( E ) abundances after translation shut - off with cycloheximide ( CHX ) ; n = 3 experiments , error bars = std . Lines indicate fit to a one - phase exponential decay . Cdc2 Cdc13 were used as long 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 ) mean CSC value for each S . pombe gene ( CSCg ) relative to number per by mass spectrometry CSC was determined using mRNA half - life data Colored dots highlight of interest . For Mad2 Bub1 , no abundance data was available . ( B ) Cumulative frequency distribution of CSCg values for - coding S . pombe genes . 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 versus . 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 regression curves . Model estimates of 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 abundances by qPCR following metabolic labeling removal of labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( = ste13 + , = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded : 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 extracts from expressing wild - type ( WT ) or codon - optimized ( co ) Mad2 - GFP or Mad3 - GFP probed with antibodies against GFP Cdc2 ( loading control ) . Lanes 3 - 5 are a 1 : 1 dilution series of extract from expressing codon - optimized version . ( B ) Immunoblot of extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad2 , Mad3 , tubulin ( loading control ) . A 1 : 1 dilution series was loaded for . ( C ) Estimates of relative to wild - type conditions from experiments such as in ( A ) ( B ) . Bars are experimental replicates , dots are technical replicates . Two - sided t - : 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 - GFP from live microscopy experiments ( a . u . = arbitrary units ) . Boxes show median interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times IQR from first third quartile , respectively . Codon - optimized significantly higher than wild type for both genes ( GLMM ) . Mad2 - GFP : n = 468 413 ; Mad2 - co - GFP : n = 206 366 ; Mad3 - GFP : n = 224 127 ; Mad3 - co - GFP : n = 160 , 450 212 . ( E ) Representative from one of experiments in ( D ) . A single Z - slice is shown . 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 mRNA versus . 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 regression curves . Model estimates of relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to three replicates per genotype .

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(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 abundances by qPCR following metabolic labeling removal of labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( = ste13 + , = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded : 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 ) 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 extracts from expressing wild - type ( WT ) or codon - optimized ( co ) Mad1 - GFP probed with antibodies against GFP Cdc2 ( loading control ) . Lanes 3 - 5 are a dilution series of extract from wild - type . ( B ) Immunoblot of extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad1 tubulin ( loading control ) . A 1 : 1 dilution series was loaded for . ( C ) Estimates of relative to wild - type conditions from experiments such as in ( A ) ( B ) . Bars are experimental replicates , dots are technical replicates . Blue lines indicate mean of experiments . Two - sided t - : 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 - GFP from live microscopy experiments ( a . u . = arbitrary units ) . Boxplots show median interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times IQR from first third quartile , respectively . Codon - optimized significantly lower than wild type ( generalized linear mixed model ) . Mad1 - GFP : n = 197 224 ; Mad1 - co - GFP : n = 80 377 . ( E ) Representative from one of experiments in ( D ) . An average projection of three Z - slices is shown ; 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 - imaging for time spent in mitosis . alp7 + gene was deleted to increase likelihood of spindle assembly checkpoint activation . Localization of Plo1 - tdTomato to spindle pole bodies was used to judge entry into exit from mitosis Exp1 : n = 73 ( WT ) 94 ( co ) ; Exp2 : n = 126 ( WT ) 152 ( co ) . Boxplots show median interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times IQR from first third quartile , respectively . Measurements for 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 co : p = 0 . 14 ( Exp1 ) 0 . 15 ( Exp2 ) by Kolmogorov - Smirnov .

