Journal: The EMBO Journal

Article Title: Niche Tet maintains germline stem cells independently of dioxygenase activity

doi: 10.1038/s44318-024-00074-9

Figure Lengend Snippet: ( A ) RNA FISH results show that Tet mRNA is expressed in the niche cells and IGS cells of the germarium. Niche cells are highlighted by antibody against Lamin C (LamC), which is enriched in the nuclear membrane of terminal filament cells and cap cells. ( B ) Tet-EGFP is expressed in the terminal filaments, cep cells, IGS cells, and follicle cells of the germarium. ( C , D ) Cap cells (CPCs), GSCs and CBs are highlighted by dashed white circles, arrows, and arrowheads, respectively (the same applies to images in the remaining Figures). bab1 ts -driven Tet knockdown causes the GSC loss but does not affect cap cell and CB numbers. ( D ) Quantification results. ( E , F ) bab1 ts -driven Tet knockdown decreases pMad expression in GSC (the ‘luc-KD ’ image is re-displayed in Fig. ). ( F ) Quantification results. ( G , H ) bab1 ts -driven Tet knockdown increases bam -GFP expression in GSC. ( H ) Quantification results. ( I , J ) bab1 ts -driven Tet knockdown decreases dpp mRNA expression in cap cells. ( J ) Quantification results. ( K , L ) bab1 ts -driven dpp overexpression can restore GSC numbers compared with Tet-KD . ( L ) Quantification results. Data information: In ( D , F , H , J and L ), data are presented as mean ± SEM. *** P ≤ 0.001, n.s., no significance (Student’s t-test). In ( D , J and L ), n = number of germaria. In ( F and H ), n = number of GSCs. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Hts antibody (1:50, DSHB), mouse monoclonal anti-Lamin C (1:5, DSHB), rabbit polyclonal anti-β-galactosidase antibody (1:100, #08559761, MP Biomedical), rat polyclonal anti-Vasa antibody (1:50, DSHB), rabbit monoclonal anti-pMad antibody (1:500, ab52903), goat polyclonal anti-GFP antibody (1:500, Rockland, #600-101-215) and chicken polyclonal anti-GFP antibody (1:200, Invitrogen, #A10262).

Techniques: Membrane, Knockdown, Expressing, Over Expression

Journal: The EMBO Journal

Article Title: Niche Tet maintains germline stem cells independently of dioxygenase activity

doi: 10.1038/s44318-024-00074-9

Figure Lengend Snippet: ( A , B ) bab1 ts -driven brm-KD , Bap170-KD , Bap180-KD , and osa-KD decrease GSC number. ( B ) Quantification results. ( C , D ) bab1 ts -driven brm-KD , Bap170-KD , and Bap180 KD decrease pMad expression in GSCs, but osa-KD has no obvious effect on pMad expression in GSCs. ( D ) Quantification results. ( E , F ) bab1 ts -driven brm-KD , Bap170-KD , and Bap180-KD decrease dpp expression in cap cells, but osa-KD has no obvious effect on dpp expression in cap cells. ( F ) Quantification results. Data information: In ( B , D and F ), data are presented as mean ± SEM. *** P ≤ 0.001, n.s., no significance (Student’s t-test). In ( B and F ), n = number of germaria. In ( D ), n = number of GSCs. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Hts antibody (1:50, DSHB), mouse monoclonal anti-Lamin C (1:5, DSHB), rabbit polyclonal anti-β-galactosidase antibody (1:100, #08559761, MP Biomedical), rat polyclonal anti-Vasa antibody (1:50, DSHB), rabbit monoclonal anti-pMad antibody (1:500, ab52903), goat polyclonal anti-GFP antibody (1:500, Rockland, #600-101-215) and chicken polyclonal anti-GFP antibody (1:200, Invitrogen, #A10262).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: Niche Tet maintains germline stem cells independently of dioxygenase activity

