N - O . of 2 week old adult retinas , showing clusters of ommatidia for a heterozygous mutant of bsk alone ( M ) or in combination with ADAM17 - / - ( N ) ; n = 3 for each . Scale bars : 10μm .

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N-O. TEM images of 2 week old adult retinas, showing clusters of ommatidia for a heterozygous mutant of bsk alone (M) or in combination with ADAM17-/- (N); n=3 for each. Scale bars: 10μm.


P . of normal , abnormal missing rhabdomeres for genotypes mentioned above ; n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t . * * p < 0 . 01 .

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P. Percentage of normal, abnormal and missing rhabdomeres for the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. **p<0.01.


A - C . of human iPSC derived microglia , labelled with 493 / 503 BODIPY ( green ) FM - dye ( red ) to mark lipids membranes respectively . iPSC derived microglia were obtained from 3 different donors , treated with either DMSO , GW , TAPI - 1 or GI . Scale bars :

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A-C. Fluorescent images of human iPSC derived microglia cells, labelled with 493/503 BODIPY (green) and FM-dye (red) to mark lipids and membranes respectively. iPSC derived microglia cells were obtained from 3 different donors, and treated with either DMSO, GW, TAPI-1 or GI. Scale bars: 10μm


Quantitation of BODIPY signal shown as integrated total lipid for corresponding to each treatment donor n = 15 for each treatment . box end points represent upper ( 75 % ) lower ( 25 % ) quartiles , whiskers define maximum 95th percentile minimum 5th percentile values respectively , central band is median , square is mean , diamond an outlier . Data were analysed using Kruskal Wallis followed by Dunn ' s for post hoc for significance due to unequal sample sizes . * * * p < 0 . 001 .

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Quantitation of the BODIPY signal shown as integrated total lipid for cells corresponding to each treatment and donor n=15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.


Quantitation of BODIPY signal shown as normalised LD ( normalised to DMSO ) for corresponding to each treatment donor mentioned = 15 for each treatment . box end points represent upper ( 75 % ) lower ( 25 % ) quartiles , whiskers define maximum 95th percentile minimum 5th percentile values respectively , central band is median , square is mean , diamond an outlier . Data were analysed using Kruskal Wallis followed by Dunn ' s for post hoc for significance due to unequal sample sizes . * * * p < 0 . 001 .

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Quantitation of the BODIPY signal shown as normalised LD size (normalised to DMSO) for cells corresponding to each treatment and donor mentioned =15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.


F - G . Q - PCR of mRNA transcripts of lipogenic genes FASN LDLR in pooled data obtained from 3 different donors of human iPSC derived microglia treated with either DMSO , GW or GI for 24 hours . n = 3 biological replicates , with 3 technical replicates for each genotype . box end points represent maximum minimum values respectively , central band is median square is mean . data were quantified for significance using Student ' s t . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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F-G. Q-PCR analysis of mRNA transcripts of lipogenic genes FASN and LDLR in pooled data obtained from 3 different donors of human iPSC derived microglia treated with either DMSO, GW or GI for 24 hours. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.


H - J . of Mitotracker MitoSOX labelling of from Donor number 2 treated for 24hours with either DMSO , GW or GI . Scale bars :

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H-J. Fluorescent images of Mitotracker and MitoSOX labelling of cells from Donor number 2 treated for 24hours with either DMSO, GW or GI. Scale bars: 10μm


K . Quantitative measurements of MitoSOX normalised to that of Mitotracker in iPSC derived microglia from three different donors , treated with either DMSO , GW or GI ; n = 10 for each treatment per donor . box end points are upper ( 75 % ) lower ( 25 % ) quartiles , whiskers define maximum 95th percentile minimum 5th percentile values respectively , central band is median square is mean . data were quantified for significance using Student ' s t . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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K. Quantitative measurements of MitoSOX intensity normalised to that of Mitotracker in iPSC derived microglia cells from three different donors, treated with either DMSO, GW or GI; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.


L - N . of C - 11 BODIPY treated microglia from Donor no . 1 , treated for 24 hours with either DMSO , GW or GI . These representative display relative amounts of peroxidated non - peroxidated lipids . Scale bars :

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L-N. Fluorescent images of C-11 BODIPY treated microglia cells from Donor no.1, treated for 24 hours with either DMSO, GW or GI. These representative images display relative amounts of peroxidated and non-peroxidated lipids. Scale bars: 10μm


O . of BODIPY C - 11 , shown as ratios of peroxidated versus non - peroxidated lipids , depicted as normalised ; n = 10 for each treatment per donor . box end points are upper ( 75 % ) lower ( 25 % ) quartiles , whiskers define maximum 95th percentile minimum 5th percentile values respectively , central band is median square is mean . data were quantified for significance using Student ' s t . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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O. Quantification of BODIPY C-11 staining, shown as ratios of peroxidated versus non-peroxidated lipids, depicted as normalised intensity; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.


Senescence - associated β - galactosidase ( SA - β - gal ) in A549 , HAFF , HepG2 H1299 after 48h transduction with either negative control lentiviruses ( shCtrl ) or those targeting GUARDIN ( shGUARDIN ) . graph shows of SA - β - gal - positive .

Senescence-associated β-galactosidase (SA-β-gal) staining in A549, HAFF, HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses (shCtrl) or those targeting GUARDIN (shGUARDIN). The graph shows percentage of SA-β-gal-positive cells.


Senescence - associated heterochromatin foci formation ( SAHF ) in A549 with either shCtrl or shGUARDIN . Representative IF with H3K9me3 antibodies ( green ) Hoechst counterstaining ( blue ) .

