Figure 4qRT - PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICimmunoblotting ( B , D ) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICqRT - PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP - L . immunoblotting ( B , D ) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP - L . qRT - PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild - type CALIC ( CALIC - full ) , CALIC - 296 - 913 or CALIC - mutHCT116 cells that had been treated with an siRNA targeting hnRNP - L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays . ChIP assays were performed using anti - hnRNP - L antibody or control mouse IgG . The promoter regions of GAPDH and Egr1 were used as negative and positive controls , respectively . Immunoblotting analysis of proteins precipitated by ChIP assays using anti - hnRNP - L antibodyChIP analysis of hnRNP - L association with the AXL promoter region in CALIC knockdown HCT116 cells . ChIP analysis of hnRNP - L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC - full . ChIRP analysis of CALIC binding to the AXL promoter . Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ . Migratory activity of AXL knockdown HCT116 cells . Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC . Immunoblotting and qRT - PCR analyses of HA - tagged AXL and CALIC expression , respectively ( left ) . α - Tubulin was used as a loading control . Representative images of gross specimens ( top ) and H & E stained sections ( bottom ) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells . Tumors were marked with yellow arrows . The dashed lines depict the boundary between normal and tumor tissues . ( Right ) Percentage of mice with lung metastases . Representative images of gross specimens of primary cecal tumors ( top ) and liver metastatic lesions ( bottom ) in mice orthotopically ( cecal ) injected with CALIC knockdown HCT116 cells . Tumors are marked with yellow arrows . ( Right ) Percentage of mice with primary tumors and liver metastases .

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Figure 4qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICimmunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICqRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.qRT-PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild-type CALIC (CALIC-full), CALIC-296-913 or CALIC-mutHCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays.ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively.Immunoblotting analysis of proteins precipitated by ChIP assays using anti-hnRNP-L antibodyChIP analysis of hnRNP-L association with the AXL promoter region in CALIC knockdown HCT116 cells.ChIP analysis of hnRNP-L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC-full.ChIRP analysis of CALIC binding to the AXL promoter. Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ.Migratory activity of AXL knockdown HCT116 cells.Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC. Immunoblotting and qRT-PCR analyses of HA-tagged AXL and CALIC expression, respectively (left). α-Tubulin was used as a loading control.Representative images of gross specimens (top) and H&E stained sections (bottom) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells. Tumors were marked with yellow arrows. The dashed lines depict the boundary between normal and tumor tissues. (Right) Percentage of mice with lung metastases.Representative images of gross specimens of primary cecal tumors (top) and liver metastatic lesions (bottom) in mice orthotopically (cecal) injected with CALIC knockdown HCT116 cells. Tumors are marked with yellow arrows. (Right) Percentage of mice with primary tumors and liver metastases.


Figure 1B . RT - PCR analysis for forebrain ( FOXG1 ) , optic cup ( VSX2 ) , dorsal forebrain ( PAX6 and EMX2 ) and ventral forebrain ( NKX2 - 1 ) identity in organoids . All expression values ( 2 - ΔCt ) calculated relative to reference gene TBP . Analysis was performed on 5 - 6 organoids from multiple independent differentiations - control ( 7 ) , dorsal ( 7 ) , extended ventral ( 9 ) and short ventral ( 5 ) . Significance values , * = < 0 . 05 , * * = < 0 . 01 , * * * = < 0 . 001 . Statistical analysis was performed using one - way ANOVA and post - hoc Tukey ' s comparison of means . The central band depicts the median , the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values . F . Magnified ( 60x ) dorsal regions from organoids fusions at day 60 ; Co - immunostaining of GFP with markers of interneuronal ( GABA ) , GABAergic ( DLX2 ) and mature neuronal ( NeuN ) identity . Scale bars , 10μm . White arrows indicate GFP + cells with positive labeling . G . Quantification of co - expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60 . Quantification was performed on a total of 3 organoid fusions ( technical replicates ) from two independent differentiations each ( biological replicates ) , with 2 regions of interest ( ROI ; 10x field of view ) in the dorsal and ventral regions of each fusion analyzed . Each dot represents the quantification of co - expression for both ROI analyzed per organoid fusion . Significance values , * = < 0 . 05 , * * = < 0 . 01 , * * * = < 0 . 001 . Statistical analysis was performed using an unpaired two - tailed Student ' s t - test . The central band displays the mean and error bars depict the SD .