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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 expressing both untagged GFP - tagged Mad1 , probed with antibodies against Mad1 tubulin ; in = input ( 2 . 5 % of extract for IP ) , sup = supernatant after IP . Bottom : Comparison between observed ( obs . ) expected ( expect . ) between Mad1 - GFP untagged Mad1 in IP given their in input ; two one experiment ( s ) , respectively . One more GFP - IP from same was unquantifiable , because no second band was visible in 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 ) Strep pull - down ( PD ) from extracts of diploid expressing Mad1 - GFP Mad1 - Strep from two endogenous loci ; probed with anti - Mad1 ; in = input ( 7 % of extract for IP / PD ) , sup = supernatant after IP / PD . Bottom : as in ( A ) , 2 experiments each . experiment shown at top two more GFP - IPs from same were unquantifiable , because no second band was visible in 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 untagged Mad1 in presence of 35S - labelled Methionine 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 His PD ( right side ) . Shown is autoradiograph after SDS - PAGE with of Mad1 - flag - His to untagged Mad1 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 ) for mRNA dimerization by single molecule mRNA FISH ; probes against mad1 + GFP . Top : Schematic of genotypes . Bottom left : of mad1 + mRNA spots in different strains . For 2 copy , a mad1 + spot was classified as mad1 + - GFP if it was colocalizing with a GFP spot , as mad1 + otherwise . Colors as indicated in schematic . Vertical solid : peak of each density plot ; dashed : theoretical position of a double - peak . Number of spots analyzed : mad1 + ( 1 copy ) = 921 , mad1 + ( 2 copy ) = 637 , mad1 + - GFP ( 2 copy ) = 982 , mad1 + - GFP ( 1 copy ) = 1699 . Bottom right : of mad1 + or mad1 + - GFP mRNA from same experiment with generalized linear mixed model fits as lines . Number of : 1 copy mad1 + = 478 , 2 copy = 327 , 1 copy 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 expressing both mad1 + - GFP mad3 + - GFP from respective endogenous locus were probed with FISH probes against mad1 + GFP mRNA . A GFP spot was classified as mad1 + - GFP if it was colocalizing with a mad1 + spot ( arrowheads ) , as mad3 + - GFP otherwise . of GFP spots was quantified . Vertical solid : peak of each density plot ; dashed : theoretical position of a double - 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 of HeLa , RPE , U2OS lines that were pre - extracted with Triton X - 100 before fixed with formaldehyde co - stained with DAPI antibodies against PCNA 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 were stained with antibodies against PML FAM111A 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 lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) , 48 h ( RPE ) , or 72h ( U2OS ) . 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.


- cycle profiles of HEK293 , RPE U2OS FAM111A - expressing lines that were induced with doxycycline for 48 h 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 lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) growth was quantitated as total on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( two - unpaired t - ) .

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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 . - cycle profiles of HEK293 FAM111A - expressing lines that were induced with doxycycline for 48 h 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 lines were induced with doxycycline for 24 h . Total from 105 was resolved by SDS - PAGE , probed in western blots for both endogenous FAM111A ( 71 kDa ) MycBioID - FAM111A ( 110 kDa ) as well as full PARP ( 116 89 kDa , respectively ) . 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 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 from ~ 105 was resolved by SDS - PAGE , probed in western blots with Myc , PARP , or Tubulin antibodies .

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

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

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

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

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

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 lines were induced with doxycycline for 24 h . Total from an equal number of was incubated with magnetic streptavidin beads , pull downs were analyzed by western blotting using GANP Myc antibodies .

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

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 lines were induced with doxycycline for 24 h , while growing in presence or absence of 50 mM supplemental biotin . Total from whole lysates was incubated with magnetic streptavidin beads . Pulldowns were analyzed by western blotting using GANP Myc antibodies .

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 B-CONTROLLED_VAR 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

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 lines were induced with doxycycline for 24 h . Total from ~ 105 was resolved by SDS - PAGE , probed in western blots using antibodies against NPC ( Mab414 ) , PARP , Caspase 3 , Myc , or Tubulin .

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

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 of SV40 LT localization 72 h after transfection into U2OS infected with control or FAM111A shRNA . were taken with a Zeiss Axio Imager . LT is shown in green , DAPI blue . Scale bar : 100 μm . B . of LT positive described in A fixed at indicated times post transfection . Five hundred or more were counted for each condition . C . of RFP marker distribution upon co - transfection with SV40 LT variants into U2OS infected with control or FAM111A shRNA . were fixed at indicated times . Five hundred or more 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 - unpaired t - ) .