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Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue).


Secretion of SASP factors IL - 6 IL - 8 in A549 with either shCtrl or shGUARDIN .

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Secretion of SASP factors IL-6 and IL-8 in A549 cells with either shCtrl or shGUARDIN.


Western blotting for p21 , p16 p15 in HepG2 after 48h transduction with shCtrl or shGUARDIN - 1 , - 2 , respectively ( left ) . Knockdown efficiencies analyzed by qPCR ( right ) .

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Western blotting for p21, p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN-1, -2, respectively (left). Knockdown efficiencies analyzed by qPCR (right).


SA - β - gal in A549 after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA ( shp21 ) . graph shows of SA - β - gal - positive .

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SA-β-gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA (shp21). The graph shows percentage of SA-β-gal-positive cells.


qPCR measurement of p21 mRNA in A549 , HepG2 , HAFF H1299 with either GUARDIN shRNA knockdown ( top ) or GUARDIN overexpression ( bottom ) .

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qPCR measurement of p21 mRNA in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).


Western blotting for p53 p21 in A549 , HepG2 , HAFF H1299 with either GUARDIN shRNA knockdown ( top ) or GUARDIN overexpression ( bottom ) .

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Western blotting for p53 and p21 in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).


qPCR assays of NOXA , TIGAR , BAX p21 mRNA in A549 with either shCtrl or shGUARDIN .

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qPCR assays of NOXA, TIGAR, BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN.


SDS - PAGE of pulldown assays using biotin - labelled sense / antisense probes against GUARDIN from whole - lysates of A549 indicating putative GUARDIN - binding ( left ) ; identities with high probabilities were determined by mass spectrometry ( right ) .

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SDS-PAGE of RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells indicating putative GUARDIN-binding proteins (left); protein identities with high probabilities were determined by mass spectrometry (right).


pulldown assays interrogating putative GUARDIN - associated identified in ( A ) from whole - lysates of A549 HAFF . BRCA1 , BARD1 served as positive controls β - actin served as negative controls .

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RNA pulldown assays interrogating putative GUARDIN-associated proteins identified in (A) from whole-cell lysates of A549 and HAFF cells. BRCA1, BARD1 served as positive controls and β-actin served as negative controls.


immunoprecipitation ( RIP ) assays against IgG / LRP130 antibodies in whole - lysates of A549 .

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RNA immunoprecipitation (RIP) assays against IgG/LRP130 antibodies in whole-cell lysates of A549 cells.


Subcellular localization of GUARDIN its co - localization with LRP130 . FISH for GUARDIN ( red ) IF for LRP130 ( green ) in A549 with either shCtrl or shGUARDIN . Nucleus were counterstained with Hoechst ( blue ) .

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Subcellular localization of GUARDIN and its co-localization with LRP130. RNA FISH for GUARDIN (red) and IF for LRP130 (green) in A549 cells with either shCtrl or shGUARDIN. Nucleus were counterstained with Hoechst (blue).


pulldown assays using biotin - labelled sense / antisense probes against GUARDIN from whole - lysates of A549 . GUARDIN were measured by RT - PCR co - precipitated LRP130 PGC1α detected by Western blotting . BRCA1 β - actin served as positive negative controls , respectively .

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RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.


RIP using IgG / PGC1α antibodies from whole - lysates of A549 . GUARDIN , LRP130 PGC1α were measured as per ( E ) .

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RIP assay using IgG/PGC1α antibodies from whole-cell lysates of A549 cells. GUARDIN, LRP130 and PGC1α levels were measured as per (E).


Two - step IP assays in whole - lysates of A549 transfected with FLAG - tagged PGC1α . First phase IPs were conducted with FLAG antibodies ( left ) following elution with FLAG peptides , eluates were further subjected to second phase IPs with LRP130 antibodies ( right ) . Samples were subjected to Western blotting qPCR for LRP130 , PGC1α GUARDIN , respectively .

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Two-step IP assays in whole-cell lysates of A549 cells transfected with FLAG-tagged PGC1α. First phase IPs were conducted with FLAG antibodies (left) and following elution with FLAG peptides, eluates were further subjected to second phase IPs with LRP130 antibodies (right). Samples were subjected to Western blotting and qPCR analysis for LRP130, PGC1α and GUARDIN, respectively.


Co - immunoprecipitation ( co - IP ) between LRP130 PGC1α in A549 after 48h transduction with shCtrl or shGUARDIN . LRP130 was precipitated samples subjected to Western blotting for LRP130 , PGC1α β - actin as loading control .

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Co-immunoprecipitation (co-IP) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130, PGC1α and β-actin as loading control.


Mammalian two - hybrid assays between pACT - LRP130 pBIND - PGC1α in A549 after 48h transduction with shCtrl or shGUARDIN . Samples were subjected to luciferase activity assays .

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Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. Samples were subjected to the luciferase activity assays.


LRP130 / PGC1α p21 expression was measured by Western blot in A549 HAFF after 48h transduction with shCtrl or shLRP130 ( top left ) or shPGC1α ( bottom left ) as indicated . qPCR assays for p21 mRNA were performed in parallel ( right panels ) .

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LRP130/PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 (top left) or shPGC1α (bottom left) as indicated. qPCR assays for p21 mRNA levels were performed in parallel (right panels).


Western blotting of LRP130 , PGC1α p21 in HAFF A549 after 48h transduction with shCtrl or shGUARDIN .

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Western blotting analysis of LRP130, PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN.