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Figure 1B. RT-PCR analysis for forebrain (FOXG1), optic cup (VSX2), dorsal forebrain (PAX6 and EMX2) and ventral forebrain (NKX2-1) identity in organoids. All expression values (2-ΔCt) calculated relative to reference gene TBP. Analysis was performed on 5-6 organoids from multiple independent differentiations - control (7), dorsal (7), extended ventral (9) and short ventral (5). Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's comparison of means. The central band depicts the median, the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values.F. Magnified (60x) dorsal regions from organoids fusions at day 60; Co-immunostaining of GFP with markers of interneuronal (GABA), GABAergic (DLX2) and mature neuronal (NeuN) identity. Scale bars, 10μm. White arrows indicate GFP+ cells with positive labeling. G. Quantification of co-expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60. Quantification was performed on a total of 3 organoid fusions (technical replicates) from two independent differentiations each (biological replicates), with 2 regions of interest (ROI; 10x field of view) in the dorsal and ventral regions of each fusion analyzed. Each dot represents the quantification of co-expression for both ROI analyzed per organoid fusion. Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using an unpaired two-tailed Student's t-test. The central band displays the mean and error bars depict the SD.


Figure 2C . UMAPs depicting the expression of GABAergic markers DLX2 and DLX5 , progenitor marker TOP2A , MGE marker SOX6 , CGE marker NR2F2 , and striatal marker ZFHX3 .

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Figure 2C. UMAPs depicting the expression of GABAergic markers DLX2 and DLX5, progenitor marker TOP2A, MGE marker SOX6, CGE marker NR2F2, and striatal marker ZFHX3.


Figure 3C . UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5 . Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis . D . UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis .

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Figure 3C. UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5. Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis. D. UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis.


Figure 4B . Illustration of saltatory tangential migration performed by an interneuron . Nucleus is marked with a red asterisk . All images taken on a spinning disc confocal microscope at 10x magnification .

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Figure 4B. Illustration of saltatory tangential migration performed by an interneuron. Nucleus is marked with a red asterisk. All images taken on a spinning disc confocal microscope at 10x magnification.


Figure 5C . Heatmap depicting the control - normalized values ( log2 - scaled ) for selected parameters ( from Figure S9A ) for the 8 receptors analyzed in 5B . Grey values indicated non - significant values . In total 3979 moving cell tracks with the following number of tracks per group : Control - 696 , Bicuculline - Methiodide - 1189 , Saclofen - 527 , TPMPA - 138 , AP - 5 - 429 , CNQX - 500 , Strychnine - 98 , Serotonin - 180 , and SR57227 - 222 , were analyzed .

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Figure 5C. Heatmap depicting the control-normalized values (log2-scaled) for selected parameters (from Figure S9A) for the 8 receptors analyzed in 5B. Grey values indicated non-significant values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed.


Figure 6C . Mean square displacement ( MSD ) in μm2 over the delay in minutes ( min ) for all cell tracks in a cluster . The curves are color - coded to denote the corresponding cluster . For visualization purposes , the MSD is shown for a time delay corresponding to 24 hours of imaging . F . Bar plot depicting the fold - change in composition of cluster groups - directed , exploratory or confined - of treatment groups relative to the control distribution . Bars are color - coded to denote the corresponding cluster group . The dotted line represents the control baseline for visualization of the fold changes between the groups .

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Figure 6C. Mean square displacement (MSD) in μm2 over the delay in minutes (min) for all cell tracks in a cluster. The curves are color-coded to denote the corresponding cluster. For visualization purposes, the MSD is shown for a time delay corresponding to 24 hours of imaging.F. Bar plot depicting the fold-change in composition of cluster groups - directed, exploratory or confined - of treatment groups relative to the control distribution. Bars are color-coded to denote the corresponding cluster group. The dotted line represents the control baseline for visualization of the fold changes between the groups.