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

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

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

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

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

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 , further incubated in presence of 10 µg of WT Plx1 - PBD for 15 min . bound were recovered by amylose beads , separated by SDS - PAGE detected by

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 in Xenopus egg extracts . purified recombinant WT or Apc - loop500 mutant APC / Cs with mutations ( 1 - T532A , 1 - T539A , 1 - T532A / T539A ( 1 - 2A ) 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 . APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , bound were analysed by SDS - PAGE immunoblotting with indicated antibodies .

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

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 was incubated with purified Plx1 in presence of [ γ32P ] - ATP at 23 ˚ C for indicated times , separated by SDS - PAGE detected by autoradiography Coomassie brilliant blue ( CBB ) .

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

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

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

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

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

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

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

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 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 , further incubated with 10 µg of WT Plx1 - PBD for 15 min . complexes were isolated by amylose beads incubated in presence ( 78 nM ) or absence of purified PP2A - B56γ at 23 ˚ C for 20 min . were recovered by amylose beads , analysed as described in ( B ) .

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

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 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

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

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

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 . 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 ) , samples taken at time points shown were analysed by SDS - PAGE immunoblotting with indicated antibodies . ' Inter ' denotes interphase .

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

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 . mutations in CDK sites on Apc3 - loop reduces phosphorylation of T532 on Apc1 . 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 . APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , phosphorylation status of T532 in Apc1 was analysed by SDS - PAGE immunoblotting with phospho - specific ( pT532 ) or Apc1 ( pan - Apc1 ) antibodies .

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

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 with anti - Vasa ( green , B - D ) or anti - GFP ( green , E ) , anti - Hts ( red ) . DAPI ( blue ) was used to visualize . ( B ) aubHN2 / + was used as a control . ( C , D ) examples of aubHN2 / QC42 germ loss , respectively . ( E ) Phenotypic rescue of aubHN2 / QC42 with UASp - GFP - Aub expressed using nos - Gal4 . arrowheads indicate GSCs ; arrow indicates GSC loss .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) anti - Hts ( red ) , 14 days after clone induction . DAPI ( blue ) was used to stain . Clonal are marked by lack of GFP . Clonal GSCs cysts are outlined with dashed . arrowheads show clonal GSCs in control . aub mutant clonal GSCs have been lost ( H , H ' ) . Scale bar : 10 μm in B - E G - H ' .

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 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 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

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

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

(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 aubHN2 clonal GSCs . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using two - Student ' s t .

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

(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 ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . Examples of mnkP6 mnkP6 aubHN2 / QC42 germaria are shown . arrowheads indicate GSCs ; arrow indicates GSC loss .

O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O

(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 ) of number of GSCs per non - tumorous germarium , of germaria with GSC tumors , in indicated genotypes . number of scored germaria ( n ) is indicated . two - Student ' s t χ2 were used in left 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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) , anti - CCR4 or anti - HA ( green ) . GSCs are shown in ( A - A ' ' ) . arrowheads in ( B - B ' ' ) indicate 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 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 absence or presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 , anti - NOT3 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 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 presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 anti - NOT3 . Inputs are extracts prior to IP .

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 by lack of GFP , either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize . 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 ) of Cbl in aubHN2 / + aubHN2 mutant GSCs using of immunostaining with 8C4 or 10F1 . was measured in arbitrary units using ImageJ software . Horizontal bars correspond to mean standard deviation . * * * p - value < 0 . 001 using two - Student ' s t .

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

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

(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 of CblL mRNA . Ovaries from 1 - day - old ( germarium to stage 8 ) wild - type , aub twin mutant , from 4 - to 7 - day - old bam mutant 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 ) CblS ( E ) mRNAs with ovarian embryonic piRNAs . Significant crosslink clusters are indicated in red at top of graphs .

O O O O O O O B-MEASURED_VAR 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

(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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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 bam promoter , with anti - GFP ( green ) anti - Cbl 8C4 ( red ) .