ChIP assays detecting binding of LRP130 / PGC1α to p21 promoter using qPCR RT - PCR ( top left right , respectively ) . IgG p53 served as a negative positive controls , respectively . Schematic illustrations of putative LRP130 / PGC1α binding sites within DNase I hypersensitive regions of p21 promoter ( bottom ) .

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ChIP assays detecting binding of LRP130/PGC1α to the p21 promoter using qPCR and RT-PCR (top left and right, respectively). IgG and p53 served as a negative and positive controls, respectively. Schematic illustrations of the putative LRP130/PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter (bottom).


qPCR assays for GUARDIN , FOXO1 , FOXO3a FOXO4 in A549 after 24h transduction with shCtrl or shGUARDIN .

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qPCR assays for GUARDIN, FOXO1, FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN.


Western blotting assays for FOXO4 p21 expression in A549 after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4 . β - actin served as loading control .

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Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4. β-actin served as loading control.


Half - life times of FOXO4 mRNA in A549 with shCtrl or shGUARDIN measured by qPCR after treating with 5 μg / ml of actinomycin ( ActD ) for indicated times .

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Half-life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg/ml of actinomycin (ActD) for the indicated times.


ChIP assays detecting binding of LRP130 / PGC1α to putative binding sites in FOXO4 promoter using qPCR ( top ) . Data were normalized to IgG negative control . Schematic illustration of LRP130 / PGC1α binding sites within DNase I hypersensitive regions of FOXO4 promoter ( bottom ) .

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ChIP assays detecting binding of LRP130/PGC1α to putative binding sites in the FOXO4 promoter using qPCR (top). Data were normalized to the IgG negative control. Schematic illustration of the LRP130/PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter (bottom).


Luciferase assays conducted in A549 transfected with LRP130 or PGC1α using pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

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Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


qPCR assays for LRP130 FOXO4 mRNA ( upper ) Western blotting for LRP130 level ( lower ) in A549 after 48h transduction with LRP130 overexpression or knockdown .

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qPCR assays for LRP130 and FOXO4 mRNA levels (upper) and Western blotting analysis for LRP130 protein level (lower) in A549 cells after 48h transduction with LRP130 overexpression or knockdown.


qPCR assays for PGC1α FOXO4 mRNA level ( upper ) Western blotting for PGC1α level ( lower ) in A549 after 48h transduction with PGC1α overexpression or knockdown .

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qPCR assays for PGC1α and FOXO4 mRNA level (upper) and Western blotting analysis for PGC1α protein level (lower) in A549 cells after 48h transduction with PGC1α overexpression or knockdown.


Luciferase activity assays in A549 transduced with shCtrl or shGUARDIN using pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

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Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


qPCR assays for GUARDIN expression in A549 treated with increasing doses rapamycin for

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qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24h


qPCR assays for GUARDIN expression in A549 treated with 100nM of rapamycin for indicated times ( B ) .

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qPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times (B).


Western blotting assays for p53 , LRP130 , FOXO4 p21 expression in A459 H1299 treated with 100nM of rapamycin or vehicle ( DMSO ) for 48h .

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Western blotting assays for p53, LRP130, FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle (DMSO) for 48h.


A549 with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in indicated combinations for 48h . Western blotting was used to measure p21 ( left ) while conducting SA - β - gal in parallel ( middle ) with of senescent calculated from SA - β - gal ( right ) .

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A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h. Western blotting was used to measure p21 levels (left) while conducting SA-β-gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA-β-gal staining (right).


Mammalian two - hybrid assays between pACT - LRP130 pBIND - PGC1α in A549 treated with DMSO or 100nM rapamycin . Samples were subjected to luciferase activity assays .

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Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells treated with DMSO or 100nM rapamycin. Samples were subjected to the luciferase activity assays.


SIX4 FOSL2 mRNA ( left ) ( right ) measured by qPCR Western blotting , respectively in A549 treated with DMSO or 100nM rapamycin . TBP served as a control .

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SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively in A549 cells treated with DMSO or 100nM rapamycin. TBP served as a control.


qPCR ( upper ) Western blotting ( lower ) assays for GUARDIN , SIX4 FOSL2 expression in A549 comparing shCtrl with shSIX4 ( left ) , shFOSL2 ( middle ) or FOSL2 ( right ) after transduction for 48h .

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qPCR (upper) and Western blotting (lower) assays for GUARDIN, SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 (left), shFOSL2 (middle) or FOSL2 (right) after transduction for 48h.


Luciferase assays in A549 transduced with shCtrl , shSIX4 or shFOSL2 using pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

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Luciferase assays in A549 cells transduced with shCtrl, shSIX4 or shFOSL2 using pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of GUARDIN promoter by RT - PCR . UPe UPk served as a positive negative controls , respectively [ 54 ] .

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ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT-PCR assay. UPe and UPk served as a positive and negative controls, respectively [54].


( a ) Confocal of HeLa expressing yellow ( YFP ) fused to Nap1 , Sintbad or Tank infected with S . Typhimurium , stained for ubiquitin ( Ub ) 2 h after infection . At least 100 were analyzed . DAPI , 4 , 6 - diamidino - 2 - phenylindole . Scale bar , 10 μm ; arrowheads , shown in insets .

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(a) Confocal micrographs of HeLa cells expressing yellow fluorescent protein (YFP) fused to Nap1, Sintbad or Tank and infected with S. Typhimurium, stained for ubiquitin (Ub) 2 h after infection. At least 100 cells were analyzed. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm; arrowheads, bacteria shown in insets.


