Journal: Cell reports

Article Title: RHOX10 drives mouse spermatogonial stem cell establishment through a transcription factor signaling cascade

doi: 10.1016/j.celrep.2021.109423

Figure Lengend Snippet: (A) Luciferase analysis of constructs harboring promoters from the indicated genes ligated upstream of the Firefly luciferase gene. All promoter constructs contain between 1 to 2 kb of sequence upstream of the TSS (see ). These reporter constructs were transiently co-transfected with an Rhox10 -expression vector into GC1 cells, which only express trace levels of endogenous Rhox10 . Data are represented as mean ± SD (n = 4). Staistical significance was determined using the two-tailed unpaired Student’s t test. *p < 0.05. (B) Left, IF analysis of testis sections from P3 Rhox10 -null (KO) or control (WT) mice co-stained with antisera against DNMT3L and GCNA1, which mark T1-ProSG and all germ cells, respectively. Cell nuclei were stained with DAPI (blue). Scale bar, 30 μm. Right, quantification of the percentage of all germ cells (GCNA1 + cells) that express either high (Bright), low (Dim), or no detectable (Neg) DNMT3L. The analysis was done blind to genotype. Statistical significance was determined using the chi-square test. Data are represented as mean ± SD (n = 3). *p < 0.05. (C) Left, IF analysis of testis sections from P3 Rhox10 -null (KO) or control (WT) mice co-stained with antisera against ETV4 and GCNA1, which stain emergent SSCs and all germ cells, respectively. Cell nuclei were stained with DAPI (blue). Scale bar, 30 μm. Right, quantification of ETV4-positive germ cells, determined as in (B). Data representation and statistical significance (n = 3) as in (B). (D) Quantification of germ cells with the indicated levels of DNMT3L-signal intensity in primary Rhox10 -null and control testicular cell cultures, analyzed as in (B), at the indicated time points after initiation of culture. Data representation and statistical significance (n = 3) as in (B). See also .

Article Snippet: Mouse: GC1 cells , ATCC , CRL-2053.

Techniques: Luciferase, Construct, Sequencing, Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Control, Staining

Journal: Cell reports

Article Title: RHOX10 drives mouse spermatogonial stem cell establishment through a transcription factor signaling cascade

doi: 10.1016/j.celrep.2021.109423

Figure Lengend Snippet: (A) Quantification of the percentage of all germ cells (GCNA1 + cells) in Rhox10 -null (KO) and control (WT) primary testicular cell cultures that express either high (Bright), low (Dim) or no detectable (Neg) DNMT3L, performed as in - . The analysis was done blind to genotype. Data are represented as mean ± SD (n = 3). Statistical significance was determined using the chi-square test. *p < 0.05. Dmrt1 , Gfrα1 , and Etv5 were transduced ectopically using lentiviral expression vectors. (B) qPCR analysis of testicular primary cultures of the indicated genotype transduced with or without a Dmrt1 -expression vector. Data representation as in (A). Statistical significance was determined using the Student’s t test (n = 3). (C) EMSA showing that RHOX10 binds to the predicted RHOX10-binding site ATTGG (reverse complement of CCAAT in ) in the Dmrt1 promoter region. The competitor is unlabeled Dmrt1 promoter probe (n = 3). (D) The Dmrt1 and mutated Dmrt1 probes used for EMSA in (C). (E) Luciferase analysis of constructs harboring either the Dmrt1 promoter or a mutated version of the Dmrt1 promoter lacking the CCAAT motif ( Dmrt1_m ) ligated upstream of the Firefly luciferase gene. These constructs were transiently co-transfected into GC1 cells with a Rhox10 -expression vector where indicated. Data representation and statistical significance (n = 3) as in (B). (F) Luciferase analysis of a construct harboring the human DMRT1 promoter ligated upstream of the Firefly luciferase gene. This construct was transiently co-transfected into TCam-2 cells with the RHOXF1 - or RHOXF2 -expression vectors, as indicated. Data representation and statistical significance (n = 3) as in (B). (G) Luciferase analysis of TCam-2 cells transiently co-transfected with the human DMRT1 promoter construct (described in F) with either a RHOXF1 - or RHOXF2 -shRNA vector (RHOXF1-KD and RHOXF2-KD, respectively). Data representation and statistical significance (n = 3) as in (B).

