( F - H ) Effect of Tyr treatment on aggregates , autophagosomes , and larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a and Dmef2 - Gal4 , UAS - GFP - Atg8a animals , shifted to restrictive temperature for 18hrs , and starved 6 hrs prior to dissection . Aggregate ( F ) and GFP - Atg8a punctae ( G ) numbers in muscles are significantly reduced with 100 mg / ml Tyr treatment in DTS but not control animals 18 hrs post - shift ( PS ) . ( H ) 100 mg / ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS . n = 12 and p - values shown relative to non - Tyr treated animals . For all measurements , SEM is shown with * p < . 05 ; * * p < . 01 ; * * * p < . 001 .

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(F-H) Effect of Tyr treatment on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and Dmef2-Gal4, UAS-GFP-Atg8a animals, shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. Aggregate (F) and GFP-Atg8a punctae (G) numbers in muscles are significantly reduced with 100 mg/ml Tyr treatment in DTS but not control animals 18 hrs post-shift (PS). (H) 100 mg/ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS. n = 12 and p-values shown relative to non-Tyr treated animals. For all measurements, SEM is shown with *p<.05; **p<.01; ***p<.001.


( I ) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS . 6 hrs starvation induces lipidated Atg8a - II in Dmef2 - G4 control with or without 100 mg / ml Tyr . After 6hrs starvation , Atg8a - II levels are reduced in DTS + 100 mg / ml Tyr relative to untreated DTS .

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(I) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-G4 control with or without 100 mg/ml Tyr. After 6hrs starvation, Atg8a-II levels are reduced in DTS + 100 mg/ml Tyr relative to untreated DTS.


( D - H ) Dmef2 - Gal4 drives expression of UAS - GFP - Atg8a in animals starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . RNAi knockdown of CG41099 ( E ) , CG7112 ( F ) , CG31935 ( G ) , and Rab3 - GAP ( H ) , blocks the formation of GFP - Atg8a labeled autophagosomes compared to knockdown of white ( D ) .

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(D-H)Dmef2-Gal4 drives expression of UAS-GFP-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. RNAi knockdown of CG41099 (E), CG7112 (F), CG31935 (G), and Rab3-GAP (H), blocks the formation of GFP-Atg8a labeled autophagosomes compared to knockdown of white (D).


( I - M ) Dmef2 - Gal4 drives expression of UAS - myc - Atg8a in animals starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . The effect of the RAB3GAPs is not via Rab3 or Rab5 function , as a Rab3 null mutation ( J ) , expression of a constitutive active form of Rab3 ( Q80L ) ( K ) or Rab5 ( Q88L ) ( L ) has no effect on muscle autophagy compared to control ( I ) . Expression of a constitutive active form of Rab39 ( Q69L ) ( M ) reduces myc - Atg8a labeled vesicles . ( N ) Quantification of autophagy phenotypes from panels D - M . SEM is indicated , with n = 8 ventral longitudinal muscles from individual animals and * * p < . 01 ; * * * p < . 001 .

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(I-M) Dmef2-Gal4 drives expression of UAS-myc-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. The effect of the RAB3GAPs is not via Rab3 or Rab5 function, as a Rab3 null mutation (J), expression of a constitutive active form of Rab3 (Q80L) (K) or Rab5 (Q88L) (L) has no effect on muscle autophagy compared to control (I). Expression of a constitutive active form of Rab39 (Q69L) (M) reduces myc-Atg8a labeled vesicles. (N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.


( N ) Quantification of autophagy phenotypes from panels D - M . SEM is indicated , with n = 8 ventral longitudinal muscles from individual animals and * * p < . 01 ; * * * p < . 001 .

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(N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.


( O ) Atg8a and Rab3 - GAP colocalize to autophagosomes . Dmef2 - Gal4 drives expression of UAS - myc - Atg8a and UAS - CFP - Rab3 - GAP in animals starved and treated with CQ for 6 hrs . In all panels green = GFP - Atg8a and red = myc - Atg8a , and blue = CFP - Rab3Gap2 .

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(O) Atg8a and Rab3-GAP colocalize to autophagosomes. Dmef2-Gal4 drives expression of UAS-myc-Atg8a and UAS-CFP-Rab3-GAP in animals starved and treated with CQ for 6 hrs. In all panels green = GFP-Atg8a and red = myc-Atg8a, and blue = CFP-Rab3Gap2.


( P ) Anti - Atg8a Western blot of third instar muscle lysate from Dmef2 - Gal4 driving the RNAi or overexpression constructs shown in D - N . Total levels of endogenous Atg8a are variable , but only knockdown of CG41099 , CG7112 , CG31935 , Rab3 - GAP , or overexpression of Rab39 ( Q69L ) block induction of lipidated Atg8a - II following 6 hrs starvation .

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(P) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4 driving the RNAi or overexpression constructs shown in D-N. Total levels of endogenous Atg8a are variable, but only knockdown of CG41099, CG7112, CG31935, Rab3-GAP, or overexpression of Rab39 (Q69L) block induction of lipidated Atg8a-II following 6 hrs starvation.


( A ) Primary cultured muscles with control lacZ RNAi , treated for 12 hrs with Rap + CQ . Note the formation of large GFP - Atg8a vesicles throughout the muscles . ( B ) GFP - Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi , treated for 12 hrs with Rap + CQ . Knockdown of Pfk ( C ) or CG9485 ( D ) causes increased accumulation of GFP - Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap + CQ . ( E ) Quantification for A - D of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . SEM is indicated with * p < . 05 ; * * p < . 01 .

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(A) Primary cultured muscles with control lacZ RNAi, treated for 12 hrs with Rap+CQ. Note the formation of large GFP-Atg8a vesicles throughout the muscles. (B) GFP-Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi, treated for 12 hrs with Rap+CQ. Knockdown of Pfk (C) or CG9485 (D) causes increased accumulation of GFP-Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap+CQ. (E) Quantification for A-D of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). SEM is indicated with *p<.05; **p<.01.


( F ) Control white RNAi muscles accumulate large autophagosomes at 1 mg / ml CQ . ( G ) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle . ( H ) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle . ( I ) Quantification of the GFP - Atg8a punctae area in muscles from white , CG9485 , and AGBE knockdowns . n = 6 larval muscles with SEM indicated and * p < . 05 .

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(F) Control white RNAi muscles accumulate large autophagosomes at 1 mg/ml CQ. (G) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle. (H) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle. (I) Quantification of the GFP-Atg8a punctae area in muscles from white, CG9485, and AGBE knockdowns.n = 6 larval muscles with SEM indicated and *p<.05.


( J ) Western blot analysis of endogenous Atg8a from third instar muscle lysate . Dmef2 - Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation , indicating reduced autophagy levels .

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(J) Western blot analysis of endogenous Atg8a from third instar muscle lysate. Dmef2-Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation, indicating reduced autophagy levels.


The MR tail is the main contributor for Nek2A ubiquitination . Ubiquitination reactions performed with APC / CCdc20 for Nek2A wild type ( Nek2AWT ) and degron mutants . Nek2AK is the KEN box mutant ( 391KEN393 / KAA ) , Nek2AK / D is the ( KEN and D box mutant ( 391KEN393 / KAA , 361RKFL364 / AKFA ) , Nek2AMR is the 443 ∆ MR mutant .