( b - e ) Normalized between luciferase activity bound to beads present in lysates . indicated purified GST fusion coupled to beads were incubated with lysates of 293ET expressing Nap1 , Sintbad or Tank , each fused to luciferase ( b )

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(b-e) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with lysates of 293ET cells expressing Nap1, Sintbad or Tank, each fused to luciferase (b)


( c ) , lysates of 293ET expressing indicated Nap1 mutants fused to

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(c), lysates of 293ET cells expressing the indicated Nap1 mutants fused to luciferase


( d ) , lysates of E . coli expressing Nap1 ( 1 - 85 ) fused to

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(d), lysates of E. coli expressing Nap1(1-85) fused to luciferase


( e ) lysates of 293ET expressing Nap1 , NEMO or Optineurin fused to

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(e) lysates of 293ET cells expressing Nap1, NEMO or Optineurin fused to luciferase


( a ) Flowthrough from nickel - agarose column , separated by SDS - PAGE stained with Coomassie blue .

(a) Flowthrough from nickel-agarose column, separated by SDS-PAGE and stained with Coomassie blue.


( c , d ) Normalized between luciferase activity bound to beads present in lysates . indicated purified GST fusion coupled to beads were incubated with bacterially expressed Nap1 ( 1 - 85 ) fused to luciferase . Reactions were complemented with lysates from 293ET that had been depleted with indicated ( α - ) antibodies ( c , top ) . Immunoblot of lysates after depletion ( c , bottom ) .

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(c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1-85) fused to luciferase. Reactions were complemented with lysates from 293ET cells that had been depleted with the indicated (α-) antibodies (c, top). Immunoblot of lysates after depletion (c, bottom).


( c , d ) Normalized between luciferase activity bound to beads present in lysates . indicated purified GST fusion coupled to beads were incubated with bacterially expressed Nap1 ( 1 - 85 ) fused to luciferase . Reactions were complemented with indicated amounts of lysates from E . coli cultures expressing myelin basic ( MBP ) fused to NDP52 ( d ) .

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(c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1-85) fused to luciferase.Reactions were complemented with the indicated amounts of lysates from E. coli cultures expressing myelin basic protein (MBP) fused to NDP52 (d).


( e ) Lysates of 293ET were immunoprecipitated ( IP ) using indicated antibodies or an isotype control . Lysates precipitates were blotted ( IB ) for presence of TBK1 , IKKε IKKβ .

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(e) Lysates of 293ET cells were immunoprecipitated (IP) using the indicated antibodies or an isotype control. Lysates and precipitates were blotted (IB) for the presence of TBK1, IKKε and IKKβ.


( f ) Lysates of 293ET were immunoprecipitated using indicated antibodies or an isotype control . Precipitates were blotted for presence of TBK1 NDP52 . Fusion used in Figure 2 are further characterized in Supplementary Figure 1 . Data in panels c - f are representative of at least two independent experiments . Ub , ubiquitin ; PCNA , proliferating antigen .

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(f) Lysates of 293ET cells were immunoprecipitated using the indicated antibodies or an isotype control. Precipitates were blotted for the presence of TBK1 and NDP52. Fusion proteins used in Figure 2 are further characterized in Supplementary Figure 1. Data in panels c-f are representative of at least two independent experiments. Ub, ubiquitin; PCNA, proliferating cell nuclear antigen.


( a ) Confocal of HeLa 2 h after infection with S . Typhimurium , stained with antibodies to Cocoa , NDP52 ubiquitin ( Ub ) . ( b ) Colocalization , at indicated time points after infection , of ubiquitin - positive S . Typhimurium with NDP52 or Cocoa .

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(a) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to Cocoa, NDP52 and ubiquitin (Ub). (b) Colocalization, at the indicated time points after infection, of ubiquitin-positive S. Typhimurium with NDP52 or Cocoa.


( c ) Confocal of HeLa 2 h after infection with S . Typhimurium , stained with antibodies to NDP52 LAMP1 . ( d ) Colocalization , at indicated time points after infection , of NDP52 - positive S . Typhimurium with LAMP1 . DAPI , 4 , 6 - diamidino - 2 - phenylindole . data are representative of at least two independent experiments . Error bars , s . d . ; three coverslips per experiment , 50 marker - positive evaluated per coverslip . Scale bars , 10 μm ; arrowheads , shown in insets .

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(c) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to NDP52 and LAMP1. (d) Colocalization, at the indicated time points after infection, of NDP52-positive S. Typhimurium with LAMP1. DAPI, 4,6-diamidino-2-phenylindole. All data are representative of at least two independent experiments. Error bars, s.d.; three coverslips per experiment, 50 marker-positive cells evaluated per coverslip. Scale bars, 10 μm; arrowheads, bacteria shown in insets.


of NDP52 - positive S . Typhimurium among ubiquitin - coated , determined by microscopy . HeLa transduced with indicated NDP52 domain - deletion mutants fused to green were infected with S . Typhimurium stained for ubiquitin 2 h after infection . ZnF , zinc finger domain . * P 0 . 01 , Student ' s t - . Mean s . d . of triplicate coverslips from two independent experiments ; more than 50 ubiquitin - positive were evaluated per coverslip .

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Percentage of NDP52-positive S. Typhimurium among ubiquitin-coated bacteria, determined by microscopy. HeLa cells transduced with the indicated NDP52 domain-deletion mutants fused to green fluorescent protein were infected with S. Typhimurium and stained for ubiquitin 2 h after infection. ZnF, zinc finger domain. *P 0.01, Student's t-test. Mean and s.d. of triplicate coverslips from two independent experiments; more than 50 ubiquitin-positive bacteria were evaluated per coverslip.


