J . Percentage of normal , abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above . n = 180 ommatidia from 3 different fly retinas for each .

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

J. Percentage of normal, abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each.


K - N . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( K ) ADAM17 - / - ; ( L ) glial specific SOD2 over - expression in an ADAM17 - / - mutant ; ( M ) glial specific SOD1 over - expression in an ADAM17 - / - mutant ; ( N ) neuronal specific SOD2 overexpression in ADAM17 - / - mutant . Scale bars : 10μm .

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

K-N. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) ADAM17-/- ; (L) glial specific SOD2 over-expression in an ADAM17-/- mutant; (M) glial specific SOD1 over-expression in an ADAM17-/- mutant; (N) neuronal specific SOD2 overexpression in ADAM17-/- mutant. Scale bars: 10μm.


O . Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above , n = 10 for each . 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 , 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 , * * p < 0 . 01 , * p < 0 . 05 .

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O. Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above, n=10 for each. 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, 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, **p<0.01, *p<0.05.


P - Q . TEM analysis of 5 week aged retinas corresponding to either ADAM17 - / - mutants or SOD2 over - expression in an ADAM17 - / - mutant . Scale bars : 10μm .

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P-Q. TEM analysis of 5 week aged retinas corresponding to either ADAM17-/- mutants or SOD2 over-expression in an ADAM17-/- mutant. Scale bars: 10μm.


R . Percentage of normal , abnormal and missing rhabdomeres in ADAM17 - / - mutant retinas , as compared to retinas overexpressing glial specific SOD2 in an ADAM17 - / - mutant background observed with TEM ; n = 180 ommatidia from 3 different fly retinas for each .

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R. Percentage of normal, abnormal and missing rhabdomeres in ADAM17-/- mutant retinas, as compared to retinas overexpressing glial specific SOD2 in an ADAM17-/- mutant background observed with TEM; n=180 ommatidia from 3 different fly retinas for each.


S . Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17 - / - . T . Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype . 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 . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 . Scale bars : 10μm .

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

S. Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17-/-. T. Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype. 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. Data were quantified for significance using Student's t test. **p<0.01. Scale bars: 10μm.


A - B . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( A ) wild type ; ( B ) ADAM17 - / - , both reared in the dark . Scale bars : 10μm .

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A-B. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A) wild type; (B) ADAM17-/-, both reared in the dark. Scale bars: 10μm.


C - F . TEM images of 4 week old retinas : ( C , D ) wildtype , or ( E , F ) ADAM17 - / - flies reared from first larval instar either in total light ( C , E ) or total dark ( D , F ) . Scale bars : 10μm .

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C-F. TEM images of 4 week old retinas: (C, D) wildtype, or (E, F) ADAM17-/- flies reared from first larval instar either in total light (C, E) or total dark (D, F). Scale bars: 10μm.


G . Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A - B , all reared in the dark . n = 10 for each . 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 and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A-B, all reared in the dark. n=10 for each. 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 and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


H . Percentage of normal , abnormal and missing rhabdomeres in the genotypes and treatments 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 .

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

H. Percentage of normal, abnormal and missing rhabdomeres in the genotypes and treatments 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.


I . Measurement of MDA levels ( in nmoles ) from 1 day old retinas of wild type or ADAM17 - / - , reared either in light or dark . Four biological replicates with 3 technical replicates for each genotype . 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 and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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I. Measurement of MDA levels (in nmoles) from 1 day old retinas of wild type or ADAM17-/- , reared either in light or dark. Four biological replicates with 3 technical replicates for each genotype. 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 and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


J . mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17 - / - mutant retinas . 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 . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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J. mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17-/- mutant retinas. 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. Data were quantified for significance using Student's t test. **p<0.01.


K - L . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( K ) heterozygous mutant of bsk alone ; or ( L ) in combination with ADAM17 - / - . Scale bars : 10μm .

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K-L. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) heterozygous mutant of bsk alone; or (L) in combination with ADAM17-/-. Scale bars: 10μm.


M . Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K - L , along with w1118 and ADAM17 - / - . n = 10 flies per genotype . 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 and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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M. Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K-L, along with w1118 and ADAM17-/-. n=10 flies per genotype. 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 and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


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 .

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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 . 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 .

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

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 . 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 :

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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 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 .

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

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 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 .

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

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 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 .

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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 . Fluorescent images of Mitotracker and MitoSOX labelling of cells 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 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 .

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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 . 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 :

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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 . 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 .

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

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 ) 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 .

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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 cells with either shCtrl or shGUARDIN . Representative IF staining with H3K9me3 antibodies ( green ) and nuclear 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 and IL - 8 in A549 cells 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 and p15 in HepG2 cells 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 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 .

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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 and H1299 cells 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 and p21 in A549 , HepG2 , HAFF and H1299 cells 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 and p21 mRNA levels in A549 cells 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 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 ) .

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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).


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 .

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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.


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

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


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 ) .

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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).


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 .

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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 assay using IgG / PGC1α antibodies from whole - cell lysates of A549 cells . GUARDIN , LRP130 and PGC1α levels 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 - 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 .

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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 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 .

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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 and pBIND - PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN . Samples were subjected to the 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 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 ) .

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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 analysis of LRP130 , PGC1α and p21 protein levels in HAFF and A549 cells 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 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 ) .

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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 and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN .

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


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 .

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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 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg / ml of actinomycin ( ActD ) for the 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 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 ) .

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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 cells transfected with LRP130 or PGC1α using the 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 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 .

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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 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 .

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 cells transduced with shCtrl or shGUARDIN using the 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.


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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 cells treated with 100nM of rapamycin for the 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 and p21 expression in A459 and H1299 cells 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 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 ) .

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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 and pBIND - PGC1α in A549 cells treated with DMSO or 100nM rapamycin . Samples were subjected to the 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 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 .

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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 ) 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 .

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

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 the GUARDIN promoter by RT - PCR assay . UPe and UPk served as a positive and 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 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 .

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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 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 )

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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 cells expressing the 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 cells 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 and stained with Coomassie blue .

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


( 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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.


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 .

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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 cells . ( D - F ) Microtubule organization in the stamen filament cells of lue1 and 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 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 .

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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 the morphologies of the stamen filament cells in wild type and 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 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 .

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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 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

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 ) .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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

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

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 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


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 .

O O O B-MEASURED_VAR O 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 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.