( B ) FLAG - tagged LGP2 fragments and HA - tagged Riplet expression vectors were transfected into HEK293FT cells . 24 h after transfection WCE were prepared . immunoprecipitation was performed with anti - FLAG antibody . Immunoprecipitates were subjected to SDS - PAGE , and the proteins were detected by western blotting with indicated antibodies . The data is a representative of two independent experiments .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

(B) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. 24 h after transfection WCE were prepared. immunoprecipitation was performed with anti-FLAG antibody. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies. The data is a representative of two independent experiments.


( C ) FLAG - tagged LGP2 and FLAG - tagged LGP2 - 4KR expression vectors were transfected into HEK293FT cells . 24 h after transfection , WCE were prepared . Immunoprecipitation was performed with anti - FLAG antibody . The data is a representative of two independent experiments .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O

(C) FLAG-tagged LGP2 and FLAG-tagged LGP2-4KR expression vectors were transfected into HEK293FT cells. 24 h after transfection, WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody. The data is a representative of two independent experiments.


( A ) RIG - I , WT LGP2 , and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc reporter plasmids . Luciferase activities were determined 24 h after transfection . The data represent the mean ± SD ( n = 3 , t - test , * p < 0 . 05 ) . WCEs were subjected to SDS - PAGE , and the proteins were detected by western blotting ( lower panels ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) RIG-I, WT LGP2, and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc reporter plasmids. Luciferase activities were determined 24 h after transfection. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). WCEs were subjected to SDS-PAGE, and the proteins were detected by western blotting (lower panels). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( B ) HE293 cells stably expressing GFP ( control ) , LGP2 , or LGP2 - 4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I : C . WCEs were prepared 24 h after transfection and stimulation , and luciferase activity was determined . The proteins were subjected to SDS - PAGE and detected by western blotting with the indicated Abs . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) HE293 cells stably expressing GFP (control), LGP2, or LGP2-4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activity was determined. The proteins were subjected to SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 cells stably expressing GFP , LGP2 , or LGP2 - 4KR were transfected with 200 ng / ml of short poly I : C ( C - E ) , Total RNA was isolated at the indicated time points . The expression of IFN - β , IP - 10 , and Ccl5 mRNA was determined by RT - qPCR and normalized to GAPDH . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293 cells stably expressing GFP, LGP2, or LGP2-4KR were transfected with 200 ng/ml of short poly I:C (C-E), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 cells stably expressing LGP2 , or LGP2 - 4KR were infected with SeV at MOI = 5 ( F - H ) , Total RNA was isolated at the indicated time points . The expression of IFN - β , IP - 10 , and Ccl5 mRNA was determined by RT - qPCR and normalized to GAPDH . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293 cells stably expressing LGP2, or LGP2-4KR were infected with SeV at MOI = 5 (F-H), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 cells stably expressing LGP2 , or LGP2 - 4KR were infected with influenza A virus ( Flu ) at MOI = 10 ( I , J ) . Total RNA was isolated at the indicated time points . The expression of IFN - β , IP - 10 mRNA was determined by RT - qPCR and normalized to GAPDH . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293 cells stably expressing LGP2, or LGP2-4KR were infected with influenza A virus (Flu) at MOI =10 (I, J). Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05 Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( K , L ) HEK293 cell clones stably expressing LGP2 or LGP2 - 4KR at lower levels were transfected with short poly I : C , and the expression of mRNAs were determined by RT - qPCR . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K, L) HEK293 cell clones stably expressing LGP2 or LGP2-4KR at lower levels were transfected with short poly I:C, and the expression of mRNAs were determined by RT-qPCR. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A ) Microarray analysis was performed as described in materials and methods . A heatmap of the microarray data of LGP2 and LGP2 - 4KR stably expressing cells stimulated with short poly I : C for 12 h .

O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A) Microarray analysis was performed as described in materials and methods. A heatmap of the microarray data of LGP2 and LGP2-4KR stably expressing cells stimulated with short poly I:C for 12 h.


B , LGP2 and LGP2 - 4KR stably expressing HEK293 cells were stimulated with 200 ng / ml of short poly I : C , and the expression of the genes at the indicated time points were determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C ) LGP2 and LGP2 - 4KR stably expressing HEK293 cells were stimulated with 200 ng / ml of short poly I : C , and the expression of the genes at the indicated time points were determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

C) LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


LGP2 and LGP2 - 4KR stably expressing A549 cells were infected with influenza A virus ( Flu ) at MOI = 10 , and the expression of the genes at the indicated time points were determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LGP2 and LGP2-4KR stably expressing A549 cells were infected with influenza A virus (Flu) at MOI = 10, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


H , I ) WT and indicated mutant HEK293 cells were stimulated with 200 ng / ml of short poly I : C for 8 h . WCEs were prepared and subjected to SDS - PAGE . The proteins were detected with indicated antibodies .

O O O O O O O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

H, I) WT and indicated mutant HEK293 cells were stimulated with 200 ng/ml of short poly I:C for 8 h. WCEs were prepared and subjected to SDS-PAGE. The proteins were detected with indicated antibodies.