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The MR tail is the main contributor for Nek2A ubiquitination. Ubiquitination reactions performed with APC/CCdc20 for Nek2A wild type (Nek2AWT) and degron mutants. Nek2AK is the KEN box mutant (391KEN393/KAA), Nek2AK/D is the (KEN and D box mutant (391KEN393/KAA, 361RKFL364/AKFA), Nek2AMR is the 443∆MR mutant.


The APC / C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A . Nek2A ubiquitination reactions performed with the APC / C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10 .

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The APC/C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A. Nek2A ubiquitination reactions performed with the APC/C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10.


The MR tail is essential for Nek2A binding to the APC / C whereas in contrast Cdc20 is dispensable . Size exclusion chromatography peak fractions of APC / C complexes with Nek2A . The input material ( 13 % of total ) is indicated ( i ) .

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The MR tail is essential for Nek2A binding to the APC/C whereas in contrast Cdc20 is dispensable. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A. The input material (13% of total) is indicated (i).


Nek2A binding does not stabilize binding of the coactivator subunit Cdc20 to the APC / C and does not compete with cyclin A for binding to the APC / C . Size exclusion chromatography peak fractions of APC / C complexes with Nek2A .

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Nek2A binding does not stabilize binding of the coactivator subunit Cdc20 to the APC/C and does not compete with cyclin A for binding to the APC/C. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A.


Nek2A ubiquitination reactions performed with either APC / CCdc20 wild type ( APC / CWT ) or mutants . APC / C2m is the N392A / E395A mutant of Apc2 . APC / C2 / 4m is the N392A / E395A / R48A / H53A / S51A mutant of Apc2 and Apc4 .

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Nek2A ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants. APC/C2m is the N392A/E395A mutant of Apc2. APC/C2/4m is the N392A/E395A/R48A/H53A/S51A mutant of Apc2 and Apc4.


Securin ubiquitination reactions performed with either APC / CCdc20 wild type ( APC / CWT ) or mutants .

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Securin ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants.


Ubiquitination reactions of either Nek2A or securin by the APC / CCdc20 and the effect of increasing concentrations of the MCC .

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Ubiquitination reactions of either Nek2A or securin by the APC/CCdc20 and the effect of increasing concentrations of the MCC.


Size exclusion chromatography peak fractions of APC / C complexes with either

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Size exclusion chromatography peak fractions of APC/C complexes with either Nek2A


Size exclusion chromatography peak fractions of APC / C complexes with Nek2A APC / CΔApc15 is the Apc15 deleted mutant APC / C ,

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Size exclusion chromatography peak fractions of APC/C complexes with Nek2A APC/CΔApc15 is the Apc15 deleted mutant APC/C,


Size exclusion chromatography peak fractions of APC / C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC . MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1

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Size exclusion chromatography peak fractions of APC/C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC. MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1


Size exclusion chromatography peak fractions of APC / C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC . MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on

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Size exclusion chromatography peak fractions of APC / C complexes securin MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1

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Size exclusion chromatography peak fractions of APC/C complexes securin MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1


Size exclusion chromatography peak fractions of APC / C complexes securin MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on

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Ubiquitination reactions of cyclin A by the APC / CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5 .

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Ubiquitination reactions of cyclin A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.


Ubiquitination reactions of Nek2A by the APC / CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5 .

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Ubiquitination reactions of Nek2A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.


Western blot of Hek293 cells showing the siRNA - mediated depletion of UbcH5 , UbcH10 , or both E2 enzymes together . Tubulin serves a loading control .

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Western blot of Hek293 cells showing the siRNA-mediated depletion of UbcH5, UbcH10, or both E2 enzymes together. Tubulin serves a loading control.


Exemplary still images from time courses between NEBD and anaphase of eGFP - Nek2A degradation in HEK cells . Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes . The chromosomes are coloured in cyan and eGFP - Nek2A in green , with the outline of the cells are indicated with dashed yellow lines . Time is given as hh : mm . Scale bar 10 μm .

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Exemplary still images from time courses between NEBD and anaphase of eGFP-Nek2A degradation in HEK cells. Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes. The chromosomes are coloured in cyan and eGFP-Nek2A in green, with the outline of the cells are indicated with dashed yellow lines. Time is given as hh:mm. Scale bar 10 μm.


Degradation profiles of eGFP - cyclin A2 ( top ) and eGFP - Nek2A ( bottom ) in HEK cells during mitosis . The timepoint of NEBD is marked at 0 minutes in the graphs . Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann - Whitney U - test Mean ± s . d . is shown . The number of cells analysed are N = 66 ( Cyclin A2 siGL2 ) , 52 ( Cyclin A2 siUbcH5 ) , 28 ( Cyclin A2 siUbcH10 ) , 27 ( Cyclin A2 siU5 / siU10 ) , 80 ( Nek2A siGL2 ) , 64 ( Nek2A siUbcH5 ) , 21 ( Nek2A siUbcH10 ) , 32 ( Nek2A siU5 / siU10 ) . All data are from at least two biological replicates .

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Degradation profiles of eGFP-cyclin A2 (top) and eGFP-Nek2A (bottom) in HEK cells during mitosis. The timepoint of NEBD is marked at 0 minutes in the graphs. Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann-Whitney U-test Mean ± s.d. is shown. The number of cells analysed are N = 66 (Cyclin A2 siGL2), 52 (Cyclin A2 siUbcH5), 28 (Cyclin A2 siUbcH10), 27 (Cyclin A2 siU5/siU10), 80 (Nek2A siGL2), 64 (Nek2A siUbcH5), 21 (Nek2A siUbcH10), 32 (Nek2A siU5/siU10). All data are from at least two biological replicates.


I , J . Cumulative cell numbers of WT and Tfcp2l1 _ KO 2i - ESCs ( I ) and FBS - ESCs ( J ) . Results are from three independent clones of WT and Tfcp2l1 _ KO ESCs .

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I, J. Cumulative cell numbers of WT and Tfcp2l1_KO 2i-ESCs (I) and FBS-ESCs (J). Results are from three independent clones of WT and Tfcp2l1_KO ESCs.


A . Real - time fatty acid oxidation ( FAO ) profile of WT and Tfcp2l1 _ KO 2i - ESCs using the FAO stress test kit in Seahorse XF . Oxygen consumption rate ( OCR ) were measured . Error bars indicate SEM ; n = 5 biological repeats . Palm - BSA , palmitate conjugated BSA , a substrate of FAO ; Oligomycin , an inhibitor of ATP synthase ( complex V ) ; FCCP , Carbonyl cyanide - 4 ( trifluoromethoxy ) phenylhydrazone , a proton gradient uncoupler ; Rotenone and Antimycin , a complex I inhibitor and a complex III inhibitor , respectively .

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A. Real-time fatty acid oxidation (FAO) profile of WT and Tfcp2l1_KO 2i-ESCs using the FAO stress test kit in Seahorse XF. Oxygen consumption rate (OCR) were measured. Error bars indicate SEM; n = 5 biological repeats. Palm-BSA, palmitate conjugated BSA, a substrate of FAO; Oligomycin, an inhibitor of ATP synthase (complex V); FCCP, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, a proton gradient uncoupler; Rotenone and Antimycin, a complex I inhibitor and a complex III inhibitor, respectively.


F . Real - time FAO profile using the FAO Stress Test in Seahorse XF . n = 4 biological repeats ; error bars indicate SEM ; * * , p - value < 0 . 01 ; NS , not significant ; t - test .

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F. Real-time FAO profile using the FAO Stress Test in Seahorse XF. n = 4 biological repeats; error bars indicate SEM; **, p-value <0.01; NS, not significant; t-test.