Article Snippet: Mouse: GC1 cells , ATCC , CRL-2053.

Techniques: Control, Expressing, Transduction, Plasmid Preparation, Binding Assay, Luciferase, Construct, Transfection, shRNA

Journal: Cell reports

Article Title: RHOX10 drives mouse spermatogonial stem cell establishment through a transcription factor signaling cascade

doi: 10.1016/j.celrep.2021.109423

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse: GC1 cells , ATCC , CRL-2053.

Techniques: Virus, Plasmid Preparation, Recombinant, cDNA Synthesis, SYBR Green Assay, Luciferase, Generated, Software

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 1. The promotion of spermatogonia activity by melatonin treatment. (A) Effects of melatonin treatment on the viability of GC-1 spg cells. The viability of spermatogonia cells were assessed by the CCK-8 method. (B, C) The enhancement of spermatogonia proliferation rates by treatments with melatonin. The Edu staining method was performed to measure GC-1 spg cell proliferation. (D, E) Inhibition of spermatogonia apoptosis by the treatments with melatonin. The per centages of GC-1 spg cells were quantitated by the flow cytometry. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Activity Assay, CCK-8 Assay, Staining, Inhibition, Flow Cytometry

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 2. Increased m6A RNA methylation levels in spermatogonia by melatonin. (A) Alterations of total m6A RNA methylation levels in GC-1 spg cells treated with 0.01, 0.1 or 1 μM mela tonin. The m6A contents in spermatogonia were quantitated by the RNA dot blot method. (B) Relative expression of major RNA methyl transferases and demethylases in GC-1 spg cells after melatonin treatments. Quantitative RT- PCR was done to assess target gene mRNA levels. (C) Significant increases of KIAA1429 protein levels in GC-1 spg cells induced by 0.01, 0.1 or 1 μM melatonin. KIAA1429 protein con tents in spermatogonia were determined via the western blot assay. GAPDH was detected as the internal standard. METTL3/4: methyltrans ferase-like protein 3/4; WTAP: Wilms’ tumor- associated protein; FTO: Fat mass and obesity- associated protein; ALKBH5: AlkB homolog 5. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Methylation, Dot Blot, Expressing, Quantitative RT-PCR, Western Blot, Wilms Tumor Assay

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 3. KIAA1429 mediates melatonin-induced spermatogonia activity changes. (A) Relative expression of KIAA1429 mRNA in spermatogonia with silenced or overexpressed KIAA1429 gene after melatonin treatment. KIAA1429 mRNA in GC-1 spg cells was detected by the quantitative RT-PCR. (B) Alterations of KIAA1429 protein levels in spermatogonia induced by KIAA1429 silencing or overexpression combined with melatonin treatment. Western blot was used to analyze KIAA1429 protein levels. (C) The changes of GC-1 spg cell viability induced by the combination of melatonin with KIAA1429 silencing or overexpression. Spermatogonia viability was assessed via CCK-8 assay. (D) Alterations of GC-1 spg cell proliferation rates caused by melatonin treatment together with KIAA1429 silencing or overexpression. Edu staining was done to analyze spermatogonia proliferation. (E) Effects of KIAA1429 silencing or overexpression on the apoptosis of GC-1 spg cells under melatonin treatment. Cell apoptosis was evaluated through the flow cytometry. si: silencing; ox: overexpression; ***P < 0.001; ###P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Over Expression, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 4. Transcriptome alterations in spermatogonia induced by KIAA1429 overexpression. (A) The scatter plots representing the differentially expressed genes in GC- 1 spg cells caused by KIAA1429 overexpression. Up-regulated and down-regulated genes in spermatogonia were showed as red and green spots respectively. (B) The functional categorization of differentially expressed genes in KIAA1429-overexpressing GC-1 spg cells. Differential gene categorization was done based on GO biological processes, cellular components and molecular functions. (C) The categorization of differentially expressed genes in KIAA1429-overexpressing GC-1 spg cells based on KEGG signaling pathways.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Over Expression, Functional Assay, Protein-Protein interactions

