A time - lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP - C - EGFP ( centromeres , green ) H2B - mCherry ( , red ) . Upper row shows maximum z projection from representative timepoints at metaphase II anaphase II . A lagging chromatid is indicated by a blue arrowhead . Number next to circles enclosing segregating chromatids denotes number of chromatids at each pole . bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots , centromeres of laggard - producing chromosome as red dots in a side view along spindle equator . One of centromeres is left behind at anaphase ( blue arrowhead ) , but will segregate equationally . Tracks of centromeres are color - coded according to speed , as indicated by colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 μm .

O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

A time-lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. A lagging chromatid is indicated by a blue arrowhead. Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole. The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots, and centromeres of laggard-producing chromosome as red dots in a side view along the spindle equator. One of the centromeres is left behind at anaphase (blue arrowhead), but will segregate equationally. Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.


Chromosome parameters obtained for laggard - producing chromosomes are shown in A - D on vertical axis , time in min on horizontal axis relative to anaphase II onset . blue lines indicate 12 laggard - producing chromosomes with correct ( equational ) chromatid segregation ; darker highlights one chromosome with chromatid non - disjunction . On schemes near charts chromosomes are red with green centromeres ; spindle equator in A spindle axis in B - C are indicated by black dashed lines . Changes in to equator plane ( shown by a black arrowed on scheme ) for 13 laggard - producing chromosomes . laggard - producing chromosomes are positioned above equator at start of period . Black dotted lines indicate average thickness of metaphase plate . Changes in to spindle axis ( shown by a black arrowed on scheme ) for 13 laggard - producing chromosomes . Black dotted indicates average radius of metaphase plate . Changes in angle to spindle axis ( highlighted in black on scheme ) for 13 laggard - producing chromosomes . Dashed on chart indicates mean angle for normally segregating chromosomes . Changes in inter - centromere distances ( shown by black arrowed on scheme ) for 13 laggard - producing chromosomes . Dashed indicates mean inter - centromere for normally segregating chromosomes .

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

Chromosome parameters obtained for laggard-producing chromosomes are shown in A-D on the vertical axis, time in min on the horizontal axis relative to the anaphase II onset. Light blue lines indicate 12 laggard-producing chromosomes with correct (equational) chromatid segregation; darker line highlights one chromosome with chromatid non-disjunction. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by black dashed lines. Changes in the distance to the equator plane (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. All laggard-producing chromosomes are positioned above the equator at the start of analysis period. Black dotted lines indicate the average thickness of the metaphase plate. Changes in the distance to spindle axis (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. Black dotted line indicates the average radius of the metaphase plate. Changes in the angle to the spindle axis (highlighted in black on the scheme) for 13 laggard-producing chromosomes. Dashed line on the chart indicates the mean angle for normally segregating chromosomes. Changes in the inter-centromere distances (shown by black arrowed line on the scheme) for 13 laggard-producing chromosomes. Dashed line indicates the mean inter-centromere distance for normally segregating chromosomes.


( a ) RT - PCR of LC3 , IL - 8 , CHOP GADD34 in U2OS subjected to glutamine deprivation ( − Q ) or exposed to TPG ( 1 μM ) . (

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

(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). (


( b ) Immunoblot of ER stress - autophagy - related in U2OS subjected to glutamine deprivation or TPG treatment . Samples are run in triplicate .

O O O B-EXP_ASSAY O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

(b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate.


( c ) Cytokine array of conditioned media from U2OS A549 grown in presence ( + Q ) or absence ( − Q ) of glutamine .

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(c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine.


( d ) IL - 8 enzyme - linked immunosorbent of conditioned media from U2OS A549 grown in presence ( + Q ) or absence ( − Q ) of glutamine . Error bars in figures represent s . d . of three biological replicates .

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

(d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.


( a ) Autophagosomes were visualized in U2OS stably expressing a GFP - LC3 reporter construct . GFP - labelled were examined after 24 h in presence ( + Q ) or absence ( − Q ) of glutamine , with or without co - addition of 100 nM CCI - 779 ( CCI ) 400 nM BafA1 . Scale bar , 10 μm . ( b ) Graphical summary of experiments performed as described in a . of with > 10 per from three independent experiments is depicted . Bars represent mean ± s . d . from three independent experiments ( > 50 per experiment ) . statistical significance ( P value ) was determined by a two - , paired Student ' s t - . * P0 . 05 .

O O O B-SUBCELLULAR O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05.


( c ) Immunoblot of U2OS subjected to glutamine deprivation with or without 400 nM BafA1 . were pretreated with BafA1 for 1 h before during exposure to glutamine - deficient medium . Autophagic activity was monitored by detection of p62 LC3 - II .

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

(c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins.


d ) U2OS mCherry - GFP - LC3 were cultured in presence ( + Q ) or absence ( − Q ) of glutamine for 18 h . Red vesicles denote autolysosomes , whereas yellow vesicles represent autophagosomes . Bars indicate numbers of yellow vesicles ( autophagosomes ) or red vesicles ( autolysosomes ) per ± s . d . ( e ) of U2OS mCherry - GFP - LC3 cultured for 18 h in presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

O O B-CELL_LINE B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


( f ) Phagophore formation in mCherry - ULK1 . Scale bar , 10 μm . ( g ) mCherry - ATG5 - expressing U2OS after 24 h in presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

(f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


( a ) Colocalization of lysosomes ( Lysotracker Green ) with phagophores ( mCherry - ULK1 mCherry - glutamine ) in glutamine - deprived U2OS . Scale bar , 25 μm .

O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O

(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm.


( b ) Colocalization of LAMP1 - positive lysosomes with GFP - LC3 - labelled autophagosomes after glutamine deprivation in absence or presence of 400 nM BafA1 in U2OS . Scale bar , 15 μm .

O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O

(b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm.


( c ) Immunofluorescence of U2OS stained for p62 expression LAMP1 following glutamine deprivation BafA1 treatment . Nuclei were stained with DAPI . Scale bar , 15 μm .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O

(c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm.


( d ) Immunofluorescence of stained with LAMP1 ( lysosomes ) Golgi marker , RCAS1 , following glutamine deprivation . Nuclei were stained with DAPI . Scale bar , 15 μm .

