(A) MARCM clones of control (FRT82B), were labeled with CD8-GFP and costained with Dpn and Mira (upper panels) or Mira and EdU (lower panels). 815; = 9, t = 947. (J) Larval NSCs in control and = 10, t = 425; = 11, t = 462. In (L), control: = 5, t = 271; = 11, t = 695. Enlarged views of the white dotted boxes in the upper panels are shown in lower panels in (G) and (J). (M) Larval brains of 0.05, *** for 0.001, and **** for 0.0001. Yellow arrows, proliferative NSCs; white arrows, quiescent NSCs. Arrowheads indicate the cellular process of quiescent NSCs. Scale bars, 10 m. The data underlying this physique can be found in S1 Data. ALH, after larval hatching; Dcr2, Dicer 2; DDB1, damaged DNA-binding protein 1; Dpn, Deadpan; EdU, 5-ethynyl-2-deoxyuridine; GFP, green fluorescent protein; MARCM, mosaic analysis of repressible cell marker; Mira, Miranda; ns, statistically nonsignificant; NSC, neural stem cell; RNAi, RNA interference; UAS, upstream activating sequence; VDRC, Vienna Drosophila Resource Center; WT, wild-type.(TIF) pbio.3000276.s001.tif (9.0M) GUID:?9C373379-62AB-470B-915D-3F99ADAA781B S2 Fig: Related to Fig 2. Cul4 functions intrinsically during NSC reactivation. (A) Larval brains (96 h ALH) in control (= 10, t = 605; = 17, t = 670. In (C), control, = 5, t = 418; = 10, t = 513. (D) Larval NSCs in control (= 7, t = 569; = 6, t = 602; = 11, t = 763. For (G), control, = 7, t = 569; = 6, t = 602; and = 12, t = 847. (H) MB NSC lineages in larval brain on amino acidCdepleted food from WT, at 24 h ALH were labeled with EdU, Dpn (an NSC marker), and Dac (marks MB neurons surrounding MB NSCs). (I) Larval brains from control (control, and overexpression of InRCA with under 0.0001, *** for 0.001, ** for 0.01, * for 0.05, and ns for 0.05. Yellow arrows, EdU+ NSCs. White arrows, NSCs without Rabbit Polyclonal to DIDO1 EdU or with process. Scale bars, 10 m. The data underlying this physique can be found in S1 Data. ALH, after larval hatching; Cul4, Cullin 4; Dac, dachshund; Dcr2, Dicer 2; loss of function in NSCs results in NSC reactivation defects. (A-F) At 24 h ALH, larval NSCs in control (stained with Mahj and NSC markers (Dpn and Mira). Blue arrows, NSCs. (H) Protein extracts from 24 h ALH whole-larvae lysate of WT and were blotted with anti-Actin (loading control) and anti-Mahj antibodies. (I) Quantification of Mahj level in (H) was obtained by normalization Entrectinib of Mahj ROD to Actin ROD, = 3. (J) At 48 h ALH, larval brains from WT, mutants, and mutants expressing either UAS-Myc-Mahj or UAS-Myc-MahjR1120/1123E driven by (Df [2R] XE-2900) transheterozygous mutants were labeled with EdU, Dpn, and Mira. (M-N) Quantification of NSCs that are EdU+ or with process of various genotypes in (L). (O) MB NSC lineages in larval brains from WT and at 24 h ALH were labeled with EdU, Dpn, and Dac. The enlarged views of white dotted boxes are shown to the right. Yellow arrowheads, MB NSCs surrounded by Dac-positive MB neurons. (P) At 0 h ALH, larval brains of 0.0001, *** for 0.001, ** for 0.01, * for 0.05, and ns for 0.05. Scale bars: 10 m. The data underlying this physique can be found in S1 Data. ALH, Entrectinib after larval hatching; BDSC, Bloomington Stock Center; DDB1-Cul4 associated factor 1; IP, immunoprecipitation; Mahj, Mahjong; Wts, Warts.(TIF) pbio.3000276.s004.tif (4.9M) GUID:?B7D45D02-40D9-4B5D-9F2E-233469F860CB S5 Fig: Related to Fig 5. Wts interacts with different components of CRL4Mahj in S2 cells. (A) Co-IP between Flag-DDB1 and HA-Wts. S2 cells were cotransfected with Flag-DDB1 and HA-Wts or respective controls. Immunoprecipitation was performed using anti-Flag antibodies, and western blot was performed using anti-Flag or anti-HA antibodies. (B) A schematic illustration for PLA. (C) Entrectinib In situ PLA assay between Flag-Wts and either Myc-Mahj, Myc-DDB1, or Myc-Cul4. S2 cells transfected with the indicated plasmids were stained with Flag, Myc, and DAPI and.