Journal: Cell Death and Differentiation

Article Title: Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway

doi: 10.1038/s41418-020-00670-2

Figure Lengend Snippet: A Mast4 genomic structure is shown. MAST4 has highly conserved kinase domain and PDZ domain. To produce the Mast4 KO mice, 71 base pairs of Mast4 gene are deleted and a frameshift is produced on exon 1 so that translation stops. B At PN 6W, Mast4 KO mice has smaller testes compared to WT mice. C The total sperm number is significantly decreased in Mast4 KO testes (n = 4). D At PN 1D, the migrating germ cells are observed in the developing seminiferous tubules. E MAST4 is expressed in the cytoplasm of the developing Sertoli cells while SOX9 is expressed in the nuclei of the Sertoli cells (arrowhead). F Similarly, strong expression patterns of AMH are localized in the developing Sertoli cells with MAST4 co-localization (arrowhead). G DDX4 is expressed in the cytoplasm of the germ cell (arrowhead), which is located in the outermost layer of seminiferous tubules. H At PN 3 W, the testes have a developed structure with differentiating spermatogonia and Leydig cells. I The expression of MAST4 is shifted to Leydig cells while SOX9 is continuously expressed in Sertoli cells (arrowhead). J MAST4 is expressed in Leydig cells with co-localization of 3β-HSD (arrowhead), while K DDX4 is expressed in spermatogonia located throughout seminiferous tubules (arrowhead). L At PN 6W, WT testes have compact seminiferous tubules with all types of germ cells. M Mast4 KO testis has seminiferous tubules with depleted germ cells or SCO morphology (arrowhead). N MAST4 is expressed in Leydig cells and spermatids although SOX9 is expressed in the nuclei of Sertoli cells in WT testes (arrowhead). O In Mast4 KO testes, SOX9 is strongly expressed in Sertoli cells located at the outermost layer of the SCO tubules along with Sertoli cells in normal seminiferous tubule. P Expression of AMH is not detected in WT pubertal testes (arrow). Q In Mast4 KO testes, AMH is expressed in the SCO tubules (arrowhead). R DDX4 is expressed mainly in differentiating spermatogonia (arrowhead). S In Mast4 KO testes, DDX4 expression is not observed in the SCO tubules (arrow). Scale bars; D–H, L, M, 100 μm; I–K, N-S, 50 μm. SC; Sertoli cell, GC; Germ cell, LC; Leydig cell, SCO; Sertoli cell-only tubule, Sp; Spermatid. **p < 0.01.

Article Snippet: Cell culture TM4 (ATCC ® CRL-1715™, USA) and Human Sertoli cell (HSerC; 10HU-149, iXCells Biotechnologies, USA) were cultured in DMEM/F-12 (#11320-033, Life Technologies, USA) supplemented with 2.5% fetal bovine serum and 5% horse serum or in Sertoli cell growth medium (MD-0091, iXCells Biotechnologies, USA), respectively, at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Produced, Expressing

Journal: Cell Death and Differentiation

Article Title: Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway

doi: 10.1038/s41418-020-00670-2

Figure Lengend Snippet: Expression analysis of testis markers between WT and Mast4 KO mice.

Article Snippet: Cell culture TM4 (ATCC ® CRL-1715™, USA) and Human Sertoli cell (HSerC; 10HU-149, iXCells Biotechnologies, USA) were cultured in DMEM/F-12 (#11320-033, Life Technologies, USA) supplemented with 2.5% fetal bovine serum and 5% horse serum or in Sertoli cell growth medium (MD-0091, iXCells Biotechnologies, USA), respectively, at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, TUNEL Assay

Journal: Cell Death and Differentiation

Article Title: Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway

doi: 10.1038/s41418-020-00670-2

Figure Lengend Snippet: A In the presence of FGF2, expression of Mast4 and Erm is increased in TM4 cells. B After inhibition of the effect of FGF signaling with SU5402, Mast4 expression is significantly decreased (n = 3). C Erm expression is also decreased similarly to Mast4 (n = 3). D In PN 6 W WT, ERM is expressed in Sertoli cells (arrowheads) along with several elongated spermatids. E In PN 6 W Mast4 KO testes, ERM expression pattern is decreased in normal seminiferous tubules (arrowhead) and rarely observed in the SCO tubules (arrow). F In TM4 cell, Erm expression is slightly decreased by MAST4 depletion (n = 3). G) Erm expression is decreased in MAST4-depleted human Sertoli cells (n = 3). H TM4 cells were treated with FGF2 for 6 h. Nuclear/cytoplasmic fractions were analyzed by western blot. Note that both basal and FGF2-induced ERM expression are decreased in MAST4-depleted TM4 cells. Scale bars; 100 μm. **p < 0.01.