J , K ) LGP2 and LGP2 - 4KR expressing vectors were transected into HeLa cells with Riplet expressing vector . 18 hr after transfection , cells were fixed , and then the PLA signals of LGP2 and endogenous MAVS were detected . Peroxisome was visualized using CellLight Peroxisome - GFP reagent . Colocalization of GFP ( peroxisome ) and the PLA signals ( red ) were observed by confocal microscopy . Scale bars represent 10 μm ( J ) . The colocalization area was normalized to the area of the PLA signals ( n = 25 , t - test , * p < 0 . 05 ) ( K ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, K) LGP2 and LGP2-4KR expressing vectors were transected into HeLa cells with Riplet expressing vector. 18 hr after transfection, cells were fixed, and then the PLA signals of LGP2 and endogenous MAVS were detected. Peroxisome was visualized using CellLight Peroxisome-GFP reagent. Colocalization of GFP (peroxisome) and the PLA signals (red) were observed by confocal microscopy. Scale bars represent 10 μm (J). The colocalization area was normalized to the area of the PLA signals (n = 25, t-test, *p < 0.05) (K). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


WT , and Riplet KO HEK293 cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 , PML , and ANKRD1 mRNA was determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT, and Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


B ) WT , LGP2 KO , HEK293 cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 , PML , and ANKRD1 mRNA was determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) WT, LGP2 KO, HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C , WT and Riplet KO A549 ( C ) cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 005 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, WT and Riplet KO A549 (C) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


D ) WT and Riplet KO MEF ( D ) cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 005 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) WT and Riplet KO MEF (D) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


E ) WT and Riplet KO BMM were infected with influenza A virus ( Flu ) at MOI = 10 . PML and SP100 mRNA expression was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O B-GENEPROD O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) WT and Riplet KO BMM were infected with influenza A virus (Flu) at MOI = 10. PML and SP100 mRNA expression was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


F ) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10 . WCE was prepared at the indicated time points and subjected to SDS - PAGE . Proteins were detected by western blotting with the indicated antibody .

O O O O B-GENEPROD O B-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

F) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10. WCE was prepared at the indicated time points and subjected to SDS-PAGE. Proteins were detected by western blotting with the indicated antibody.


G , LGP2 or LGP2 - 4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV ( G ) IFN - β mRNA levels at indicated time points were determined by RT - qPCR . ( n = 3 , two - way ANOVA , * p < 0 . 05 , ns : not significant ( p > 0 . 05 ) ) . Right graph in panel G is an enlargement of Y - axis of the left graph ( dotted line ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV (G) IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Right graph in panel G is an enlargement of Y-axis of the left graph (dotted line). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


H ) LGP2 or LGP2 - 4KR stably expressing WT or Riplet KO HEK293 cells were stimulated with 200 ng / ml of short poly I : C ( H ) . IFN - β mRNA levels at indicated time points were determined by RT - qPCR . ( n = 3 , two - way ANOVA , * p < 0 . 05 , ns : not significant ( p > 0 . 05 ) ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H) LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C (H). IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A - F ( A , B , D - F ) RNA levels determined by RT - qPCR . Results are presented as mean ± SD , n = 10 . P - values are indicated on the top of each plot . ( C ) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner . Spearman correlation coefficient , P - values , and sample numbers are marked on the top left of the plot .

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F (A, B, D-F) RNA levels determined by RT-qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.


A - D Endometrial stromal cells from patients # 166 and # 80 were each transfected with a mixture of siCon ( control siRNA ) and microRNA inhibitor control ( iCon ) , siH19 and iCon , or siH19 and iLet7 ( let - 7 - specific inhibitor ) . RNA and proteins were analyzed at 48 h post - transfection . Combined results from the two patient cells are presented .

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O

A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). RNA and proteins were analyzed at 48 h post-transfection. Combined results from the two patient cells are presented.


A - D Endometrial stromal cells from patients # 166 and # 80 were each transfected with a mixture of siCon ( control siRNA ) and microRNA inhibitor control ( iCon ) , siH19 and iCon , or siH19 and iLet7 ( let - 7 - specific inhibitor ) . Western blot gels from # 166 cells are shown in ( C ) . Quantitation of Western blots combining both patient cells are shown in ( D ) .

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O

A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). Western blot gels from #166 cells are shown in (C). Quantitation of Western blots combining both patient cells are shown in (D).


E - H Endometrial stromal cells from patients # 98 and # 212S were each transfected with empty vector or pH19 . RNA and proteins were analyzed 48 h post - transfection . Combined results from the two patient cells are presented .

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O

E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented.


E - H Endometrial stromal cells from patients # 98 and # 212S were each transfected with empty vector or pH19 . RNA and proteins were analyzed 48 h post - transfection . Combined results from the two patient cells are presented . Western blot gels from # 98 cells are shown in ( G ) . Quantitation of Western blots combining both patient cells are shown in ( H ) .

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O

E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #98 cells are shown in (G). Quantitation of Western blots combining both patient cells are shown in (H).


A - F The indicated cells were transfected with siCon , siH19 , empty vector , or pH19 . RNA , cell viability ( as indicated by viable cell numbers ) , and caspase activity ( as a readout for apoptosis ) were analyzed 48 h post - transfection . Combined results from two patient cells in each group are presented .

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A-F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.


G E2 stimulates H19 expression in endometrial stromal cells . Endometrial stromal cells # 166 and # 98 were stimulated with E2 ( + ) or vehicle ( − ) for 48 h , followed by RT - qPCR analysis of RNA extracted from the cells . Results combining two patient cells are shown .

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G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.


( B ) P2 MRI images ( axial , sagittal and coronal plane ) showing ventriculomegaly ( red asterisk ) , slightly thickened cortex and bilateral enlarged gyri ( white asterisk ) .

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(B) P2 MRI images (axial, sagittal and coronal plane) showing ventriculomegaly (red asterisk), slightly thickened cortex and bilateral enlarged gyri (white asterisk).


( D ) FIBCD1 expression in various human visceral tissues and brain regions ( inset ) . Expression is plotted relative to the tissue with lowest detectable expression ( trachea ; inset , choroid plexus ) . n represents technical replicates ( n = 2 ) .

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(D) FIBCD1 expression in various human visceral tissues and brain regions (inset). Expression is plotted relative to the tissue with lowest detectable expression (trachea; inset, choroid plexus). n represents technical replicates (n = 2).