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

(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 ) anti - Cbl 8C4 ( red ) . DAPI ( blue ) was used to visualize . 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 extracts from wild - type Cbl mutant ovaries showing CblL ( left panel ) CblS ( right panel ) revealed with 8C4 10F1 antibodies , respectively . A low level of CblS remained , while no CblL was present in CblEY11427 / MB05683 combination . 10F1 antibody recognizes very poorly CblL in western blot .

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(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 CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes fusomes . arrowheads indicate GSCs ; 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 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

(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.


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

O O O O O B-CONTROLLED_VAR 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 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. (I) Quantification of germaria with increased number of spectrosomes


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes fusomes . arrowheads indicate GSCs ; arrow indicates increased number of spectrosomes . ( J ) of spectrosomes per germarium . number of scored germaria ( n ) is indicated in ( I ) . * * * p - value < 0 . 001 using χ2 in ( I ) , two - Student ' s t in ( J ) .

O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O 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-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. (J) quantification of spectrosomes per germarium. The number of scored germaria (n) is indicated in (I). ***p-value <0.001 using the χ2 test in (I), and the two-tailed Student's t test in (J).


( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblL ( K ) arrows indicate a cyst in GSC niche ( K , outlined )

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

(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblL (K)White arrows indicate a cyst in the GSC niche (K, outlined)


( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblS ( L ) with anti - Vasa ( green ) anti - Hts ( red ) . ( blue ) was revealed with DAPI . arrows indicatethe loss of GSCs germ ( L ) .

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(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblS (L) with anti-Vasa (green) and anti-Hts (red). DNA (blue) was revealed with DAPI. White arrows indicatethe loss of GSCs and germ cells (L).


( K - M ) Cbl induces GSC differentiation . ( M ) of germaria with 0 to 1 GSC in indicated genotypes . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using χ2 . Scale bar : 10 μm in A - C ' , G , H , K , L .

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(K-M) Cbl induces GSC differentiation.(M) Quantification of germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test. Scale bar: 10 μm in A-C', G, H, K, L.


( A - B ) Immunostaining of germaria from aubHN2 / QC42 ( A ) with anti - Vasa ( green ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . arrow indicates lack of GSCs ; arrowheads indicate GSCs . Scale bar : 10 μm .

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(A-B) Immunostaining of germaria from aubHN2/QC42 (A) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.


( A - B ) Immunostaining of germaria from aubHN2 / QC42 ; CblMB / + ( B ) with anti - Vasa ( green ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . arrow indicates lack of GSCs ; arrowheads indicate GSCs . Scale bar : 10 μm .

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(A-B) Immunostaining of germaria from aubHN2/QC42; CblMB/+ (B) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.


( C ) of mutant germaria with 0 to 1 GSC in indicated genotypes . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 , using χ2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mutant germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001, ** p-value <0.01, * p-value <0.05, using the χ2 test.


( b ) N2a coexpressing YFP - PrP HLA - A2 - CFP were subjected to 6 mM trypsin for 100 s . were taken before after trypsin treatment as indicated . Note that signal of YFP - PrP was destroyed by protease , whereas HLA - A2 - CFP ' s signal remained unaffected .

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(b) N2a cells coexpressing YFP-PrP and HLA-A2-CFP were subjected to 6 mM trypsin for 100 s. Images were taken before and after trypsin treatment as indicated. Note that the membrane signal of YFP-PrP was destroyed by the protease, whereas HLA-A2-CFP's membrane signal remained unaffected.


( d ) Digitonin treatment permeabilizes plasma releases first later nucleoplasmicGFP . N2a expressing GFP were treated with 20 μM digitonin . were taken before after digitonin application at indicated time points . Scale bars , 10 μm .

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(d) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP. N2a cells expressing GFP were treated with 20 μM digitonin. Images were taken before and after digitonin application at the indicated time points. Scale bars, 10 μm.