Article Snippet: Cell culture TM4 (ATCC ® CRL-1715™, USA) and Human Sertoli cell (HSerC; 10HU-149, iXCells Biotechnologies, USA) were cultured in DMEM/F-12 (#11320-033, Life Technologies, USA) supplemented with 2.5% fetal bovine serum and 5% horse serum or in Sertoli cell growth medium (MD-0091, iXCells Biotechnologies, USA), respectively, at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Inhibition, Western Blot

Journal: Cell Death and Differentiation

Article Title: Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway

doi: 10.1038/s41418-020-00670-2

Figure Lengend Snippet: A Mast4 genomic structure is shown. MAST4 has highly conserved kinase domain and PDZ domain. To produce the Mast4 KO mice, 71 base pairs of Mast4 gene are deleted and a frameshift is produced on exon 1 so that translation stops. B At PN 6W, Mast4 KO mice has smaller testes compared to WT mice. C The total sperm number is significantly decreased in Mast4 KO testes (n = 4). D At PN 1D, the migrating germ cells are observed in the developing seminiferous tubules. E MAST4 is expressed in the cytoplasm of the developing Sertoli cells while SOX9 is expressed in the nuclei of the Sertoli cells (arrowhead). F Similarly, strong expression patterns of AMH are localized in the developing Sertoli cells with MAST4 co-localization (arrowhead). G DDX4 is expressed in the cytoplasm of the germ cell (arrowhead), which is located in the outermost layer of seminiferous tubules. H At PN 3 W, the testes have a developed structure with differentiating spermatogonia and Leydig cells. I The expression of MAST4 is shifted to Leydig cells while SOX9 is continuously expressed in Sertoli cells (arrowhead). J MAST4 is expressed in Leydig cells with co-localization of 3β-HSD (arrowhead), while K DDX4 is expressed in spermatogonia located throughout seminiferous tubules (arrowhead). L At PN 6W, WT testes have compact seminiferous tubules with all types of germ cells. M Mast4 KO testis has seminiferous tubules with depleted germ cells or SCO morphology (arrowhead). N MAST4 is expressed in Leydig cells and spermatids although SOX9 is expressed in the nuclei of Sertoli cells in WT testes (arrowhead). O In Mast4 KO testes, SOX9 is strongly expressed in Sertoli cells located at the outermost layer of the SCO tubules along with Sertoli cells in normal seminiferous tubule. P Expression of AMH is not detected in WT pubertal testes (arrow). Q In Mast4 KO testes, AMH is expressed in the SCO tubules (arrowhead). R DDX4 is expressed mainly in differentiating spermatogonia (arrowhead). S In Mast4 KO testes, DDX4 expression is not observed in the SCO tubules (arrow). Scale bars; D–H, L, M, 100 μm; I–K, N-S, 50 μm. SC; Sertoli cell, GC; Germ cell, LC; Leydig cell, SCO; Sertoli cell-only tubule, Sp; Spermatid. **p < 0.01.

Article Snippet: Cell culture TM4 (ATCC ® CRL-1715™, USA) and Human Sertoli cell (HSerC; 10HU-149, iXCells Biotechnologies, USA) were cultured in DMEM/F-12 (#11320-033, Life Technologies, USA) supplemented with 2.5% fetal bovine serum and 5% horse serum or in Sertoli cell growth medium (MD-0091, iXCells Biotechnologies, USA), respectively, at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Produced, Expressing

Journal: Cell Death and Differentiation

Article Title: Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway

doi: 10.1038/s41418-020-00670-2

Figure Lengend Snippet: Expression analysis of testis markers between WT and Mast4 KO mice.

Article Snippet: Cell culture TM4 (ATCC ® CRL-1715™, USA) and Human Sertoli cell (HSerC; 10HU-149, iXCells Biotechnologies, USA) were cultured in DMEM/F-12 (#11320-033, Life Technologies, USA) supplemented with 2.5% fetal bovine serum and 5% horse serum or in Sertoli cell growth medium (MD-0091, iXCells Biotechnologies, USA), respectively, at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, TUNEL Assay

