Growth curves of the MCF7 ( left ) or T47D ( right ) cells upon HNRNPC knock - down and JAK - STAT inhibition with Ruxolitinib ( 5uM ) . Error bars represent mean ± SD . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock-down and JAK-STAT inhibition with Ruxolitinib (5uM). Error bars represent mean ± SD. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. Each sample has 3 replicates. Error bars represent mean ± SD.


Expression levels of ISGs ( A ) upon HNRNPC silencing in the cells with lentiviral shRNA - mediated stable knock - down of DDX58 or IFIH1 . Each sample has 3 replicates . Error bars represent mean ± SD .

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Expression levels of ISGs (A) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.


Expression levels of IFNβ concentration in the media ( B ) upon HNRNPC silencing in the cells with lentiviral shRNA - mediated stable knock - down of DDX58 or IFIH1 . Each sample has 3 replicates . Error bars represent mean ± SD .

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Expression levels of IFNβ concentration in the media (B) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of the DDX58 - and IFIH1 - deficient MCF7 ( left ) and T47D ( right ) cells upon HNRNPC knock - down . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the DDX58- and IFIH1-deficient MCF7 (left) and T47D (right) cells upon HNRNPC knock-down. Each sample has 3 replicates. Error bars represent mean ± SD.


Xenograft tumor models with DDX58 - deficient MCF7 cells . Images , weights , and growth records of the xenograft tumor models in female NSG mice established from the DDX58 - deficient MCF7 cells upon lentivirus - mediated stable knock - down of LMNA or HNRNPC . Each group has 6 mice . The error bars represent the ± SEM .

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Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the DDX58-deficient MCF7 cells upon lentivirus-mediated stable knock-down of LMNA or HNRNPC. Each group has 6 mice. The error bars represent the ± SEM.


Xenograft tumor models with DDX58 - deficient MCF7 cells . Images , weights , and growth records of the xenograft tumors derived from DDX58 - deficient MCF7 cells , which were subjected to periodic siRNA injection , starting from 2 weeks after transplantation of the cells . Each group has 4 mice . The error bars represent the ± SEM .

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Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumors derived from DDX58-deficient MCF7 cells, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.


Immunofluorescence analysis of the dsRNA in MCF7 cells after knock - down of HNRNPC , with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) staining ( blue ) and anti - dsRNA antibody J2 ( green ) . Cells transfected with poly I : C was included as a positive control of dsRNA , and the cells treated with RNase III was used as a negative control . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC . The size of scale bar is 10um .

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Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10um.


Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC , identified in the dsRNA - enriched libraries with anti - dsRNA J2 pull - down or in the control libraries including the IgG control and the input control for dsRNA pull - down . Each bar represents the overlapping dsRNA species from two replicate experiments . Among each set of the dsRNA regions , the dsRNAs from Alus were marked in grey .

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Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in grey.


MA plot showing all the dsRNA regions identified in the dsRNA - enriched libraries . For each dsRNA region , mean of the read counts ( log2 ) for two replicates of siHNRNPC and siNC cells was shown on the x - axis , and the average fold change ( log2 ) in siHNRNPC vs . siNC on the y - axis . The up - regulated dsRNA regions were marked in red and down - regulated regions in black .

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MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black.


( A ) Internalization of apoptotic or necrotic GFP - expressing RAW264 . 7 cells . ( B ) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres ( B ) in primary M2 ( IL - 4 ) and M0 macrophages . Cytochalasin D ( Cyto ) ( 6 µM ) was used as an inhibitor of phagocytosis , 1 h before phagocytosis . Data are shown as means of relative fluorescence units ( RFU ) ± standard error of the mean ( SEM ) .

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(A) Internalization of apoptotic or necrotic GFP-expressing RAW264.7 cells. (B) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL-4) and M0 macrophages. Cytochalasin D (Cyto) (6 µM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM).


( C - E ) Real - time fluorescence assays for intraphagosomal proteolysis ( C ) , acidification ( D ) and lipolysis ( E ) show substantially increased proteolysis , acidification and lipolysis in the phagosomes of M2 ( IL - 4 ) macrophages . The kinetics of proteolysis , acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence . Beads were added to macrophages at 0 min . Shaded area in ( C ) & ( D ) represents SEM . ( E ) is a representative of three independent experiments . Leupeptin ( 100nM ) and Bafilomycin ( 100nM ) treatment serve as negative controls in ( C ) and ( D ) , respectively .

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(C-E) Real-time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2(IL-4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. Shaded area in (C) & (D) represents SEM. (E) is a representative of three independent experiments. Leupeptin (100nM) and Bafilomycin (100nM) treatment serve as negative controls in (C) and (D), respectively.


( B ) Volcano plot of the phagosome proteome data . 1 , 766 proteins were quantified of which 121 proteins were significantly up - regulated and 62 proteins were down - regulated in M2 ( IL - 4 ) macrophages . Selected proteins are indicated .

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(B) Volcano plot of the phagosome proteome data. 1,766 proteins were quantified of which 121 proteins were significantly up-regulated and 62 proteins were down-regulated in M2(IL-4) macrophages. Selected proteins are indicated.


( C ) Heatmap of proteomics data shows high reproducibility between biological replicates . Selected proteins are highlighted .

(C) Heatmap of proteomics data shows high reproducibility between biological replicates. Selected proteins are highlighted.


( A ) Immunoblot ( IB ) analysis showing the recruitment of TAK1 , MKK7 , JNK and TAB1 / TAB2 to the phagosome in M2 ( IL - 4 ) macrophages , while MKK4 is not recruited . Rab7a , a phagosomal marker , was used as a loading control for phagosomes and vimentin was used as a loading control for total cell lysates . The white line in the JNK blot shows other bands were cut out .

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(A) Immunoblot (IB) analysis showing the recruitment of TAK1, MKK7, JNK and TAB1/TAB2 to the phagosome in M2(IL-4) macrophages, while MKK4 is not recruited. Rab7a, a phagosomal marker, was used as a loading control for phagosomes and vimentin was used as a loading control for total cell lysates. The white line in the JNK blot shows other bands were cut out.


( B ) Quantitation of three independent IB experiments by imageJ of non - saturated blots for TAK1 , MKK7 , TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2 ( IL - 4 ) macrophages . Error bars represent SEM . * p < 0 . 01 , * * p < 0 . 001 ( Student ' s T - test )

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(B) Quantitation of three independent IB experiments by imageJ of non-saturated blots for TAK1, MKK7, TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2(IL-4) macrophages. Error bars represent SEM. *p<0.01, **p<0.001 (Student's T-test)


( C ) IB showing enrichment of K63 polyubiquitylated proteins on the phagosome of M2 ( IL - 4 ) macrophages compared to M0 macrophages .

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(C) IB showing enrichment of K63 polyubiquitylated proteins on the phagosome of M2(IL-4) macrophages compared to M0 macrophages.


Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1 , TAB2 , TAK1 and MKK7 to the phagosome of M2 ( IL - 4 ) macrophages , indicating a K63 - polyubiquitylation dependent translocation for these proteins .

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Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1, TAB2, TAK1 and MKK7 to the phagosome of M2(IL-4) macrophages, indicating a K63-polyubiquitylation dependent translocation for these proteins.


( B ) Selected ubiquitylated phagosomal proteins identified by TUBE - MS approach .

(B) Selected ubiquitylated phagosomal proteins identified by TUBE-MS approach.


( D ) Tab2 - TUBE pull - downs from phagosome extracts of M2 ( IL - 4 ) macrophages treated with the K63 specific deubiquitylase ( DUB ) AMSH - LP or the unspecific DUB USP2 .

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(D) Tab2-TUBE pull-downs from phagosome extracts of M2(IL-4) macrophages treated with the K63 specific deubiquitylase (DUB) AMSH-LP or the unspecific DUB USP2.


MSR1 immunoprecipitation from M2 ( IL - 4 ) macrophage phagosomes shows that TAB1 , TAB2 , TAK1 and MKK7 bind to polyubiquitylated MSR1 . TAK1 shows a specific pattern of post - translational modifications indicative of its activation .

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MSR1 immunoprecipitation from M2(IL-4) macrophage phagosomes shows that TAB1, TAB2, TAK1 and MKK7 bind to polyubiquitylated MSR1. TAK1 shows a specific pattern of post-translational modifications indicative of its activation.


( A ) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL .

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(A) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL.


( B ) Msr1 + / + ( WT ) and Msr1 - / - ( KO ) M0 macrophages and M2 ( IL - 4 ) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL ( 50 μg / ml , 30 min ) were analysed for the phosphorylated and the total forms of JNK1 / 2 and MSR1 . ITGAM ( CD11b ) serves as a loading control . Both fucoidan and oxLDL activate JNK in a MSR1 - dependent manner .

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(B) Msr1+/+ (WT) and Msr1-/- (KO) M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL (50 μg/ml, 30 min) were analysed for the phosphorylated and the total forms of JNK1/2 and MSR1. ITGAM (CD11b) serves as a loading control. Both fucoidan and oxLDL activate JNK in a MSR1-dependent manner.


( C ) IL4 - activated MSR1 knock - out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg / ml Fucoidan for 1 hr . Mutation of the ubiquitylation site K27 abolishes MSR1 signalling .

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(C) IL4-activated MSR1 knock-out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg/ml Fucoidan for 1 hr. Mutation of the ubiquitylation site K27 abolishes MSR1 signalling.


( D ) qPCR data of Tnfa , Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2 ( IL - 4 ) BMDMs shows an MSR1 - dependent increase of pro - inflammatory cytokines in response to MSR1 ligation by fucoidan .

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(D) qPCR data of Tnfa, Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2(IL-4) BMDMs shows an MSR1-dependent increase of pro-inflammatory cytokines in response to MSR1 ligation by fucoidan.


( E ) Inhibition of JNK by JNK - IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation , showing that it is JNK - dependent .

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(E) Inhibition of JNK by JNK-IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation, showing that it is JNK-dependent.


( F ) Flow cytometry analysis of cell surface markers in WT and MSR1 - / - M0 macrophages and M2 ( IL - 4 ) macrophages untreated or stimulated with the MSR1 ligands fucoidan ( 50 μg / ml , 24 h ) . Data shows MSR1 - dependent increase of the early activation markers CD54 , CD69 and CD86 and a decrease of the M2 marker CD301b / Mgl2 . CD11b serves as a control .

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(F) Flow cytometry analysis of cell surface markers in WT and MSR1-/- M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan (50 μg/ml, 24 h). Data shows MSR1-dependent increase of the early activation markers CD54, CD69 and CD86 and a decrease of the M2 marker CD301b/Mgl2. CD11b serves as a control.


( A ) Immunohistochemistry analysis of MSR1 in patient ovarian , uterus and sarcoma cancers showing tumour - associated macrophages .

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(A) Immunohistochemistry analysis of MSR1 in patient ovarian, uterus and sarcoma cancers showing tumour-associated macrophages.


( B ) Pull - downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour .

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(B) Pull-downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour.


( B ) Schematic of marker - less GFP - tagging at the endogenous locus and representative live cell images of Mad1 - , Mad2 - , and Mad3 - GFP strains ( average intensity projections ) .

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(B) Schematic of marker-less GFP-tagging at the endogenous locus and representative live cell images of Mad1-, Mad2-, and Mad3-GFP strains (average intensity projections).


( C ) Representative images of single molecule mRNA FISH ( smFISH ) staining of S . pombe using probes against GFP ( red ) . DNA was stained with DAPI ( blue ) . The gamma - value was adjusted to make the cytoplasm visible ; cell shapes are outlined in blue . ( D ) Frequency distribution of mRNA numbers per cell determined by smFISH ; n = number of cells . Curves show fit to a Poisson distribution .

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(C) Representative images of single molecule mRNA FISH (smFISH) staining of S. pombe using probes against GFP (red). DNA was stained with DAPI (blue). The gamma-value was adjusted to make the cytoplasm visible; cell shapes are outlined in blue. (D) Frequency distribution of mRNA numbers per cell determined by smFISH; n = number of cells. Curves show fit to a Poisson distribution.


( E ) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP - tagged gene or the endogenous , untagged gene . Curves show fit to a Poisson distribution . The difference for mad1 + is statistically significant , that for mad2 + is not A lower mRNA number for untagged mad1 + was also observed in an independent strain .

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(E) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP-tagged gene or the endogenous, untagged gene. Curves show fit to a Poisson distribution. The difference for mad1+ is statistically significant, that for mad2+ is not A lower mRNA number for untagged mad1+ was also observed in an independent strain.


( F ) Co - staining by smFISH using probes against mad1 + and GFP either in a strain expressing mad1 + - GFP as a positive control or in a strain expressing wild - type mad1 + and mad2 + - GFP . Cytoplasmic mad1 + ( green ) or GFP mRNA spots ( magenta ) were quantified as co - localizing or not with the respective other . For the mad1 + - GFP strain , 544 cells and a total of 1 , 641 mad1 spots and 1 , 839 GFP spots were analyzed ; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm . For the mad1 + mad2 + - GFP strain , 571 cells and a total of 1 , 107 mad1 spots and 1 , 537 GFP spots were analyzed ; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm .

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(F) Co-staining by smFISH using probes against mad1+ and GFP either in a strain expressing mad1+-GFP as a positive control or in a strain expressing wild-type mad1+ and mad2+-GFP. Cytoplasmic mad1+ (green) or GFP mRNA spots (magenta) were quantified as co-localizing or not with the respective other. For the mad1+-GFP strain, 544 cells and a total of 1,641 mad1 spots and 1,839 GFP spots were analyzed; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm. For the mad1+ mad2+-GFP strain, 571 cells and a total of 1,107 mad1 spots and 1,537 GFP spots were analyzed; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm.