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

(d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm.


( e ) U2OS were treated for 24 h with culture medium containing indicated . lysates were immunoblotted to detect changes in S6K phosphorylation p62 . (

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

(e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. (


( f ) Immunofluorescence for LAMP1 ( green ) mTOR ( red ) in U2OS subjected to glutamine deprivation ( 24 h ) , in presence of 100 nM WYE - 125132 ( WYE - 132 ) . − AA denotes deprived of . Scale bar , 15 μm . Error bars in figures represent s . d . of three biological replicates . Ess . , essential .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.


( a ) Immunoblot of autophagy - related in U2OS expressing a control TALEN ( U2OS - TAL2 - C1 ) or two independent subcloned TALEN lines targeting ATG7 ( U2OS - TAL2 - A7 , U2OS - TAL2 - A8 ) . p62 long short refer to either long or short exposure times during film development .

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

(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development.


( b ) U2OS expressing an ATG7 - TALEN ( TAL2 - ATG7 ) or control TALEN ( TAL2 - CTL ) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine - replete medium . were then stained with SRB absorbance was read at 540 nM . Graph shows average results from five clonal sub - lines for each condition . statistical significance ( P value ) was determined by a two - , paired Student ' s t - . P0 . 05 . ( c ) Representative of SRB - stained clones in

O O O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in b


d ) IL - 8 enzyme - linked immunosorbent ( ELISA ) of U2OS - TAL2 - CTL - C1 ( control ) U2OS - TAL2 - ATG7 - A8 ( ATG7 knockout ) following 24 h glutamine deprivation .

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

d) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.


( e ) IL - 8 U2OS of U2OS parental starved of glutamine for indicated times with or without BafA1 . Error bars in figures represent s . d . of three biological replicates . OD , optical density .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-CELL_LINE O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) immunofluorescence microscopy of U2OS stained for LAMP1 Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE - 125132 . Scale bar , 15 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.


( b ) Cytokine array of conditioned medium from U2OS subjected to glutamine deprivation in presence of 1 μM BrefA or 100 nM WYE - 125132 .

O O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.


( c ) IL - 8 enzyme - linked immunosorbent in U2OS after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 .

(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.


( d ) RT - PCR of IL - 8 mRNA expression in U2OS after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . Error bars in figures represent s . d . of three biological replicates . OD , optical density .

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

(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) Immunoblot of autophagy , ER stress - mTOR - related , JNK in U2OS after 24 h glutamine deprivation with or without CCI - 779 .

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

(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.


( b ) RT - PCR of IL - 8 mRNA expression in U2OS transfected with two different siRNAs targeting JNK ( JNK # 1 JNK # 2 ) subjected to 24 h glutamine deprivation .

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

(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.


( c ) IL - 8 enzyme - linked immunosorbent ( ELISA ) of conditioned media from experiment described in b .

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

(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.


( d ) Immunoblot of JNK expression in JNK siRNA - treated .

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

(d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells.


e ) RT - PCR of IL - 8 expression in U2OS after 24 h glutamine deprivation with or without JNK inhibitor SP600125 ( JNKi ) .

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

e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).


( f ) IL - 8 ELISA of conditioned medium from experiment described in e .

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

(f) IL-8 ELISA of conditioned medium from the experiment described in e.


( g ) IL - 8 ELISA of U2OS transfected with two different siRNAs targeting IRE1 ( IRE - A IRE - B ) subjected to glutamine deprivation for 24 h .

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

(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.


( h ) RT - PCR of IL - 8 mRNA expression from experiment described in g . ( i ) RT - PCR of CHOP mRNA expression from experiment described in g . ( j ) RT - PCR of IRE1 mRNA expression from experiment described in g .

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

(h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g.


( k ) Immunoblot of experiment described in g showing phosphorylation of JNK . Error bars in figures represent s . d . of three biological replicates . OD , optical density .

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

(k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) IL - 8 enzyme - linked immunosorbent ( ELISA ) on U2OS starved of glutamine or treated with BPTES ( 10 μM ) for 24 h .

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(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.


( b ) RT - PCR of IL - 8 CHOP mRNA expression in U2OS starved of glutamine for 24 h co - treated with DMαKG .

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(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.


( c ) Metabolite of U2OS starved of glutamine , treated with BPTES or glutamine - starved co - treated with DMαKG .

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(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.


( d ) IL - 8 ELISA of U2OS starved of glutamine or treated with BPTES for 24 h co - treated with DMαKG .

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(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.


( e ) Immunoblot of pS6K pJNK in U2OS starved of glutamine for 24 h co - treated with DMαKG . Error bars in figures represent s . d . of three biological replicates . OD , optical density .

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(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


Representative density plots of Ca2 + flux from conjugated OptoCAR ‑ T over time at 37ºC . Conjugated were stimulated with dimerizer addition for 0 ( F ) , 5 ( G ) , 10 ( H ) , 15 ( I ) or 30 ( J ) minutes prior to disrupting signaling by illuminating .

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Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.


OptoCAR ‑ T were conjugated with ligand - presenting activated as in upper regime shown in ( A ) . median of ppERK distribution was plotted with time , after subtraction of background . Bars show mean ± ( n = 3 ) . An equivalent experiment was performed as in ( B ) but following middle regime shown in ( A ) , with sample illumination after 5 minutes for remainder of dataset . Indicated half - life was calculated from time taken for median ppERK to decrease to 50 % of maximum . Bars show mean ± ( n = 4 ) . An equivalent experiment was performed as in ( B ) but following lower regime shown in ( A ) , with sample illumination after 5 minutes for 3 minutes . Bars show mean ± ( n = 3 ) .

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OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).


Representative Western blot showing dynamics of FOS phosphorylation ( p - FOSS32 ) on - modulated control . Conjugated OptoCAR ‑ T were stimulated for a defined period after dimerizer addition ( denoted above blots ) before either being left in ( ) or illuminated ( blue ) . Full blot image with total normalization control shown in Figure EV3A . Representative Western blot showing dynamics of FOS expression ( FOSTotal ) on - modulated control , from same dataset as in ( A ) . Full blot image with total normalization control shown in Figure EV3B .

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Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A. Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.


