one control sample, all samples were above the threshold of quantification. The expression of Stra8 was significantly increased in the 3,30, and 300 nM isotretinoin dose groups (p-values all <0.0001). (D) Expression of Plzf in PND 10 derived CIVMs determined by RT-qPCR. All samples were above the threshold of quantification. The expression of Plzf in the 30 and 300nM isotretinoin dose groups were significantly lower than control (p-values <0.0001).

Article Snippet: RT-qPCR was used to target the rat genes Stra8 (Rn01747849_m1) and Plzf (Rn01418644_m1).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Differential retinoic acid responses across testicular development in vitro

doi: 10.64898/2025.12.30.696417

Figure Lengend Snippet: The expression of Plzf and Stra8 quantified by RT-qPCR at day in vitro 4 and day in vitro 15 in PND 5 tissue CIVMs and PND 10 tissue CIVMs in control and media supplementation conditions. Color indicates day in vitro , dashed line divides plot by PND 5 CIVMs and PND 10 CIVMs. Points that are “X” indicate samples that were below the limit of quantification (cycle threshold of 37). Data displayed as expression relative to beta-actin (housekeeping gene (HK), (2^ [gene – HK])).

Article Snippet: RT-qPCR was used to target the rat genes Stra8 (Rn01747849_m1) and Plzf (Rn01418644_m1).

Techniques: Expressing, Quantitative RT-PCR, In Vitro, Control

Journal: PLoS ONE

Article Title: Tzfp Represses the Androgen Receptor in Mouse Testis

doi: 10.1371/journal.pone.0062314

Figure Lengend Snippet: (A) Overview of the Tzfp protein with the BTB/POZ and zink finger domains (top), and of the wild-type and mutated Tzfp allele upon insertional mutagenesis (middle). The wild-type and mutated Tzfp transcript is also included (bottom). (B) Genotyping gel showing fragments indicating samples from wild-type, heterozygous and Tzfp GTi/GTi mice. (C) Gene expression analysis of Mll4 and Plzf in wild-type and Tzfp GTi/GTi testis reveals no significant difference between wild-type and Tzfp GTi/GTi testis. (D) Crosses between heterozygous animals reveal a sex-ratio distortion and fewer than expected homozygous mutant pups. The χ2 -test was used to determine significance. (E) Weight distribution of testis and epididymus in wild-type and Tzfp GTi/GTi mouse reveal a small, but not significant reduction in testis weight in the Tzfp GTi/GTi mice.

Article Snippet: Plzf: Mm01176865_m1.

Techniques: Mutagenesis, Gene Expression