( A ) Cellular protein noise ( coefficient of variation , CV = std / mean ) in live - cell microscopy images of S . pombe ; n = 7 images ( Nmt1 - GFP ) , 11 ( Mad1 - GFP ) , 19 ( Mad2 - GFP ) , 10 ( Mad3 - GFP ) ; single images had 16 - 79 GFP - positive and 6 - 94 GFP - negative ( control ) cells . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Mad1 , Mad2 , and Mad3 all showed significantly lower noise than Nmt1 ( Wilcoxon rank sum test ; all p < 0 . 001 ) .

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(A) Cellular protein noise (coefficient of variation, CV = std / mean) in live-cell microscopy images of S. pombe; n = 7 images (Nmt1-GFP), 11 (Mad1-GFP), 19 (Mad2-GFP), 10 (Mad3-GFP); single images had 16-79 GFP-positive and 6-94 GFP-negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all p < 0.001).


( B ) Simulations of stochastic gene expression noise from selected mRNA / protein half - life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell , and a mean protein number of 6000 per cell . The x - axis of each graph shows time , the y - axis shows mRNA number per cell ( blue ) or protein number per cell ( black ) . ( C ) Theoretical prediction for the coefficient of variation ( CV = std / mean ) of the protein number per cell , assuming different mRNA and protein half - lives , using the same underlying model as in B . Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3 . 5 , and a mean protein number per cell of 6000 ( approx . 100 nM ) .

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(B) Simulations of stochastic gene expression noise from selected mRNA/protein half-life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6000 per cell. The x-axis of each graph shows time, the y-axis shows mRNA number per cell (blue) or protein number per cell (black). (C) Theoretical prediction for the coefficient of variation (CV = std / mean) of the protein number per cell, assuming different mRNA and protein half-lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6000 (approx. 100 nM).


( D ) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Lines are regression curves from generalized linear mixed model fits , excluding the measurements at t = 0 in order to accommodate for non - instantaneous labeling by 4tU . Act1 + and ecm33 + were used as long and short half - life controls , respectively ; qPCR was performed for the endogenous mRNAs . Half - lives ( 95 % confidence interval ) : mad1 + 5 . 6 min ( 4 . 3 - 8 . 4 ) , mad2 + 7 . 7 min ( 6 . 2 - 10 . 4 ) , mad3 + 5 . 2 min ( 4 . 3 - 6 . 9 ) , act1 + 61 . 8 min ( 37 . 2 - 172 . 3 ) , ecm33 + 5 . 0 min ( 4 . 5 - 5 . 7 ) .

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(D) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for non-instantaneous labeling by 4tU. Act1+ and ecm33+ were used as long and short half-life controls, respectively; qPCR was performed for the endogenous mRNAs. Half-lives (95 % confidence interval): mad1+ 5.6 min (4.3 - 8.4), mad2+ 7.7 min (6.2 - 10.4), mad3+ 5.2 min (4.3 - 6.9), act1+ 61.8 min (37.2 - 172.3), ecm33+ 5.0 min (4.5 - 5.7).


( E ) Protein abundances after translation shut - off with cycloheximide ( CHX ) ; n = 3 experiments , error bars = std . Lines indicate fit to a one - phase exponential decay . Cdc2 and Cdc13 were used as long and short half - life controls , respectively .

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(E) Protein abundances after translation shut-off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one-phase exponential decay. Cdc2 and Cdc13 were used as long and short half-life controls, respectively.


( A ) The mean CSC value for each S . pombe gene ( CSCg ) relative to protein number per cell by mass spectrometry CSC was determined using the mRNA half - life data Colored dots highlight proteins of interest . For Mad2 and Bub1 , no protein abundance data was available . ( B ) Cumulative frequency distribution of the CSCg values for protein - coding S . pombe genes . The position of spindle assembly checkpoint genes is highlighted .

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(A) The mean CSC value for each S. pombe gene (CSCg) relative to protein number per cell by mass spectrometry CSC was determined using the mRNA half-life data Colored dots highlight proteins of interest. For Mad2 and Bub1, no protein abundance data was available. (B) Cumulative frequency distribution of the CSCg values for protein-coding S. pombe genes. The position of spindle assembly checkpoint genes is highlighted.


( D ) Scatter plots of whole cell RNA counts versus cell length . Solid lines are regression curves from generalized linear mixed model fits ( gray : wild type , black : codon - optimized or ste13 ∆ ) . Dashed lines : 95 % bootstrap confidence bands for the regression curves . Model estimates of the ratio relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to five replicates per genotype .

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(D) Scatter plots of whole cell RNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to five replicates per genotype.


( E ) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( dark = ste13 + , light = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded area : 95 % bootstrap confidence band for ste13 + ; dashed lines : 95 % bootstrap confidence band for ste13 ∆ . Half - life estimates are included with 95 % bootstrap confidence intervals in brackets .

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(E) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.


( A ) Immunoblot of S . pombe protein extracts from cells expressing wild - type ( WT ) or codon - optimized ( co ) Mad2 - GFP or Mad3 - GFP probed with antibodies against GFP and Cdc2 ( loading control ) . Lanes 3 - 5 are a 1 : 1 dilution series of the extract from cells expressing the codon - optimized version . ( B ) Immunoblot of protein extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad2 , Mad3 , and tubulin ( loading control ) . A 1 : 1 dilution series was loaded for quantification . ( C ) Estimates of the protein concentration relative to wild - type conditions from experiments such as in ( A ) and ( B ) . Bars are experimental replicates , dots are technical replicates . Two - sided t - tests : p = 0 . 03 ( Mad2 - co ) , 0 . 004 ( Mad3 - co ) , 0 . 82 ( Mad2 ste13 ∆ ) , 0 . 15 ( Mad3 ste13 ∆ ) .

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(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad2-GFP or Mad3-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a 1:1 dilution series of the extract from cells expressing the codon-optimized version. (B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad2, Mad3, and tubulin (loading control). A 1:1 dilution series was loaded for quantification. (C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Two-sided t-tests: p = 0.03 (Mad2-co), 0.004 (Mad3-co), 0.82 (Mad2 ste13∆), 0.15 (Mad3 ste13∆).


( D ) Whole - cell GFP concentration from individual live cell fluorescence microscopy experiments ( a . u . = arbitrary units ) . Boxes show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Codon - optimized concentration significantly higher than wild type for both genes ( GLMM ) . Mad2 - GFP : n = 468 and 413 ; Mad2 - co - GFP : n = 206 and 366 ; Mad3 - GFP : n = 224 and 127 ; Mad3 - co - GFP : n = 160 , 450 and 212 cells . ( E ) Representative images from one of the experiments in ( D ) . A single Z - slice is shown . Cells are outlined in gray .