RT - qPCR of relative mRNA for four representative genes following OptoCAR - mediated T activation . OptoCAR ‑ T conjugates were activated in ( signaling competent ) state for a period of 3 hours before - mediated disruption of signaling ( open circles ) or maintained for a further 60 minutes in ( filled circles ) . relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3 - 4 hours . datasets are presented as gray lines , with bars showing mean ± ( n = 3 ) . Raw cRQ plots for datasets are provided in Appendix Fig S2 RQ values can be found in Dataset EV1 .

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RT-qPCR analysis of relative mRNA levels for four representative genes following OptoCAR-mediated T cell activation. OptoCAR‑T cell conjugates were activated in the dark (signaling competent) state for a period of 3 hours before light-mediated disruption of signaling (open circles) or maintained for a further 60 minutes in the dark (filled circles). The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3-4 hours. Individual datasets are presented as gray lines, with bars showing mean ± SEM (n=3). Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1.


Single pulses of varying duration were applied to OptoCAR ‑ T geometric mean of GFP expression was measured for lengths 24 hours after initiation of signaling , with illumination started once signal had ended . values have been corrected for background GFP from non - activated OptoCAR ‑ T shown relative to output at 24 hours . A representative dataset is provided in Figure EV4F . Bars show mean ± ( n = 3 ) Equivalent datasets as in ( B ) for geometric mean of CD69 upregulation . A representative dataset is provided in Figure EV4F . Bars show mean ± ( n = 3 )

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Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)


cRQ values from RT - qPCR of indicated . Total was extracted CD137 mRNA measured relative to that from HEK293T . Conjugated OptoCARCD19 ‑ T were also assayed either before or after 24 hours stimulation in state . values from three biological replicates are shown , with mean depicted by bar plot .

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The cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot.


Determination of maximum growth rate ( Vmax ) using a label - free - proliferation . Representative growth curves of WT NUP58 mutant clone F are plotted using a semi - log scale displaying total numbers of over time . Growth rates were calculated across entire duration of experiment using a sliding window of three timepoints .

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Determination of maximum growth rate (Vmax) using a label-free cell count-based proliferation assay. Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi-log scale displaying total numbers of cells over time. Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints.


Western blot for NUP58 expression in mutant clones ( B - E ) control ( clone A WT ) . Whole - lysate hybridized with customized antibody 1 ( Ab1 , top panel ) antibody 2 ( Ab2 , bottom panel ) . Actin was used as loading control .

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Western blot for NUP58 protein expression in mutant clones (B-E) and control cells (clone A and WT). Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Actin was used as loading control.


Representation of highest growth rates ( Vmax ) for each technical replicate for each indicated . Each dot represents a technical replicate ( WT n = 35 , C1 n = 27 , C2 n = 28 , C3 n = 34 , C4 n = 32 , C5 n = 35 , C6 n = 38 ) of one independent experimental replicate performed .

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Representation of the highest growth rates (Vmax) for each technical replicate for each indicated cell line. Each dot represents a technical replicate (WT n=35, C1 n=27, C2 n=28, C3 n=34, C4 n=32, C5 n=35, C6 n=38) of one independent experimental replicate performed.


Western blot for NUP58 expression in mutant clones control . Whole - lysate hybridized with customized antibody 1 ( Ab1 , top panel ) antibody 2 ( Ab2 , bottom panel ) . Blots were loaded with wild - type HAP1 ( WT ) , empty vector ( EV ) , non - targeting siRNA ( siNT ) NUP58 targeting siRNA ( siNUP58 ) , followed by NUP58 mutant clones ( C1 / C2 / C3 / C4 in left panels , C5 / C6 in right panels ) . Actin was used as loading control . Asterisks indicate possible NUP58 alternative isoforms .

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Western blot for NUP58 protein expression in mutant clones and control cells. Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Blots were loaded with wild-type HAP1 cell line (WT), empty vector (EV), non-targeting siRNA (siNT) and NUP58 targeting siRNA (siNUP58), followed by NUP58 mutant clones (C1 / C2 / C3 / C4 in left panels, C5 / C6 in right panels). Actin was used as loading control. Asterisks indicate possible NUP58 alternative isoforms.


viability expressed as number of viable colonies following Cas9 - induced gene editing of NUP58 in WT in a stably overexpressing KPNB1 - KPNA4 - eGFP ( KAPs ) .

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Cellular viability expressed as number of viable colonies following Cas9-induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1-KPNA4-eGFP (KAPs).


Improvement in active import upon passaging . Dots represent analysed ( WT P3 n = 16 , WT P19 n = 25 , C5 P1 n = 24 , C5 P3 n = 27 , C5 P19 n = 23 ) of one independent experimental replicate performed .

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Improvement in active nuclear import upon cell passaging. Dots represent analysed cells (WT P3 n=16, WT P19 n=25, C5 P1 n=24, C5 P3 n=27, C5 P19 n=23) of one independent experimental replicate performed.


NUP58 expression in C5 at P1 P19 . Blots were performed using Antibody 1 ( Ab1 , top ) or Antibody 2 ( Ab2 , bottom ) . Actin was used as loading control . band corresponding to full - NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk .

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NUP58 protein expression in C5 at P1 and P19. Blots were performed using Antibody 1 (Ab1, top) or Antibody 2 (Ab2, bottom). Actin was used as loading control. The band corresponding to the full-length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk.


viability following Cas9 - induced gene editing of native NUP58 ( left ) or NUP85 ( right ) in WT ( black circles ) in a overexpressing NUP58 mutant isoform ( grey circles ) .

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Cellular viability following Cas9-induced gene editing of native NUP58 (left) or NUP85 (right) in WT cell line (black circles) and in a cell line overexpressing NUP58 mutant isoform (grey circles).


Diagram at top represents G - banding pattern of chromosome 13 highlights chromosome locations of tested genes ( NUP58 , GRK1 , ADPRHL1 ) . Graph at bottom reports qPCR results for indicated genes preformed on gDNA of indicated samples at indicated passage number .

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Diagram at the top represents G-banding pattern of chromosome 13 and highlights chromosome locations of tested genes (NUP58, GRK1, ADPRHL1). Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number.