G . Percentage of dead cells measured by live - cell propidium iodide staining detecting spontaneous cell death in 2i - ESCs . n = 3 biological repeats ; error bars indicate SEM ; p - values were calculated using two - sample t - test . H . Cumulative cell number of WT , Tfcp2l1 _ KO , and Tfcp2l1 _ KO with Cpt1a overexpression ( OE ) 2i - ESCs . Error bars indicate SEM ; n = 3 biological repeats ; p - values were calculated using two - sample t - test .

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G. Percentage of dead cells measured by live-cell propidium iodide staining detecting spontaneous cell death in 2i-ESCs. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test. H. Cumulative cell number of WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using two-sample t-test.


C . Relative levels of metabolites in glycolysis and the TCA cycle . n = 9 biological repeats ; upper whiskers , maximum - the third quartile ; lower whiskers , the first quartile - minimum ; boxes , the third quartile - the first quartile ; the central line indicates the median ; p - values were calculated using two - sample t - test .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative levels of metabolites in glycolysis and the TCA cycle.n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.


D . Live - cell propidium iodide staining to measure cell death in WT , Tfcp2l1 _ KO , and Tfcp2l1 _ KO with Cpt1a overexpression ( OE ) 2i - ESCs after 5 mM 2 - DG treatment for 24 hours . n = 3 biological repeats ; error bars indicate SEM ; p - value were calculated using two - sample t - test .

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D. Live-cell propidium iodide staining to measure cell death in WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs after 5 mM 2-DG treatment for 24 hours. n=3 biological repeats; error bars indicate SEM; p-value were calculated using two-sample t-test.


F . Relative levels of metabolites in glutamine metabolism . n = 9 biological repeats ; upper whiskers , maximum - the third quartile ; lower whiskers , the first quartile - minimum ; boxes , the third quartile - the first quartile ; the central line indicates the median ; p - values were calculated using two - sample t - test .

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F. Relative levels of metabolites in glutamine metabolism. n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.


D . 0 . 5 million of 2i - ESCs with indicated genotypes were seeded and treated with 200 nM INK - 128 on day0 . Cell numbers were counted on day2 , 4 , and 6 . n = 4 biological repeats ; error bars indicate SEM ; p - values were calculated using two - sample t - test .

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D. 0.5 million of 2i-ESCs with indicated genotypes were seeded and treated with 200 nM INK-128 on day0. Cell numbers were counted on day2, 4, and 6. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.


E . Immunoblotting of WT and Tfcp2l1 _ KO 2i - ESCs untreated or treated with 200 nM INK - 128 .

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E. Immunoblotting of WT and Tfcp2l1_KO 2i-ESCs untreated or treated with 200 nM INK-128.


F . Real - time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1 _ KO 2i - ESCs in the presence and absence of 200 nM INK - 128 . n = 3 biological repeats ; error bars indicate SEM ; p - values were calculated using two - sample t - test .

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F. Real-time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1_KO 2i-ESCs in the presence and absence of 200 nM INK-128. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.


D . Kaplan - Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK - 128 . n , number of blastocysts of each genotype . p - value was calculated using the log rank test . E . Kaplan - Meier curve of ex vivo WT blastocysts cultured in the M2 medium in the presence of 200 nM of INK - 128 with or without 50 µM of etomoxir ( Eto ) and / or 100 µM of octanoate , as indicated . p - values were calculated using the log rank test .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

D. Kaplan-Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK-128. n, number of blastocysts of each genotype. p-value was calculated using the log rank test. E. Kaplan-Meier curve of ex vivo WT blastocysts cultured in the M2 medium in the presence of 200 nM of INK-128 with or without 50 µM of etomoxir (Eto) and/or 100 µM of octanoate, as indicated. p-values were calculated using the log rank test.


( D and E ) Growth comparisons of bir1 ∆ - ad2 strains that contain ( 18 strains ) or do not contain ( 28 strains ) confirmed suppressor mutations as measured by area of colony growth after serial dilution . The mean ( red line ) is shown . 10 - fold serial dilutions were made on YPAD plates containing either 0 . 1 % DMSO ( D ) or 10µg / mL of benomyl ( E ) .

O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD 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-EXP_ASSAY O O O O O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O

(D and E) Growth comparisons of bir1∆-ad2 strains that contain (18 strains) or do not contain (28 strains) confirmed suppressor mutations as measured by area of colony growth after serial dilution. The mean (red line) is shown. 10-fold serial dilutions were made on YPAD plates containing either 0.1% DMSO (D) or 10µg/mL of benomyl (E).


( A ) Serial dilutions on YPAD media at the permissive ( 25 ˚ C ) or restrictive ( 35 ˚ C ) temperatures for the ipl1 - 321 mutation . The suppressor mutations from the Dam1c ( blue ) were the only mutants that rescued viability at the restrictive temperature .

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(A) Serial dilutions on YPAD media at the permissive (25˚C) or restrictive (35˚C) temperatures for the ipl1-321 mutation. The suppressor mutations from the Dam1c (blue) were the only mutants that rescued viability at the restrictive temperature.


( B ) Top : representative images showing Bub3 - mNeonGreen localization to kinetochores ( Nuf2 - mRuby3 ) , indicating spindle assembly checkpoint activation in wild - type yeast . Scale bar is 2µm . Bottom : quantification of the proportion of prometaphase cells with Bub3 - mNeonGreen foci . Only the mutations in Dam1 complex ( blue ) show a significant increase in SAC activity . Data are from at least 5 independent experiments . Means and standard deviations are shown .

O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O 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) Top: representative images showing Bub3-mNeonGreen localization to kinetochores (Nuf2-mRuby3), indicating spindle assembly checkpoint activation in wild-type yeast. Scale bar is 2µm. Bottom: quantification of the proportion of prometaphase cells with Bub3-mNeonGreen foci. Only the mutations in Dam1 complex (blue) show a significant increase in SAC activity. Data are from at least 5 independent experiments. Means and standard deviations are shown.


( B ) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on the indicated media are shown . dam1 ( S20D ) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1 .

O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O

(B) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. dam1(S20D) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1.


( B ) Left : representative images showing the localization of Dad3 - mNeonGreen ( Dam1c member ) and Nuf2 - mRuby3 ( kinetochore marker ) in prometaphase cells . Scale bar is 2µm . Right : quantification of the intensity of Dad3 - mNeonGreen at kinetochores / spindles in prometaphase cells . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments .

O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(B) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in prometaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at kinetochores/spindles in prometaphase cells. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.


( C ) Left : representative images showing the localization of Dad3 - mNeonGreen ( Dam1c member ) and Nuf2 - mRuby3 ( kinetochore marker ) in anaphase cells . Scale bar is 2µm . Right : quantification of the intensity of Dad3 - mNeonGreen at the spindle in anaphase cells . The intensity was measured in the middle of the spindle using a perpendicular line - scan . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments .

O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(C) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in anaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at the spindle in anaphase cells. The intensity was measured in the middle of the spindle using a perpendicular line-scan. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.


( D ) Quantification of the intensity of Dad3 - mNeonGreen at kinetochores in anaphase cells . Kinetochore position was based on Nuf2 - mRuby3 localization . The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle ( Fig 5C ) . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments . The mean ( red line ) is shown .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O 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) Quantification of the intensity of Dad3-mNeonGreen at kinetochores in anaphase cells. Kinetochore position was based on Nuf2-mRuby3 localization. The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle (Fig 5C). Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments. The mean (red line) is shown.