( b ) FPP of N2a expressing RFP type I CD3δ - CFP . were taken before after 1 - min treatment with 20 μM digitonin 4 mM trypsin as indicated . Scale bar , 10 μm .

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(b) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ-CFP. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


( b ) NRK expressing CD3δ - CFP YFP - CD3δ were subjected to FPP . were taken before after 1 - min treatment with 20 μM digitonin 4 mM trypsin as indicated . Scale bar , 10 μm .

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(b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


c ) Biochemical protease protection of NRK expressing YFP - CD3δ , CD3δ - CFP GFP . Transfected were subjected to SDS - PAGE Western blot before ( lane 1 ) after separation of ( lane 2 ) ( lane 3 ) fractions by ultracentrifugation . Equal amounts of fraction were treated with trypsin in absence ( lane 4 ) or presence ( lane 5 ) of Triton X - 100 . extracts were analyzed by immunoblotting with monoclonal anti - GFP antibody JL - 8 .

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c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.


( a ) Cartoon of FPP illustrating localization of Golgi marker GalT - YFP ( red ) ER - bound CD3δ - CFP ( green ) , position of tag relative to its respective . ( b ) N2a coexpressing GalT - YFP CD3δ - CFP were subjected to FPP , as in Figure

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(a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing GalT-YFP and CD3δ-CFP were subjected to the FPP assay, as in Figure 2b


c ) Cartoon illustrating localization of mitochondrial EYFP - Mito ( red ) Cd3δ - CFP ( green ) . ( d ) N2a coexpressing EYFP - Mito CD3δ - CFP were subjected to FPP , as in b . Scale bars , 10 μm .

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c) Cartoon illustrating localization of mitochondrial EYFP-Mito (red) and Cd3δ-CFP (green). (d) N2a cells coexpressing EYFP-Mito and CD3δ-CFP were subjected to the FPP assay, as in b. Scale bars, 10 μm.


( a ) Cartoon of FPP illustrating localization of RFP - SKL ( red ) Pex3p - GFP ( green ) position of tag relative to its respective . ( b ) N2a coexpressing RFP - SKL Pex3p - GFP were subjected to FPP . were taken before after 1 - min treatment with 20 μM digitonin 100 - s treatment with 4 mM trypsin as indicated . Scale bar , 10 μm .

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(a) Cartoon of the FPP assay illustrating the cellular localization of RFP-SKL (red) and Pex3p-GFP (green) and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing RFP-SKL and Pex3p-GFP were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin as indicated. Scale bar, 10 μm.


( a ) Cartoon of FPP illustrating localization of position of tag relative to its respective . GAPDH - RFP is shown in red CFP - LC3 is shown in green . ( b ) NRK stably expressing CFP - LC3 were transiently transfected to express GAPDH - RFP subjected to FPP . were taken before after 1 - min treatment with 20 μM digitonin 100 - s treatment with 4 mM trypsin . inset shows overlap of GAPDH - RFP CFP - LC3 signals ( 4 × magnification of central ( outlined by a box ) of . Scale bar , 10 μm .

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(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.


A . Left column , an active summer squirrel ; right column , a torpid squirrel .

A. Left column, an active summer squirrel; right column, a torpid squirrel.


B . of quadriceps is unchanged by hibernation as seen in haematoxylin eosin ( H & amp ; amp ; E ) stained sections ( scale bar 90 µm ) .

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B. The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin (H&amp;E) stained sections (scale bar 90 µm).


C . Dystrophin was performed to outline sarcolemma to determine distribution of minimum Feret ' s diameter .

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C. Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret's diameter.


D . Average ± SD of minimum Feret ' s diameter in quadriceps ( p = 0 . 26 ) tibialis anterior ( p = 0 . 33 ) is not significantly different between summer hibernation .

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D. Average ± SD of minimum Feret's diameter in quadriceps (p = 0.26) and tibialis anterior (p = 0.33) muscles is not significantly different between summer and hibernation.