Representative of NUP58 gene - specific FISH hybridization for control HAP1 lines ( WT P3 - WT P19 ) mutant C5 at P19 . Each dot represents a NUP58 locus of cluster thereof . Scale bar , 31 . 3

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Representative images of NUP58 gene-specific FISH hybridization for control HAP1 cell lines (WT P3-WT P19) and mutant C5 at P19. Each dot represents a NUP58 locus of cluster thereof. Scale bar, 31.3 μm


A - O UAS . RNAi lines were driven with eyeless . Gal4gmr . Gal4 for expression during eye development or nubbin - Gal4 for expression during wing development . ( A , B ) Control adult Drosophilaeye ( A ) wing ( B ) . ( C , D ) α - spectrin RNAi results in overgrowth of eye ( C ) wing ( D ) . ( E , F ) βH - spectrin / karst RNAi results in overgrowth of eye ( E ) wing ( F ) . ( G , H ) β - spectrin RNAi does not affect eye ( G ) or wing ( H ) . ( I , J ) kibra RNAi results in overgrowth of eye ( I ) wing ( J ) . ( K , L ) α - spectrin , kibra double RNAi results in stronger overgrowth of eye ( K ) wing ( L ) . ( M , N ) βH - spectrin / wing , kibra double RNAi results in stronger overgrowth of eye ( M ) wing ( N ) . ( O ) of wing sizes by pixel , 5 wings per genotype were measured . Error bars show standard deviation . P - RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes . eyes mutant eyes ( Q ) overgrow slightly compared to controls ( P ) , while α - spectrin mutant eyes expressing α - spectrin RNAi ( R ) overgrow strongly compared to controls ( P ) . Data information : Scale bars , 250 μm .

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A-O UAS.RNAi lines were driven with eyeless.Gal4gmr.Gal4 for expression during eye development or nubbin-Gal4 for expression during wing development. (A, B) Control adult Drosophilaeye (A) and wing (B). (C, D) α-spectrin RNAi results in overgrowth of the eye (C) and wing (D). (E, F) βH-spectrin/karst RNAi results in overgrowth of the eye (E) and wing (F). (G, H) β-spectrin RNAi does not affect eye size (G) or wing size (H). (I, J) kibra RNAi results in overgrowth of the eye (I) and wing (J). (K, L) α-spectrin, kibra double RNAi results in stronger overgrowth of the eye (K) and wing (L). (M, N) βH-spectrin/wing, kibra double RNAi results in stronger overgrowth of the eye (M) and wing (N). (O) Quantification of female wing sizes by pixel area, 5 wings per genotype were measured. Error bars show standard deviation.P-RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes. eyes mutant eyes (Q) overgrow slightly compared to controls (P), while α-spectrin mutant eyes expressing α-spectrin RNAi (R) overgrow strongly compared to controls (P).Data information: Scale bars, 250 μm.


A - F eyeless FLP MARCM system was used to generate mutant eyes that also express UAS . RNAi targeting Spectrin cytoskeleton . Pupalretinas were examined at 42 - 46 h after puparium formation ( APF ) . Cellmembranes were marked with Dlg . ( A ) Control pupalretina showing cone surrounded by interommatidial . ( B ) kibra mutant pupalretina displaying additional interommatidial . ( C ) Pupalretina expressing α - spectrin RNAi showing additional interommatidial . ( D ) kibra mutant pupalretinas expressing α - spectrin RNAi showing many additional interommatidial . ( E , F ) Clones of α - spectrin mutant ( GFP negative ) ( E ) βH - spectrin / karst mutant ( GFP negative ) ( F ) show extra interommatidial . Scale bars , 20 μm .

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A-F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS.RNAi targeting the Spectrin cytoskeleton. Pupalretinas were examined at 42-46 h after puparium formation (APF). Cellmembranes were marked with Dlg staining. (A) Control pupalretina showing cone cells surrounded by interommatidial cells. (B) kibra mutant pupalretina displaying additional interommatidial cells. (C) Pupalretina expressing α-spectrin RNAi showing additional interommatidial cells. (D) kibra mutant pupalretinas expressing α-spectrin RNAi showing many additional interommatidial cells. (E, F) Clones of α-spectrin mutant cells (GFP negative) (E) and βH-spectrin/karst mutant cells (GFP negative) (F) show extra interommatidial cells. Scale bars, 20 μm.


G - K UAS . RNAi lines were driven with hh . Gal4 UAS . GFP for expression in posterior compartment contained ex . lacZ reporter transgene . ( G ) Control wing showing a normal ex . lacZ expression pattern , which is low at dorsal - ventral boundary but high in proximal regions of wing disc . ( H ) RNAi knock - down of α - Spectrin results in a mild up - regulation of ex . lacZ in posterior compartment . ( I ) RNAi knock - down of kibra results in a mild up - regulation of ex . lacZ in posterior compartment . ( J ) Dual RNAi knock - down of both α - spectrin kibra results in enhanced up - regulation of ex . lacZ in posterior compartment . ( K ) RNAi knock - down of hippo results in an up - regulation of ex . lacZ in posterior compartment . Scale bars , 50 μm .

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G-K UAS.RNAi lines were driven with hh.Gal4 UAS.GFP for expression in the posterior compartment and contained the ex.lacZ reporter transgene. (G) Control wing showing a normal ex.lacZ expression pattern, which is low at the dorsal-ventral boundary but high in the proximal regions of the wing disc. (H) RNAi knock-down of α-Spectrin results in a mild up-regulation of ex.lacZ in the posterior compartment. (I) RNAi knock-down of kibra results in a mild up-regulation of ex.lacZ in the posterior compartment. (J) Dual RNAi knock-down of both α-spectrin and kibra results in enhanced up-regulation of ex.lacZ in the posterior compartment. (K) RNAi knock-down of hippo results in an up-regulation of ex.lacZ in the posterior compartment. Scale bars, 50 μm.


A Co - IP of V5 - tagged Expanded with FLAG - tagged N - terminal region of Karst / βH - Spectrin , but not other Karst truncation constructs .

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A Co-IP of V5-tagged Expanded with the FLAG-tagged N-terminal region of Karst/βH-Spectrin, but not other Karst truncation constructs.


B , C Expanded co - localises with Karst at apical of wing imaginal disc ( apical view , B ; cross section , C ) .