( A ) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on the indicated media are shown . The met30 - 6 and cdc4 - 1 alleles are temperature sensitive . Two independent clones of each of these alleles are shown . The serial dilutions were performed at the permissive temperature ( 20 ˚ C ) .

O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O 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) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. The met30-6 and cdc4-1 alleles are temperature sensitive. Two independent clones of each of these alleles are shown. The serial dilutions were performed at the permissive temperature (20˚C).


( B ) The amount of mNeonGreen - Sli15 ( CPC member ) located at kinetochores ( Nuf2 - mCherry ) was measured in prometaphase . Sgo1 was depleted by placing it under a galactose - inducible promoter and switching to glucose - containing media ( YPAD ) . Representative images are on the left . Scale bar is 2µm . Averages from 3 independent experiments are shown .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O 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) The amount of mNeonGreen-Sli15 (CPC member) located at kinetochores (Nuf2-mCherry) was measured in prometaphase. Sgo1 was depleted by placing it under a galactose-inducible promoter and switching to glucose-containing media (YPAD). Representative images are on the left. Scale bar is 2µm. Averages from 3 independent experiments are shown.


( C ) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores . Inner centromeres were identified as the regions directly between two centromeres ( ACA staining ) . Images from an example WT cell are shown to the right . Scale bar is 5µm long . Each data point is an individual chromosome . More than 15 cells for each cell line were measured across two independent experiments . The mean ( red line ) is shown .

O O O O O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O 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-SUBCELLULAR O 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) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores. Inner centromeres were identified as the regions directly between two centromeres (ACA staining). Images from an example WT cell are shown to the right. Scale bar is 5µm long. Each data point is an individual chromosome. More than 15 cells for each cell line were measured across two independent experiments. The mean (red line) is shown.


( B ) Notch signaling effector Hes5 is expressed high in NSCs while Eomes ( Tbr2 ) and Btg2 are expressed high in both NSCs and BPs at the mRNA level . Each dot defines the mean and lines define the Standard Deviation ( SD ) . 3 - 4 biological replicates were collected for each time point .

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(B) Notch signaling effector Hes5 is expressed high in NSCs while Eomes (Tbr2) and Btg2 are expressed high in both NSCs and BPs at the mRNA level. Each dot defines the mean and lines define the Standard Deviation (SD). 3-4 biological replicates were collected for each time point.


( C ) Heatmap validating the known cell - type specific marker gene expression from RNA sequencing data . ( E ) Heatmap illustrating the novel marker genes identified from the RNA sequencing data , as signature genes for NSCs , BPs and NBNs . NSCs - Neural stem cells , BPs - Basal progenitors , NBNs - Newborn neurons , E - Embryonic day , PN - Post natal , VZ - Ventricular zone , SVZ - Subventricular zone , IZ - Intermediate Zone . Expression values on the heatmaps are log2 ( transcripts per million ) .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-EXP_ASSAY O O O O O O O O O O O O

(C) Heatmap validating the known cell-type specific marker gene expression from RNA sequencing data. (E) Heatmap illustrating the novel marker genes identified from the RNA sequencing data, as signature genes for NSCs, BPs and NBNs. NSCs- Neural stem cells, BPs-Basal progenitors, NBNs-Newborn neurons, E-Embryonic day, PN-Post natal, VZ-Ventricular zone, SVZ- Subventricular zone, IZ-Intermediate Zone. Expression values on the heatmaps are log2 (transcripts per million).


( D ) Expression profiles of PC1 and PC2 along developmental time ( top ) and expression profiles of the top three genes with highest negative and positive projection on PC1 ( first and second column ) and highest negative and positive projection on PC2 ( third and fourth column ) for NSCs . Data Information : In D , ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O

(D) Expression profiles of PC1 and PC2 along developmental time (top) and expression profiles of the top three genes with highest negative and positive projection on PC1 (first and second column) and highest negative and positive projection on PC2 (third and fourth column) for NSCs. Data Information: In D, (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( H ) Expression profiles of PC1 and PC2 across all samples with time from left to right and NSCs in green , BPs in red , and NBNs in purple ( top ) , and the gene expression profiles of top three genes with highest negative and positive projection on PC1 ( first and second column ) and highest negative and positive projection on PC2 ( third and fourth column ) . Data Information : In H , ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O

(H) Expression profiles of PC1 and PC2 across all samples with time from left to right and NSCs in green, BPs in red, and NBNs in purple (top), and the gene expression profiles of top three genes with highest negative and positive projection on PC1 (first and second column) and highest negative and positive projection on PC2 (third and fourth column). Data Information: In H, (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( L ) As in panel D but now for BPs . Data Information : In ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

O 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-TISSUE O O O O O O O O O O O O O

(L) As in panel D but now for BPs. Data Information: In (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( P ) As in panels D and L but now for NBNs . Data Information : In P ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O

(P) As in panels D and L but now for NBNs. Data Information: In P (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( B ) Heatmap illustrating the dynamics of expression of cortical layering markers in NSCs at population level . In B , , heatmaps are based on z - score of log2 ( TPM ) expression values .

O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(B) Heatmap illustrating the dynamics of expression of cortical layering markers in NSCs at population level. In B, , heatmaps are based on z-score of log2(TPM) expression values.


( C ) Examples of expression dynamics of deep layer markers Tbr1 , Ctip2 and upper layer markers Satb2 , Cux2 in NSCs , BPs and NBNs , profiles at population level ( left ) and single cell level ( right ) . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point .

O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O 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) Examples of expression dynamics of deep layer markers Tbr1, Ctip2 and upper layer markers Satb2, Cux2 in NSCs, BPs and NBNs, profiles at population level (left) and single cell level (right). Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( D ) Experimental validation of NSCs isolated at E13 . 5 using Hes5 : : GFP transgenic embryos , showing no detectable protein for Tbr1 , Ctip2 and Satb2 . NSCs do express Brn2 ( Pou3f2 ) in vitro and in vivo at protein level . Scale bar = 20μm .

O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(D) Experimental validation of NSCs isolated at E13.5 using Hes5::GFP transgenic embryos, showing no detectable protein for Tbr1, Ctip2 and Satb2. NSCs do express Brn2 (Pou3f2) in vitro and in vivo at protein level. Scale bar = 20μm.


( E ) Heatmap illustrating the dynamics of expression of cortical layering markers in BPs at population level . ( G ) Heatmap illustrating the dynamics of expression of cortical layering markers in NBNs at population level . heatmaps are based on z - score of log2 ( TPM ) expression values .

O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(E) Heatmap illustrating the dynamics of expression of cortical layering markers in BPs at population level. (G) Heatmap illustrating the dynamics of expression of cortical layering markers in NBNs at population level. heatmaps are based on z-score of log2(TPM) expression values.


( F ) Experimental validation of BPs isolated at E16 . 5 using Tbr2 : : GFP transgenic embryos , showing no detectable protein for Tbr1 , Ctip2 and Satb2 . Scale bar = 20μm . ( H ) Experimental validation of NBNs isolated at E16 . 5 using Tbr2 : : GFP transgenic embryos , showing protein expression for Tbr1 , Ctip2 , Satb2 and Brn2 ( Pou3f2 ) . Scale bar = 20μm .

O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O

(F) Experimental validation of BPs isolated at E16.5 using Tbr2::GFP transgenic embryos, showing no detectable protein for Tbr1, Ctip2 and Satb2. Scale bar = 20μm. (H) Experimental validation of NBNs isolated at E16.5 using Tbr2::GFP transgenic embryos, showing protein expression for Tbr1, Ctip2, Satb2 and Brn2(Pou3f2). Scale bar = 20μm .