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 5. The roles of KIAA1429-induced PI3K/AKT activation in the regulation of spermatogonia activity by melatonin treatment. (A) Relative abundances of PI3K/ AKT signaling proteins in KIAA1429-overexpressing GC-1 spg cells without melatonin treatments. Signaling protein levels in spermatogonia were detected by western blot using GAPDH as the internal standard. (B) Relative abundances of PI3K/AKT signaling proteins in KIAA1429-overexpressing GC-1 spg cells under melatonin and LY294002 treatments. Signaling protein levels in spermatogonia were detected by western blot using GAPDH as the internal standard. (C) Effects of LY294002 on the viability of KIAA1429-overexpressing GC-1 spg cells under melatonin treatment. CCK-8 assay was used to assess spermatogonia viability. (D) Influences of LY294002 treatment on the proliferation rates of KIAA1429-overexpressing GC-1 spg cells treated with melatonin. Spermatogonia proliferation was quantitated by the Edu staining method. (E) Alterations of KIAA1429-overexpressing GC-1 spg cell apoptosis caused by LY294002 under melatonin treatment. PI3K: Phosphatidylinositol 3–kinase; AKT: Protein kinase B; *P < 0.05; **P < 0.01; ***P < 0.001; ###P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Activation Assay, Activity Assay, Western Blot, CCK-8 Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Localization of outer segment-resident proteins in Cep290fl/fl;Cre+ mice. Localization of RHO (A), PRPH2 (B), ROM1 (C), ABCA4 (D), PDE6B (E), GUCY2D (F), ATP8A2 (G), and CNGA1 (H) was probed in Cep290+/fl;Cre+ (at P20) and Cep290fl/fl;Cre+ (at P20 and P40) retinas. I, retinal sections of Ift88+/fl;Cre+ (at P22) and Ift88fl/fl;Cre+ (at P22 and P40) mice were immunostained with anti-RHO antibody and shown for comparisons with Cep290fl/fl;Cre+ mice. Red brackets indicate outer segments. Sections were counterstained with DAPI to show nuclei (blue). At least three animals, both male and female, were used per group, and representative images are shown. Scale bar represents 50 μm. J, length of the outer segment in Cep290fl/fl;Cre+ and Ift88fl/fl;Cre+ retinas. Lengths of the outer segment were measured in areas 0.5–1.0 mm away from the optic nerve. Data are from five animals per genotype (two sections per animal). Mean and standard deviation (S.D.; error bar) are shown. Asterisk indicates statistical significance (two-tailed, two-sample t test assuming unequal variances; p < 0.01).

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( 68 )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Localization of inner segment membrane proteins in Cep290fl/fl;Cre+ mice. Retinal sections from Cep290+/fl;Cre+ (at P20) and Cep290fl/fl;Cre+ (at P20 and P40) mice were labeled with IMPG2 (A), HCN1 (B), and ATP1A3 (C) antibodies (green). Outer segments were delineated with ABCA4 or RHO (A), ATP8A2 (B), and GUCY2D (C) antibodies (red). Nuclei were counterstained with DAPI. D and E, localization of endomembrane proteins SYP (D) and LAMP1 (E) was examined using respective antibodies. At least three animals were used per group, and representative images are shown. Scale bar represents 50 μm. F, quantification of inner segment membrane protein mislocalization to the outer segment. Others are the same as in Fig. 4.

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( 68 )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Membrane, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Disruption of compartmentalized protein localization in Cep290rd16/rd16 mice. Localization of outer segment-resident proteins (PRPH2 (A), RHO (B and D), ROM1 (C), ABCA4 (E), GUCY2D (F), ATP8A2 (G), PDE6B (H), and CNGA1 (H); all in red except CNGA1, which is in green) and inner segment membrane proteins (STX3 (A), STXBP1 (B), SNAP25 (C), IMPG2 (D), VAMP2 (E), ATP1A3 (F), and HCN1 (G); all in green) was examined by indirect immunofluorescence microscopy. Scale bar represents 25 μm.

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( 68 )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Disruption, Membrane, Immunofluorescence, Microscopy