( A ) Immunofluorescent images of control and neuronal ( Nsyb ) CG10359 ( dFibcd1 ) RNAi - mediated knockdown D . melanogaster , 3rd instar larvae NMJ ( NMJ6 / 7 ) stained with anti - horseradish peroxidase antibodies . Empty control and RNAi - mediated knockdown of CG10359 ( dFibcd1 - i ) lines # 1 , 2 and 3 shown . Scale bar = 20µm . Representative images of 3 independent experiments . Quantification of ( A ) , control and CG10359 knockdown lines NMJ neuron bouton number ( B ) n ( empty ) = 12 ; n ( line # 1 ) = 20 ; n ( line # 2 ) = 11 ; n ( line # 3 ) = 14 . Data information : each data point represents an individual NMJ Data is represented as mean and error bars represent SEM . p values were calculated using 2 - way ANOVA ( panels B * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunofluorescent images of control and neuronal (Nsyb) CG10359 (dFibcd1) RNAi-mediated knockdown D. melanogaster, 3rd instar larvae NMJ (NMJ6/7) stained with anti-horseradish peroxidase antibodies. Empty control and RNAi-mediated knockdown of CG10359 (dFibcd1-i) lines #1, 2 and 3 shown. Scale bar = 20µm. Representative images of 3 independent experiments. Quantification of (A), control and CG10359 knockdown lines NMJ neuron bouton number (B) n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


Quantification of NMJ neuron axon branch points ( C ) . n ( empty ) = 12 ; n ( line # 1 ) = 20 ; n ( line # 2 ) = 11 ; n ( line # 3 ) = 14 . Data information : each data point represents an individual NMJ Data is represented as mean and error bars represent SEM . p values were calculated using 2 - way ANOVA ( panels C ) , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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Quantification of NMJ neuron axon branch points (C). n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels C), * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( E ) Representative coronal section images of Golgi - Cox staining of Fibcd1 WT and KO hippocampi ( left ) , Neurolucida tracing of hippocampal CA1 pyramidal neurons ( middle ) and apical dendrites with spines ( right ) . Scale bars as indicated .

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(E) Representative coronal section images of Golgi-Cox staining of Fibcd1 WT and KO hippocampi (left), Neurolucida tracing of hippocampal CA1 pyramidal neurons (middle) and apical dendrites with spines (right). Scale bars as indicated.


( F - I ) Quantifications of ( F ) dendritic spine density , ( G ) total length of apical as well as basal dendrites , ( H ) dendritic nodes in apical as well as basal dendrites and ( I ) number of dendrites ( n = 5 ) . Data information : for panels F - I , each data point represents an individual mouse . Data is represented as mean and error bars represent SEM . p values were calculated using unpaired Student ' s t - test ( panels F - I , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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(F-I) Quantifications of (F) dendritic spine density, (G) total length of apical as well as basal dendrites, (H) dendritic nodes in apical as well as basal dendrites and (I) number of dendrites (n = 5). Data information: for panels F-I, each data point represents an individual mouse. Data is represented as mean and error bars represent SEM. p values were calculated using unpaired Student's t-test (panels F-I, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( B ) Immunoblot analysis ( left ) and quantification of signal intensity ( right ) of Fibcd1 WT ( blue ) versus Fibcd1 KO littermates ( red ) adult hippocampi with antibodies against CS - 0S , CS - 4S , CS - 6S and actin as a loading control . Each lane represents an independent animal ( n = 3 ) . Protein marker sizes are indicated . Data information : For panel B , each data point represent hippocampal protein isolates from an individual mouse , Data are shown as mean values + / - SEM . p values were calculated using paired Student ' s t - test ( panels B * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Immunoblot analysis (left) and quantification of signal intensity (right) of Fibcd1 WT (blue) versus Fibcd1 KO littermates (red) adult hippocampi with antibodies against CS-0S, CS-4S, CS-6S and actin as a loading control. Each lane represents an independent animal (n = 3). Protein marker sizes are indicated. Data information: For panel B, each data point represent hippocampal protein isolates from an individual mouse, Data are shown as mean values +/- SEM. p values were calculated using paired Student's t-test (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( F ) Confocal images depicting internalisation of FITC - tagged CS - 4S by FIBCD1 - overexpressing HEK293T lines compared to untransduced cells and unstained cells . Left , representative images ; right , quantification . Data is plotted as total puncta per condition ( n = 5 ) . Cells are further stained with CellMask Orange ( cellular membrane ) and Hoechst ( nuclei ) . Scale bar = 50µm . Data information : for panels F , each data point represents an individual cell preparation . Data are shown as mean values + / - SEM . p values were calculated using 1 - way ANOVA ( panels F , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Confocal images depicting internalisation of FITC-tagged CS-4S by FIBCD1-overexpressing HEK293T lines compared to untransduced cells and unstained cells. Left, representative images; right, quantification. Data is plotted as total puncta per condition (n = 5). Cells are further stained with CellMask Orange (cellular membrane) and Hoechst (nuclei). Scale bar = 50µm. Data information: for panels F, each data point represents an individual cell preparation. Data are shown as mean values +/- SEM. p values were calculated using 1-way ANOVA (panels F, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( A ) Left , representative image of immunofluorescent staining ( MAP2 , red ; DAPI , blue ) of primary hippocampal cultures at 2 days in vitro ( DIV ) , plated on + / - CSPG coating with and without prior digestion with ChABC , as indicated . Right , quantification of DIV2 images , showing the number of protruding cells per field normalised to untreated condition . n ( Fibcd1 WT ) = 4 ; n ( Fibcd1 KO ) = 3 . Scale bar = 250µm . ( B ) Left , representative images of DIV14 neurons , same conditions as in ( A ) . Right , quantification of DIV14 images , representing the percentage of cells per field that are clumped . n ( Fibcd1 WT ) = 3 ; n ( Fibcd1 KO ) = 2 . Scale bar = 250µm . Data information : For panels A and B each data point represents an individual preparations of primary cell culture . Data are represented as mean and error bars represent SEM . p values were calculated by 1 - way ANOVA . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Left, representative image of immunofluorescent staining (MAP2, red; DAPI, blue) of primary hippocampal cultures at 2 days in vitro (DIV), plated on +/- CSPG coating with and without prior digestion with ChABC, as indicated. Right, quantification of DIV2 images, showing the number of protruding cells per field normalised to untreated condition. n(Fibcd1 WT) = 4; n(Fibcd1 KO) = 3. Scale bar = 250µm. (B) Left, representative images of DIV14 neurons, same conditions as in (A). Right, quantification of DIV14 images, representing the percentage of cells per field that are clumped. n(Fibcd1 WT) = 3; n(Fibcd1 KO) = 2. Scale bar = 250µm. Data information: For panels A and B each data point represents an individual preparations of primary cell culture. Data are represented as mean and error bars represent SEM. p values were calculated by 1-way ANOVA. * = p≤0.05; ** =p≤0.01; *** =p≤0.001.