( D ) Expression profile of selected target or modulator of key signaling pathways : BMP , Wnt , Shh and Notch signaling . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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) Expression profile of selected target or modulator of key signaling pathways: BMP, Wnt, Shh and Notch signaling. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( B ) Expression profile of selected stem cell maintenance markers Hes1 , Hey1 and Id4 . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point . ( C ) Expression profile of selected neurogenic markers Neurog2 , Neurod2 , and Neurod6 . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point . ( D ) Expression profile of selected gliogenic markers Olig1 , Olig2 and Id1 . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point .

O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O 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-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O 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-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD 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) Expression profile of selected stem cell maintenance markers Hes1, Hey1 and Id4. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point. (C) Expression profile of selected neurogenic markers Neurog2, Neurod2, and Neurod6. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point. (D) Expression profile of selected gliogenic markers Olig1, Olig2 and Id1. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


C , D Growth rates as measured by the change in optical density ( OD595 ) over time for wild type ( C ) and Torin1 - resistant ( D ) cells upon 5 µM of Torin1 treatment ( at T = 0 min ) . Respective strains were treated with the DMSO vehicle alone to control for potential effects of the solvent on cell growth . Logarithmic values of the OD595 increase are plotted against time in minutes . Data aggregated from 3 independent experiments with error bars representing the standard deviations ( SD ) . Linear regressions were fitted using the curve fitting function on Prism9 . Ratios in the rate of mass doubling ( as calculated from gradient of regression lines detailed in Methods ) of Torin1 treated cells to DMSO control were 0 . 34 in wild type ( C ) and 0 . 97 for Torin1 - resistant ( D ) cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

C, D Growth rates as measured by the change in optical density (OD595) over time for wild type (C) and Torin1-resistant (D) cells upon 5 µM of Torin1 treatment (at T = 0 min). Respective strains were treated with the DMSO vehicle alone to control for potential effects of the solvent on cell growth. Logarithmic values of the OD595 increase are plotted against time in minutes. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD). Linear regressions were fitted using the curve fitting function on Prism9. Ratios in the rate of mass doubling (as calculated from gradient of regression lines detailed in Methods) of Torin1 treated cells to DMSO control were 0.34 in wild type (C) and 0.97 for Torin1-resistant (D) cells.


E Rate of protein synthesis as measured by the amount of HPG incorporation after 20 mins of differing time intervals ( 0 - 60 mins ) of 5 µM Torin1 for wild type ( green ) and tor1 ∆ ( yellow ) . Values are normalised to T = 0 minutes . Exponential one - phase decay non - linear regressions were fitted using the curve fitting function on Prism9 . Data aggregated from 3 independent experiments with error bars representing the standard deviations ( SD ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Rate of protein synthesis as measured by the amount of HPG incorporation after 20 mins of differing time intervals (0 - 60 mins) of 5 µM Torin1 for wild type (green) and tor1∆ (yellow). Values are normalised to T = 0 minutes. Exponential one-phase decay non-linear regressions were fitted using the curve fitting function on Prism9. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD).


C - F Phosphorylation changes of the conserved phosphosites , S235 and S236 , on Rps601 ( C ) and Rps602 ( D - F ) , over 85 minutes of Torin1 ( 5 µM ) treatment wild type ( green ) and Torin1 - resistant ( brown ) cells . M - values ( in brackets ) represent the different multiplicities of the same phosphosites . Lines were fitted with the curve fitting function on Prism9 using the the non - linear regressions exponential one - phase decay ( C , D ) or plateau followed by one - phase decay ( E , F ) functions respectively . Grey dashed lines indicate 2 - fold threshold .

O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - F Phosphorylation changes of the conserved phosphosites, S235 and S236, on Rps601(C) and Rps602 (D - F), over 85 minutes of Torin1 (5 µM) treatment wild type (green) and Torin1-resistant (brown) cells. M-values (in brackets) represent the different multiplicities of the same phosphosites. Lines were fitted with the curve fitting function on Prism9 using the the non-linear regressions exponential one-phase decay (C, D) or plateau followed by one-phase decay (E, F) functions respectively. Grey dashed lines indicate 2-fold threshold.


A Relative protein expression levels of the 3 S6 kinase homologues , Psk1 , Sck1 and Sck2 , plotted against time of Torin1 addition . Respective proteins are represented by the different coloured lines as detailed in the legend .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

A Relative protein expression levels of the 3 S6 kinase homologues, Psk1, Sck1 and Sck2, plotted against time of Torin1 addition. Respective proteins are represented by the different coloured lines as detailed in the legend.


B Relative phosphorylation level of the T632 phosphosite identified on Sck1 in the 40 - minute time course study . Phosphorylation levels of both wild type ( green ) and tor1 ∆ ( yellow ) are presented relative to the value for wild type cells at T = 0 minutes . Grey dashed line indicates 2 - fold threshold .

O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O 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 Relative phosphorylation level of the T632 phosphosite identified on Sck1 in the 40-minute time course study. Phosphorylation levels of both wild type (green) and tor1∆ (yellow) are presented relative to the value for wild type cells at T = 0 minutes. Grey dashed line indicates 2-fold threshold.


A , B Phosphosites identified on ( A ) Tif212 and ( B ) Erf1 respectively showing 2 - fold phosphorylation change within 20 minutes of Torin1 ( 5 µM ) addition . Both wild type ( green ) and tor1 ∆ ( yellow ) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells ( T = 0 minutes ) . Grey dashed lines indicate 2 - fold threshold .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

A, B Phosphosites identified on (A) Tif212 and (B) Erf1 respectively showing 2-fold phosphorylation change within 20 minutes of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T=0 minutes). Grey dashed lines indicate 2-fold threshold.


E , F Changes in rates of protein synthesis of the tif471 _ 18A mutant compared to the wild type control ( tif471 + ) upon Torin1 ( 5 µM ) treatment . Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17 % for the tif471 _ 18A phosphomutant and 4 . 6 % for the tif471 + control relative to starting levels in the respective strains . The two graphs represent the untransformed ( E ) and log2 transformed ( F ) rates respectively . Error bars represent the standard deviation ( SD ) of data aggregated from 3 independent experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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, F Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471+) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471+ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log2 transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.


A . Spindle in a fixed HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented horizontally with respect to the imaging plane , as shown in the scheme . Images of different z - slices ( central plane of the spindle z = 0 , two images below , z = - 4 µm and z = - 2 µm , and above , z = + 3 µm and z = + 5 µm ) , maximum projection of a z - stack ( max z ) , and 3D projections ( 3D ) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown . Additional z - slices of this spindle are shown in Fig EV1A . B . Spindle in a fixed HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented vertically with respect to the imaging plane , as shown in the scheme . Legend as in A . Additional z - slices of this spindle are shown in Fig EV1B . C . Correlation between the number of PRC1 - labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal ( black ) and vertical ( blue ) spindles of fixed HeLa cells in metaphase . Data points represent individual spindles , lines show linear fits . D . Pie charts showing the fraction of PRC1 - labeled bundles associated with kinetochore pairs ( blue ) , PRC1 - labled bundles not associated with kinetochores ( green ) and kinetochores not associated with PRC1 bundles ( magenta ) in horizontal ( top row ) and vertical spindles ( bottom row ) from cells with up to 70 chromosomes ( left column ) and more than 70 chromosomes ( right column ) . Horizontal spindles contained a total of 1786 kinetochore - PRC1 pairs ( n = 29 cells ) and vertical spindles 1133 kinetochore - PRC1 pairs ( n = 16 cells ) . E , F . Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1 - labeled bundles . G . Number of kinetochore pairs coupled with PRC1 - labeled bundles per unit area of the cross section of the central part of horizontal ( black ) and vertical ( blue ) spindles as a function of the number of kinetochore pairs coupled with PRC1 - labeled bundles in the spindle .