A . Western blot of quadriceps muscle from summer active ( S ) hibernating ( H ) squirrels using antibodies against indicated . An increased abundance of P − Foxo3a ( serine − 253 ) is accompanied by a decrease in P − Akt ( serine − 478 ) . Corresponding densitometry of P − Akt P − Foxo3a as a function of total Akt Foxo3a .

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A. Western blot of quadriceps muscle from summer active (S) and hibernating (H) squirrels using antibodies against the proteins indicated. An increased abundance of P−Foxo3a (serine−253) is accompanied by a decrease in P−Akt (serine−478). Corresponding densitometry of P−Akt and P−Foxo3a as a function of total Akt and Foxo3a.


B . Relative mRNA for four Foxo3a downstream targets show no significant changes during hibernation .

B. Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation.


C . Western blots of mTOR downstream targets shows significant upregulation of P − P70 P − 4E − BP1 during hibernation .

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C. Western blots analysis of mTOR downstream targets shows significant upregulation of P−P70 and P−4E−BP1 during hibernation.


A . Western blot analyses densitometry of quadriceps from non − hibernating hibernating squirrels demonstrates significant increases in total SGK1 phosphorylated SGK1 . Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation .

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A. Western blot analyses and densitometry of quadriceps muscles from non−hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1. Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation.


B . of phosphorylated Foxo3a in S315 T32 are significantly increased during hibernation . Densitometric of P ‐ Foxo3a as a function of Foxo3a .

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B. Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation. Densitometric analysis of P‐Foxo3a as a function of Foxo3a.


A . of tibialis anterior muscle as revealed by haematoxylin - eosin shows increased variation in muscle fiber with numerous atrophic muscle ( scale bar 90 µm ) .

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A.The morphology of tibialis anterior muscle as revealed by haematoxylin-eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers (scale bar 90 µm).


B . distribution mean minimum Feret ' s diameter in tibialis anterior muscle ( p = 0 . 02 ) .

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B. Percentage distribution and mean minimum Feret's diameter in tibialis anterior muscle (p = 0.02).


C . Increased of phosphorylated Akt ( S478 ) in skeletal of sgk1 − / − mice .

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C. Increased levels of phosphorylated Akt (S478) in skeletal muscles of sgk1−/− mice.


D . sgk1 − / − mice exhibit decreased running after 36 days of exposure to a running wheel , ( p = 0 . 01 ; n = 6 per group ) .

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D. sgk1−/− mice exhibit decreased running distance after 36 days of exposure to a running wheel, (p = 0.01; n = 6 animals per group).


E . Isometric force from soleus of WT sgk1 - / - mice during repetitive electrical stimulations for 500 ms . Amplitudes of isometric force from soleus at two stimulation frequencies . Specific force is plotted in cases . Mean values ± SD from n = 9 ( sgk1 − / − ) n = 10 ( WT ) independent ( p = 0 . 01 ) .

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E. Isometric force from soleus muscles of WT and sgk1-/- mice during repetitive electrical stimulations for 500 ms. Amplitudes of isometric force from soleus muscles at two stimulation frequencies. Specific force is plotted in all cases. Mean values ± SD from n = 9 (sgk1−/−) and n = 10 (WT) independent animals (p = 0.01).


A . H & amp ; amp ; E of tibialis anterior sections from 2 ‐ month old control transgenic mice shows no differences ( scale bar 90 µm ) .

A. H&amp;E staining of tibialis anterior sections from 2‐month old control and transgenic mice shows no differences (scale bar 90 µm).


B . Morphometric analyses of minimum Feret ' s diameter of TA muscle reveal no changes in fiber distribution or mean fiber .

B. Morphometric analyses of minimum Feret's diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size.


C . Western blots densitometric of muscle from transgenic control mice . Downstream targets of mTOR signalling cascade ( p70S6K 4EBP1 ) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice .

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C. Western blots and densitometric analysis of muscle from transgenic and control mice. Downstream targets of the mTOR signalling cascade (p70S6K and 4EBP1) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice.


