Figure 7Loss of cyclin F induced E2F7 / 8 - dependent Homologous Recombination deficiency . HeLa cells that were stably transformed with pDR - GFP were transfected with siRNA as indicated . 48h after the initial transfection , cells were harvested for Flow - cytometry . GFP positive cells were gated ( Fig EV5A ) . Relative HR efficiency were adjusted to the scramble siRNA condition . Data represent averages ± SEM ( n = 3 ) ; * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Loss of cyclin F induced E2F7 / 8 - dependent γ - H2AX accumulation . HeLa cells were transfected with indicated siRNA for 24 hours , and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ - H2AX . DAPI was used to stain the cell nucleus . Relative intensity of γ - H2AX was quantified by Image J software , and 150 cells were quantified for each condition . Red bars represent averages ; * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Scale bar 20 μm . Loss of cyclin F increased DNA damage recovery time before cell division . RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours , and then treated with HU for 16 hours . At the beginning of the imaging , only the single cells with at least one 53BP1 foci were traced , till the time frame that no 53BP1 foci was observed . The mitotic progression of the cells was defined as the duration from damage recovery to cell division . Histogram shows the damage recovery time ( green ) and the mitotic progression ( black ) of 50 cells for each condition . Chi - square analysis was performed to test the statistical significance ( P < 0 . 01 ) . Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7 / 8 . The cumulative curves represent the add - up number of cells that overcome the DNA damage lesions , for a time frame of 24 hours . For each condition , 50 cells were quantified . Log - rank tests were performed to analyze the statistical significance .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.


Figure 1Wild - type and LGP2 - KO HEK293 cells in 24 - well plates were transfected with 200 ng / ml of short poly I : C IFN - β and IP - 10 mRNA expression was determined by RT - qPCR . The data represent the mean ± SD ( n = 3 , t - test , * p < 0 . 05 ) . LGP2 KO2 and KO3 are independently isolated LGP2 KO clones . 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 . Wild - type and LGP2 - KO HEK293 cells in 24 - well plates were infected with SeV . IFN - β and IP - 10 mRNA expression was determined by RT - qPCR . The data represent the mean ± SD ( n = 3 , t - test , * 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 ) HEK293 cells in 24 - well plates were transfected with LGP2 ( 0 . 1 , 0 . 3 , or 0 . 6 μg / well ) and RIG - I ( 0 . 1 μg / well ) expression vectors as indicated . 24 h after transfection , cells were stimulated with 200 ng of short poly I : C for 4 hr . Total RNAs were extracted , and the expression of IFN - β mRNA was determined by RT - qPCR and normalized to GAPDH ( n = 3 , t - test , * 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 . ( G ) HEK293FT cells were transfected with FLAG - tagged LGP2 and HA - tagged ubiquitin expression vectors . Whole cell extracts ( WCE ) were prepared 24 h after transfection . Immunoprecipitation was performed with anti - FLAG antibody ( IP : FLAG ) and the proteins were detected by western blot analysis with the indicated antibodies . ( H ) FLAG - tagged LGP2 was transfected into HEK293 cells . Cells were then infected with SeV at MOI = 10 for 24 h . Immunoprecipitation was performed with anti - FLAG antibody , and the proteins were detected with the indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Wild-type and LGP2-KO HEK293 cells in 24-well plates were transfected with 200 ng/ml of short poly I:C IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). LGP2 KO2 and KO3 are independently isolated LGP2 KO clones. 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.Wild-type and LGP2-KO HEK293 cells in 24-well plates were infected with SeV. IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *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) HEK293 cells in 24-well plates were transfected with LGP2 (0.1, 0.3, or 0.6 μg/well) and RIG-I (0.1 μg/well) expression vectors as indicated. 24 h after transfection, cells were stimulated with 200 ng of short poly I:C for 4 hr. Total RNAs were extracted, and the expression of IFN-β mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, t-test, *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.(G) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged ubiquitin expression vectors. Whole cell extracts (WCE) were prepared 24 h after transfection. Immunoprecipitation was performed with anti-FLAG antibody (IP: FLAG) and the proteins were detected by western blot analysis with the indicated antibodies.(H) FLAG-tagged LGP2 was transfected into HEK293 cells. Cells were then infected with SeV at MOI = 10 for 24 h. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected with the indicated antibodies.