O O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O 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-SUBCELLULAR O O O B-GENEPROD O O O O O B-SUBCELLULAR O

A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z=0, two images below, z=-4 µm and z=-2 µm, and above, z=+3 µm and z=+5 µm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A.B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in A. Additional z-slices of this spindle are shown in Fig EV1B.C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits.D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1786 kinetochore-PRC1 pairs (n=29 cells) and vertical spindles 1133 kinetochore-PRC1 pairs (n=16 cells).E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles.G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle.


A . Central plane of the spindle in a live HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented vertically with respect to the imaging plane . B . Examples of trajectories , with respect to the spindle ' s center of the mass , of individual PRC1 - GFP ( green ) and corresponding mRFP - CENP - B ( magenta ) signals from the spindle in A that moved together for at least 200 s . Dots represent starting points of trajectories , t = 0 s . Trajectories finish at t = 200 s . Grey circle represents the center of mass of the spindle . C . Pie chart showing the fraction of PRC1 - labeled fibers and sister kinetochores that moved together for at least 60 s ( blue , n = 226 pairs in 5 cells ) . A small fraction of PRC1 - labeled fibers ( green , n = 32 in 5 cells ) and kinetochores ( magenta , n = 16 in 5 cells ) did not move together .

O O O O O O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR 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-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

A. Central plane of the spindle in a live HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane.B. Examples of trajectories, with respect to the spindle's center of the mass, of individual PRC1-GFP (green) and corresponding mRFP-CENP-B (magenta) signals from the spindle in A that moved together for at least 200 s. Dots represent starting points of trajectories, t=0 s. Trajectories finish at t=200 s. Grey circle represents the center of mass of the spindle.C. Pie chart showing the fraction of PRC1-labeled fibers and sister kinetochores that moved together for at least 60 s (blue, n=226 pairs in 5 cells). A small fraction of PRC1-labeled fibers (green, n=32 in 5 cells) and kinetochores (magenta, n=16 in 5 cells) did not move together.


D . Correlation coefficients between trajectories ( with respect to the spindle ' s center of the mass ) of a kinetochore pair and the trajectories of the PRC1 - labeled fiber coupled to the kinetochore pair ( green ) , the nearest neighbor PRC1 - labeled fiber ( blue ) , the next nearest neighbor ( black ) and a randomly chosen PRC1 - labeled fiber ( grey ) ; n , the number of trajectories from 3 cells .

O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

D. Correlation coefficients between trajectories (with respect to the spindle's center of the mass) of a kinetochore pair and the trajectories of the PRC1-labeled fiber coupled to the kinetochore pair (green), the nearest neighbor PRC1-labeled fiber (blue), the next nearest neighbor (black) and a randomly chosen PRC1-labeled fiber (grey); n, the number of trajectories from 3 cells.


E . Examples of scenarios observed in dynamic interplay of PRC1 - labeled bundles ( green ) and kinetochores ( magenta ) from the spindle in A . Row 1 : PRC1 - labeled bundle and kinetochore move together until they separate at 130 s . Row 2 : kinetochore is free of any PRC1 - labeled bundle until it appears at 130 s . Row 3 : kinetochore and PRC1 - labeled bundle move together . Row 4 : kinetochore moves with PRC1 - labeled bundle which merges with neighboring bundle at 169 s . Row 5 : two kinetochores move together with a single PRC1 - labeled bundle .

O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O

E. Examples of scenarios observed in dynamic interplay of PRC1-labeled bundles (green) and kinetochores (magenta) from the spindle in A. Row 1: PRC1-labeled bundle and kinetochore move together until they separate at 130 s. Row 2: kinetochore is free of any PRC1-labeled bundle until it appears at 130 s. Row 3: kinetochore and PRC1-labeled bundle move together. Row 4: kinetochore moves with PRC1-labeled bundle which merges with neighboring bundle at 169 s. Row 5: two kinetochores move together with a single PRC1-labeled bundle.


A . Top row : MG132 arrested HeLa cells stably expressing tubulin - GFP ( green ) and immunostained for endogenous PRC1 ( AlexaFluor555 shown in magenta ) in 5 individual z - images . Enlargements of the boxed region ( top : merge , middle : GFP , bottom : AlexaFluor555 ) below the first image show the bridging fiber determined as the line connecting two ends of the sister k - fibers in green channel and localization of endogenous PRC1 ( magenta ) . Corresponding maximum projections are shown to the right ( max z , left : merge , middle : GFP , right : AlexaFluor555 ) . B . Quantification of tubulin - GFP bridging fibers ( green bar ) immunostained for endogenous PRC1 ( magenta bar ) .

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A. Top row: MG132 arrested HeLa cells stably expressing tubulin-GFP (green) and immunostained for endogenous PRC1 (AlexaFluor555 shown in magenta) in 5 individual z-images. Enlargements of the boxed region (top: merge, middle: GFP, bottom: AlexaFluor555) below the first image show the bridging fiber determined as the line connecting two ends of the sister k-fibers in green channel and localization of endogenous PRC1 (magenta). Corresponding maximum projections are shown to the right (max z, left: merge, middle: GFP, right: AlexaFluor555).B. Quantification of tubulin-GFP bridging fibers (green bar) immunostained for endogenous PRC1 (magenta bar).


C . Images show pole - to - pole tracking ( white curve ) of tubulin signal ( top , green ) , which was used to measure the endogenous PRC1 signal along the same contour ( middle , magenta ) , and the corresponding scheme ( bottom ) from a HeLa cell expressing tubulin - GFP and immunostained for PRC1 . Images of the same spindle without ( left ) and with the tracked contour ( right ) are shown . The graph shows normalized pole - to - pole intensity profiles ( each intensity profile was scaled so that one pole is at x = 0 and the other at x = 1 ) of endogenous PRC1 ( grey lines ) acquired in tubulin - GFP HeLa cells immunostained for PRC1 . Black line shows the mean value .

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C. Images show pole-to-pole tracking (white curve) of tubulin signal (top, green), which was used to measure the endogenous PRC1 signal along the same contour (middle, magenta), and the corresponding scheme (bottom) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (each intensity profile was scaled so that one pole is at x=0 and the other at x=1) of endogenous PRC1 (grey lines) acquired in tubulin-GFP HeLa cells immunostained for PRC1. Black line shows the mean value.


D . Images show pole - to - pole tracking ( white curve ) of PRC1 - GFP signal ( top , merge ; middle , GFP ; bottom , scheme ) from a HeLa cell expressing PRC1 - GFP and mRFP - CENP - B . Images of the same spindle without ( left ) and with the tracked contour ( right ) are shown . The graph shows normalized pole - to - pole intensity profiles ( legend as in C ) of PRC1 - GFP acquired in HeLa cells expressing PRC1 - GFP and mRFP - CENP - B .

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D. Images show pole-to-pole tracking (white curve) of PRC1-GFP signal (top, merge; middle, GFP; bottom, scheme) from a HeLa cell expressing PRC1-GFP and mRFP-CENP-B. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (legend as in C) of PRC1-GFP acquired in HeLa cells expressing PRC1-GFP and mRFP-CENP-B.