( a , b ) Confocal of HeLa expressing YFP - Nap1 , YFP - Sintbad or YFP - Tank ( a ) or YFP - TBK1 ( K38M ) , a catalytically inactive mutant ( b ) , stained with NDP52 antiserum 2 h after infection with S . Typhimurium . data represent at least two independent experiments . DAPI , 4 , 6 - diamidino - 2 - phenylindole . Scale bars , 10 μm ; arrowheads , shown in insets .

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(a,b) Confocal micrographs of HeLa cells expressing YFP-Nap1, YFP-Sintbad or YFP-Tank (a) or YFP-TBK1(K38M), a catalytically inactive mutant (b), stained with NDP52 antiserum 2 h after infection with S. Typhimurium. All data represent at least two independent experiments. DAPI, 4,6-diamidino-2-phenylindole. Scale bars, 10 μm; arrowheads, bacteria shown in insets.


( a ) Lysates of transfected with indicated siRNAs blotted for NDP52 , TBK1 , IKKε proliferating antigen ( PCNA ) . Control , a nonspecific band detected by IKKε antibody .

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(a) Lysates of cells transfected with the indicated siRNAs blotted for NDP52, TBK1, IKKε and proliferating cell nuclear antigen (PCNA). Control, a nonspecific band detected by the IKKε antibody.


( b , c ) Numbers of recovered from HeLa transfected with indicated siRNAs infected with S . Typhimurium . At indicated time points after infection , were lysed counted on basis of their ability to form colonies on agar plates . Mean s . d . of triplicate HeLa cultures duplicate colony .

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(b,c) Numbers of bacteria recovered from HeLa cells transfected with the indicated siRNAs and infected with S. Typhimurium. At the indicated time points after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts.


( d ) Numbers of recovered from HCT116 expressing indicated shRNAs infected with S . Typhimurium . At indicated time points were lysed counted by flow cytometry . Mean s . d . of triplicate cultures .

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(d) Numbers of bacteria recovered from HCT116 cells expressing the indicated shRNAs and infected with S. Typhimurium. At the indicated time points cells were lysed and bacteria counted by flow cytometry. Mean and s.d. of triplicate cultures.


( e ) HeLa transfected with indicated siRNAs were infected with S . Typhimurium expressing green ( GFP ) . At indicated time points after infection , were scored according to number of they contained .

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(e) HeLa cells transfected with the indicated siRNAs were infected with S. Typhimurium expressing green fluorescent protein (GFP). At the indicated time points after infection, cells were scored according to the number of bacteria they contained.


( f ) HeLa transfected with indicated siRNAs were infected with S . Typhimurium expressing GFP . At indicated time points after infection , were stained for ubiquitin infected were scored if they contained ubiquitylated . data represent at least two independent experiments . * P 0 . 05 , Student ' s t - .

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(f) HeLa cells transfected with the indicated siRNAs were infected with S. Typhimurium expressing GFP. At the indicated time points after infection, cells were stained for ubiquitin and infected cells were scored if they contained ubiquitylated bacteria. All data represent at least two independent experiments. *P 0.05, Student's t-test.


( a - d ) of HeLa stably expressing green - LC3 , infected with S . Typhimurium . ( a ) Confocal of stained 1 h after infection with NDP52 antiserum . Scale bar , 10 μm ; arrowheads , shown in insets . ( b ) Colocalization of NDP52 with LC3 - positive . ( c , d ) of LC3 - positive S . Typhimurium 1 h after infection in transfected with indicated siRNAs . In c , were stained for ubiquitin ( Ub ) ; in d , red ( RFP ) - expressing S . Typhimurium were used . Means of duplicate from four independent experiments ; * P 0 . 05 , Student ' s t - .

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(a-d) Analysis of HeLa cells stably expressing green fluorescent protein-LC3, infected with S. Typhimurium. (a) Confocal micrographs of cells stained 1 h after infection with NDP52 antiserum. Scale bar, 10 μm; arrowheads, bacteria shown in insets. (b) Colocalization of NDP52 with LC3-positive bacteria. (c,d) Counts of LC3-positive S. Typhimurium 1 h after infection in cells transfected with the indicated siRNAs. In c, cells were stained for ubiquitin (Ub); in d, red fluorescence protein (RFP)-expressing S. Typhimurium were used. Means of duplicate counts from four independent experiments; *P 0.05, Student's t-test.


( e ) Binding of NDP52 to GST - LC3 GST - ubiquitin . Beads carrying indicated purified GST fusion were incubated with lysates of E . coli expressing NDP52 . Bound were eluted with glutathione stained with Coomassie blue after SDS - PAGE .

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(e) Binding of NDP52 to GST-LC3 and GST-ubiquitin. Beads carrying the indicated purified GST fusion proteins were incubated with lysates of E. coli expressing NDP52. Bound proteins were eluted with glutathione and stained with Coomassie blue after SDS-PAGE.


( f ) Normalized of luciferase - LC3 binding to beads . indicated purified GST coupled to beads were incubated with bacterially expressed LC3 fused to luciferase indicated volumes of lysed E . coli expressing NDP52 . Data in a , b , e f are representative of at least two independent experiments .

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(f) Normalized ratio of luciferase-LC3 binding to beads. The indicated purified GST proteins coupled to beads were incubated with bacterially expressed LC3 fused to luciferase and the indicated volumes of lysed E. coli expressing NDP52. Data in a,b,e and f are representative of at least two independent experiments.


of wild type nine microtubule - associated gene mutants . Comparison of filament stigma of wild type nine microtubule - associated gene mutants .

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Phenotypes of wild type and nine microtubule-associated gene mutants. Comparison of the filament and stigma phenotypes of wild type and nine microtubule-associated gene mutants.