( A ) Live imaging of MTOC dynamics during meiosis I . Z - projection images ( top ) show MTOCs ( mNG - Cep192 , green ) and chromosomes ( H2B - mCherry , magenta ) . Central MTOCs are magnified ( squares ) . 3D - reconstructed spindles are aligned with the spindle axis ( middle ) . Central MTOCs are indicated with arrowheads . Time in h : mm . The kymograph ( bottom ) shows projected signals on the spindle axis for all time points .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O

(A) Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points.


( B ) Density maps of MTOCs ( left ) and chromosomes ( right ) along the spindle axis in a representative oocyte . The color represents the percentage of MTOCs or chromosome volumes , respectively , coded from dark ( 0 % ) to white ( 50 % or more of the total volume ) . ( C ) Average density maps of 21 oocytes mapped as in ( B ) .

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(B) Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume). (C) Average density maps of 21 oocytes mapped as in (B).


( D ) Dynamics of MTOC sorting and chromosome congression . Ratio of MTOCs ( left ) or chromosomes ( right ) in the polar region versus those in the middle region of the spindle are plotted over time .

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(D) Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time.


( E ) Dynamics of spindle elongation . The spindle was visualized with EGFP - Map4 The length / width ratio of the spindle was measured after 3D reconstruction ( n = 13 oocytes from 3 independent experiments ) .

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(E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments).


( F ) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle . Oocytes were fixed and stained for MTOCs ( pericentrin , green ) , chromosomes ( Hoechst33342 , magenta ) and microtubules ( α - tubulin , grey ) . n = 21 , 28 , 24 oocytes . A representative oocyte fixed at 5 hours after NEBD is shown . Arrowheads indicate central MTOCs .

O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O

(F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.


( A ) Live imaging of MTOC and kinetochore dynamics . MTOCs ( mNG - Cep192 , green ) and kinetochores ( tdTomato - CENP - C , magenta ) in B6D2F1 oocytes are shown . Time in h : mm after NEBD . Circles indicate central MTOCs , which are positioned in the middle region of the spindle later than 4 hours after NEBD . Arrows indicate displacement of the MTOCs . Scale bar , 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(A) Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 hours after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm.


( B ) Tracking analysis of central MTOCs . In the left graph , the positions of central MTOCs along the spindle axis are shown over time . MTOC positions were used to calculate distance to the spindle equator . In the right graph , lines showing temporal changes in MTOC - equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator . Horizontal lines at 7 and at − 7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle .

O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O

(B) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle.


( C ) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle . trajectories of central MTOCs ( n = 25 MTOCs of 8 oocytes from 3 independent experiments ) were analyzed and their temporal changes in MTOC - equator distance are shown ( Left graph ) . Central MTOCs were categorized into two groups : ( 1 ) ones that came from polar regions of the spindle ( n = 21 , middle graph ) and ( 2 ) others that stayed in the middle region throughout the period before reaching a position closest to the equator ( n = 4 , right graph ) .

O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. trajectories of central MTOCs (n=25 MTOCs of 8 oocytes from 3 independent experiments) were analyzed and their temporal changes in MTOC-equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n=21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n=4, right graph).


( E ) Destination of central MTOCs . The tracks of central MTOCs that originated from polar regions were used ( n = 21 MTOCs ) . The pole of origin was defined as Pole 1 , while the other pole was defined as Pole 2 . MTOC positions along the spindle axis ( positive values for those closer to Pole 1 ) over time are shown . Horizontal lines at 7 and at − 7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle . Note that central MTOCs moved back to the original pole ( Pole 1 ) or switched their positions to the opposite pole ( Pole 2 ) . ( F ) Destination of central MTOCs that originated from a polar region was categorized as in ( E ) .

O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

(E) Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n=21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2). (F) Destination of central MTOCs that originated from a polar region was categorized as in (E).


( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , Ndc80 - 9D or Ndc80 - 9A . Z - projection and 3D - reconstructed images show MTOCs ( mNeonGreen - Cep192 , green ) and chromosomes ( H2B - mCherry , magenta ) . Time in h : mm after NEBD . Scale bar , 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O

(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A. Z-projection and 3D-reconstructed images show MTOCs (mNeonGreen-Cep192, green) and chromosomes (H2B-mCherry, magenta). Time in h:mm after NEBD. Scale bar, 10 μm.


( B ) Ndc80 - 9D is defective for MTOC sorting . The density map of MTOCs along the spindle axis in Zp3 - Cre Ndc80f / f oocytes expressing Ndc80 - WT , Ndc80 - 9D or Ndc80 - 9A . The color represents the percentage of MTOC volume coded from dark ( 0 % ) to white ( 50 % or more of the total volume ) .