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: Total cellular RNAs were extracted from human Sertoli cells after a 24 h treatment with DMSO (0 μM) or 7–10 μM CBD. Gene expression profiles (n=4/group) were analyzed by mRNA-seq. (A) A heatmap shows concentration-dependent changes of representative DNA replication-related genes. Relative expression of E2F1 (B), E2F7 (C), MCM3 (D), PRIM1 (E), POLE2 (F), and POLD1 (G) was determined using real-time PCR. The bar graphs represent means ± standard deviation (SD) (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques: Gene Expression, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: (A) A heatmap shows concentration-dependent changes of representative cell cycle-related genes. (B and C) Relative expression of CDC25A (B) and CDKN1A (C) was measured using real-time PCR in CBD-treated human Sertoli cells. The bar graphs show means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques: Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: (A) A heatmap shows concentration-dependent changes of representative DNA repair-related genes. (B-E) Relative expression of EXO1 (B), MRE11 (C), NEIL3 (D), and FANCB (E) was measured using real-time PCR in CBD-treated human Sertoli cells. The bar graphs show means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques: Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: (A) A heatmap shows concentration-dependent changes of representative genes in the p53 signaling pathway. (B-E) Western blot analysis of phospho-p53 (Ser15), p53, and CDKN1A/p21 was conducted in CBD-treated human Sertoli cells. GAPDH was used as an internal control. Representative Western blots are shown in (B). Quantification of relative band intensity is shown in (C-E). The bar graphs represent means ± SD (n=3). The intensity of each protein band was normalized to its individual GAPDH band. *, significantly different from the DMSO control.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques: Concentration Assay, Western Blot, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: (A) A heatmap shows concentration-dependent changes of representative genes in SASP. (B) Relative gene expression of IL-1A (left panel) and IL-6 (right panel) was measured using real-time PCR in human Sertoli cells after a 24 h CBD treatment. (C) Western blot analysis of IL-6 was conducted in human Sertoli cells after a 24 h CBD treatment. GAPDH was used as an internal control. Representative Western blots are shown in (C, left panel). Quantification of relative band intensity is shown in (C, right panel). The bar graphs represent means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques: Concentration Assay, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: (A) Relative gene expression of CDKN2A/p16 was measured using real-time PCR in human Sertoli cells after a 24 h CBD treatment. (B) Western blot analysis of CDKN2A/p16 was conducted in human Sertoli cells after a 24 h CBD treatment. GAPDH was used as an internal control. Representative Western blots are shown in left panel. Quantification of relative band intensity is shown in right panel. The bar graphs represent means ± SD (n=3). The intensity of each protein band was normalized to its individual GAPDH band. *, significantly different from the DMSO control. (C) The line graph shows the cumulative population doubling of human Sertoli cells treated with DMSO (0 μM) or 10 μM CBD for 12 days. (D) The bar graph shows the relative fluorescence intensity of SA-β-gal in DMSO (0 μM)- or 10 μM CBD-treated human Sertoli cells. The bar graphs represent means ± SD (n=3). *, significantly different from the DMSO control at each time point. #, significant time-related linear trend in 10 μM CBD treatment group. (E) Human Sertoli cells were stained for SA-β-gal (blue-green) after a 12-day treatment with DMSO (0 μM) or 10 μM CBD. Bright-field images are representative of three independent experiments. Scale bar is 300 μm.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Control, Fluorescence, Staining

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: CBD treatment activates p53 in human Sertoli cells, increases downstream p21, which are important in inhibiting DNA replication-, DNA repair-, and cell cycle-related gene transcription. The increase of p16 can contribute to prolonged cell cycle arrest in human Sertoli cells leads to cellular senescence.

Article Snippet: Cell cultures Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA).

Techniques:

Journal: BMC Veterinary Research

Article Title: Comparison of the sensitivity of three cell cultures to ORFV

doi: 10.1186/s12917-018-1760-1

Figure Lengend Snippet: Growth curves of bovine Sertoli cells cultured in vitro at different temperature. The blue, red, and green curves represent the growth behavior of bovine Sertoli cells at 37 °C, 38.5 °C, and 39.5 °C, respectively

Article Snippet: After the isolation and purification of the culture, bovine Sertoli cells were finally obtained and submitted to the China Center for Type Culture collection (CCTCC) at Wuhan University (CCTCC accession number: C201438) for preservation.

Techniques: Cell Culture, In Vitro

Journal: BMC Veterinary Research

Article Title: Comparison of the sensitivity of three cell cultures to ORFV

doi: 10.1186/s12917-018-1760-1

Figure Lengend Snippet: Identification results of the 3rd passage of the bovine Sertoli cells after isolation and purification by HE staining, and indirect immunofluorescence staining. HE staining assay (nucleus was indicated by 1 arrow) ( a ); FasL protein (indicated by 1 arrow) detection in the bovine Sertoli cells ( b ) and primary neonatal bovine testicular cells by immunohistochemical assay ( c ). Negative control ( d ). Bovine Sertoli cells and primary neonatal bovine testicular cells cultured for 96 h were visualized with bright-field and fluorescent microscopy, and the resulting images were merged. Ninety-five percent of the bovine Sertoli cells and 78% of the primary neonatal bovine testicular cells exhibited fluorescence, respectively

Article Snippet: After the isolation and purification of the culture, bovine Sertoli cells were finally obtained and submitted to the China Center for Type Culture collection (CCTCC) at Wuhan University (CCTCC accession number: C201438) for preservation.

Techniques: Isolation, Purification, Staining, Immunofluorescence, Immunohistochemical staining, Negative Control, Cell Culture, Microscopy, Fluorescence

Journal: BMC Veterinary Research

Article Title: Comparison of the sensitivity of three cell cultures to ORFV

doi: 10.1186/s12917-018-1760-1

Figure Lengend Snippet: Sensitivities of the 3 cell cultures to ORFV

Article Snippet: After the isolation and purification of the culture, bovine Sertoli cells were finally obtained and submitted to the China Center for Type Culture collection (CCTCC) at Wuhan University (CCTCC accession number: C201438) for preservation.

Techniques: Virus

Journal: BMC Veterinary Research

Article Title: Comparison of the sensitivity of three cell cultures to ORFV

doi: 10.1186/s12917-018-1760-1

Figure Lengend Snippet: Bovine Sertoli cell growth properties and virus replication

Article Snippet: After the isolation and purification of the culture, bovine Sertoli cells were finally obtained and submitted to the China Center for Type Culture collection (CCTCC) at Wuhan University (CCTCC accession number: C201438) for preservation.

Techniques: Virus