D . Western blot analyses densitometry of phosphorylated FOXO3a total FOXO3a of tibialis anterior from control transgenic mice ( n = 4 ) show an increase of phosphorylation at S315 T32 .

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D. Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice (n = 4) show an increase of phosphorylation at S315 and T32.


A . H & amp ; amp ; E of tibialis anterior sections reveals no changes in muscle architecture ( scale bar 90 µm ) .

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A. H&amp;E staining of tibialis anterior sections reveals no changes in muscle architecture (scale bar 90 µm).


B . Morphometric demonstrates a decrease in muscle fiber of wild − type control mice compared with sgk1tg transgenic ( p = 0 . 0015 ) .

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B. Morphometric analysis demonstrates a decrease in muscle fiber size of the wild−type control mice compared with sgk1tg transgenic littermates (p = 0.0015).


C . Western blot densitometry shows that of phosphorylated Foxo3a at S315 T32 are increased in sgk1tg .

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C. Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.


A . Transfection of constitutively active Sgk1 ( CA ) into immobilized tibialis anterior muscle ( green ) reveals increased fiber diameter when compared to control , EGFP only transfected muscle of immobilized tibialis anterior ( 100 µm ) . Laminin γ − 1 ( red ) outlines basement blue marks nuclei ( DAPI ) .

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A. Transfection of constitutively active Sgk1 (CA) into immobilized tibialis anterior muscle (green) reveals increased fiber size diameter when compared to control, EGFP only transfected muscle fibers of immobilized tibialis anterior muscles (100 µm). Laminin γ−1 staining (red) outlines the basement membrane and blue staining marks nuclei (DAPI).


B . Representation of minimum Feret ' s diameter average ± SD ( control immobilized ; n = 9 - 10 per group ; 800 - 950 were measured ) ( * p = 0 . 029 , * p = 0 . 034 , * p = 0 . 047 , p = 0 . 43 , respectively ) .

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B. Representation of the minimum Feret's diameter average ± SD (control and immobilized; n = 9-10 per group; 800-950 fibers were measured) (*p = 0.029, *p = 0.034, *p = 0.047, p = 0.43, respectively).


C . distribution of minimum Feret ' s diameter of tibialis anterior muscle immobilized transfected with eGFP ( GFP ) , wild − type Sgk1 ( WT ) , kinase dead Sgk1 ( KD ) constitutively active Sgk1 ( CA ) compared to non − immobilized control TA muscle ( black dotted ) .

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C. Percentage distribution of the minimum Feret's diameter of tibialis anterior muscle immobilized and transfected with eGFP (GFP), wild−type Sgk1 (WT), kinase dead Sgk1 (KD) and constitutively active Sgk1 (CA) compared to non−immobilized control TA muscle (black dotted line).


( B ) Representative confocal show distribution of P4M2x reporter in HEK293 - AT1 before after addition of GSK - A1 ( 30 nM ) . Note signal disappearing from PM strongly highlighting endosomes Golgi . Scale bars are 20

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(B) Representative confocal images show the distribution of the P4M2x reporter in HEK293-AT1 cells before and after the addition of GSK-A1 (30 nM). Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi. Scale bars are 20 µ


( C ) Representative result from a BRET experiment detecting PI4P in various endosomes ( Rab7 , Rab5 Rab11 ) following addition of GSK - A1 . graph shows normalized BRET values where averages of triplicate measurements of A1 - treated were divided by averages of triplicates obtained in DMSO - treated controls . Note that these signals originate from thousands of .

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(C) Representative result from a BRET experiment detecting PI4P in various endosomes (Rab7, Rab5 and Rab11) following addition of GSK-A1. The graph shows normalized BRET values where the averages of triplicate measurements of A1-treated cells were divided by the averages of triplicates obtained in DMSO-treated controls. Note that these signals originate from thousands of cells.


( D ) Similar BRET experiment showing Rab7 - pool of PI4P both in control HEK293 - AT1 in two clones of with PI4K2A deletion ( # 20 # 26 ) . Means ± S . E . M . are shown from three experiments performed in triplicates .