Figure 1Growth curves of the MCF7 ( left ) and T47D cells ( right ) upon gene silencing with siRNAs . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC , siLMNA : siRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD . Relative protein levels of HNRNPC upon siRNA - mediated silencing , quantified from Western blots of 3 replicates . The error bars represent the ± SD . Growth curves of the Tet - on Crispr - Cas9 - MCF7 ( left ) and Tet - on Crispr - Cas9 - T47D cells ( right ) . Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs . sgNC : non - targeting sgRNA as a negative control , sgHN - 1 : sgRNA sequence 1 for HNRNPC , sgHN - 2 : sgRNA sequence 2 for HNRNPC , sgLMNA : sgRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD . Relative protein levels of HNRNPC upon CRISPR - mediated gene knock - down , quantified from Western blots of 3 replicates . The error bars represent the ± SD . Anchorage independent cell growth assays of the HNRNPC - deficient MCF7 ( top ) and T47D ( bottom ) cells . Relative protein levels of HNRNPC upon shRNA - mediated silencing , quantified from Western blots of 3 replicates . The error bars represent the ± SD . Images , weights , and growth records of the xenograft tumor models in female NSG mice established from the MCF7 cells with lentivirus - mediated stable gene knock - down . Each group has 6 mice . The error bars represent the ± SEM . Images , weights , and growth records of the MCF7 - derived xenograft tumors , 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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Figure 1Growth curves of the MCF7 (left) and T47D cells (right) upon gene silencing with siRNAs. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.Relative protein levels of HNRNPC upon siRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.Growth curves of the Tet-on Crispr-Cas9-MCF7 (left) and Tet-on Crispr-Cas9-T47D cells (right). Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs. sgNC: non-targeting sgRNA as a negative control, sgHN-1: sgRNA sequence 1 for HNRNPC, sgHN-2: sgRNA sequence 2 for HNRNPC, sgLMNA: sgRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.Relative protein levels of HNRNPC upon CRISPR-mediated gene knock-down, quantified from Western blots of 3 replicates. The error bars represent the ± SD.Anchorage independent cell growth assays of the HNRNPC-deficient MCF7 (top) and T47D (bottom) cells.Relative protein levels of HNRNPC upon shRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the MCF7 cells with lentivirus-mediated stable gene knock-down. Each group has 6 mice. The error bars represent the ± SEM.Images, weights, and growth records of the MCF7-derived xenograft tumors, 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.


Figure 2Volcano plots showing the differential expression of the genes after HNRNPC knock - down in the MCF7 ( A ) and T47D ( B ) cells . The ISGs with significant differential expression were marked as red dots . The cutoffs ( dashed line ) were set at the log2 fold - change > 1 . 5 or < - 1 . 5 and the P - value < 0 . 001 . qPCR measurements of the expressions of ISGs upon knock - down of HNRNPC in MCF7 and T47D cells using siRNAqPCR measurements of the expressions of ISGs upon knock - down of HNRNPC in MCF7 and T47D cells using CRISPR - Cas911 TFs involved in the interferon response and signaling , which were identified by MARINa as master TFs upon HNRNPC knock - down . In each row , all genes were sorted ( from left to right ) by their differential expressions in siHNRNPC vs . control cells . The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars . All the TFs were sorted by the P - values ( FDR - corrected ) from the MARINa analysis . Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right .

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Figure 2Volcano plots showing the differential expression of the genes after HNRNPC knock-down in the MCF7 (A) and T47D (B) cells. The ISGs with significant differential expression were marked as red dots. The cutoffs (dashed line) were set at the log2 fold-change > 1.5 or < -1.5 and the P-value < 0.001.qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using siRNAqPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using CRISPR-Cas911 TFs involved in the interferon response and signaling, which were identified by MARINa as master TFs upon HNRNPC knock-down. In each row, all genes were sorted (from left to right) by their differential expressions in siHNRNPC vs. control cells. The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars. All the TFs were sorted by the P-values (FDR-corrected) from the MARINa analysis. Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right.