O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

(B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume).


( C ) MTOCs do not accumulate at the spindle poles in Ndc80 - 9D - expressing oocytes . Ratio of MTOCs ( green ) or chromosomes ( magenta ) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD ( n = 19 , 19 and 22 oocytes , respectively , from at least 3 independent experiments ) .

O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-CELL_TYPE O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

(C) MTOCs do not accumulate at the spindle poles in Ndc80-9D-expressing oocytes. Ratio of MTOCs (green) or chromosomes (magenta) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD (n=19, 19 and 22 oocytes, respectively, from at least 3 independent experiments).


( D ) Ndc80 - 9A decreases central MTOCs . The number of central MTOCs ( MTOCs that were positioned in the middle region of the spindle , was determined at each time point ( n = 19 , 21 oocytes , respectively , from at least 3 independent experiments ) .

O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

(D) Ndc80-9A decreases central MTOCs. The number of central MTOCs (MTOCs that were positioned in the middle region of the spindle , was determined at each time point (n=19, 21 oocytes, respectively, from at least 3 independent experiments).


( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , - 9D or - 9A . Z - projection and 3D - reconstructed images show microtubules ( EGFP - Map4 , green ) and chromosomes ( H2B - mCherry , magenta ) . Scale bar , 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A. Z-projection and 3D-reconstructed images show microtubules (EGFP-Map4, green) and chromosomes (H2B-mCherry, magenta). Scale bar, 10 μm.


( B ) Ndc80 - 9D is defective for limiting spindle elongation . The aspect ratio ( length / width ) of 3D - reconstructed spindles was measured over time ( n = 18 , 18 and 18 oocytes , respectively , from 3 independent experiments ) .

O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O

(B) Ndc80-9D is defective for limiting spindle elongation. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


( C ) Ndc80 - 9D causes excessive spindle elongation . Spindle lengths measured after 3D reconstruction are shown ( n = 18 , 18 and 18 oocytes , respectively , from 3 independent experiments ) .

O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O

(C) Ndc80-9D causes excessive spindle elongation. Spindle lengths measured after 3D reconstruction are shown (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


( A ) K - fibers are connected to MTOCs and extend to spindle poles . Oocytes 6 hours after NEBD were fixed following cold treatment . Microtubules ( α - tubulin , green ) , MTOCs ( pericentrin , magenta ) , kinetochores ( ACA , red ) and DNA ( Hoechst333342 ) are shown . Five oocytes from 2 independent experiments were analyzed . Scale bar , 10 μm .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O

(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm.


( B ) A majority of MTOCs are attached to K - fibers . The volumes of individual MTOCs were measured after 3D reconstruction . The sum volume of MTOCs attached to K - fibers relative to the total volume of MTOCs was calculated for each oocyte ( n = 5 oocytes from 2 independent experiments ) .

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(B) A majority of MTOCs are attached to K-fibers. The volumes of individual MTOCs were measured after 3D reconstruction. The sum volume of MTOCs attached to K-fibers relative to the total volume of MTOCs was calculated for each oocyte (n=5 oocytes from 2 independent experiments).


( C ) K - fibers extend to spindle poles . The length of K - fibers and distance between kinetochores and a spindle pole ( KT - to - Pole distance ) were measured . K - fiber length relative to the KT - to - Pole distance was calculated ( n = 90 K - fibers of 5 oocytes from 2 independent experiments ) .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O O O O O

(C) K-fibers extend to spindle poles. The length of K-fibers and distance between kinetochores and a spindle pole (KT-to-Pole distance) were measured. K-fiber length relative to the KT-to-Pole distance was calculated (n=90 K-fibers of 5 oocytes from 2 independent experiments).


( B ) Wing size variation in absolute centroid size ( CS ) and relative CS ( adjusted for body size ) .

O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(B) Wing size variation in absolute centroid size (CS) and relative CS (adjusted for body size).


( C ) Reproducibility of RNA - seq experiment . Pairwise Spearman correlation coefficients between RNA levels showed higher correlations among biological replicates than among non - replicates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(C) Reproducibility of RNA-seq experiment. Pairwise Spearman correlation coefficients between RNA levels showed higher correlations among biological replicates than among non-replicates.


( D ) Comparable variability of RNA and protein levels for genes quantified at both levels .

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(D) Comparable variability of RNA and protein levels for genes quantified at both levels.


( E ) RNAs of the genes quantified at both RNA and protein levels have the largest means and smallest variabilities among the whole RNAs quantified .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(E) RNAs of the genes quantified at both RNA and protein levels have the largest means and smallest variabilities among the whole RNAs quantified.


( F ) Weak correlation between RNA and protein levels . The median Spearman correlation coefficient was 0 . 1 and only 18 % of genes compared showed a positive correlation with statistical significance ( p < 0 . 05 ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Weak correlation between RNA and protein levels. The median Spearman correlation coefficient was 0.1 and only 18 % of genes compared showed a positive correlation with statistical significance (p < 0.05).


( B ) Association of RNAs with the whole wing size variation that encompasses both sexes . Volcano plot of p - values against the slope of the fitted lines . The horizontal lines indicate 10 and 20 % FDR thresholds .

O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Association of RNAs with the whole wing size variation that encompasses both sexes. Volcano plot of p-values against the slope of the fitted lines. The horizontal lines indicate 10 and 20 % FDR thresholds.


( C ) Association of RNAs with between - line wing size variation ( absolute / relative CS , adjusted for sex ) .

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(C) Association of RNAs with between-line wing size variation (absolute/ relative CS, adjusted for sex).


( E ) Association of canonical growth regulator transcripts with wing size ( absCS and relCS ) . P - values from the tests for association type 2 ( ANCOVA ) and type 3 ( ANOVA on sex ) are plotted . The dashed and dotted lines indicate 10 % and 20 % FDR thresholds , respectively . Note that canonical growth genes mostly exhibit type - 3 association .