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(D) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxes show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly higher than wild type for both genes (GLMM). Mad2-GFP: n = 468 and 413; Mad2-co-GFP: n = 206 and 366; Mad3-GFP: n = 224 and 127; Mad3-co-GFP: n = 160, 450 and 212 cells. (E) Representative images from one of the experiments in (D). A single Z-slice is shown. Cells are outlined in gray.


( B ) Scatter plots of whole cell mRNA counts versus cell length . Solid lines are regression curves from generalized linear mixed model fits ( gray : wild type , black : codon - optimized or ste13 ∆ ) . Dashed lines : 95 % bootstrap confidence bands for the regression curves . Model estimates of the ratio relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to three replicates per genotype .

(B) Scatter plots of whole cell mRNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to three replicates per genotype.


( C ) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( dark = ste13 + , light = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded area : 95 % bootstrap confidence band for ste13 + ; dashed lines : 95 % bootstrap confidence band for ste13 ∆ . Half - life estimates are included with 95 % bootstrap confidence intervals in brackets .

(C) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.


( D ) Comparison between mean CSC values for selected genes ( CSCg ) and mRNA half - life measured with or without deletion of ste13 + .

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(D) Comparison between mean CSC values for selected genes (CSCg) and mRNA half-life measured with or without deletion of ste13+.


( A ) Immunoblot of S . pombe protein extracts from cells expressing wild - type ( WT ) or codon - optimized ( co ) Mad1 - GFP probed with antibodies against GFP and Cdc2 ( loading control ) . Lanes 3 - 5 are a dilution series of the extract from wild - type cells . ( B ) Immunoblot of protein extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad1 and tubulin ( loading control ) . A 1 : 1 dilution series was loaded for quantification . ( C ) Estimates of the protein concentration relative to wild - type conditions from experiments such as in ( A ) and ( B ) . Bars are experimental replicates , dots are technical replicates . Blue lines indicate the mean of all experiments . Two - sided t - tests : p = 0 . 005 ( Mad1 - co , n = 4 experimental replicates ) ; p = 0 . 16 ( Mad1 ste13 ∆ , n = 2 ) .

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(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad1-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a dilution series of the extract from wild-type cells. (B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad1 and tubulin (loading control). A 1:1 dilution series was loaded for quantification. (C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Blue lines indicate the mean of all experiments. Two-sided t-tests: p = 0.005 (Mad1-co, n = 4 experimental replicates); p = 0.16 (Mad1 ste13∆, n = 2).


( D ) Whole - cell GFP concentration from individual live cell fluorescence microscopy experiments ( a . u . = arbitrary units ) . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Codon - optimized concentration significantly lower than wild type ( generalized linear mixed model ) . Mad1 - GFP : n = 197 and 224 ; Mad1 - co - GFP : n = 80 and 377 cells . ( E ) Representative images from one of the experiments in ( D ) . An average projection of three Z - slices is shown ; cells are outlined in gray .

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(D) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly lower than wild type (generalized linear mixed model). Mad1-GFP: n = 197 and 224; Mad1-co-GFP: n = 80 and 377 cells. (E) Representative images from one of the experiments in (D). An average projection of three Z-slices is shown; cells are outlined in gray.


( F ) Live - cell imaging for time spent in mitosis . The alp7 + gene was deleted to increase the likelihood of spindle assembly checkpoint activation . Localization of Plo1 - tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis Exp1 : n = 73 ( WT ) and 94 cells ( co ) ; Exp2 : n = 126 ( WT ) and 152 cells ( co ) . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Measurements for individual cells are shown in addition ( gray circles if measurement was exact , red triangles if end of mitosis was not captured because imaging ended ) . Difference between WT and co : p = 0 . 14 ( Exp1 ) and 0 . 15 ( Exp2 ) by Kolmogorov - Smirnov test .

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(F) Live-cell imaging for time spent in mitosis. The alp7+ gene was deleted to increase the likelihood of spindle assembly checkpoint activation. Localization of Plo1-tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis Exp1: n = 73 (WT) and 94 cells (co); Exp2: n = 126 (WT) and 152 cells (co). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Measurements for individual cells are shown in addition (gray circles if measurement was exact, red triangles if end of mitosis was not captured because imaging ended). Difference between WT and co: p = 0.14 (Exp1) and 0.15 (Exp2) by Kolmogorov-Smirnov test.


( A ) Top : Immunoprecipitation ( IP ) with anti - GFP or anti - Mad1 from extracts of haploid S . pombe cells expressing both untagged and GFP - tagged Mad1 , probed with antibodies against Mad1 and tubulin ; in = input ( 2 . 5 % of extract for IP ) , sup = supernatant after IP . Bottom : Comparison between the observed ( obs . ) and the expected ( expect . ) ratio between Mad1 - GFP and untagged Mad1 in the IP given their ratio in the input ; two and one experiment ( s ) , respectively . One more GFP - IP from the same strain was unquantifiable , because no second band was visible in the IP .

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(A) Top: Immunoprecipitation (IP) with anti-GFP or anti-Mad1 from extracts of haploid S. pombe cells expressing both untagged and GFP-tagged Mad1, probed with antibodies against Mad1 and tubulin; in = input (2.5 % of extract for IP), sup = supernatant after IP. Bottom: Comparison between the observed (obs.) and the expected (expect.) ratio between Mad1-GFP and untagged Mad1 in the IP given their ratio in the input ; two and one experiment(s), respectively. One more GFP-IP from the same strain was unquantifiable, because no second band was visible in the IP.


( C ) Top : Anti - GFP immunoprecipitation ( IP ) and Strep pull - down ( PD ) from extracts of diploid cells expressing Mad1 - GFP and Mad1 - Strep from the two endogenous loci ; membrane probed with anti - Mad1 ; in = input ( 7 % of extract for IP / PD ) , sup = supernatant after IP / PD . Bottom : as in ( A ) , 2 experiments each . The experiment shown at the top and two more GFP - IPs from the same strain were unquantifiable , because no second band was visible in the IP .

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(C) Top: Anti-GFP immunoprecipitation (IP) and Strep pull-down (PD) from extracts of diploid cells expressing Mad1-GFP and Mad1-Strep from the two endogenous loci; membrane probed with anti-Mad1; in = input (7 % of extract for IP/PD), sup = supernatant after IP/PD. Bottom: as in (A), 2 experiments each. The experiment shown at the top and two more GFP-IPs from the same strain were unquantifiable, because no second band was visible in the IP.