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B, C Expanded co-localises with Karst at the apical membrane of wing imaginal disc cells (apical view, B; cross section, C).


D Control nubbin . Gal4 expressing wing . E Overexpression of Ex activates Hippo signalling to produce a small wing . F RNAi knock - down of α - Spectrin results in an overgrown wing . G Overexpression of Ex blocks effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I Overexpression of Ex blocks effect of βH - spectrin / karst RNAi .

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D Control nubbin.Gal4 expressing wing.E Overexpression of Ex activates Hippo signalling to produce a small wing.F RNAi knock-down of α-Spectrin results in an overgrown wing.G Overexpression of Ex blocks the effect of α-Spectrin RNAi.H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.I Overexpression of Ex blocks the effect of βH-spectrin/karst RNAi.


apical α - βHSpectrincytoskeleton may be mechanosensory in wing imaginal discA Schematic diagram of central compression circumferencial stretching in third instarwing pouch . B - D Third instarwing imaginal disc stained for beta - galactosidase expressed from expanded . lacZ reporter gene ( B ) , Crb ( C ) Kst - YFP ( D ) . of shown below . Note inverse correlation of Crb Kst - YFP with ex . lacZ . Scale bars , 50 μm . E , F Zoom of Kst - YFP under compression ( E ) under stretch ( F ) . Scale bars , 10 μm . G Schematic diagram of effect of force on apical spectrin cytoskeleton leading to declustering of Crumbs complexes reduced Hippo signalling .

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The apical α-βHSpectrincytoskeleton may be mechanosensory in the wing imaginal discA Schematic diagram of central compression and circumferencial stretching in the third instarwing pouch.B-D Third instarwing imaginal disc stained for beta-galactosidase expressed from the expanded.lacZ reporter gene (B), Crb (C) and Kst-YFP (D). Quantification of line intensity shown below. Note inverse correlation of Crb and Kst-YFP with ex.lacZ. Scale bars, 50 μm.E, F Zoom of Kst-YFP cells under compression (E) and under stretch (F). Scale bars, 10 μm.G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling.


B Control nub . Gal4 wing . C , D Expression of UAS . CrbExTM - GFP with Gal4 . Gal4 ( C ) or UAS . Wts with nub . Gal4 ( D ) results in a small wing . E RNAi knock - down of α - Spectrin results in an overgrown wing . F , G Expression of UAS . CrbExTM - GFP ( F ) or UAS . Wts ( G ) blocks effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I , J Expression of UAS . CrbExTM - GFP ( I ) or UAS . Wts ( J ) blocks effect of α - Spectrin RNAi . K , L RNAi knock - down of Wts results in a strongly overgrown wing ( K ) expression of UAS . CrbExTM - GFP does not block effect of wts RNAi ( L ) . M , N RNAi knock - down of Ajuba results in a small wing ( M ) , expression of UAS . CrbExTM - GFP enhances effect of ajuba RNAi ( N ) . O of various wing sizes . Error bars show standard deviation .

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B Control nub.Gal4 wing.C, D Expression of UAS.CrbExTM-GFP with Gal4.Gal4 (C) or UAS.Wts with nub.Gal4 (D) results in a small wing.E RNAi knock-down of α-Spectrin results in an overgrown wing.F, G Expression of UAS.CrbExTM-GFP (F) or UAS.Wts (G) blocks the effect of α-Spectrin RNAi.H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.I, J Expression of UAS.CrbExTM-GFP (I) or UAS.Wts (J) blocks the effect of α-Spectrin RNAi.K, L RNAi knock-down of Wts results in a strongly overgrown wing (K) and expression of UAS.CrbExTM-GFP does not block the effect of wts RNAi (L).M, N RNAi knock-down of Ajuba results in a small wing (M), and expression of UAS.CrbExTM-GFP enhances the effect of ajuba RNAi (N).O Quantification of various wing sizes. Error bars show standard deviation.


P , Q Localisation of Wts - GFP ( P ) E - cadherin ( Q ) in third instar wing imaginal disc .

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P, Q Localisation of Wts-GFP (P) and E-cadherin (Q) in the third instar wing imaginal disc.


R of in ( P ) ( Q ) . No increased Wts - GFP is observed in proximal regions where are subject to stretching .

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R Quantification of line intensity in (P) and (Q). No increased Wts-GFP intensity is observed in proximal regions where cells are subject to stretching.


A Control egg chamber stained for DAPI to mark nuclei . B Overexpression of Yki3SA stimulates follicle proliferation ( MARCM clone , GFP positive ) . C - F Mutation of βH - spectrin / karst ( C ) or crb ( D ) does not increase follicle proliferation ( GFP negative clone ) , while mutation of α - spectrin ( E ) or β - spectrin ( F ) stimulates follicle proliferation ( GFP negative clone ) .

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A Control egg chamber stained for DAPI to mark nuclei.B Overexpression of Yki3SA stimulates follicle cell proliferation (MARCM clone, GFP positive).C-F Mutation of βH-spectrin/karst (C) or crb (D) does not increase follicle cell proliferation (GFP negative clone), while mutation of α-spectrin (E) or β-spectrin (F) stimulates follicle cell proliferation (GFP negative clone).


G Control egg chamber stained for Cut , a marker of proliferating that is normally down - regulated after stage 6 of oogenesis . H - K Mutation of βH - spectrin / karst ( H ) or crb ( I ) does not increase Cut expression in follicle ( GFP negative clone ) , while mutation of α - spectrin ( J ) or β - spectrin ( K ) stimulates Cut expression in posterior follicle ( GFP negative clone ) . L Control egg chamber showing ex . lacZ reporter gene expression . M - P Mutation of βH - spectrin / karst ( M ) or crb ( N ) does not increase ex . lacZ expression in follicle ( GFP negative clone ) , while mutation of α - spectrin ( O ) or β - spectrin ( P ) stimulates ex . lacZ expression in posterior follicle ( GFP negative clone ) .