Microtubule organization in wild type stamen filament . ( D - F ) Microtubule organization in stamen filament of lue1 ktn80 . 1234 . Arrows indicate microtubule crossovers . Dotted lines indicate abnormal microtubules .

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Microtubule organization in wild type stamen filament cells. (D-F) Microtubule organization in the stamen filament cells of lue1 and ktn80.1234. Arrows indicate microtubule crossovers. Dotted lines indicate abnormal microtubules.


Time - lapse showing that KTN1 is associated with microtubule severing at crossover sites in wild type filament . GFP - labeled KTN1 particles are pseudo - colored in red , microtubules ( labeled with mCherry - TUB6 ) are shown in green . Yellow arrows indicate KTN1 at microtubule crossover sites . Asterisks indicate location at which a microtubule has been severed at a crossover site . Time - lapse showing that KTN1 cannot be recruited to crossover sites no severing was observed in ktn80 . 1234 filament . Yellow open arrows indicate microtubule crossovers , which remain stable for a long time .

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Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in wild type filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site. Time-lapse images showing that KTN1 cannot be recruited to crossover sites and no severing was observed in ktn80.1234 filament cells. Yellow open arrows indicate microtubule crossovers, which remain stable for a long time.


Comparison of morphologies of stamen filament in wild type qui - 2 .

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Comparison of the morphologies of the stamen filament cells in wild type and qui-2.


EMSA showing that BZR1 binds to E - box of KTN80 . 4 promoter regions in vitro . Nonlabelled ( free ) native probe in 10 - to 100 - fold molar excess relative to biotin - labeled native probe was used as a cold competitor . mutant probe was used as a negative control .

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EMSA showing that BZR1 binds to the E-box of the KTN80.4 promoter regions in vitro. Nonlabelled (free) native probe in 10- to 100-fold molar excess relative to the biotin-labeled native probe was used as a cold competitor. The mutant probe was used as a negative control.


Time - lapse showing that KTN1 is associated with microtubule severing at crossover sites in ( A ) wild type ( B qui - 2 filament . GFP - labeled KTN1 particles are pseudo - colored in red , microtubules ( labeled with mCherry - TUB6 ) are shown in green . Yellow arrows indicate KTN1 at microtubule crossover sites . Asterisks indicate location at which a microtubule has been severed at a crossover site in A

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Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in (A) wild type (B qui-2 filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at the microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site in A and B


Time - lapse showing that KTN1 is associated with microtubule severing at crossover sites C ) qui - 2 filament . GFP - labeled KTN1 particles are pseudo - colored in red , microtubules ( labeled with mCherry - TUB6 ) are shown in green . Yellow arrows indicate KTN1 at microtubule crossover sites . or location at which a microtubule has not been severed at a crossover site in C .

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Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites C) qui-2 filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at the microtubule crossover sites. or the location at which a microtubule has not been severed at a crossover site in C.


( A - D ) cultures from D . melanogaster expressing GFP - Atg8a under control of Dmef2 - Gal4 , stained with Phalloidin ( actin ) in red . Inset are magnifications of areas denoted by dotted . ( A ) In untreated control cultures , GFP - Atg8a localizes throughout muscle cytoplasm nuclei . ( B ) Addition of rapamycin ( Rap ) for 12 hrs results in formation of small GFP - Atg8a labeled . ( C ) Addition of chloroquine ( CQ ) for 12 hrs results in accumulation of large GFP - Atg8a labeled vesicles that localize around nucleus between myofibers . ( D ) Addition of both CQ Rap for 12hrs , triggers formation of greater numbers of enlarged vesicles in than either drug alone . ( E ) Knockdown of autophagy gene , Atg1 , prevents formation of GFP - Atg8a vesicles in CQ + Rap treated cultures . ( F ) of mean of GFP - Atg8a to total muscle per well ( n = 4 ) . Error bars indicate with * * p < . 01 ; * * * p < . 001 .

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(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the muscle cytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.


( A - D ) cultures from D . melanogaster expressing GFP - Atg8a under control of Dmef2 - Gal4 , stained with Phalloidin ( actin ) in red . Inset are magnifications of areas denoted by dotted . ( A ) In untreated control cultures , GFP - Atg8a localizes throughout musclecytoplasm nuclei . ( B ) Addition of rapamycin ( Rap ) for 12 hrs results in formation of small GFP - Atg8a labeled . ( C ) Addition of chloroquine ( CQ ) for 12 hrs results in accumulation of large GFP - Atg8a labeled vesicles that localize around nucleus between myofibers . ( D ) Addition of both CQ Rap for 12hrs , triggers formation of greater numbers of enlarged vesicles in musclesthan either drug alone . ( E ) Knockdown of autophagy gene , Atg1 , prevents formation of GFP - Atg8a vesicles in CQ + Rap treated cultures . ( F ) of mean of GFP - Atg8a to total muscle per well ( n = 4 ) . Error bars indicate with * * p < . 01 ; * * * p < . 001 .

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(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the musclecytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.


( E ) Control RNAi starved treated with CQ accumulate GFP - Atg8a , but very few colocalize with ubiquitin ( Ubi ) . ( F ) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits formation of GFP - Atg8a . remainder colocalize with ubiquitin . ( G ) of mean GFP - Atg8a Mander ' s overlap coefficient for GFP - Atg8a Ubi for genotypes shown in E F . Error bars indicate for n = 8 ventral longitudinal from , with p - values for Rpn1 RNAi relative to RNAi control ( * * * p < . 001 ) .

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(E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001).