E . Example of length measurement of the immunostained PRC1 signal ( LPRC1 ) in HeLa cell expressing tubulin - GFP and immunostained for PRC1 .

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E. Example of length measurement of the immunostained PRC1 signal (LPRC1) in HeLa cell expressing tubulin-GFP and immunostained for PRC1.


F . Comparison of the length LPRC1 between PRC1 - GFP signal ( green , corresponds to the PRC1 fused to GFP from a PRC1 - GFP cell line ) and PRC1 - AlexaFluor555 ( magenta , corresponds to the endogenous immunostained PRC1 ) ( p = 0 . 10 ) .

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F. Comparison of the length LPRC1 between PRC1-GFP signal (green, corresponds to the PRC1 fused to GFP from a PRC1-GFP cell line) and PRC1-AlexaFluor555 (magenta, corresponds to the endogenous immunostained PRC1) (p=0.10).


G . Cross section signal intensity of the immunostained endogenous PRC1 ( magenta line ) from a HeLa cell expressing tubulin - GFP and immunostained for PRC1 . Area under the peak is defined as Icross , measured at the position of the blue line as in scheme . Horizontal lines mark the background signal ( black line ) , vertical lines delimit the area ( grey ) where the signal was measured .

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G. Cross section signal intensity of the immunostained endogenous PRC1 (magenta line) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Area under the peak is defined as Icross, measured at the position of the blue line as in scheme. Horizontal lines mark the background signal (black line), vertical lines delimit the area (grey) where the signal was measured.


A . Images of HeLa cells stably expressing tubulin - GFP ( green ) and transiently mRFP - CENP - B ( magenta ) treated with control siRNA ( left images ) and siRNA targeting PRC1 ( right images ) . Enlargements of the boxed region , shown to the right of the image of the whole spindle , are focused on kinetochores and bridging fiber ( top : merge ; middle : GFP , bottom : scheme ) .

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A. Images of HeLa cells stably expressing tubulin-GFP (green) and transiently mRFP-CENP-B (magenta) treated with control siRNA (left images) and siRNA targeting PRC1 (right images). Enlargements of the boxed region, shown to the right of the image of the whole spindle, are focused on kinetochores and bridging fiber (top: merge; middle: GFP, bottom: scheme).


B . Western blot showing PRC1 protein from unlabeled cells , tubulin - GFP HeLa cell line and PRC1 - GFP HeLa cell line . Detailed measurements are in Expanded View 4 .

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B. Western blot showing PRC1 protein from unlabeled cells, tubulin-GFP HeLa cell line and PRC1-GFP HeLa cell line. Detailed measurements are in Expanded View 4.


C . Mean ratio of signal intensities of the bridging fiber ( Ib , measured at the position of a blue line as in scheme ) and sum of the bridging and k - fiber ( Ibk , measured at the position of the orange line as in scheme ) , measured in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) ( p = 0 . 0003 ) . D . Mean interkinetochore distance ( dk , see scheme ) measured in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) ( p = 0 . 0001 ) .

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C. Mean ratio of signal intensities of the bridging fiber (Ib, measured at the position of a blue line as in scheme) and sum of the bridging and k-fiber (Ibk, measured at the position of the orange line as in scheme), measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0003).D. Mean interkinetochore distance (dk, see scheme) measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0001).


E . Images of individual spindles of HeLa cells expressing tubulin - GFP and immunostained for PRC1 obtained from control cells ( top ) and PRC1 siRNA treated cells ( bottom ) . All cells were imaged with the same imaging parameters . Only AlexaFluor555 channel is shown .

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E. Images of individual spindles of HeLa cells expressing tubulin-GFP and immunostained for PRC1 obtained from control cells (top) and PRC1 siRNA treated cells (bottom). All cells were imaged with the same imaging parameters. Only AlexaFluor555 channel is shown.


F . Mean signal intensity I of the immunostained PRC1 in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) in HeLa cells expressing tubulin - GFP and immunostained for PRC1 ( p = 0 . 0001 ) .

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F. Mean signal intensity I of the immunostained PRC1 in control cells (grey bar) and PRC1 siRNA treated cells (black bar) in HeLa cells expressing tubulin-GFP and immunostained for PRC1 (p=0.0001).


G . Images of HeLa cells stably expressing PRC1 - GFP treated with control ( left image ) and siRNA targeting PRC1 ( right image ) immunostained for PRC1 ( magenta ) . Graph showing the length of the immunostained PRC1 signal , LPRC1 ( p = 0 . 5167 ) , signal intensity , I ( p = 0 . 0003 ) and Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1 - GFP and immunostained for PRC1 . H . Images of HeLa cells stably expressing PRC1 - GFP ( green ) treated with control ( left image ) and siRNA targeting PRC1 ( right image ) immunostained for PRC1 . Graph showing the length of the PRC1 - GFP signal , LPRC1 ( p = 0 . 3407 ) , signal intensity , I ( p = 0 . 0001 ) , and Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1 - GFP and immunostained for PRC1 .

O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O 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-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O

G. Images of HeLa cells stably expressing PRC1-GFP treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1 (magenta). Graph showing the length of the immunostained PRC1 signal, LPRC1 (p=0.5167), signal intensity, I (p=0.0003) and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.H. Images of HeLa cells stably expressing PRC1-GFP (green) treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1. Graph showing the length of the PRC1-GFP signal, LPRC1 (p=0.3407), signal intensity, I (p=0.0001), and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.


( B ) qRT - PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non ‐ skipped α1S form : 18 ± 6 % ( * * P ⩽ 0 . 001 , n = 19 ) and 10 ± 7 % ( * * P ⩽ 0 . 001 n = 19 ) ( black bars ) of the total α1S mRNA ( grey bars ) at 2 and 6 months post injection in TA of AAV1 ‐ ( U7 ‐ ESE ) and AAV1 ‐ ( U7 ‐ SA ) : ΔDHPR or AAV1 ‐ ( U7 ‐ Ctrl ) : c as a control , respectively .

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(B) qRT-PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non‐skipped α1S form: 18±6% (**P⩽0.001, n=19) and 10±7% (**P⩽0.001 n=19) (black bars) of the total α1S mRNA (grey bars) at 2 and 6 months post injection in TA of AAV1‐(U7‐ESE) and AAV1‐(U7‐SA):ΔDHPR or AAV1‐(U7‐Ctrl): c as a control, respectively.


( C ) Six months post injection , lysates from TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were immuno ‐ blotted for α1S or α - actin for four mice . Graph depicts mean ± s . e . m . of relative expression of α1S subunit determined by densitrometry and nomalized to the α - actin expression for each muscle . Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice , * * P ⩽ 0 . 001 , n = 4 .

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(C) Six months post injection, lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for α1S or α-actin for four mice. Graph depicts mean±s.e.m. of relative expression of α1S subunit determined by densitrometry and nomalized to the α-actin expression for each muscle. Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice, **P⩽0.001, n=4.


( D ) Longitudinal cryo ‐ sections from TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ α1S subunit ( red ) and anti ‐ laminin ( green ) antibodies , nuclei were visualized by Dapi ( blue ) and imaged by confocal microscopy . Bars represent 20 μm .

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(D) Longitudinal cryo‐sections from TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red) and anti‐laminin (green) antibodies, nuclei were visualized by Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( A ) Six months post injection , TAs from eight mice were dissected and weighed .