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G Control egg chamber stained for Cut, a marker of proliferating cells that is normally down-regulated after stage 6 of oogenesis.H-K Mutation of βH-spectrin/karst (H) or crb (I) does not increase Cut expression in follicle cells (GFP negative clone), while mutation of α-spectrin (J) or β-spectrin (K) stimulates Cut expression in posterior follicle cells (GFP negative clone).L Control egg chamber showing ex.lacZ reporter gene expression.M-P Mutation of βH-spectrin/karst (M) or crb (N) does not increase ex.lacZ expression in follicle cells (GFP negative clone), while mutation of α-spectrin (O) or β-spectrin (P) stimulates ex.lacZ expression in posterior follicle cells (GFP negative clone).


Q Wild - type follicle showing normal hexagonal packing of epithelial membranes . R Mutation of β - spectrin disrupts normal hexagonal packing , suggesting a role in maintaining tension . S Clone of β - spectrin mutant ( GFP negative ) showing differential tension at interface with wild - type ( GFP positive ) .

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Q Wild-type follicle cells showing normal hexagonal packing of epithelial membranes.R Mutation of β-spectrin disrupts the normal hexagonal packing, suggesting a role in maintaining membrane tension.S Clone of β-spectrin mutant cells (GFP negative) showing differential membrane tension at the interface with wild-type cells (GFP positive).


Basolateralα - β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A . Gal4UAS . GFP adult midgut stained for phospho - histone H3 to mark mitotic . B - E RNAi knock - down of βH - spectrin / karst ( B ) or crumbs ( C ) does not increase cellproliferation , while RNAi knock - down of β - spectrin ( D ) or α - spectrin ( E ) strongly stimulates cellproliferation . F Overexpression of Yki strongly stimulates cellproliferation . G of number of PH3 - positive mitotic . N > 13 samples . Statistical performed using two - Student ' s t - . Error bars show standard deviation . * * * P - value ≤ 0 . 001 . H - K A Hippo reporter DIAP1 - HRE - GFP is normally expressed in sporadic . Its expression is not affected by RNAi knock - down of βH - spectrin / karst ( I ) , but is up - regulated in many by RNAi knock - down of α - spectrin ( J ) or by overexpression of Yki ( K ) . Data information : Scale bars , 100 μm .

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Basolateralα-β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A.Gal4UAS.GFP adult midgut stained for phospho-histone H3 to mark mitotic stem cells.B-E RNAi knock-down of βH-spectrin/karst (B) or crumbs (C) does not increase stem cellproliferation, while RNAi knock-down of β-spectrin (D) or α-spectrin (E) strongly stimulates stem cellproliferation.F Overexpression of Yki strongly stimulates stem cellproliferation.G Quantification of the number of PH3-positive mitotic cells. N > 13 samples. Statistical analysis performed using the two-tailed Student's t-test. Error bars show standard deviation. ***P-value ≤ 0.001.H-K A Hippo reporter DIAP1-HRE-GFP is normally expressed in sporadic stem cells. Its expression is not affected by RNAi knock-down of βH-spectrin/karst (I), but is up-regulated in many stem cells by RNAi knock-down of α-spectrin (J) or by overexpression of Yki (K).Data information: Scale bars, 100 μm.


A Control Caco - 2 epithelial plated a low density show strong YAP localisation . B Control Caco - 2 epithelial plated at high density show relocalisation of YAP to cytoplasm . C , D siRNA knock - down of SPTAN1 ( C ) or SPTBN1 ( D ) prevents relocalisation of YAP to cytoplasm .

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A Control Caco-2 epithelial cells plated a low density show strong nuclear YAP localisation.B Control Caco-2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm.C, D siRNA knock-down of SPTAN1 (C) or SPTBN1 (D) prevents relocalisation of YAP to the cytoplasm.


E Quantification of YAP localisation in (B-D).


F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal of YAP phosphorylation , of LATS1 phosphorylation .

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F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation, and of LATS1 phosphorylation.


G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target .

G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins.


B . Immunoblot of parental wild - type ( WT ) UPF3B mutant lines showing of on right . In 3BΔ2BD , a smaller UPF3B with deletion of 20 - 155 ( UPF3BΔ20 - 155 ) is expressed . Relative Expression ( Rel Exp ) of this deletion as compared to full - UPF3B along with standard error of mean ( ) are indicated below lane 2 . UPF3B antibody recognizes antigen outside deleted region in 3BΔ2BD . HNRNPA1 is used as a loading control . ND = UPF3B not determined .

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B. Immunoblot of parental wild-type (WT) and UPF3B mutant cell lines showing levels of proteins on the right. In 3BΔ2BD, a smaller UPF3B protein with deletion of amino acids 20-155 (UPF3BΔ20-155) is expressed. Relative Expression (Rel Exp) of this deletion protein as compared to the full-length UPF3B along with standard error of mean (SEM) are indicated below lane 2. UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD. HNRNPA1 is used as a loading control. ND = UPF3B levels not determined.


C . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms PTC - isoforms from same set of genes . X - axis represents fold change in 3BΔ2BD versus WT each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown .

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C. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from the same set of genes. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.


D . Bar plots from isoform specific RT - qPCR showing average fold change ( y - axis ) of PTC + PTC - isoforms from genes indicated on bottom in WT two UPF3B mutant identified in legend on top right . For each isoform , in mutant are compared to in WT ( set to 1 ) .

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D. Bar plots from isoform specific RT-qPCR analysis showing average fold change (y-axis) of PTC+ and PTC- isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right. For each isoform, levels in mutant cells are compared to the levels in WT cells (set to 1).


E . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms NMD - isoforms . X - axis represents fold change in 3BΔ2BD versus WT each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown . F . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms PTC - isoforms from same set of genes . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown . G . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms NMD - isoforms . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown .

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E. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. F. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from same set of genes. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. G. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.


A . Immunoblots showing of on right in input or FLAG immunoprecipitates ( IP ) from WT UPF3B mutant expressing endogenously FLAG - tagged UPF1 as indicated above each lane . presence of RNase A during FLAG - IP is indicated above each lane .

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A. Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates (IP) from WT and UPF3B mutant cells expressing endogenously FLAG-tagged UPF1 protein as indicated above each lane. The presence of RNase A during FLAG-IP is indicated above each lane.


B . Immunoblots showing of ( right ) in input IP with normal rabbit IgG ( IgG - IP ) or antibody targeting EIF4A3 ( EIF4A3 - IP ) from WT UPF3B mutant .

