( E ) Effect of ( PR ) 20 ( 10 μM , 16 h ) in the number of Cajal Bodies ( CB ) identified by immunofluorescence with anti - COILIN and anti - SMN antibodies ( n = 3 ) . Data represent mean values ± SD . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test . Representative images from these analyses are provided in each case . Scale bar ( white ) indicates 2 - 5 μm .

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(E) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( B , C ) Percentage of GAPDH or 5 . 8 rRNA levels quantified by qPCR in reactions containing increasing doses of ( PR ) 20 ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test .

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(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.


( D ) HTM - mediated analysis of 5 - ethynyl - deoxyuridine ( EdU ) levels per nucleus found in U2OS cells exposed to 10 μM ( PR ) 20 for 4 h . EdU was added 30 ´ prior to fixation . Representative images from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( white ) indicates 5 μm .

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(D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( E ) HTM - mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM ( PR ) 20 for the indicated times . Note the accumulation of cells with persistent DNA damage ( γH2AX ) 10 h after IR in the presence of the DPR . Representative images from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( white ) indicates 5 μm .

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(E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( D ) Immunofluorescence of Cy3 - labelled salmon protamine ( red ) and the nucleolar factor UBF1 ( green ) in U2OS cells treated with 30 μM Cy3 - protamine for 4 h . Scale bar ( white ) indicates 2 . 5 μm .

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(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.


( E , F ) HTM - mediated quantification of the EU levels per nucleus ( E ) and O - propargyl - puromycin ( OPP ) levels per cell ( F ) in U2OS cells exposed to 30 μM protamine for 16 ( E ) or 24 ( F ) h . EU and OPP were added 30 ´ prior to fixation . Black lines indicate median values ( * * * * , p < 0 . 0001 ; t - test ) .

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(E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).


( D ) Immunofluorescence of HA - ( PR ) 20 ( green ) and the nucleolar factor UBF1 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test .

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(D) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( E ) Immunofluorescence of HA - ( PR ) 20 ( green ) and Cy5 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 4 μM of Cy5 - labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test .

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(E) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( F ) Clonogenic survival assay of U2OS cells exposed to 7 . 5 μM with the indicated peptides or protamine ( PROT . ) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0 . 5 μM Heparin .

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(F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.


( G ) Immunofluorescence of HA - ( PR ) 20 ( green ) and the nucleolar factor UBF1 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with heparin ( 0 . 5 μM ) . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test .

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(G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( A ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without 2 μM of a non - coding 38nt oligonucleotide . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . Note that besides its impact on overall survival , the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide . ( B , C ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( B ) and NSC34DIFF ( C ) treated as in ( A ) ( n = 3 ) . Data represent mean values ± SEM . ( Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.


( D ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without heparin ( 0 . 5 μM ) . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . ( E , F ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( E ) and NSC34DIFF ( F ) treated as in ( D ) ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . S

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(D) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S


Protein levels of E2F7 and E2F8 during cell cycle progression . HeLa cells were synchronized by a double thymidine block and released into fresh medium . Cells were harvested at the indicated time points and an asynchronous ( AS ) condition was used as control . Protein levels were measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . The asterisk indicates the E2F7 - specific band .

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Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.


Decreased stability of E2F7 and E2F8 in nocodazole - arrested cells . HeLa cells were either treated with DMSO or nocodazole ( 50 ng / ml ) for 16 hours . Cells were harvested and lysed for immunoblotting . Protein expression of cyclin B1 was used as a marker for G2 or M , and γ - tubulin was used as loading control .

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Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.


Selective Cullin - RING inhibitor MLN4924 rescued the degradation of E2F7 / 8 under nocodazole - arrested condition . HeLa cells were treated with DMSO , nocodazole or nocodazole plus MLN4924 ( 0 . 1 μM ) for 16 hours . Cells were harvested and lysed for immunoblotting . cyclin B1 expression was used as a marker for G2 or M cell cycle progression , and γ - tubulin was used as loading control .

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Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.


Increased half - life of E2F7 / 8 by MLN4924 treatment . HeLa cells were either treated with cycloheximide ( CHX , 50 ug / ml ) with or without MLN4924 ( 0 . 1 μM ) . Protein levels of E2F7 and E2F8 were determined by immunoblotting ( left panel ) . Asterisk indicates the E2F7 - specific band . cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression , γ - tubulin was used as loading control . Quantifications ( right panels ) were performed based on two independent experiments . Bar and error bars represent mean ± SEM .

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Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.


C - terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F . The residues on each motif were mutated to alanines ( R to A , I / L to A ) with site - direct mutagenesis PCR . HEK293 cells were transfected with either EGFP - tagged empty vector ( EGFP ) , wild - types E2F7 / 8 ( WT ) or alanine mutants . Nocodazole ( 50 ng / ml ) was added 32 hours after transfection , and MG132 ( 1 μg / ml ) was added 5 hours before harvesting at 48 hours post transfection . Cells were harvested and lysed for immunoprecipitation using anti - EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP .

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C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.


Schematic view showing the truncated mutants and ML / AA mutant of cyclin F ( Left ) . HEK cells were transfected with indicated constructs and co - immunoprecipitation was performed using Flag resin ( Right ) .

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Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).


Wild - type or mutant versions of EGFP - tagged E2F7 / 8 were co - transfected with either empty vector or Flag - tagged cyclin F in HEK293 cells . Nocodazole was added to cells 8h before harvest . 48 hours after transfection , cells were collected and lysed for immunoblotting .

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Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.


Knockdown of cyclin F stabilized E2F7 and E2F8 . HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA . Cells were harvested at 48 hours post transfection . Protein levels of E2F7 / 8 were analyzed by immunoblotting . Asterisk indicates the specific band of E2F7 detection .

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Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.


Cyclin F targets atypical E2Fs during G2 / M . HeLa cells were transfected with either scrambled siRNA ( scr ) or cyclin F siRNA ( sicyclin F ) for 24 hours . Then cells were synchronized by double thymidine block and released into fresh medium after the second block . Cells were harvested at the indicated timepoints after the release . Protein expression was measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . Asterisk indicates the specific detection of endogenous E2F7 .

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Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.


Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo . HEK293 cells were transfected with HA - E2F7 / 8 , with or without Flag - cyclin F , and with HA - tagged ubiquitin . 5 hours before harvest , cells were treated with MG132 . 48 hours after transfection , HEK cells were harvested and lysed for immunoprecipitation pull - down assay with anti - HA resin followed by immunoblotting .

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Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.


Schematic view of the experimental setting for the HU - synchronized live cell imaging . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . 16 hours before imaging , cells were synchronized at the G1 / S border by HU ( 2 mM ) treatment . Representative images from different channels are shown , and white arrows in Differential Interference Contrast DIC channel indicate the traced cell . Scale bar : 10µm .

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Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.


Quantification of the number of Ctrl ( left panel ) and 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or sicyclin F that completed mitosis after HU release . For each condition , 100 cells were monitored by live cell imaging . Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance .

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Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.


Schematic view of the experimental setting for live imaging of asynchronous cells . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . At the start of the imaging , G1 cells ( red : mKO2 - Cdt1 > mAG1 - Geminin , 50 cells per condition ) were enrolled and subsequently monitored though their entire cell cycle until mitosis . The black dot represents mitosis .

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Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.


Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8 . Number of Ctrl ( left panel ) or 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown . For each condition , 50 cells at G1 were monitored by live cell imaging . Each cell was followed until it successfully progressed through S and G2 phase , finished mitosis and divided into two daughter cells , for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance .

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Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.


Loss of cyclin F delays the progression from G1 / S transition to mitosis . Histogram shows the time from G1 / S ( mAG1 - Geminin intensity increases to higher than 10 % of the maximum value in three consecutive imaging frames ) to completed mitosis . Only cells that finished mitosis were enrolled in this quantification . Student t - test was used to test the statistical significance , asterisks indicates the P value < 0 . 01 . Bar and error bars represent mean ± SEM .

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Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.


Depletion of cyclin F stalls the cell cycle at late S / G2 . After 24 hours of live cell imaging , the cell cycle progression from panel E was quantified . Histogram shows the percentage of cells at each stage ( at 24h ) over the whole population ( 50 cells per condition ) .

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Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).


Disruption of cyclin F binding site increased stability of E2F7 . E2F7WT and E2F7R894A constructs were integrated in to HeLa / TO system . Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium , and cells were collected every 3 hours for immunoblotting .

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Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.


qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels . Bar and error bars represent mean ± SEM .

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qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.


Over - expression of E2F7R894A delays cell cycle progression through G2 - M phase . HeLa / TO cells expressing either wild - type or mutant version of E2F7 were arrested with 16h of HU , and then cells were released into fresh medium with or without doxycycline . Live cell imaging was performed to trace the G2 - M progression of HeLa / TO cells .

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Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.


Heatmap showing differentially expressed genes after cyclin F knockdown , and rescued by additional E2F7 / 8 depletion . Highlighted genes are all involved in DNA repair . Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions .

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Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.


qPCR showing the RNA expression of atypical E2F - target genes that are involved in DNA replication or DNA repair . HeLa cells were transfected for 48 hours with siRNAs as indicated . Cells were incubated with nocodazole for 16 hours before harvesting . Data represent averages ± SEM ( n = 3 ) ; * P < 0 . 05 or * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant .

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qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.


Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions . HeLa cells were treated with nocodazole for 16 hours prior to harvesting .

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Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.


Loss 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 .

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Loss 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 .

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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 ) .

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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 .

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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.


Wild - 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 .

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Wild-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 .

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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 .

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(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 .

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(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 .

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(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.


A ) 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 .

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A) 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 .

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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 .

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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 .

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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.


A ) 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 .

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A) 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 .

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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 .

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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 .

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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 .

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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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(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.


( 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 .

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(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 .

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(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 .

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(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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O 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-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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-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 .

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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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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.