( H , I ) Autophagosome number is reduced , but autophagic flux is not significantly altered in starved Rpn1 vs . RNAi expressing GFP - mCherry - Atg8 . ( J ) Flux measurement is shown on right as of mCherry to GFP - mCherry ( n = 6 ) . is indicated .

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(H, I) Autophagosome number is reduced, but autophagic flux is not significantly altered in starved Rpn1 vs. white RNAi muscles expressing GFP-mCherry-Atg8. (J) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP-mCherry (n = 6). SEM is indicated.


( K ) Anti - Atg8a Western blot of third instar muscle lysate from Dmef2 - Gal4 ; UAS - RNAi . In fed RNAi control , endogenous Atg8a appears as a single band ( Atg8a - I ) . 6 hrs starvation induces lipid modification of Atg8a appearance of a second band ( Atg8a - II ) . Knockdown of Atg1 or Rpn1 inhibits Atg8a modification , indicating reduced autophagy .

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(K) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4; UAS-RNAi animals. In fed white RNAi control, endogenous Atg8a appears as a single band (Atg8a-I). 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band (Atg8a-II). Knockdown of Atg1 or Rpn1 inhibits Atg8a modification, indicating reduced autophagy levels.


( A - D ) Inhibition of proteasome by overexpression of a sensitive proteasome mutant ( DTS ) . Dmef2 - Gal4 , UAS - GFP - Atg8a control UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a were shifted to restrictive then starved CQ treated 6 hrs prior to dissection . 18 hrs post - shift ( PS ) , both control ( A ) DTS ( B ) accumulate GFP - Atg8a , but latter also accumulate ubiquitin ( Ubi ) . 48 hrs PS , control ( C ) maintain strong autophagy induction , but DTS mutant ( D ) have reduced GFP - Atg8a accumulation , similar to Rpn1 knockdown phenotype . ( E ) of mean GFP - Atg8a Mander ' s overlap coefficient for GFP - Atg8a Ubi for genotypes shown in A - D ( is indicated for n = 8 ventral longitudinal from with p - values of DTS relative to control ) . green = Dmef2 - Gal4 , UAS - GFP - Atg8a ; green = UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a .

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(A-D) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant (DTS). Dmef2-Gal4, UAS-GFP-Atg8a control and UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection. 18 hrs post-shift (PS), both control (A) and DTS muscles (B) accumulate GFP-Atg8a punctae, but the latter also accumulate ubiquitin (Ubi) aggregates. 48 hrs PS, control muscles (C) maintain strong autophagy induction, but DTS mutant muscles (D) have reduced GFP-Atg8a punctae accumulation, similar to the Rpn1 knockdown phenotype. (E) Quantification of the mean GFP-Atg8a punctae and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in A-D (SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p-values of DTS relative to control). Dark green = Dmef2-Gal4, UAS-GFP-Atg8a; Light green = UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a.


( F ) Western blot of endogenous Atg8a from third instar muscle lysate . 6 hrs starvation induces lipidated Atg8a - II in Dmef2 - Gal4 control at both 18 hrs 48 hrs PS , but not in DTS at 48 hrs PS .

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(F) Western blot analysis of endogenous Atg8a from third instar muscle lysate. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-Gal4 control at both 18 hrs and 48 hrs PS, but not in DTS at 48 hrs PS.


( A - C ) Time course of effect of shift on , autophagosomes , larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a starved 6 hrs prior to . ( A ) number of ubiquitin peaks at 18 - 24 hrs PS , decreases significantly from 18 to 48 hrs PS ( n = 10 p - values shown relative to 18 hrs PS ) . ( B ) number of GFP - Atg8a peaks at 12 - 24 hrs PS decreases significantly from 18 to 48 hrs PS ( n = 10 p - values shown relative to 18 hrs PS ) . ( C ) Larval locomotion , measured as time to crawl 3cm , gradually significantly worsens from 18 to 48 hrs PS ( n = 6 p - values shown relative to 0 hrs PS .

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(A-C) Time course of effect of temperature shift on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals starved 6 hrs prior to analysis. (A) The number of ubiquitin aggregates peaks at 18-24 hrs PS, and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (B) The number of GFP-Atg8a punctae peaks at 12-24 hrs PS and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (C) Larval locomotion, measured as the time to crawl 3cm, gradually and significantly worsens from 18 to 48 hrs PS (n = 6 and p-values shown relative to 0 hrs PS.


( D - E ) tyramine ( Tyr ) treatment reduces GFP - Atg8a in larval muscle . UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a were placed on 100mg / ml Tyr ( D ) or control food ( E ) , shifted to restrictive for 18hrs , starved 6 hrs prior to dissection .

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(D-E) tyramine (Tyr) treatment reduces GFP-Atg8a punctae in larval muscle. UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals were placed on 100mg/ml Tyr (D) or control food (E), shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection.