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B. Immunoblots showing levels of proteins (right) in input and IP with normal rabbit IgG (IgG-IP) or antibody targeting EIF4A3 (EIF4A3-IP) from WT and UPF3B mutant cells.


C , D . CDF plots of ( C ) PTC + PTC - isoforms , ( D ) NMD + NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in WT . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot . E , F . CDF plots of ( E ) PTC + PTC - isoforms , ( F ) NMD + NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot . G , H . CDF plots of UPF3B - dependent ( G ) - independent ( H ) PTC + isoforms their respective PTC - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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C, D. CDF plots of (C) PTC+ and PTC-isoforms, (D) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in WT cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. E, F. CDF plots of (E) PTC+ and PTC-isoforms, (F) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. G, H. CDF plots of UPF3B-dependent (G) and -independent (H) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


I . CDF plots of UPF3B - dependent UPF3B - independent NMD + isoforms as compared to NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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I. CDF plots of UPF3B-dependent and UPF3B-independent NMD+ isoforms as compared to NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


J . Bar plot showing average fold change as measured by isoform specific RT - qPCR of PTC + PTC - isoform from genes indicated on bottom in WT two independent clones of 3AKO , 3BΔ2BD , 3BKO , 3DKO . Relative from each replicate are shown by circles . Error bars indicate standard errors of means .

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J. Bar plot showing average fold change as measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and two independent clones of 3AKO, 3BΔ2BD, 3BKO, and 3DKO cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means.


B . Immunoblot showing of EJC UPF ( on right ) in input or FLAG - IP samples from 3DKO # 2 stably expressing different FLAG - tagged indicated above each lane . D . Immunoblot showing EJC / UPF ( on right ) in input or FLAG - IP samples from 3DKO # 2 stably expressing FLAG - tagged given above each lane ( hUPF3B = human UPF3B ; hUPF3B - h3AC = human UPF3B with C - terminal domain from human UPF3A ; hUPF3B - m3AC = human UPF3B with C - terminal domain from mouse UPF3A ) .

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

B. Immunoblot showing levels of EJC and UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing different FLAG-tagged proteins indicated above each lane. D. Immunoblot showing EJC/UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing FLAG-tagged proteins given above each lane (hUPF3B = human UPF3B; hUPF3B-h3AC = human UPF3B protein with C-terminal domain from human UPF3A; hUPF3B-m3AC = human UPF3B protein with C-terminal domain from mouse UPF3A).


A . Northern blots showing of β - globin reporter mRNAs in wild - type HeLa Tet - off UPF3B knockout HeLa Tet - off . β39 is a tetracycline ( Tet ) - inducible reporter with a PTC at codon 39 whose are shown at different timepoints after transcriptional shut - off ( chase ) as indicated above each lane . β - GAP is a stable , constitutively - expressed , longer β - globin mRNA used as transfection control . overexpressed ( OE ) in each condition are indicated on top reporter mRNA half - lives ( t1 / 2 ) along with standard errors of means are on bottom .

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

A. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom.


C . Northern blot showing steady - state of β39 NMD reporter β - GAP control in HeLa Tet - off UPF3B knockout upon transient overexpression of wild - type UPF3 or different UPF3A chimeric indicated above each lane . Below each lane , relative fold - change ( Rel FC ) indicates β39 reporter ( normalized to β - GAP control ) as compared to normalized β39 reporter in UPF3B expressing .

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

C. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells.


D . Immunoblot showing of EJC or HNRNPA1 in input or EIF4A3 - IP from 3DKO # 2 stably expressing different UPF3 or EGFP as a control as indicated above each lane . Relative IP of FLAG - tagged are quantified against EIF4A3 .

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

D. Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3-IP from 3DKO#2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane. Relative IP of FLAG-tagged proteins are quantified against EIF4A3.


A , B . CDF plots of ( A ) PTC + PTC - isoforms , ( B ) NMD + NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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A, B. CDF plots of (A) PTC+ and PTC-isoforms, (B) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


D , E . CDF plots of UPF3A / 3B - independent ( UPF3A / 3B - indep . , D ) - dependent ( UPF3A / 3B - dep . , E ) PTC + isoforms their respective PTC - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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

D, E. CDF plots of UPF3A/3B-independent (UPF3A/3B-indep., D) and -dependent (UPF3A/3B-dep., E) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


F . CDF plots of UPF3A / 3B - dependent ( UPF3A / 3B - dep . ) , UPF3A / 3B - independent ( UPF3A / 3B - indep . ) NMD + isoforms NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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

F. CDF plots of UPF3A/3B-dependent (UPF3A/3B-dep.), UPF3A/3B-independent (UPF3A/3B-indep.) NMD+ isoforms and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


A , B . Western blots showing of EJC or HNRNPA1 ( control ) in input , IgG IP or EIF4A3 IP following overexpression ( OE ) of CASC3 wild - type ( WT ) EJC binding deficient ( HDAA ) mutant in ( A ) HeLa Tet - off , ( B ) 3BKO # 3 HeLa Tet - off . Ramps above lanes indicate expression of CASC3 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_LINE B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-CELL_LINE B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O

A, B. Western blots showing levels of EJC proteins or HNRNPA1 (control) in input, IgG IP or EIF4A3 IP following overexpression (OE) of CASC3 wild-type (WT) and EJC binding deficient (HDAA) mutant proteins in (A) HeLa Tet-off cells, and (B) 3BKO#3 HeLa Tet-off cells. Ramps above lanes indicate expression levels of the CASC3 proteins.


C . Western blots showing of EJC / UPF HNRNPA1 in input FLAG followed by EIF4A3 tandem IP from HCT116 expressing FLAG - tagged endogenous indicated above each lane . Quantifications of UPF3B CASC3 enrichment from two replicates are shown at bottom .

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C. Western blots showing levels of EJC/UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG-tagged endogenous protein indicated above each lane. Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom.


D . Meta - exon plot showing read distributions within 100 nucleotide ( nt ) window from exon 3 ′ end in RIPiT - Seq replicates of MAGOH : EIF4A3 , UPF3B : EIF4A3 , CASC3 : EIF4A3 . black vertical indicates - 24 nt position .