Figure 2A ) HEK293 cells were transfected with FLAG - RIG - I or FLAG - LGP2 expressing vector . Cells were then infected with SeV , and whole cell extracts were collected at indicated time points . Immunoprecipitation was performed with anti - FLAG antibody . The proteins were subjected to SDS - PAGE and detected by western blotting . B ) A549 cells transfected with HA - tagged ubiquitin were stimulated with 200 ng / ml of short poly I : C for 6 h . PLA was performed with anti - HA , anti - RIG - I , and anti - LGP2 antibodies . The number of PLA signals were counted ( t - test , * p < 0 . 05 , n = 20 ) . Each dot represents the number of PLA signals of each cell . Data information : Scale bars represent 10 μm . 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 ) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr . PLA was performed with anti - LGP2 and anti - K63 - Ub antibodies . The number of PLA signals were counted ( t - test , * p < 0 . 05 , n = 33 ) . Each dot represents the number of PLA signals of each cell . Data information : Scale bars represent 10 μm . 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 - F ) HEK293 cells were transfected with 200 ng / ml of short poly I : C . Cells were fixed at the indicated time points , and PLA was performed with anti - RIG - I , anti - LGP2 , and anti - K63 - Ub antibodies ( D ) . The number of PLA signals were counted ( E , F ) ( t - test , * p < 0 . 05 , n = 30 ) . Each dot represents the number of PLA signals of each cell . Data information : Scale bars represent 10 μm . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) HEK293 cells were transfected with FLAG-RIG-I or FLAG-LGP2 expressing vector. Cells were then infected with SeV, and whole cell extracts were collected at indicated time points. Immunoprecipitation was performed with anti-FLAG antibody. The proteins were subjected to SDS-PAGE and detected by western blotting.B) A549 cells transfected with HA-tagged ubiquitin were stimulated with 200 ng/ml of short poly I:C for 6 h. PLA was performed with anti-HA, anti-RIG-I, and anti-LGP2 antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 20). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. 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) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr. PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 33). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. 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-F) HEK293 cells were transfected with 200 ng/ml of short poly I:C. Cells were fixed at the indicated time points, and PLA was performed with anti-RIG-I, anti-LGP2, and anti-K63-Ub antibodies (D). The number of PLA signals were counted (E, F) (t-test, *p < 0.05, n = 30). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. 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.


Figure 3A ) HEK293FT cells were transfected with FLAG - tagged LGP2 and HA - tagged Riplet expression vectors . 24 h after transfection , cells were lysed , and WCE were prepared . Immunoprecipitation was performed with anti - FLAG antibody , and the proteins were detected by western blotting with the indicated antibodies . B ) HEK293FT cells were transfected with FLAG - tagged LGP2 expression vector . 24 h after transfection , immunoprecipitation was performed with anti - FLAG antibody and control IgG . Immunoprecipitates were subjected to SDS - PAGE , and the proteins were detected by western blotting with indicated antibodies . C ) HEK293FT cells were transfected with FLAG - tagged LGP2 , Riplet , and HA - tagged ubiquitin expression vectors , and immunoprecipitation was performed with anti - FLAG antibody . D ) HEK293FT cells were transfected with FLAG - tagged LGP2 , Riplet , and myc - tagged K63 - only - Ub expression vectors . 24 h after transfection , immunoprecipitation was performed with anti - FLAG antibody . E ) WT and Riplet KO HEK293 cells were transfected with FLAG - tagged LGP2 and myc - tagged K63 - only - Ub expression vectors , and immunoprecipitation was performed with anti - FLAG antibody . ( F , G ) WT and Riplet KO HEK293 cells were transfected with 200 ng / ml of short poly I : C for 6 h , and PLA was performed with anti - LGP2 and anti - K63 - Ub antibodies . The number of PLA signals were counted ( t - test , * p < 0 . 05 , n = 100 ) . Each dot represents the number of PLA signals in each cell . Scale bars represent 10 μm . 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 .

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Figure 3A) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged Riplet expression vectors. 24 h after transfection, cells were lysed, and WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected by western blotting with the indicated antibodies.B) HEK293FT cells were transfected with FLAG-tagged LGP2 expression vector. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody and control IgG. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies.C) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and HA-tagged ubiquitin expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.D) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and myc-tagged K63-only-Ub expression vectors. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody.E) WT and Riplet KO HEK293 cells were transfected with FLAG-tagged LGP2 and myc-tagged K63-only-Ub expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.(F, G) WT and Riplet KO HEK293 cells were transfected with 200 ng/ml of short poly I:C for 6 h, and PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 100). Each dot represents the number of PLA signals in each cell. Scale bars represent 10 μm. 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.


Figure 4 ( A ) Liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) of LGP2 in Riplet - expressing cells . LC - MS / MS data was quantified using LFQ method . The chromatographic profiles for each ubiquitination site are shown in the right panels . Schematic representation of ubiquitination sites of RIG - I and LGP2 are shown in the upper panel . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of LGP2 in Riplet-expressing cells. LC-MS/MS data was quantified using LFQ method. The chromatographic profiles for each ubiquitination site are shown in the right panels. Schematic representation of ubiquitination sites of RIG-I and LGP2 are shown in the upper panel.(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.


Figure 5 ( 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.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.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.


Figure 6A ) 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 . 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 . 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 . 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 .

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Figure 6A) 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.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.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.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.


Figure 7WT , 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7WT, 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.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.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.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.


Figure 1A - 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 .

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Figure 1A-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.


Figure 2A - 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 ) . 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 ) .

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Figure 2A-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).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).


Figure 3A - 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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Figure 3A-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.


Figure 1 ( 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 ) .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).


Figure 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 . 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 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.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.(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.


Figure 4 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 1 ( 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 ) . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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). (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).(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.


Figure 2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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).


Figure 3 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).(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).


Figure 4 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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).(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).


Figure 5 ( 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 ) . ( 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 ) .

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Figure 5(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).(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).


Figure 1 ( 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 . ( 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 . ( 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 ) .

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Figure 1(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.(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.(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).


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

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


Figure 3 ( 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 .

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Figure 3(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.


Figure 4 ( 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 .

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Figure 4(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.


Figure 5 ( 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 . ( 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 . ( 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 .

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Figure 5(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.(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.(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.


Figure 1 ( 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 ) . ( 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 ) .

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Figure 1 (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).  (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).


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 .

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Figure 3 (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.


Figure 4 ( 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 ) . ( 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 ) . ( 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 .

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Figure 4(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).  (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).(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.