O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Association of canonical growth regulator transcripts with wing size (absCS and relCS). P-values from the tests for association type 2 (ANCOVA) and type 3 (ANOVA on sex) are plotted. The dashed and dotted lines indicate 10% and 20% FDR thresholds, respectively. Note that canonical growth genes mostly exhibit type-3 association.


( F ) Sexually dimorphic expression pattern of canonical growth genes . Plots of RNA levels confirm type - 3 association with wing size .

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(F) Sexually dimorphic expression pattern of canonical growth genes. Plots of RNA levels confirm type-3 association with wing size.


( G ) Exceptional growth genes that are associated with between - line size variation ( association type 2 ) .

O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

(G) Exceptional growth genes that are associated with between-line size variation (association type 2).


( C ) The ratio of compartment sizes ( P area divided by A area ) is plotted . The 14 genes were knocked down using multiple ( 2 or 3 ) RNAi lines per gene . The x - axis indicates RNAi case IDs that represent the genes knocked down and the RNAi line type ( KK , GD , or TP as TRiP ) . Detailed information is provided in Dataset EV6 . The horizontal line indicates the size ratio in the negative control cases . Wilcoxon rank sum tests evaluated the statistical significance against controls ( * : p < 0 . 05 , * * * : p < 0 . 001 , n . s . : not significant , cont . : control cases ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The ratio of compartment sizes (P area divided by A area) is plotted. The 14 genes were knocked down using multiple (2 or 3) RNAi lines per gene. The x-axis indicates RNAi case IDs that represent the genes knocked down and the RNAi line type (KK, GD, or TP as TRiP). Detailed information is provided in Dataset EV6. The horizontal line indicates the size ratio in the negative control cases. Wilcoxon rank sum tests evaluated the statistical significance against controls (*:p <0.05, ***:p<0.001, n.s.: not significant, cont.: control cases).


( D ) Adult wings with RNAi manipulation in the P compartment ( shaded area ) . Note that the boundary lines between A and P compartments , which are relatively straight in the control cases , bent towards the P compartment due to the reduced growth and size in the P compartment .

O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O

(D) Adult wings with RNAi manipulation in the P compartment (shaded area). Note that the boundary lines between A and P compartments, which are relatively straight in the control cases, bent towards the P compartment due to the reduced growth and size in the P compartment.


( A ) Cis - association of SNP variation with RNA expression for females and males separately . P - values for eQTLs at FDR < 20 % are plotted against the effect size of the SNPs . The top SNPs are labeled with the corresponding gene names .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

(A) Cis-association of SNP variation with RNA expression for females and males separately. P-values for eQTLs at FDR < 20% are plotted against the effect size of the SNPs. The top SNPs are labeled with the corresponding gene names.


( D and E ) Cis - association of SNP variation with protein expression for females and males . P - value for each pQTL is plotted against the effect size of the SNP . pQTLs at FDRs of 20 % and 30 % are depicted for females and males , respectively . The corresponding proteins are designated with distinct colors . The circle indicates that the pQTL is also eQTL for the same gene .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) Cis-association of SNP variation with protein expression for females and males. P-value for each pQTL is plotted against the effect size of the SNP. pQTLs at FDRs of 20% and 30% are depicted for females and males, respectively. The corresponding proteins are designated with distinct colors. The circle indicates that the pQTL is also eQTL for the same gene.


( B ) Selection of mediator RNAs and the linked eQTLs . RNAs co - associated with SNPs and wing size traits are further tested whether the SNPs are also associated with wing size traits ( absCS and relCS ) for each sex . The dotted lines indicate a significance threshold ( p = 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Selection of mediator RNAs and the linked eQTLs. RNAs co-associated with SNPs and wing size traits are further tested whether the SNPs are also associated with wing size traits (absCS and relCS) for each sex. The dotted lines indicate a significance threshold (p=0.05).


( C and D ) RNAi of CG34015 in the wing P - compartment significantly reduces the size in both sexes . The images are taken from female wings . Statistical significance was evaluated by Wilcoxon test .

O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O

(C and D) RNAi of CG34015 in the wing P-compartment significantly reduces the size in both sexes. The images are taken from female wings. Statistical significance was evaluated by Wilcoxon test.


( E ) Genome - wide association of CG34015 expression . Manhattan plot shows a strong association of CG34015 RNA with X chromosome in both sexes . P - values are plotted against ordered SNPs along the genome . eQTLs associated with CG34015 are designated as red dots .

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(E) Genome-wide association of CG34015 expression. Manhattan plot shows a strong association of CG34015 RNA with X chromosome in both sexes. P-values are plotted against ordered SNPs along the genome. eQTLs associated with CG34015 are designated as red dots.


( F ) Zoomed plot around the CG34015 gene region on X chromosome . eQTLs linked to CG34015 are mostly located upstream of the CG34015 gene region . The red arrow indicates the transcriptional direction .

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(F) Zoomed plot around the CG34015 gene region on X chromosome. eQTLs linked to CG34015 are mostly located upstream of the CG34015 gene region. The red arrow indicates the transcriptional direction.


( G ) SNP variation of CG34015 - linked eQTLs . SNP states at each eQTL are depicted for each line using different colors ( grey dots : reference SNP , blue dots : alternate SNP with a negative effect on RNA level ) . Lines are ordered from left to right as CG34015 RNA levels increase . Note that the eQTLs are highly linked with each other .

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(G) SNP variation of CG34015-linked eQTLs. SNP states at each eQTL are depicted for each line using different colors (grey dots: reference SNP, blue dots: alternate SNP with a negative effect on RNA level). Lines are ordered from left to right as CG34015 RNA levels increase. Note that the eQTLs are highly linked with each other.