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D. Meta-exon plot showing read distributions within the 100 nucleotide (nt) window from the exon 3′ end in RIPiT-Seq replicates of MAGOH:EIF4A3, UPF3B:EIF4A3, and CASC3:EIF4A3. The black vertical line indicates the -24 nt position.


F . Scatter plot comparing log2 - transformed fold change in occupancy of CASC3 : EIF4A3 EJC as compared to MAGOH : EIF4A3 EJC ( x - axis ) UPF3B : EIF4A3 EJC compared to MAGOH : EIF4A3 EJC . Each dot represents a gene where gene - level occupancy of each EJC composition was quantified at canonical position for EJC footprints . Pearson correlation coefficient is shown on top left .

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

F. Scatter plot comparing log2-transformed fold change in occupancy of CASC3:EIF4A3 EJC as compared to MAGOH:EIF4A3 EJC (x-axis) and UPF3B:EIF4A3 EJC compared to MAGOH:EIF4A3 EJC. Each dot represents a gene where gene-level occupancy of each EJC composition was quantified at the canonical position for EJC footprints. Pearson correlation coefficient is shown on the top left.


G . Box plots comparing occupancy of a specific EJC composition on mRNA ( indicated on y - axis ; normalized by gene expression from - Seq ) between genes of different NMD efficiency . Up , no , down stands for upregulation , no obvious change or downregulation of a representative PTC + isoform for each gene ( see material methods ) that is used to reflect overall NMD efficiency for each gene . In box plots , central band represents median , boxes represent interquartile range ( IQR ) , whiskers indicate maximum ( top ) minimum ( bottom ) values ( 1 . 5 - times highest or lowest IQR values , respectively ) , circles represent outliers . p - values are from Wilcoxon signed - rank comparing two indicated distributions .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plots comparing occupancy of a specific EJC composition on mRNA (indicated on y-axis; normalized by gene expression from RNA-Seq) between genes of different NMD efficiency. Up, no, and down stands for upregulation, no obvious change or downregulation of a representative PTC+ isoform for each gene (see material and methods) that is used to reflect overall NMD efficiency for each gene. In the box plots, central band represents the median, boxes represent the interquartile range (IQR), whiskers indicate maximum (top) and minimum (bottom) values (1.5-times the highest or lowest IQR values, respectively), and circles represent the outliers. p-values are from Wilcoxon signed-rank test comparing the two indicated distributions.


H . Box plots as in G comparing specific EJC composition occupancy relative to EJC occupancy ( normalized by MAGOH : EIF4A3 RIPiT - Seq ) between genes of different NMD efficiency . Up , no , down , central band , boxes , whiskers are as in G .

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

H. Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy (normalized by MAGOH:EIF4A3 RIPiT-Seq) between genes of different NMD efficiency. Up, no, down, central band, boxes, and whiskers are as in G.


I . Bar plots showing fold changes measured by isoform specific RT - qPCR of PTC + PTC - isoform from genes indicated on bottom in WT CASC3 - KO HCT116 . Relative from each replicate are shown by circles . Error bars indicate standard error of means .

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

I. Bar plots showing fold changes measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and CASC3-KO HCT116 cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard error of means.


viability using CellTiter - Glo of BN175 treated with indicated agents for 24 hrs 48 hrs ( n = 4 biological replicates ) . viability values are displayed as a of relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) . SM represents SM - 164 . Error bars represent SD statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

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Cell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


TNF - induced - II immuno - precipitation . BN175 were treated with indicated agents for 8 hrs . FADD immuno - precipitation was performed followed by western blot ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) .

B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O

TNF-induced complex-II immuno-precipitation. BN175 cells were treated with the indicated agents for 8 hrs. FADD immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).


Proximity ligation between Ripk1 Casp - 8 performed in BN175 upon treatment with indicated agents for 8 hrs ( n = 3 biological replicates , shown are representative ) . Scale bar represents 10 μm . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) .

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Proximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs (n = 3 biological replicates, shown are representative images). Scale bar represents 10 μm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).


DEVDase activity of BN175 treated with indicated drugs for 6 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) . Error bars represent SD statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

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

DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


viability using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 Casp - 8 knockouts ( KO ) treated with indicated agents for 18 48 hrs ( n = 3 biological replicates ) . viability values are displayed as a of relative untreated control . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed to compare mean value of each treatment to treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

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

Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


TNF - induced - II immuno - precipitation . HT1080 were treated with indicated agents for 8 hrs . CASP - 8 immuno - precipitation was performed followed by western blot ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) .

B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O

TNF-induced complex-II immuno-precipitation. HT1080 cells were treated with the indicated agents for 8 hrs. CASP-8 immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml).


measured by Celigo of PI - positive HT1080 , were treated with indicated agents for 24 hrs . ( n = 3 biological replicates ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD an unpaired t - was performed , * * * * P ≤ 0 . 0001 .

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

Cell death analysis measured by Celigo of PI-positive HT1080 cells, cells were treated with the indicated agents for 24 hrs. (n = 3 biological replicates). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and an unpaired t-test was performed, **** P ≤ 0.0001.


by Celigo of PI - positive HT1080 treated with indicated agents for 72 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed , * * * * P ≤ 0 . 0001 .

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Cell death analysis by Celigo of PI-positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed, **** P ≤ 0.0001.


TNF - induced - II immuno - precipitation . A375 were treated with indicated agents for 6 hrs . Caspase - 8 immuno - precipitation was performed followed by western blot . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) ( n = 3 biological replicates , shown is a representative experiment ) .

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TNF-induced complex-II immuno-precipitation. A375 cells were treated with the indicated agents for 6 hrs. Caspase-8 immuno-precipitation was performed followed by western blot analysis. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) (n = 3 biological replicates, shown is a representative experiment).


by Celigo of PI - positive A375 treated with indicated agents for 12 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

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

Cell death analysis by Celigo of PI-positive A375 treated with the indicated agents for 12 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


viability using CellTiter - Glo of A375 treated with indicated drugs for 24 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48hrs ( n = 3 biological replicates ) . viability values are displayed as a of relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed to compare mean value of each treatment to treated A375 siCTRL , * * * * P ≤ 0 . 0001 .

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

Cell viability analysis using CellTiter-Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL, **** P ≤ 0.0001.