( D ) In vitro translation ( IVT ) of Mad1 - flag - His and untagged Mad1 in the presence of 35S - labelled Methionine and Cysteine , followed by Mad1 immunoprecipitation ( IP ) or His pull - down ( PD ) ; in = input ( 9 . 5 % of extract for IP / PD ) , sup = supernatant after IP / PD . An IVT with only untagged Mad1 was used to check for specificity of the His PD ( right side ) . Shown is the autoradiograph after SDS - PAGE with quantification of the Mad1 - flag - His to untagged Mad1 ratio in select lanes .

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(D) In vitro translation (IVT) of Mad1-flag-His and untagged Mad1 in the presence of 35S-labelled Methionine and Cysteine, followed by Mad1 immunoprecipitation (IP) or His pull-down (PD); in = input (9.5 % of extract for IP/PD), sup = supernatant after IP/PD. An IVT with only untagged Mad1 was used to check for specificity of the His PD (right side). Shown is the autoradiograph after SDS-PAGE with quantification of the Mad1-flag-His to untagged Mad1 ratio in select lanes.


( E ) Test for mRNA dimerization by single molecule mRNA FISH ; probes against mad1 + and GFP . Top : Schematic of genotypes . Bottom left : Intensity of cytoplasmic mad1 + mRNA spots in the different strains . For the 2 copy strain , a mad1 + spot was classified as mad1 + - GFP if it was colocalizing with a GFP spot , and as mad1 + otherwise . Colors as indicated in the schematic . Vertical solid line : peak of each density plot ; dashed line : theoretical position of a double - intensity peak . Number of spots analyzed : mad1 + ( 1 copy strain ) = 921 , mad1 + ( 2 copy strain ) = 637 , mad1 + - GFP ( 2 copy strain ) = 982 , mad1 + - GFP ( 1 copy strain ) = 1699 . Bottom right : Counts of cytoplasmic mad1 + or mad1 + - GFP mRNA from the same experiment with generalized linear mixed model fits as lines . Number of cells : 1 copy strain mad1 + = 478 , 2 copy strain = 327 , 1 copy strain mad1 + - GFP = 466 .

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(E) Test for mRNA dimerization by single molecule mRNA FISH; probes against mad1+ and GFP. Top: Schematic of genotypes. Bottom left: Intensity of cytoplasmic mad1+ mRNA spots in the different strains. For the 2 copy strain, a mad1+ spot was classified as mad1+-GFP if it was colocalizing with a GFP spot, and as mad1+ otherwise. Colors as indicated in the schematic. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+ (1 copy strain) = 921, mad1+ (2 copy strain) = 637, mad1+-GFP (2 copy strain) = 982, mad1+-GFP (1 copy strain) = 1699. Bottom right: Counts of cytoplasmic mad1+ or mad1+-GFP mRNA from the same experiment with generalized linear mixed model fits as lines. Number of cells: 1 copy strain mad1+ = 478, 2 copy strain = 327, 1 copy strain mad1+-GFP = 466.


( F ) Experiment similar to ( E ) , except that cells expressing both mad1 + - GFP and mad3 + - GFP from the respective endogenous locus were probed with FISH probes against mad1 + and GFP mRNA . A GFP spot was classified as mad1 + - GFP if it was colocalizing with a mad1 + spot ( arrowheads ) , and as mad3 + - GFP otherwise . The intensity of GFP spots was quantified . Vertical solid line : peak of each density plot ; dashed line : theoretical position of a double - intensity peak . Number of spots analyzed : mad1 + - GFP = 987 , mad3 + - GFP = 1299 .

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(F) Experiment similar to (E), except that cells expressing both mad1+-GFP and mad3+-GFP from the respective endogenous locus were probed with FISH probes against mad1+ and GFP mRNA. A GFP spot was classified as mad1+-GFP if it was colocalizing with a mad1+ spot (arrowheads), and as mad3+-GFP otherwise. The intensity of GFP spots was quantified. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+-GFP = 987, mad3+-GFP = 1299.


B . Confocal microscopy images of HeLa , RPE , and U2OS cell lines that were pre - extracted with Triton X - 100 before fixed with formaldehyde and co - stained with DAPI and antibodies against PCNA and FAM111A . Scale bars : 10

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B. Confocal microscopy images of HeLa, RPE, and U2OS cell lines that were pre-extracted with Triton X-100 before fixed with formaldehyde and co-stained with DAPI and antibodies against PCNA and FAM111A. Scale bars: 10 μm


C . U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy . Scale bars : 10 μm .

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C. U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy. Scale bars: 10 μm.


HEK293 , RPE , or U2OS / MycBioID - FAM111A cell lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) , 48 h ( RPE ) , or 72h ( U2OS ) . The colonies were fixed then stained with crystal violet .

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HEK293, RPE, or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293), 48 h (RPE), or 72h (U2OS). The colonies were fixed then stained with crystal violet.


Cell - cycle profiles of HEK293 , RPE and U2OS FAM111A - expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry .

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Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.


C . HEK293 cell lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) .

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C. HEK293 cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293) Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3). *p<0.05, **p<0.01, ****p<0.0001 (two-tailed unpaired t-test).


D . Cell - cycle profiles of HEK293 FAM111A - expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry .

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D. Cell-cycle profiles of HEK293 FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.


A . HEK293 or U2OS / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from 105 cells was resolved by SDS - PAGE , and probed in western blots for both endogenous FAM111A ( 71 kDa ) and MycBioID - FAM111A ( 110 kDa ) as well as full length and cleaved PARP ( 116 and 89 kDa , respectively ) . The Tubulin western blot is a sample loading control .

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A. HEK293 or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from 105 cells was resolved by SDS-PAGE, and probed in western blots for both endogenous FAM111A (71 kDa) and MycBioID-FAM111A (110 kDa) as well as full length and cleaved PARP (116 and 89 kDa, respectively). The Tubulin western blot is a sample loading control.


B . HEK293 / MycBioID - FAM111A cell lines with no treatment ( - Dox ) , or treated with doxycycline , along with DMSO or 100 μM Z - VAD - FMK for 24 h ( total inhibition of PARP cleavage was also observed when 50 μM Z - VAD - FMK was used ) . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots with Myc , PARP , or Tubulin antibodies .

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B. HEK293/MycBioID-FAM111A cell lines with no treatment (- Dox), or treated with doxycycline, along with DMSO or 100 μM Z-VAD-FMK for 24 h (total inhibition of PARP cleavage was also observed when 50 μM Z-VAD-FMK was used). Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots with Myc, PARP, or Tubulin antibodies.


C . HEK293 cell lines were treated with doxycycline and Z - VAD - FMK for 24 h . The colonies were fixed then stained with crystal violet . Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) .

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C. HEK293 cell lines were treated with doxycycline and Z-VAD-FMK for 24 h. The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3).


C . Cells were treated with doxycycline along with DMSO or Z - VAD - FMK for 24 h . They were fixed with formaldehyde , and stained with NPC antibody Mab414 and DAPI . The images were captured by confocal microscopy . Scale bar : 10 μm .