doi: 10.1038/s44318-024-00074-9

Figure Lengend Snippet: ( A ) Domains of the Tet protein and the design of wild-type Tet ( Tet WT , RNAi-resistant) and enzyme-dead Tet ( Tet ED , RNAi-resistant) transgenic flies. Codons in the shRNA (BDSC #62280) target region are replaced with synonymous codons that do not change the amino acids. For the Tet ED , HSHRDL in core catalytic region is mutated to HSARAL. ( B , C ) Tet WT and Tet ED overexpression (OE) do not affect the GSC number. Both Tet WT -OE and Tet ED -OE can significantly rescue the GSC loss caused by Tet-KD . ( C ) Quantification results. ( D , E ) Tet WT -OE and Tet ED -OE do not change pMad expression in GSCs. Both Tet WT -OE and Tet ED -OE can significantly rescue the decreased pMad expression caused by Tet- KD . ( E ) Quantification results ( n = GSC numbers). ( F , G ) Tet WT -OE and Tet ED -OE have no obvious effect on dpp expression in cap cells. Both Tet WT -OE and Tet ED -OE can significantly rescue the deceased dpp mRNA expression caused by Tet-KD . ( G ) Quantification results. Data information: In ( C , E and G ), data are presented as mean ± SEM. *** P ≤ 0.001, n.s., no significance (Student’s t-test). In ( C and G ), n = number of germaria. In ( E ), n = number of GSCs. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Hts antibody (1:50, DSHB), mouse monoclonal anti-Lamin C (1:5, DSHB), rabbit polyclonal anti-β-galactosidase antibody (1:100, #08559761, MP Biomedical), rat polyclonal anti-Vasa antibody (1:50, DSHB), rabbit monoclonal anti-pMad antibody (1:500, ab52903), goat polyclonal anti-GFP antibody (1:500, Rockland, #600-101-215) and chicken polyclonal anti-GFP antibody (1:200, Invitrogen, #A10262).

Techniques: Transgenic Assay, shRNA, Over Expression, Expressing

Journal: The EMBO Journal

Article Title: Niche Tet maintains germline stem cells independently of dioxygenase activity

doi: 10.1038/s44318-024-00074-9

Figure Lengend Snippet: ( A ) Diagram shows Flag-Tet associated proteins identified by co-IP and mass spectrometry, including PBAP components, Bap55, Bap111, and Act5C. ( B , C ) Co-IP experiments show that Flag-tagged Tet C-terminal fragment (Flag-Tet-C) can bring down Bap55-HA in S2 cells. Moreover, Flag-Tet-CF1 and Flag-Tet-CF4 can bring down Bap55-HA in S2 cells. ( D , E ) bab1 ts -driven Bap55 and Bap111 knockdowns cause the GSC loss but do not affect cap cell and CB numbers. ( E ) Quantification results. ( F – I ) bab1 ts -driven Bap55 and Bap111 knockdowns reduce pMad ( F ) expression in GSCs and decrease dpp mRNA ( H ) expression in cap cells. ( G , I ) Quantification results. Data information: In ( E , G and I ), data are presented as mean ± SEM. ** P ≤ 0.01, *** P ≤ 0.001, n.s., no significance (Student’s t-test). In ( E and I ), n = number of germaria. In ( G ), n = number of GSCs. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Hts antibody (1:50, DSHB), mouse monoclonal anti-Lamin C (1:5, DSHB), rabbit polyclonal anti-β-galactosidase antibody (1:100, #08559761, MP Biomedical), rat polyclonal anti-Vasa antibody (1:50, DSHB), rabbit monoclonal anti-pMad antibody (1:500, ab52903), goat polyclonal anti-GFP antibody (1:500, Rockland, #600-101-215) and chicken polyclonal anti-GFP antibody (1:200, Invitrogen, #A10262).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing

Journal: The EMBO Journal

Article Title: Niche Tet maintains germline stem cells independently of dioxygenase activity

doi: 10.1038/s44318-024-00074-9

Figure Lengend Snippet: ( A , B ) Co-IP experiments show that Flag-tagged Tet C-terminus (Flag-Tet-C) can bring down Stat92E-HA in S2 cells. Moreover, Flag-Tet-CF1 and Flag-Tet-CF4 (two smaller regions in Tet-C) can bring down Stat92E-HA in S2 cells. ( C ) Coomassie blue staining on purified Myc-Tet-C and HA-Stat92E from bacteria, and Strep-Bap55 from insect cells. ( D – F ) In vitro interaction assay shows that Myc-Tet-C directly interacts with HA-Stat92E ( D ) and Strep-Bap55 ( E ). Strep-Bap55 brings down HA-Stat92E only in the presence of Myc-Tet-C, but not Strep-Bap55 alone ( F ). (*Indicates that these bands may be a result of protein instability and degradation) ( G , H ) bab1 ts -driven Bap55-Stat92E overexpression can significantly rescue the GSC loss caused by Tet-KD , but overexpression of Bap55 or Stat92E alone cannot. ( H ) Quantification results. ( I , J ) bab1 ts -driven Bap55-Stat92E overexpression can significantly rescue the decreased pMad expression caused by Tet-KD , but overexpression of Bap55 or Stat92E alone cannot. ( J ) Quantification results. ( K , L ) bab1 ts -driven Bap55-Stat92E overexpression can significantly rescue the reduced dpp mRNA expression caused by Tet-KD , but overexpression of Bap55 or Stat92E alone cannot. ( L ) Quantification results. Data information: In ( H , J , and L ), data are presented as mean ± SEM. *** P ≤ 0.001, n.s., no significance (Student’s t-test). In ( H and L ), n = number of germaria. In ( J ), n = number of GSCs. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Hts antibody (1:50, DSHB), mouse monoclonal anti-Lamin C (1:5, DSHB), rabbit polyclonal anti-β-galactosidase antibody (1:100, #08559761, MP Biomedical), rat polyclonal anti-Vasa antibody (1:50, DSHB), rabbit monoclonal anti-pMad antibody (1:500, ab52903), goat polyclonal anti-GFP antibody (1:500, Rockland, #600-101-215) and chicken polyclonal anti-GFP antibody (1:200, Invitrogen, #A10262).

Techniques: Co-Immunoprecipitation Assay, Staining, Purification, Bacteria, In Vitro, Over Expression, Expressing

Journal: The EMBO Journal

Article Title: Niche Tet maintains germline stem cells independently of dioxygenase activity

doi: 10.1038/s44318-024-00074-9

Figure Lengend Snippet: ( A , B ) bab1 ts -driven hTET3 - OE has no obvious impact on the GSC number in the wild-type germarium but can significantly rescue the GSC loss caused by Tet-KD . ( B ) Quantification results. ( C , D ) hTET3 - OE had no obvious effect on pMad expression in wild-type GSCs but it can significantly rescue the decreased pMad expression caused by Tet-KD . ( D ) Quantification results. ( E , F ) hTET3 - OE had no significant effect on dpp expression in wild-type cap cells but it can significantly rescue the reduced dpp mRNA expression in the Tet-KD cap cells. ( F ) Quantification results. ( G ) Schematic diagram explaining how Tet bridges the PBAP complex and Stat92E independent of its enzymatic activity to activate dpp expression in the niche, thus promoting GSC self-renewal. Data information: In ( B , D and F ), data are presented as mean ± SEM. *** P ≤ 0.001, n.s., no significance (Student’s t-test). In ( B and F ), n = number of germaria. In ( D ), n = number of GSCs. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Hts antibody (1:50, DSHB), mouse monoclonal anti-Lamin C (1:5, DSHB), rabbit polyclonal anti-β-galactosidase antibody (1:100, #08559761, MP Biomedical), rat polyclonal anti-Vasa antibody (1:50, DSHB), rabbit monoclonal anti-pMad antibody (1:500, ab52903), goat polyclonal anti-GFP antibody (1:500, Rockland, #600-101-215) and chicken polyclonal anti-GFP antibody (1:200, Invitrogen, #A10262).

Techniques: Expressing, Activity Assay