Figure 3Concentrations of IFNβ , measured by ELISA , in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC , siLMNA : siRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD . The normal MCF7 ( left ) and T47D ( right ) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA - mediated gene knock - down . Expressions of the ISGs in these normal cells were measured with qPCR . Each sample has 3 replicates . Error bars represent mean ± SD . Different doses of the IFNβ antibody were added to the media of the siRNA - transfected MCF7 ( left ) or T47D ( right ) cells right before the media were transferred to the wild type cells . The wild type MCF7 or T47D cells were then cultured in these media for 48 hours , and the expressions of ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD . Growth curves of the MCF7 ( left ) and T47D ( right ) cells cultured in the media collected from the siRNA - transfected cells . Each sample has 3 replicates . Error bars represent mean ± SD .

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Figure 3Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA-mediated gene knock-down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.Different doses of the IFNβ antibody were added to the media of the siRNA-transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild type cells. The wild type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.


Figure 4In the siRNA - transfected MCF7 ( left ) or T47D cells ( right ) , the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization The expressions of the ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD . In the siRNA - transfected MCF7 ( left ) or T47D cells ( right ) , the interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization The expressions of the ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD . In the siRNA - transfected MCF7 ( left ) or T47D cells ( right ) , the interferon signaling pathway was blocked at different stages by means of JAK - STAT inhibition ( C ) . The expressions of the ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD . Growth curves of the MCF7 ( D ) or T47D cells ( E ) upon HNRNPC knock - down but with the interferon response blocked with the IFNβ antibody . Each sample has 3 replicates . Error bars represent mean ± SD . 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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Figure 4In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of JAK-STAT inhibition (C). The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock-down but with the interferon response blocked with the IFNβ antibody. Each sample has 3 replicates. Error bars represent mean ± SD.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.


Figure 5Expression 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 . 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 . 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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Figure 5Expression 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.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.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.


Figure 6Immunofluorescence 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 . 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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Figure 6Immunofluorescence 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.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.


Figure 1 ( 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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Figure 1(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.


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 ) ( 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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Figure 3(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)(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.


Figure 4 ( 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 . 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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Figure 4(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.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.


Figure 5 ( 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 . ( 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 . ( 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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Figure 5(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.(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.(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.


Figure 6 ( 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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Figure 6(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.


Figure 1 ( 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 . ( 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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Figure 1(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.(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.


Figure 2 ( 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 ) . ( 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 .

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

Figure 2(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).(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.


Figure 3 ( 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 . ( 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 .

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

Figure 3(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.(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.


Figure 4 ( 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 .

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

Figure 4(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.


Figure 5 ( 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 . ( D ) Comparison between mean CSC values for selected genes ( CSCg ) and mRNA half - life measured with or without deletion of ste13 + .

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

Figure 5(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.(D) Comparison between mean CSC values for selected genes (CSCg) and mRNA half-life measured with or without deletion of ste13+.


Figure 6 ( 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 . ( 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 .

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

Figure 6(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.(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.


Figure 7 ( 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 . ( 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 . ( 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 .

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

Figure 7(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.(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.(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.


Figure 1B . 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 μmC . U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy . Scale bars : 10 μm .

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Figure 1B. 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 μmC. U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy. Scale bars: 10 μm.


Figure 2HEK293 , 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 . 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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Figure 2HEK293, 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.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.


Figure 3A . 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 . 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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Figure 3A. 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.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).


Figure 4C . 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 . 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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Figure 4C. 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.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.


Figure 5A . 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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Figure 5A. 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.


Figure 1B . 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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Figure 1B. 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 Coomassie


Figure 2A . 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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Figure 2A. 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.


Figure 3C . 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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Figure 3C. 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.


Figure 4A . 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 ) .

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

Figure 4A. 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).