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(D) Similar BRET experiment showing the Rab7-pool of PI4P both in control HEK293-AT1 cells and in two clones of cells with PI4K2A deletion (#20 and #26). Means ± S.E.M. are shown from three experiments performed in triplicates.


( A ) BRET measurement of PI4P in Rab7 compartment in parental PI4K2A K / O after expression of HA - tagged PI4K2A or its kinase - dead mutant . Means ± S . E . M . are shown from three experiments performed in triplicates .

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(A) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K/O cells after expression of HA-tagged PI4K2A or its kinase-dead mutant. Means ± S.E.M. are shown from three experiments performed in triplicates.


( B ) Western blot shows that level of PI4K2A expression was closely matched with that of wild - type .

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(B) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild-type cells.


( C ) Distribution of GFP - Rab7 in parental ( left panels ) two clones of PI4K2A K / O . Representative pictures show tubulation in K / O reminiscent of those seen in wild - type expressing GTP - locked Rab7Q67L mutant . Scale bars are 10 µm .

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(C) Distribution of GFP-Rab7 in parental (left panels) and two clones of PI4K2A K/O cells. Representative pictures show tubulation in the K/O cells reminiscent of those seen in wild-type cells expressing the GTP-locked Rab7Q67L mutant. Scale bars are 10 µm.


( B ) Representative confocal pictures showing these changes in live . were transfected with indicated constructs imaged before after addition of rapamycin ( 100 nM ) for 10 min . Note that Rab7 recruiter itself is dissociating from membranes after PI ( 4 , 5 ) P2 generation . Left panels show some enlarged . Scale bars : 20 µm .

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(B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5)P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm.


( C ) of changes in BRET experiments . Here BRET sensor contained PLCδ1PH - fused to Sluc Venus targeted with Rab7 expressed from a single vector it was co - transfected with Rab7 - targeted FRB ( tagged with iRFP ) FKBP12 - fused PIP5Kγ in which CFP was mutated to eliminate its ( CFP * ) . Recruitment of PIP5Kγ but not its kinase - dead version caused an increase in BRET signal indicating increased PI ( 4 , 5 ) P2 in this compartment ( means ± S . E . M . , from three separate experiments performed in triplicates ) .

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(C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5)P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates).


( D ) PI ( 4 , 5 ) P2 increases were larger when Venus part of BRET sensor was targeted with GTP - locked form of Rab7 ( Q67L ) was negligible with GDP - locked form of Rab7 ( N125I ) in BRET construct . Also note that Rab7 wild - type - BRET signal slowly returned toward green trace consistent with Rab7 falling off from both in BRET recruiting constructs ( means ± S . E . M . , from three separate experiments performed in triplicates ) .

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(D) PI(4,5)P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates).


( E ) response of PI4K2A K / O was significantly reduced Means ± S . E . M . are shown from three experiments performed in triplicates .

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(E) The response of PI4K2A K/O cells was significantly reduced Means ± S.E.M. are shown from three experiments performed in triplicates.


( F ) Areas below curves calculated from time of rapamycin addition for each of three separate experiments ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical ( * P = 0 . 0478 0 . 0287 for # 20 # 26 clones , respectively ) .

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(F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).


( A ) Representative confocal of HEK293 - AT1 , which show that treatment with OSW1 ( 20 nM ) , a drug that inhibits cholesterol - PI4P transport , OSBP , causes accumulation of PI4P in Golgi in endosomes ( upper panels ) . Note that accumulation is much reduced in endosome but less so in Golgi in PI4K2A K / O . Scale bars : 20

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(A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm


( B ) of these changes by BRET . PI4P - Rab7 BRET sensor construct was transfected into respective lines treated with OSW1 ( 20 nM ) for indicated times . BRET ratios were expressed relative to those of DMSO treated . Data information : Means ± S . E . M . are shown from three experiments performed in triplicates .

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(B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells. Data information: Means ± S.E.M. are shown from three experiments performed in triplicates.