( H and I ) Segregation of CG34015 RNA levels and wing size ( absCS ) by the variation of an eQTL ( X _ 16344746 _ SNP ) . P - values are shown . ( J ) Association of the eQTL ( X _ 16344746 _ SNP ) with population - wide , wing size variation among 143 DGRP lines . P - value and R2 are shown for each sex .

O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(H and I) Segregation of CG34015 RNA levels and wing size (absCS) by the variation of an eQTL (X_16344746_SNP). P-values are shown. (J) Association of the eQTL (X_16344746_SNP) with population-wide, wing size variation among 143 DGRP lines. P-value and R2 are shown for each sex.


( A ) Flow cytometry analysis of CD34 + CD1a - thymocytes cultured in FTOCs , in the presence of a control or anti - Notch1 blocking antibody . Numbers in dot plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( B , C ) ( B ) Mean frequency and ( C ) absolute cell numbers of human CD4 + CD8β + DP ( black shades ) , CD3 + TCRαβ + ( blue shades ) and CD3 + TCRγδ + ( red shades ) thymocytes developed in corresponding cultures from A ( n = 6 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a non - parametric paired Wilcoxon test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Flow cytometry analysis of CD34+CD1a- thymocytes cultured in FTOCs, in the presence of a control or anti-Notch1 blocking antibody. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B, C) (B) Mean frequency and (C) absolute cell numbers of human CD4+CD8β+ DP (black shades), CD3+TCRαβ+ (blue shades) and CD3+TCRγδ+ (red shades) thymocytes developed in corresponding cultures from A (n=6 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric paired Wilcoxon test).


( A ) Flow cytometry analysis of CD34 + CD1a - thymocytes cultured in FTOCs , in the presence of a control or anti - Notch1 blocking antibody . Numbers in dot plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(A) Flow cytometry analysis of CD34+CD1a- thymocytes cultured in FTOCs, in the presence of a control or anti-Notch1 blocking antibody. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.


( D ) Flow cytometry analysis of CD34 + CD1a - thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and / or Jag2 ( Cre + ) as indicated above the dot plots . Numbers in dot plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( E , F ) ( E ) Mean frequency and ( F ) absolute cell numbers of CD3 + TCRαβ + ( blue ) and CD3 + TCRγδ + ( red ) thymocytes developed in corresponding cultures from D ( n = 5 - 7 independent experiments , depending on the mouse genotype , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Flow cytometry analysis of CD34+CD1a- thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and/or Jag2 (Cre+) as indicated above the dot plots. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (E, F) (E) Mean frequency and (F) absolute cell numbers of CD3+TCRαβ+ (blue) and CD3+TCRγδ+ (red) thymocytes developed in corresponding cultures from D (n=5-7 independent experiments, depending on the mouse genotype, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).


( D ) Flow cytometry analysis of CD34 + CD1a - thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and / or Jag2 ( Cre + ) as indicated above the dot plots . Numbers in dot plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(D) Flow cytometry analysis of CD34+CD1a- thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and/or Jag2 (Cre+) as indicated above the dot plots. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.


( A ) Line plots depicting fluctuations in gene expression levels from vsn normalized probe intensities across human T cell development for both αβ - and γδ - lineages ( n = 2 biological replicates and error bars indicate SEM ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

(A) Line plots depicting fluctuations in gene expression levels from vsn normalized probe intensities across human T cell development for both αβ- and γδ-lineages (n=2 biological replicates and error bars indicate SEM).


( D - E ) Scatterplot displaying the up ( red ) or down ( blue ) regulation of probes ( log2 fold change > = 2 ) identified by sequential pairwise comparisons across either αβ ( D ) or γδ ( E ) development .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Scatterplot displaying the up (red) or down (blue) regulation of probes (log2 fold change >= 2) identified by sequential pairwise comparisons across either αβ (D) or γδ (E) development.


( C - D ) Scatterplot displaying the up ( red ) or down ( blue ) regulation of miRNA ( log2 fold change > = 2 ) identified by sequential pairwise comparisons starting from CB towards either the αβ ( C ) or γδ ( D ) developmental lineages .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Scatterplot displaying the up (red) or down (blue) regulation of miRNA (log2 fold change >= 2) identified by sequential pairwise comparisons starting from CB towards either the αβ (C) or γδ (D) developmental lineages.


( A ) Line plots displaying averaged NOTCH1 , MYC and HES1 expression across T cell development ( n = 2 biological replicates and error bars indicate SEM ) .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

(A) Line plots displaying averaged NOTCH1, MYC and HES1 expression across T cell development (n=2 biological replicates and error bars indicate SEM).


( B ) Heatmap displaying transcript wise scaled expression of NOTCH1 , HES1 , MYC and miRNAs from ex vivo isolated subsets correlating ( spearman rho > = 0 . 5 ) with NOTCH1 gene expression Red arrows indicate all 6 members of the miR - 17 ~ 92 cluster ( n = 2 ) .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(B) Heatmap displaying transcript wise scaled expression of NOTCH1, HES1, MYC and miRNAs from ex vivo isolated subsets correlating (spearman rho >= 0.5) with NOTCH1 gene expression Red arrows indicate all 6 members of the miR-17~92 cluster (n=2).


( C ) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions ( OP9 - control , OP9 - JAG1 , OP9 - JAG2 , OP9 - DLL1 and OP9 - DLL4 ) and time points ( day 0 , 5 and 10 ) .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O B-CELL_LINE O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O

(C) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions (OP9-control, OP9-JAG1, OP9-JAG2, OP9-DLL1 and OP9-DLL4) and time points (day 0, 5 and 10).


( D ) NOTCH1 correlating miRNAs ( spearman rho > = 0 . 3 ) of in vitro cultured thymocytes across multiple culturing conditions and timepoints . The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR - 17 ~ 92 cluster ( n = 2 ) .