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(A) Six months post injection, TAs from eight mice were dissected and weighed.


( B ) The internal diameters ( shortest diameter ) from all fibres throughout the total muscle section were recorded and analysed . Muscles from five different animals were examined . The bar graph presents mean ± s . e . m . of the number of myofibres by fibre diameter class for TAΔDHPR ( black ) and TACtrl ( grey ) .

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(B) The internal diameters (shortest diameter) from all fibres throughout the total muscle section were recorded and analysed. Muscles from five different animals were examined. The bar graph presents mean±s.e.m. of the number of myofibres by fibre diameter class for TAΔDHPR (black) and TACtrl(grey).


( C ) Transversal sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with haematoxylin and eosin , bars represent 100 μm .

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(C) Transversal sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with haematoxylin and eosin, bars represent 100 μm.


( D ) To quantify fibrosis , transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software ( marked in blue ) , data were normalized with total surface of each muscle ( orange line surrounding the sections ) , bars represent 500 μm . ( E ) Quantification is presented in bar graph and showed 4 . 1 ± 0 . 1 fold increase of fibrosis in TAΔDHPR ( ΔDHPR ) compared to TACtrl ( c ) ( * * P0 . 001 , n = 4 ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O

(D) To quantify fibrosis, transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software (marked in blue), data were normalized with total surface of each muscle (orange line surrounding the sections), bars represent 500 μm. (E) Quantification is presented in bar graph and showed 4.1±0.1 fold increase of fibrosis in TAΔDHPR (ΔDHPR) compared to TACtrl (c) (**P0.001, n=4).


( A ) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT - PCR . Results are expressed as mean ± s . e . m . , * * P0 . 001 , n = 4 .

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(A) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT-PCR. Results are expressed as mean±s.e.m., **P0.001, n=4.


( B ) Transversal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ nNOS ( red ) , anti ‐ laminin ( green ) antibodies , nuclei with Dapi ( blue ) and imaged by confocal microscopy . Bars represent 20 μm .

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(B) Transversal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐nNOS (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( C ) The tissues from extracts were analysed by western blot with FoxO3a ‐ P antibody and normalized with α ‐ actin antibody . Graph depicts mean ± s . e . m . of relative expression of FoxO3a ‐ P determined by densitometry and nomalized to the α ‐ actin expression for each muscle , * P ⩽ 0 . 005 , n = 3 .

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(C) The tissues from extracts were analysed by western blot with FoxO3a‐P antibody and normalized with α‐actin antibody. Graph depicts mean±s.e.m. of relative expression of FoxO3a‐P determined by densitometry and nomalized to the α‐actin expression for each muscle, *P⩽0.005, n=3.


( D ) Longitudinal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ FoxO3a ( red ) , anti ‐ laminin ( green ) antibodies , nuclei with Dapi ( blue ) and imaged by confocal microscopy . Bars represent 20 μm .

(D) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐FoxO3a (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( E ) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT - PCR . Bnip3 , CathepsinL , LC3 and PI3KIII expression ( noted in red ) were significantly increased in TAΔDHPR compared with the controlateral TACtrl , * * P0 . 001 , n = 4 .

O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O

(E) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT-PCR. Bnip3, CathepsinL, LC3 and PI3KIII expression (noted in red) were significantly increased in TAΔDHPR compared with the controlateral TACtrl, **P0.001, n=4.


( A ) Ultrathin sections were imaged by electron microscopy , M , mitochondria ; C , collagen fibres ; T , T ‐ tubule . Arrows show double ‐ membrane vesicules called auphagosomes .

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(A) Ultrathin sections were imaged by electron microscopy, M, mitochondria; C, collagen fibres; T, T‐tubule. Arrows show double‐membrane vesicules called auphagosomes.


( B ) Transversal cryo ‐ sections were stained with anti ‐ LC3b ( red ) , anti ‐ dystrophin ( green ) antibodies , nuclei with Dapi ( blue ) and imaged by confocal microscopy . Upper panel : TACtrl ( Ctrl ) and lower panel : TAΔDHPR ( ΔDHPR ) . Bars represent 20 μm .

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(B) Transversal cryo‐sections were stained with anti‐LC3b (red), anti‐dystrophin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Upper panel: TACtrl (Ctrl) and lower panel: TAΔDHPR (ΔDHPR). Bars represent 20 μm.


( C ) Myofibres isolated from control ( Ctrl ) or 6 months post ‐ injected FDB ( ΔDHPR ) muscles were processed for immuno ‐ fluorescent labelling for P62 ( green ) and LC3b ( red ) and imaged by confocal microscopy . Scale bars , 10 μm .

O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(C) Myofibres isolated from control (Ctrl) or 6 months post‐injected FDB (ΔDHPR) muscles were processed for immuno‐fluorescent labelling for P62 (green) and LC3b (red) and imaged by confocal microscopy. Scale bars, 10 μm.


( A ) Ultrathin sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were imaged by electron microscopy . Cis , terminal cisternae ; T , tubule ; SR , sarcoplasmic reticulum . Bars : 500 nm .

O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

(A) Ultrathin sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were imaged by electron microscopy. Cis, terminal cisternae; T, tubule; SR, sarcoplasmic reticulum. Bars: 500 nm.


( B ) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno ‐ fluorescent labelling for RyR1 ( green ) , SERCA ( red ) , Dapi ( blue ) and imaged by confocal microscopy . Scale bars , 10 μm .

O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(B) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for RyR1 (green), SERCA (red), Dapi (blue) and imaged by confocal microscopy. Scale bars, 10 μm.


( A ) Lysates from TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were immuno ‐ blotted for RyR1 or α ‐ actin . ( n = 4 ) . Graph depicts mean ± s . e . m . of relative expression of RyR1 determined by densitometry and nomalized to the α ‐ actin expression for each muscle . Results were expressed in protein levels of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice , * P ⩽ 0 . 005 , n = 4 .

O O O O O B-TISSUE O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O

(A) Lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for RyR1 or α‐actin. (n=4). Graph depicts mean±s.e.m. of relative expression of RyR1 determined by densitometry and nomalized to the α‐actin expression for each muscle. Results were expressed in protein levels of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice, *P⩽0.005, n=4.


( B ) Longitudinal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ α1S subunit ( red ) , anti ‐ RyR1 ( green ) antibodies and imaged by confocal microscopy . Bars = 20 μm .

O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(B) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red), anti‐RyR1 (green) antibodies and imaged by confocal microscopy. Bars=20 μm.


Localization of α 1S subunit . Whole skeletal muscle fibres enzymatically isolated from Ctrl ( A - D ) or ΔDHPR ( E - H ) FDB muscles 6 months post injection were processed for immuno ‐ fluorescent labelling for α1 S subunit ( red ) and laminin ( green ) . Scale bars , 10 μm ; Arrows indicate α1 S subunit expression on sarcolemma ; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections ( Supplementary Movies S5A and S5B ) . ( D , H ) Present projections of confocal Z ‐ series ( step between each frame is 1 μm ) along XZ and YZ planes as indicated .

O O B-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Localization of α 1S subunit. Whole skeletal muscle fibres enzymatically isolated from Ctrl (A-D) or ΔDHPR (E-H) FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for α1 S subunit (red) and laminin (green). Scale bars, 10 μm; Arrows indicate α1 S subunit expression on sarcolemma; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections (Supplementary Movies S5A and S5B). (D, H) Present projections of confocal Z‐series (step between each frame is 1 μm) along XZ and YZ planes as indicated.