( F - H ) Effect of Tyr treatment on , autophagosomes , larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a Dmef2 - Gal4 , UAS - GFP - Atg8a , shifted to restrictive for 18hrs , starved 6 hrs prior to dissection . Aggregate ( F ) GFP - Atg8a ( G ) numbers in are significantly reduced with 100 mg / ml Tyr treatment in DTS but not control 18 hrs post - shift ( PS ) . ( H ) 100 mg / ml Tyr treatment significantly worsens larval locomotion in both control DTS 18 hrs PS . n = 12 p - values shown relative to non - Tyr treated . For measurements , 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 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 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 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 ) , Rab3 - GAP ( H ) , blocks formation of GFP - Atg8a labeled autophagosomes compared to knockdown of ( 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 starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . effect of 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 ) of autophagy from panels D - M . is indicated , with n = 8 ventral longitudinal from * * 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 ) of autophagy from panels D - M . is indicated , with n = 8 ventral longitudinal from * * 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 Rab3 - GAP colocalize to autophagosomes . Dmef2 - Gal4 drives expression of UAS - myc - Atg8a UAS - CFP - Rab3 - GAP in starved treated with CQ for 6 hrs . In panels green = GFP - Atg8a red = myc - Atg8a , 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 RNAi or overexpression constructs shown in D - N . Total 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 ) cultured with control lacZ RNAi , treated for 12 hrs with Rap + CQ . Note formation of large GFP - Atg8a vesicles throughout . ( B ) GFP - Atg8a vesicles are reduced in cultured 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 cultured treated for 12 hrs with Rap + CQ . ( E ) for A - D of mean of GFP - Atg8a to total muscle per well ( n = 4 ) . 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 RNAi accumulate large autophagosomes at 1 mg / ml CQ . ( G ) RNAi knockdown of CG9485 increases autophagosome accumulation in muscle . ( H ) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in muscle . ( I ) of GFP - Atg8a in from , CG9485 , AGBE knockdowns . n = 6 larval with indicated * 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 of endogenous Atg8a from third instar muscle lysate . Dmef2 - Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation , indicating reduced autophagy .

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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.


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


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


MR tail is essential for Nek2A binding to APC / C whereas in contrast Cdc20 is dispensable . exclusion chromatography peak fractions of APC / C complexes with Nek2A . 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 coactivator subunit Cdc20 to APC / C does not compete with cyclin A for binding to APC / C . 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 N392A / E395A mutant of Apc2 . APC / C2 / 4m is N392A / E395A / R48A / H53A / S51A mutant of Apc2 Apc4 .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O

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 .

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

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


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

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

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


exclusion chromatography peak fractions of APC / C complexes with either

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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

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

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 ) eGFP - Nek2A ( bottom ) in HEK during mitosis . timepoint of NEBD is marked at 0 minutes in graphs . Asterisks indicate values that are significantly different from same time point of siGL2 control as determined by a Mann - Whitney U - Mean ± s . d . is shown . number of 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 ) . data are from at least two biological replicates .

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

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

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 B-CONTROLLED_VAR O O O O

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

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

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 FAO Stress in Seahorse XF . n = 4 biological repeats ; error bars indicate ; * * , p - value < 0 . 01 ; NS , not significant ; t - .

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 treated with 200 nM INK - 128 on day0 . numbers were counted on day2 , 4 , 6 . n = 4 biological repeats ; error bars indicate ; p - values were calculated using two - sample t - .

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

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 Tfcp2l1 _ KO 2i - ESCs untreated or treated with 200 nM INK - 128 .

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

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


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

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

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 log rank . E . Kaplan - Meier curve of ex vivo WT blastocysts cultured in M2 medium in presence of 200 nM of INK - 128 with or without 50 µM of etomoxir ( Eto ) / or 100 µM of octanoate , as indicated . p - values were calculated using log rank .

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

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 E ) Growth comparisons of bir1 ∆ - ad2 strains that contain ( 18 strains ) or do not contain ( 28 strains ) confirmed suppressor mutations as measured by of colony growth after serial dilution . mean ( red ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(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 permissive ( 25 ˚ C ) or restrictive ( 35 ˚ C ) temperatures for ipl1 - 321 mutation . suppressor mutations from Dam1c ( blue ) were only mutants that rescued viability at restrictive .

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

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

O O O O O 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 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

(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 indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on 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 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 O O O O O O O O O O B-CONTROLLED_VAR 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 showing localization of Dad3 - mNeonGreen ( Dam1c member ) Nuf2 - mRuby3 ( kinetochore marker ) in prometaphase . Scale bar is 2µm . Right : of of Dad3 - mNeonGreen at kinetochores / spindles in prometaphase . Each point on graph represents a single measurement of Dad3 - mNeonGreen ; up to 10 measurements per replicate . Data are from 5 independent experiments .

O O O O O O O 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 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 B-MEASURED_VAR O 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 showing localization of Dad3 - mNeonGreen ( Dam1c member ) Nuf2 - mRuby3 ( kinetochore marker ) in anaphase . Scale bar is 2µm . Right : of of Dad3 - mNeonGreen at spindle in anaphase . was measured in middle of spindle using a perpendicular - scan . Each point on graph represents a single measurement of Dad3 - mNeonGreen ; up to 10 measurements per replicate . Data are from 5 independent experiments .

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

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 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

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

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

(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 ) 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 switching to glucose - containing media ( YPAD ) . Representative are on left . Scale bar is 2µm . Averages from 3 independent experiments are shown .

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(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 at inner centromere of prometaphase kinetochores . Inner centromeres were identified as regions directly between two centromeres ( ACA ) . from an example WT are shown to right . Scale bar is 5µm long . Each data point is an chromosome . More than 15 for each were measured across two independent experiments . mean ( red ) is shown .

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(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 ) Btg2 are expressed high in both NSCs BPs at mRNA level . Each dot defines mean lines define 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 ) validating known - type specific marker gene expression from sequencing data . ( E ) illustrating novel marker genes identified from sequencing data , as signature genes for NSCs , BPs NBNs . NSCs - Neural , BPs - Basal progenitors , NBNs - Newborn neurons , E - Embryonic day , PN - Post natal , VZ - Ventricular zone , SVZ - Subventricular zone , IZ - Intermediate Zone . Expression values on heatmaps are log2 ( transcripts per million ) .

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(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).