O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(D) NOTCH1 correlating miRNAs (spearman rho >= 0.3) of in vitro cultured thymocytes across multiple culturing conditions and timepoints. The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR-17~92 cluster (n=2).


( E ) CHIPseq data for Notch1 and RBPj binding at the miR - 17 ~ 92 locus in CUTTL1 cells . Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O O O O O O O O O O

(E) CHIPseq data for Notch1 and RBPj binding at the miR-17~92 locus in CUTTL1 cells. Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample.


( B ) Transfection of miR - 17 Pre - miR miRNA Precursor , but not of miR - 92a , specifically targets the wild - type ( black bars ) 3 ' UTR of BCL11B in a 3 ' UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls ( transfection of no miRNA or a control miRNA ) . Mutation of the predicted miR - 17 binding sites in the 3 ' UTR BCL11B reporter construct prevents miR - 17 dependent targeting ( grey bars ) . Graphs show mean firefly luciferase values ( from 3 ' UTR reporter constructs ) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA ( n = 5 independent experiments , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a paired t - test ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Transfection of miR-17 Pre-miR miRNA Precursor, but not of miR-92a, specifically targets the wild-type (black bars) 3'UTR of BCL11B in a 3'UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls (transfection of no miRNA or a control miRNA). Mutation of the predicted miR-17 binding sites in the 3'UTR BCL11B reporter construct prevents miR-17 dependent targeting (grey bars). Graphs show mean firefly luciferase values (from 3'UTR reporter constructs) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA (n=5 independent experiments, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).


( C ) Quantitative RT - PCR gene expression analysis of BCL11B in control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes after 24h of culture on coated Fc - DLL1 plates , relative to the geometric mean of ACTIN and GAPDH and relative to the control - transduced sample ( n = 3 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes after 24h of culture on coated Fc-DLL1 plates, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=3 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( D ) Quantitative real - time RT - PCR gene expression analysis of BCL11B in control ( black bar ) or miR - 17 sponge ( white bar ) transduced CD34 + thymocytes , relative to the geometric mean of ACTIN and GAPDH ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative real-time RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 sponge (white bar) transduced CD34+ thymocytes, relative to the geometric mean of ACTIN and GAPDH (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( A ) Gene expression pattern of BCL11B ( red dots ) and HES1 ( blue squares ) during human T cell development , derived from microarray data as shown in Figure 2 . ( n = 2 biological replicates and error bars indicate SEM ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(A) Gene expression pattern of BCL11B (red dots) and HES1 (blue squares) during human T cell development, derived from microarray data as shown in Figure 2. (n=2 biological replicates and error bars indicate SEM).


Quantitative RT - PCR expression analysis of BCL11B in human after 5 days , respectively , of coculture on OP9 stromal cells expressing different Notch ligands . Graphs show mean expression , relative to the geometric mean of ACTIN and GAPDH and relative to the OP9 - DLL1 cultured cells ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a paired t - test ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of BCL11B in human after 5 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).


Quantitative RT - PCR expression analysis of BCL11B in human CD34 + thymocyte progenitors after 2 days , respectively , of coculture on OP9 stromal cells expressing different Notch ligands . Graphs show mean expression , relative to the geometric mean of ACTIN and GAPDH and relative to the OP9 - DLL1 cultured cells ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a paired t - test ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).


( D ) Quantitative RT - PCR expression analysis of BCL11B in human CD34 + thymocyte progenitors after 2 days of coculture on OP9 - DLL1 stromal cells in the presence of 5 µM DAPT ( GSI ) . Graphs show mean expression , relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O B-CELL_LINE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


Quantitative RT - PCR expression analysis of miR - 17 in human CB CD34 + Lin - after 5 days , respectively , of coculture on OP9 stromal cells expressing different Notch ligands Graphs show mean expression , relative to the geometric mean of SNORD61 , SNORD95 and RNU6 - 2 and relative to the OP9 - DLL1 cultured cells ( n = 2 independent experiments with different donors ; error bars indicate SEM - the two data points are shown ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM B-TISSUE B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of miR-17 in human CB CD34+Lin- after 5 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands Graphs show mean expression, relative to the geometric mean of SNORD61, SNORD95 and RNU6-2 and relative to the OP9-DLL1 cultured cells (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).


Quantitative RT - PCR expression analysis of miR - 17 in human CD34 + thymocyte progenitors after 5 and 2 days , respectively , of coculture on OP9 - DLL1 stromal cells in the presence of 5 µM DAPT ( GSI ) . Graphs show mean expression , relative to the sample cultured in the presence of control DMSO ( F ) ( n = 2 independent experiments with different donors ; error bars indicate SEM - the two data points are shown ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR expression analysis of miR-17 in human CD34+ thymocyte progenitors after 5 and 2 days, respectively, of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the sample cultured in the presence of control DMSO (F) (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).


( G ) Quantitative RT - PCR gene expression analysis of BCL11B in control ( black bar ) , GATA3 shRNA or TCF7 shRNA ( grey bars ) transduced CD34 + thymocytes after 48h of coculture on OP9 - DLL1 stromal cells , relative to the geometric mean of ACTIN and GAPDH and relative to the control - transduced sample ( n = 6 independent experiments with different donors for GATA3 shRNA , n = 4 independent experiments with different donors for TCF7 shRNA , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-CELL_LINE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(G) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar), GATA3 shRNA or TCF7 shRNA (grey bars) transduced CD34+ thymocytes after 48h of coculture on OP9-DLL1 stromal cells, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=6 independent experiments with different donors for GATA3 shRNA, n=4 independent experiments with different donors for TCF7 shRNA, error bars indicate SEM and * indicates p<0.05 in a paired t-test).