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C. Cells were treated with doxycycline along with DMSO or Z-VAD-FMK for 24 h. They were fixed with formaldehyde, and stained with NPC antibody Mab414 and DAPI. The images were captured by confocal microscopy. Scale bar: 10 μm.


D . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from an equal number of cells was incubated with magnetic streptavidin beads , and pull downs were analyzed by western blotting using GANP and Myc antibodies .

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D. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from an equal number of cells was incubated with magnetic streptavidin beads, and pull downs were analyzed by western blotting using GANP and Myc antibodies.


E . HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h , while growing in the presence or absence of 50 mM supplemental biotin . Total protein from whole cell lysates was incubated with magnetic streptavidin beads . Pulldowns were analyzed by western blotting using GANP and Myc antibodies .

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E. HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h, while growing in the presence or absence of 50 mM supplemental biotin. Total protein from whole cell lysates was incubated with magnetic streptavidin beads. Pulldowns were analyzed by western blotting using GANP and Myc antibodies.


F . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots using antibodies against NPC ( Mab414 ) , PARP , cleaved Caspase 3 , Myc , or Tubulin .

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F. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots using antibodies against NPC (Mab414), PARP, cleaved Caspase 3, Myc, or Tubulin.


A . Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA . Images were taken with a Zeiss Axio Imager . LT is shown in green , and DAPI blue . Scale bar : 100 μm . B . Quantification of LT positive cells described in A and fixed at indicated times post transfection . Five hundred or more cells were counted for each condition . C . Quantification of RFP marker distribution upon co - transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA . Cells were fixed at the indicated times . Five hundred or more cells were counted for each condition . In B & C , values are mean ± s . d . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) .

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A. Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA. Images were taken with a Zeiss Axio Imager. LT is shown in green, and DAPI blue. Scale bar: 100 μm. B. Quantification of LT positive cells described in A and fixed at indicated times post transfection. Five hundred or more cells were counted for each condition. C. Quantification of RFP marker distribution upon co-transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA. Cells were fixed at the indicated times. Five hundred or more cells were counted for each condition. In B & C, values are mean ± s.d. of independent experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired t-test).


D . Western blot analysis of control or FAM111A - depleted U2OS cells transfected with SV40 LT variants . Cells were collected and lysed at indicated timepoints after SV40 transfection , immunoblotted with FAM111A , LT , or PARP antibodies . Cdc2 - PSTAIR was used as a loading control .

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D. Western blot analysis of control or FAM111A-depleted U2OS cells transfected with SV40 LT variants. Cells were collected and lysed at indicated timepoints after SV40 transfection, immunoblotted with FAM111A, LT, or PARP antibodies. Cdc2-PSTAIR was used as a loading control.


B . Binding assay using MBP - fused Apc1 - loop500 fragments . MBP - fused Apc1 - loop500 WT or its derivatives ( T532A , T539A , 2A ( T532A / T539A ) and S558A ) was incubated with anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining .

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B. Binding assay using MBP-fused Apc1-loop500 fragments. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, 2A (T532A/T539A) and S558A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining.


D . Specific binding of Plx1 - PBD to Apc1 - loop500 . MBP - fused Apc1 - loop500 WT or its derivatives 2A ( T532A / T539A ) was incubated in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated in the presence of 10 µg of WT Plx1 - PBD for 15 min . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by

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D. Specific binding of Plx1-PBD to Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives 2A (T532A/T539A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated in the presence of 10 µg of WT Plx1-PBD for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie


A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant WT or Apc - loop500 mutant APC / Cs with mutations ( 1 - T532A , 1 - T539A , 1 - T532A / T539A ( 1 - 2A ) and 1 - S558A ) was incubated with APC / C depleted ( ΔAPC ) interphase extract ( Inter ) or ΔAPC anaphase extracts supplemented with non - degradable cyclin B ( Ana ) at 23 ˚ C for 1 h . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies .

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A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant WT or Apc-loop500 mutant APC/Cs with mutations (1-T532A, 1-T539A, 1-T532A/T539A (1-2A) and 1-S558A) was incubated with APC/C depleted (ΔAPC) interphase extract (Inter) or ΔAPC anaphase extracts supplemented with non-degradable cyclin B (Ana) at 23˚C for 1 h. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies.


E . In vitro phosphorylation by Plx1 . Apc3 - loop fragment , α - Casein , N - terminus of Cdc20 ( N159 ) or MBP protein was incubated with purified Plx1 in the presence of [ γ32P ] - ATP at 23 ˚ C for indicated times , separated by SDS - PAGE and detected by autoradiography and Coomassie brilliant blue ( CBB ) staining .

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E. In vitro phosphorylation by Plx1. Apc3-loop fragment, α-Casein, N-terminus of Cdc20 (N159) or MBP protein was incubated with purified Plx1 in the presence of [γ32P]-ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by autoradiography and Coomassie brilliant blue (CBB) staining.


C . Plx1 - binding assay using MBP - fused Apc1 - loop500 fragments with mutations in B56 - binding site . The Plx1 - binding to Apc1 - loop500 WT or its derivatives ( L557A / V560A or E562A ) was analysed as described in Fig 1B . D . Quantification of ( C ) . The bar graph is quantification of bound Plx1 . The intensities of WT were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

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C. Plx1-binding assay using MBP-fused Apc1-loop500 fragments with mutations in B56-binding site. The Plx1-binding to Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was analysed as described in Fig 1B. D. Quantification of (C). The bar graph is quantification of bound Plx1. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


E . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . MBP - fused Apc1 - loop500 WT or its derivatives ( T532A , T539A , T532A / T539A ( 2A ) and S558A ) was incubated with the 35S - labelled Flag - B56γ in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or Coomassie brilliant blue ( CBB ) staining . The recovery of WT ( x1 / 2 and x1 ) was run as a standard . F . Quantification of ( E ) . The bar graph is quantification of bound B56γ . The intensities of WT were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

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E. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, T532A/T539A (2A) and S558A) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The recovery of WT (x1/2 and x1) was run as a standard. F. Quantification of (E). The bar graph is quantification of bound B56γ. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


A . Plx1 and PP2A - B56 competes for Apc1 - loop500 . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including the B56 - binding motif ) was incubated with the 35S - labelled Flag - B56γ in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated with indicated amounts of WT Plx1 - PBD or Plx1 - PBD Pincer mutant ( Pin ) for 15 min . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or Coomassie brilliant blue ( CBB ) staining .

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A. Plx1 and PP2A-B56 competes for Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining.


C . Dephosphorylation of Apc1 - loop500 by PP2A - B56 in the presence of Plx1 - PBD . MBP - fused Apc1 - loop500 WT or its derivatives ( L557A / V560A or E562A ) was incubated in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated with 10 µg of WT Plx1 - PBD for 15 min . The complexes were isolated by amylose beads and incubated in the presence ( 78 nM ) or absence of purified PP2A - B56γ at 23 ˚ C for 20 min . The proteins were recovered by amylose beads , and analysed as described in ( B ) .

