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