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C. Dephosphorylation of Apc1-loop500 by PP2A-B56 in the presence of Plx1-PBD. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, and analysed as described in (B).


A . Phosphorylation kinetics of T532 , T539 and S558 on Apc1 - loop500 in anaphase extracts . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including the B56 - binding motif ) was incubated with Xenopus egg extracts in the presence of non - degradable cyclin B at 23 ˚ C . The samples taken as indicated were analysed by SDS - PAGE and immunoblotting using phospho - specific antibodies ( pT532 , pT539 and pS558 ) or MBP antibody . To monitor CDK activity , Apc3 antibody was used ( bottom ) . The signals of a phospho - specific antibody and MBP antibody on the same membrane were detected in the 800 - and 700 - nm channels , respectively . The two channels were pseudo - coloured ( green ; pT532 , pT539 and pS558 , red ; MBP ) and overlaid in the bottom panel ( Overlay ) . The signal of pS558 was not detected in ∆ 11 mutant because ∆ 11 mutation includes S558 .

O O O O O O O O O O O B-MEASURED_VAR O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

A. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 in anaphase extracts. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with Xenopus egg extracts in the presence of non-degradable cyclin B at 23˚C. The samples taken as indicated were analysed by SDS-PAGE and immunoblotting using phospho-specific antibodies (pT532, pT539 and pS558) or MBP antibody. To monitor CDK activity, Apc3 antibody was used (bottom). The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was not detected in ∆11 mutant because ∆11 mutation includes S558.


A . Cdc20 binding assay in Xenopus egg extracts . The Cdc20 - binding activities of WT APC / C and Apc - loop500 - mutant APC / Cs with mutations ( 1 - L557A / V560A or 1 - E562A ) were analysed as described in Fig 2A . B . Quantification of ( A ) . The bar graph is quantification of bound Cdc20 . The intensities of WT control at Ana - 45 min were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O 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 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

A. Cdc20 binding assay in Xenopus egg extracts. The Cdc20-binding activities of WT APC/C and Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) were analysed as described in Fig 2A. B. Quantification of (A). The bar graph is quantification of bound Cdc20.The intensities of WT control at Ana-45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


D . APC / C dephosphorylation after inactivation of CDK . The purified recombinant WT or Apc - loop500 - mutant APC / Cs with mutations ( 1 - L557A / V560A or 1 - E562A ) was incubated with APC / C depleted ( ΔAPC ) in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1h ( Ana ) . After addition of p27 ( 0 . 3 µM ) , the samples taken at time points shown were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ' Inter ' denotes interphase .

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D. APC/C dephosphorylation after inactivation of CDK. The purified recombinant WT or Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) was incubated with APC/C depleted (ΔAPC) in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1h (Ana). After addition of p27 (0.3 µM), the samples taken at time points shown were analysed by SDS-PAGE and immunoblotting with indicated antibodies. 'Inter' denotes interphase.


A . The mutations in CDK sites on Apc3 - loop reduces phosphorylation of T532 on Apc1 . The purified recombinant WT or a mutant APC / C with mutations at nine CDK sites in Apc3 - loop ( 3 - 9A ) was incubated with APC / C - depleted ( ΔAPC ) anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 60 min . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , and the phosphorylation status of T532 in Apc1 was analysed by SDS - PAGE and immunoblotting with phospho - specific ( pT532 ) or Apc1 ( pan - Apc1 ) antibodies .

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A. The mutations in CDK sites on Apc3-loop reduces phosphorylation of T532 on Apc1. The purified recombinant WT or a mutant APC/C with mutations at nine CDK sites in Apc3-loop (3-9A) was incubated with APC/C-depleted (ΔAPC) anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 60 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the phosphorylation status of T532 in Apc1 was analysed by SDS-PAGE and immunoblotting with phospho-specific (pT532) or Apc1 (pan-Apc1) antibodies.


( B - E ) Immunostaining of germaria from 7 day - old females with anti - Vasa ( green , B - D ) or anti - GFP ( green , E ) , and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . ( B ) aubHN2 / + was used as a control . ( C , D ) examples of aubHN2 / QC42 germ cell loss and tumor , respectively . ( E ) Phenotypic rescue of aubHN2 / QC42 with UASp - GFP - Aub expressed using nos - Gal4 . White arrowheads indicate GSCs ; the white arrow indicates GSC loss .

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(B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss.


( F ) Quantification of mutant germaria with 0 to 1 GSC , or with GSC tumors shown in ( B - E ) . The number of scored germaria ( n ) is indicated on the right graph . * * * p - value < 0 . 001 , ns : non significant , using the χ2 test .

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

(F) Quantification of mutant germaria with 0 to 1 GSC, or with GSC tumors shown in (B-E). The number of scored germaria (n) is indicated on the right graph. *** p-value <0.001, ns: non significant, using the χ2 test.


( G - H ' ) Germaria containing control ( G , G ' ) or aubHN2 mutant ( H , H ' ) clonal GSCs stained with anti - GFP ( green ) and anti - Hts ( red ) , 14 days after clone induction . DAPI ( blue ) was used to stain DNA . Clonal cells are marked by the lack of GFP . Clonal GSCs and cysts are outlined with dashed line . White arrowheads show clonal GSCs in the control . aub mutant clonal GSCs have been lost ( H , H ' ) . Scale bar : 10 μm in B - E and G - H ' .

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(G-H') Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). Scale bar: 10 μm in B-E and G-H'.


( I ) Quantification of germaria containing at least one clonal GSC at 7 , 14 and 21 days after clonal induction .

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(I) Quantification of germaria containing at least one clonal GSC at 7, 14 and 21 days after clonal induction.


( J ) Division rate of wild - type and aubHN2 clonal GSCs . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

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

(J) Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. *** p-value <0.001 using the two-tailed Student's t test.


( A , B ) Immunostaining of germaria with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of mnkP6 and mnkP6 aubHN2 / QC42 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss .

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(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss.


( C ) Quantification of the number of GSCs per non - tumorous germarium , and of germaria with GSC tumors , in the indicated genotypes . The number of scored germaria ( n ) is indicated . The two - tailed Student ' s t test and the χ2 test were used in the left and right panels , respectively . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. The two-tailed Student's t test and the χ2 test were used in the left and right panels, respectively. *** p-value <0.001, ** p-value <0.01, * p-value <0.05.


( D - F ) Immunostaining of germaria from 7 day - old females with anti - Vasa or anti - GFP ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . aubHN2 / QC42 ; nos - Gal4 / + was used as a negative control . Examples of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - Aub germarium ( E ) , and of lack of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - AubAA germarium ( F ) . White arrowheads indicate GSCs ; white arrows indicate GSC loss in ( D ) and germ cell loss in ( F ) . Scale bar : 10 μm in A , B and D - F .

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

(D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F.


( G ) Quantification of mutant germaria with 0 to 1 GSC shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , ns : non - significant , using the χ2 test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of mutant germaria with 0 to 1 GSC shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001, ns: non-significant, using the χ2 test.


( A - B ' ' ) Immunostaining of wild - type ( A - A ' ' ) or nos - Gal / UASp - CCR4 - HA ( B - B ' ' ) germaria with anti - Aub ( red ) , and anti - CCR4 or anti - HA ( green ) . GSCs are shown in ( A - A ' ' ) . White arrowheads in ( B - B ' ' ) indicate cytoplasmic accumulation of CCR4 - HA colocalized with Aub in GSCs .

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(A-B'') Immunostaining of wild-type (A-A'') or nos-Gal/UASp-CCR4-HA (B-B'') germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A-A''). White arrowheads in (B-B'') indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs.


( C ) Co - immunoprecipitation ( IP ) of NOT1 , NOT3 and CCR4 with GFP - Aub in ovaries . Wild - type ( WT , mock IP ) or nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) ovarian extracts were immunoprecipitated with anti - GFP , either in the absence or the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 , anti - NOT3 and anti - CCR4 . Inputs are extracts prior to IP .

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(C) Co-immunoprecipitation (IP) of NOT1, NOT3 and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3 and anti-CCR4. Inputs are extracts prior to IP.


( D ) Co - IP of NOT1 and NOT3 with GFP - Aub or GFP - AubAA in ovaries . Wild - type ( WT , mock IP ) , nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) or nos - Gal4 / UASp - GFP - AubAA ( GFP - AubAA ) ovarian extracts were immunoprecipitated with anti - GFP in the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 and anti - NOT3 . Inputs are extracts prior to IP .

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(D) Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub) or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1 and anti-NOT3. Inputs are extracts prior to IP.


( E - G ) Genetic interaction between aub and twin in Vasa self - renewal . Immunostaining of germaria with anti - DNA ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of aubHN2 / + , twinDG24102 and aubHN2 / + ; twinDG24102 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss . Scale bar : 10 μm in A - B ' ' and E - G .

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(E-G) Genetic interaction between aub and twin in Vasa self-renewal. Immunostaining of germaria with anti-DNA (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B'' and E-G.


( H ) Quantification of mutant germaria with no GSC in 3 , 7 and 14 day - old females of the genotypes shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of mutant germaria with no GSC in 3, 7 and 14 day-old females of the genotypes shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test.


( B - C ' ' ) Immunostaining of mosaic germaria with anti - GFP ( green ) , to identify clonal cells by the lack of GFP , and either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize DNA . White arrowheads indicate aubHN2 / + control GSCs ; yellow arrowheads indicate clonal mutant aubHN2 GSCs . Scale bar : 10 μm .

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(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.


( D - E ) Quantification of Cbl protein levels in aubHN2 / + and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1 . Fluorescence intensity was measured in arbitrary units using the ImageJ software . Horizontal bars correspond to the mean and standard deviation . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

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(D-E) Quantification of Cbl protein levels in aubHN2/+ and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1. Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. *** p-value <0.001 using the two-tailed Student's t test.


( F ) RNA immunoprecitation ( IP ) with anti - GFP antibody in wild - type ( mock IP ) and nos - Gal4 / UASp - GFP - Aub ovarian extracts . Cbl mRNA was quantified using RT - qPCR . Normalization was with RpL32 mRNA . Mean of three biological replicates . The error bar represents standard error to the mean . * p - value < 0 . 05 using the two - tailed Student ' s t test .

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(F) RNA immunoprecitation (IP) with anti-GFP antibody in wild-type (mock IP) and nos-Gal4/UASp-GFP-Aub ovarian extracts. Cbl mRNA was quantified using RT-qPCR. Normalization was with RpL32 mRNA. Mean of three biological replicates. The error bar represents standard error to the mean. * p-value <0.05 using the two-tailed Student's t test.


( G ) ePAT assay of CblL mRNA . Ovaries from 1 - day - old ( germarium to stage 8 ) wild - type , aub and twin mutant females , and from 4 - to 7 - day - old bam mutant females were used .

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(G) ePAT assay of CblL mRNA. Ovaries from 1-day-old (germarium to stage 8) wild-type, aub and twin mutant females, and from 4- to 7-day-old bam mutant females were used.


( D , E ) Coverage of CblL ( D ) and CblS ( E ) mRNAs with ovarian and embryonic piRNAs . Significant crosslink clusters are indicated in red at the top of the graphs .

O O O O O O O B-MEASURED_VAR 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

(D, E) Coverage of CblL (D) and CblS (E) mRNAs with ovarian and embryonic piRNAs. Significant crosslink clusters are indicated in red at the top of the graphs.


O O O O O O O O O O O O B-MEASURED_VAR 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


( A - E ) Cbl expression in germaria . ( A ) Immunostaining of wild - type germaria with anti - Cbl 8C4 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(A-E) Cbl expression in germaria. (A) Immunostaining of wild-type germaria with anti-Cbl 8C4.


( A - E ) Cbl expression in germaria . ( B - B ' ' ) Immunostaining of bamP - GFP germaria that express GFP under the bam promoter , with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) .

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(A-E) Cbl expression in germaria. (B-B'') Immunostaining of bamP-GFP germaria that express GFP under the bam promoter, with anti-GFP (green) and anti-Cbl 8C4 (red).


( A - E ) Immunostaining expression in germaria . ( C - C ' ) Immunostaining of germaria expressing GFP - Par1 to label spectrosomes with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) . DAPI ( blue ) was used to visualize DNA . White arrowheads indicate GSCs ; yellow arrowheads indicate cystoblasts .

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(A-E) Immunostaining expression in germaria. (C-C') Immunostaining of germaria expressing GFP-Par1 to label spectrosomes with anti-GFP (green) and anti-Cbl 8C4 (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; yellow arrowheads indicate cystoblasts.


( F ) Western blots of protein extracts from wild - type and Cbl mutant ovaries showing CblL ( left panel ) and CblS ( right panel ) revealed with 8C4 and 10F1 antibodies , respectively . A low level of CblS remained , while no CblL was present in the CblEY11427 / MB05683 combination . The 10F1 antibody recognizes very poorly CblL in western blot .

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

(F) Western blots of protein extracts from wild-type and Cbl mutant ovaries showing CblL (left panel) and CblS (right panel) revealed with 8C4 and 10F1 antibodies, respectively. A low level of CblS remained, while no CblL was present in the CblEY11427/MB05683 combination. The 10F1 antibody recognizes very poorly CblL in western blot.


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes .

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

(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes.