( A ) Expression of RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 4 )

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(A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)


( B ) Phospho - RagC positively regulates wing compartment size in Drosophila . Over - expression ( OE ) of dRagC WT , 3A , or 3E in wing posterior compartment using en - Gal4 driver and the posterior compartment sizes were measured in each genotype ( ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Bonferroni multiple comparisons , n ≥ 63 flies per condition )

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(B) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition)


( C ) Empty vector ( EV ) , RagC WT , 3A or 3E were transiently expressed in stable RagC knockdown HEK - 293E cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 2 for C2 and n = 4 for other conditions )

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(C) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions)


( D ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Lysates were analyzed by Western blot for phosphorylation of ULK1 and total

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(D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1


( E ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . Lysates were analyzed by Western blot for levels of LC3 - II and 14 - 3 - 3 . Graphs show mean ± S . E . M . of quantitative analyses of LC3 - II blotting after normalization for 14 - 3 - 3 . * p < 0 . 05 , two tailed Student ' s t test , n = 4

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(E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4


( F ) GFP - LC3 was co - expressed with empty vector , RagC WT , 3A or 3E in HEK - 293E cells followed 48 hr later by serum deprivation for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . LC3 and RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm

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(F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( G ) Quantitative analysis of GFP puncta ( F ) ( * * * * p < 0 . 0001 , two tailed Student ' s t test ; n ≥ 21 ) . Mean value is shown as a horizontal bar

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(G) Quantitative analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar


( A ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were transiently expressed in HEK - 293E cells . RagC and lysosome maker , Lamp1 , were monitored by confocal fluorescence microscopy . Scale bar , 10 μm

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(A) Empty vector (EV), HA-RagC WT, 3A or 3E were transiently expressed in HEK-293E cells. RagC and lysosome maker, Lamp1, were monitored by confocal fluorescence microscopy. Scale bar, 10 μm


( B ) Empty vector ( EV ) , HA - RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK - 293E cells . mTOR and HA - RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm

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(B) Empty vector (EV), HA-RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK-293E cells. mTOR and HA-RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( C ) Empty vector ( EV ) , Flag - RagC WT , 3A or 3E were transiently expressed with HA - RagA in HEK - 293E cells . Total cell lysates and amounts of mTOR , Raptor and HA - RagA in the Flag - RagC immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * p < 0 . 05 , two tailed Student ' s t test , n ≥ 3 )

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(C) Empty vector (EV), Flag-RagC WT, 3A or 3E were transiently expressed with HA-RagA in HEK-293E cells. Total cell lysates and amounts of mTOR, Raptor and HA-RagA in the Flag-RagC immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (* p<0.05, two tailed Student's t test, n ≥ 3)


( D ) Expression of HA - RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells . Total cell lysates ( TCL ) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * * * p < 0 . 001 , Student ' s t test , n = 3 )

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(D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3)


Figure 1 . Identification of non - canonical p53 binding sites in human subtelomeres . ( A and B ) ChIP - seq tracks for p53 in camptothecin ( CPT ) treated or untreated HCT116 cells were aligned with RNAPII , CTCF , RAD21 , and SMC1 for human chromosome18q ( A ) or 13q ( B ) subtelomeres . Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top . Subtelomeric DNA paralogy segment tracks , internal telomere - like sequence tracks ( red CCCTAA ) , and annotated RefSeq gene tracks are indicated above the ChIP - seq tracks . ( C ) Alignment of human subtelomeric elements containing non - canonical p53 recognition sequences . The position of the RepeatMasker - defined LTR10c element is shown ; also note the ( TG ) n microsatellite repeat adjacent to the p53 binding site . ( D ) Schematic organization of non - canonical p53 binding sites found in human subtelomeres .

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Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG)n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.


Stress - induced p53 binding at human subtelomeres . ( A ) Western blot of HCT116 p53 - / - and p53 + / + cells treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hrs . Cell lysates were assayed for total p53 , phospho - Ser15 p53 ( p - p53 ) , γH2AX , or GAPDH .

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Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH.


( B ) ChIP - qPCR was assayed in HCT116 p53 - / - or p53 + / + cells treated with DMSO or etoposide as in panel A , and assayed by ChIP with antibodies to p53 ( Ab6 ) or control IgG , followed by qPCR for p53 binding site in subtelomere 18q ( top panel ) or 13q ( lower panel ) .

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(B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel).


( C ) HCT116 p53 - / - and p53 + / + cells were cultured in DMEM with 10 % FBS or Hank ' s buffered saline solution ( HBSS ) lacking FBS and then assayed by ChIP with either IgG control ( grey ) or two different p53 - specific antibodies Ab6 ( red ) or FL ( black ) . ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q , 13q , or p21 promoter .

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(C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank's buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter.


. ( D ) Doxycyclin ( Dox ) - inducible p53 H1299 cells were treated without ( - ) or with ( + ) Dox in media containing 10 % serum or in serum free HBSS , and then subject to ChIP assay with control IgG or p53 - specific Ab6 antibody . ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 ( top panel ) , p53 - negative control IGX1A ( middle panel ) , or p21 positive control ( lower panel ) . Error bars represent SD , and * indicates p value < . 05 . p - values were determined by Student t - test .

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. (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.


. Transcriptional activity of subtelomeric p53 - response elements . ( A ) Luciferase test constructs containing either empty test vector ( top ) , p53 - response elements from 18q subtelomere ( middle ) or MDM2 promoter ( lower ) were assayed in HCT p53 - / - cells transfected with two independent preparations of empty vector or p53 WT expression vector .

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. Transcriptional activity of subtelomeric p53-response elements. (A) Luciferase test constructs containing either empty test vector (top), p53-response elements from 18q subtelomere (middle) or MDM2 promoter (lower) were assayed in HCT p53-/- cells transfected with two independent preparations of empty vector or p53 WT expression vector.


( B ) RT - qPCR measure of p21 ( top panel ) or PARD6G ( lower panel ) in HCT116 p53 - / - or p53 + / + cells after treatment with DMSO or 50 µM etoposide .

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(B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide.


( C ) RT - qPCR analysis of TERRA transcripts from subtelomere 13q ( upper ) or 2q ( lower ) .

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(C) RT-qPCR analysis of TERRA transcripts from subtelomere 13q (upper) or 2q (lower).


( D ) RT - qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 ( top panel ) or pr2 ( lower panel ) from HCT116 p53 + / + cells cultured with serum ( grey bars ) or in HBSS lacking serum ( black bars ) . Error bars represent SD and * indicates p value < . 05 using student t - test comparing treated and untreated samples .

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(D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.


. p53 - dependent cell viability and telomere protection in response to DNA damage . ( A ) Western blot of total p53 , γH2AX , and GAPDH in HCT116 p53 - / - or p53 + / + cells treated with 1 µM or 50 µM etoposide for 24 hrs .

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. p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs.


s . ( B ) Cell cycle analysis of HCT116 p53 - / - or p53 + / + cells untreated or treated with 50 µM etoposide for 24 hrs . Cell cycle was determined by FACS analysis of propidium iodide stained cells .

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s. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells.


( C ) Cell clonal survival assay for HCT116 p53 - / - or p53 + / + cells treated with DMSO or 1 , 1 . 5 or 2 µM etoposide for 24 hr prior to replating .

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(C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating.


. ( D ) Quantification of clonal survival assay relative total cell numbers ( top panel ) or normalized to DMSO controls ( lower panel ) .

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. (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel).


E ) TIF assay for HCT116 p53 + / + or p53 - / - cells treated with 1 µM etoposide for 3 hrs . Telomere DNA FISH with TelC probe ( red ) , γH2AX ( green ) , Dapi ( blue ) .

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E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue).


( F ) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53 + / + ( black ) or p53 - / - ( red ) cell .

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(F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell.


G ) Telomere length assay showing p53 - / - or p53 + / + cells treated with either DMS0 , 1 or 50 µM etoposide . Ethidium bromide ( EtBr ) stained agarose gels ( left panel ) , Southern blot hybridized with Tel C probe ( middle panel ) or with α Satellite DNA promote ( right panel ) . Quantification of telomereDNA signal relative to invariant bands in α Sat . Error bars represent SD and * indicates p values < . 05 determined by student t - test ( TLA ) , or chi - square ( TIF assay ) .

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G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomereDNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay).


( H ) Two - dimensional agarose gel analysis of telomeric DNA from HCT p53 + / + or or p53 - / - treated with 50 µM etoposide for 24 hrs . Ethidium gel ( top ) and Southern blot probed with TelG probe ( lower panel ) . Single stranded telomere DNA ( red arrow ) and recombination / replication - associated structures ( blue arrow ) are indicated .

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(H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.


Figure 5 . p53 - dependent histone modification changes in response to DNA damage stress . HCT116p53 - / - ( left panels ) or p53 + / + ( right panels ) were treated with DMSO ( black ) or 50 µM etoposide ( red ) for 24 hrs and assayed by ChIP - qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere ( panel A ) or 13qsubtelomere ( panel B ) or for control regions ( p21 promoter , GKN1 / 2 , PARKIN ) as indicated below . ChIP - for p53 , H3K9Ac , H3K27Ac , γH2AX , or IgG control were quantified as % input . Error bars indicates SD and * indicates p value < . 05 using student t - test comparing ChIP values in p53 + / + relative to p53 - / - cells for specific primer pairs .

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Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere (panel A) or 13qsubtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP- for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs.


( C ) Dot blot analysis of telomere repeat or Alu repeat DNA from p53 + / + or p53 - / - HCT116 cells treated with DMSO or etoposide ( ETP ) ( as described in panel A ) , that were subject to ChIP with antibodies to IgG , p53 , γH2AX , TRF2 , or TRF1 . Quantitation of three independent ChIP - assays for TRF1 and TRF2 is shown in the panel to the right . (

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(C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. (


( E ) ChIP - qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q ( left ) or 13q ( right ) .

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(E) ChIP-qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q (left) or 13q (right).


( A ) HCT116 p53 + / + or p53 - / - cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53 , γH2AX , H3K9Ac , panH3 , and GAPDH at times 0 , 8 , 16 , or 24 hr post - treatment .

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

(A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment.


( B ) ChIP - qPCR for γH2AX ( upper panels ) or IgG control ( lower panels ) for cells treated as indicated in panel A . ( C - D ) RT - qPCR analysis for cells treated as shown in panel A .

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(B) ChIP-qPCR for γH2AX (upper panels) or IgG control (lower panels) for cells treated as indicated in panel A. (C-D) RT-qPCR analysis for cells treated as shown in panel A.


C - D ) RT - qPCR analysis for cells treated as shown in panel A . ( C ) RT - PCR for the p21 ( top ) , or PARD6G mRNA ( lower ) . ( D ) RT - PCR for primer pairs spanning subtelomere18q as indicated below . Lower panel shows the No - RT control for 18qsubtelomere primers . RT - qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value < . 05 using student t - test comparing etoposide ( 24 hr ) relative to untreated for specific primer pairs .

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C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere18q as indicated below. Lower panel shows the No-RT control for 18qsubtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs.


( E ) Meta - data analysis of published GRO - Seq and ChIP Seq data sets . Gro - Seq Minus ( red / maroon ) or Plus ( purple / pink ) RNA strand from Control or Nutlin - 3a treated MCF7 cells was mapped to human 18q subtelomere . Histone H3K27ac , H3K4me1 , and H3K4me3ChIP - Seq data sets from untreated MCF7 are also shown for these subtelomeric regions . Right panel shows a zoom in of approximately 1000bp ( light blue on left panel ) surrounding the p53 response element within the subtelomere .

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

(E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere.


( F ) Summary of GRO - Seq and ChIP - Seq metadata analyses from human subtelomeres with p53 binding sites . Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself .

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

(F) Summary of GRO-Seq and ChIP-Seq metadata analyses from human subtelomeres with p53 binding sites. Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself.


( B ) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hr . Blots were probed with pan - p53 , phospho - p53 , γH2AX , or GAPDH ( as loading control )

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

(B) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO (D) or 50 µM etoposide (E) for 24 hr. Blots were probed with pan-p53, phospho-p53, γH2AX, or GAPDH (as loading control)


( C - E ) RT - qPCR for p21 ( C ) , PARDG6 ( D ) , or 18q subtelomeric RNA ( E ) in 18q WT or18q Δp53 treated with DMSO ( black ) or 50 μM etoposide ( red ) for 24 hr . ( F ) ChIP - qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B - E .

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

(C-E) RT-qPCR for p21 (C), PARDG6 (D), or 18q subtelomeric RNA (E) in 18q WT or18q Δp53 treated with DMSO (black) or 50 μM etoposide (red) for 24 hr. (F) ChIP-qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B-E.


A Double immunofluorescence for GFP ( green ) and the BP marker TBR2 ( yellow ) , combined with DAPI staining ( white ) , of a 57 days - old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and TBR2 + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells that are TBR2 + in 57 days - old chimpanzee cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 9 control and 9 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * * , P < 0 . 001 ( two - sided Student ' s t - test ) .

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

A Double immunofluorescence for GFP (green) and the BP marker TBR2 (yellow), combined with DAPI staining (white), of a 57 days-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells that are TBR2+ in 57 days-old chimpanzee cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 9 control and 9 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).


C Triple immunofluorescence for GFP ( green ) , the cycling cell marker Ki67 ( magenta ) and TBR2 ( yellow ) , combined with DAPI staining ( white ) , of a 59 days - old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + , Ki67 + and TBR2 + triple - positive cells . Scale bar , 50 µ D Quantification of the proportion of GFP + cells in the SVZ / NL that are Ki67 + and TBR2 + double - positive in 59 days - old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 8 control and 8 ARHGAP11B - transfected cerebral organoids of four independent batches each ; error bars indicate SD ; * , P < 0 . 05 ( one - sided Wilcoxon rank sum test ) .

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

C Triple immunofluorescence for GFP (green), the cycling cell marker Ki67 (magenta) and TBR2 (yellow), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+, Ki67+ and TBR2+ triple-positive cells. Scale bar, 50 µ D Quantification of the proportion of GFP+ cells in the SVZ/NL that are Ki67+ and TBR2+ double-positive in 59 days-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 8 control and 8 ARHGAP11B-transfected cerebral organoids of four independent batches each; error bars indicate SD; *, P <0.05 (one-sided Wilcoxon rank sum test).


A Double immunofluorescence for GFP ( green ) and the radial glia marker HOPX ( yellow ) , combined with DAPI staining ( white ) , of a 59 days - old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and HOPX + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells in the SVZ / NL that are HOPX + in 59 days - old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 10 control and 10 ARHGAP11B - transfected cerebral organoids of three independent batches each ; error bars indicate SD ; * * , P < 0 . 01 ( one - sided Wilcoxon rank sum test ) . C Double immunofluorescence for GFP ( green ) and HOPX ( yellow ) , combined with DAPI staining ( white ) , of a 61 days - old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and HOPX + double - positive cells . Scale bar , 50 µm . D Quantification of the proportion of GFP + cells in the SVZ / NL that are HOPX + in 61 days - old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 5 control and 7 ARHGAP11B - transfected cerebral organoids of three independent batches each ; error bars indicate SD ; * , P < 0 . 05 ( two - sided Student ' s t - test ) .

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

A Double immunofluorescence for GFP (green) and the radial glia marker HOPX (yellow), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL that are HOPX+ in 59 days-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids of three independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test). C Double immunofluorescence for GFP (green) and HOPX (yellow), combined with DAPI staining (white), of a 61 days-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are HOPX+ in 61 days-old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 5 control and 7 ARHGAP11B-transfected cerebral organoids of three independent batches each; error bars indicate SD; *, P<0.05 (two-sided Student's t-test).


A Double immunofluorescence for GFP ( green ) and the neuron marker Hu ( magenta ) , combined with DAPI staining ( white ) , of a 61 days - old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and Hu + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells that are Hu + in 61 days - old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 7 control and 11 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * , P < 0 . 01 ( two - sided Student ' s t - test ) .

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 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 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 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 B-CONTROLLED_VAR 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

A Double immunofluorescence for GFP (green) and the neuron marker Hu (magenta), combined with DAPI staining (white), of a 61 days-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and Hu+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells that are Hu+ in 61 days-old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 7 control and 11 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (two-sided Student's t-test).


C Double immunofluorescence for GFP ( green ) and the deep - layer neuron marker CTIP2 ( magenta ) , combined with DAPI staining ( white ) , of a 59 days - old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and CTIP2 + double - positive cells . Scale bar , 50 µm . D Quantification of the proportion of GFP + cells in the SVZ / NL that are CTIP2 + in 59 - days old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 10 control and 10 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * * , P < 0 . 001 ( two - sided Student ' s t - test ) .

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

C Double immunofluorescence for GFP (green) and the deep-layer neuron marker CTIP2 (magenta), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and CTIP2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are CTIP2+ in 59-days old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).


E Double immunofluorescence for GFP ( green ) and the upper - layer neuron marker SATB2 ( magenta ) , combined with DAPI staining ( white ) , of a 66 days - old chimpanzee cerebral organoid 15 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and SATB2 + double - positive cells . Scale bar , 50 µm . F Quantification of the proportion of GFP + cells in the SVZ / NL that are SATB2 + in 66 days - old chimpanzee cerebral organoids 15 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 6 control and 4 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * , P < 0 . 01 ( one - sided Wilcoxon rank sum test ) .

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

E Double immunofluorescence for GFP (green) and the upper-layer neuron marker SATB2 (magenta), combined with DAPI staining (white), of a 66 days-old chimpanzee cerebral organoid 15 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and SATB2+ double-positive cells. Scale bar, 50 µm. F Quantification of the proportion of GFP+ cells in the SVZ/NL that are SATB2+ in 66 days-old chimpanzee cerebral organoids 15 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 6 control and 4 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test).


A Double immunofluorescence for GFP ( green ) and the thymidine analog BrdU ( magenta , combined with DAPI staining ( white ) , of 57 days - old human ( top panels ) and chimpanzee ( bottom panels ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( upper panels ) or ARHGAP11A220 expression plasmid ( lower panels ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and BrdU + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells in the SVZ / NL that are BrdU + in 57 days - old human ( blue ) and chimpanzee ( red ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( dark blue and dark red ) or ARHGAP11A220 expression plasmid ( light blue and light red ) . Data are the mean of 12 human control , 12 human ARHGAP11A220 - transfected , 9 chimpanzee control and 8 chimpanzee ARHGAP11A220 - transfected cerebral organoids ( grown from one human and one chimpanzee iPSC line , respectively ) of six ( human ) and four ( chimpanzee ) independent batches each ; error bars indicate SD ; n . s . , not significant ; * * , P < 0 . 01 ( Kruskal Wallis test ) .

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

A Double immunofluorescence for GFP (green) and the thymidine analog BrdU (magenta, combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and BrdU+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL that are BrdU+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 12 human control, 12 human ARHGAP11A220-transfected, 9 chimpanzee control and 8 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of six (human) and four (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; **, P <0.01 (Kruskal Wallis test).


C Double immunofluorescence for GFP ( green ) and TBR2 ( yellow ) , combined with DAPI staining ( white ) , of 57 days - old human ( top panels ) and chimpanzee ( bottom panels ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( upper panels ) or ARHGAP11A220 expression plasmid ( lower panels ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and TBR2 + double - positive cells . Scale bar , 50 µm . D Quantification of the proportion of GFP + cells in the SVZ / NL that are TBR2 + in 57 days - old human ( blue ) and chimpanzee ( red ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( dark blue and dark red ) or ARHGAP11A220 expression plasmid ( light blue and light red ) . Data are the mean of 9 human control , 9 human ARHGAP11A220 - transfected , 10 chimpanzee control and 10 chimpanzee ARHGAP11A220 - transfected cerebral organoids ( grown from one human and one chimpanzee iPSC line , respectively ) of four ( human ) and three ( chimpanzee ) independent batches each ; error bars indicate SD ; n . s . , not significant ; * , P < 0 . 05 ( Kruskal Wallis test ) .

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C Double immunofluorescence for GFP (green) and TBR2 (yellow), combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are TBR2+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 9 human control, 9 human ARHGAP11A220-transfected, 10 chimpanzee control and 10 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of four (human) and three (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05 (Kruskal Wallis test).


A Double immunofluorescence for GFP ( green ) and the bRG marker PTPRZ1 ( magenta ) , combined with DAPI staining ( white ) , of 60 days - old human forebrain organoids , generated from ARHGAP11A plus ARHGAP11B double - knockout clone 16 . Images were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids ( ARHGAP11A + B - rescue , referred to as Control ) , ARHGAP11B expression plasmid only ( ARHGAP11B - rescue , referred to as ARHGAP11A - KO ) , or ARHGAP11A expression plasmid only ( ARHGAP11A - rescue , referred to as ARHGAP11B - KO ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and PTPRZ1 + double - positive cells . Scale bar , 50 µm B Quantification of the proportion of GFP + cells in the SVZ / NL that are PTPRZ1 + in 60 days - old ARHGAP11A plus ARHGAP11B double - knockout ( 11A + 11B KO ) human forebrain organoids . Organoids were generated from clone 16 ( left ) and clone j ( right ) , and data were obtained 10 days after the indicated electroporation with GFP expression plasmid plus either ARHGAP11A ( 11A ) plus ARHGAP11B ( 11B ) expression plasmids ( referred to as Control , as this constitutes an ARHGAP11A + B double - rescue in the 11A + 11B KO organoids ; dark blue ) , ARHGAP11B ( 11B ) expression plasmid only ( referred to as 11A - KO , as this constitutes a selective ARHGAP11B rescue in the 11A + 11B KO organoids ; light blue ) , or ARHGAP11A ( 11A ) expression plasmid only ( referred to as 11B - KO , as this constitues a selective ARHGAP11A rescue in the 11A + 11B KO organoids ; dark red ) . For clone 16 , data are the mean of 8 control , 8 11A - KO and 8 11B - KO human forebrain organoids of two independent batches each ; for clone j , data are the mean of 9 control , 10 11A - KO and 8 11B - KO human forebrain organoids of three independent batches each ; error bars indicate SD ; n . s . , not significant ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ( Kruskal Wallis test )

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A Double immunofluorescence for GFP (green) and the bRG marker PTPRZ1 (magenta), combined with DAPI staining (white), of 60 days-old human forebrain organoids, generated from ARHGAP11A plus ARHGAP11B double-knockout clone 16. Images were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids (ARHGAP11A+B-rescue, referred to as Control), ARHGAP11B expression plasmid only (ARHGAP11B-rescue, referred to as ARHGAP11A-KO), or ARHGAP11A expression plasmid only (ARHGAP11A-rescue, referred to as ARHGAP11B-KO). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and PTPRZ1+ double-positive cells. Scale bar, 50 µm B Quantification of the proportion of GFP+ cells in the SVZ/NL that are PTPRZ1+ in 60 days-old ARHGAP11A plus ARHGAP11B double-knockout (11A+11B KO) human forebrain organoids. Organoids were generated from clone 16 (left) and clone j (right), and data were obtained 10 days after the indicated electroporation with GFP expression plasmid plus either ARHGAP11A (11A) plus ARHGAP11B (11B) expression plasmids (referred to as Control, as this constitutes an ARHGAP11A+B double-rescue in the 11A+11B KO organoids; dark blue), ARHGAP11B (11B) expression plasmid only (referred to as 11A-KO, as this constitutes a selective ARHGAP11B rescue in the 11A+11B KO organoids; light blue), or ARHGAP11A (11A) expression plasmid only (referred to as 11B-KO, as this constitues a selective ARHGAP11A rescue in the 11A+11B KO organoids; dark red). For clone 16, data are the mean of 8 control, 8 11A-KO and 8 11B-KO human forebrain organoids of two independent batches each; for clone j, data are the mean of 9 control, 10 11A-KO and 8 11B-KO human forebrain organoids of three independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05; **, P <0.01; ***, P <0.001 (Kruskal Wallis test)


( A - F ) Atg1 ‐ induced myosin II activation depends on the kinase activity of Atg1 . Third ‐ instar wing imaginal discs from ptc ‐ GAL4 UAS ‐ GFP controls or flies expressing indicated transgenes were stained with phospho ‐ MRLC ( blue ) and TRITC ‐ labelled phalloidin ( red ) . Low level of phospho ‐ MRLC staining was observed in controls ( A ) and cells overexpressing kinase ‐ deficient Atg1 ‐ KR ( C ) , whereas a robust increase in phospho ‐ MRLC was found in cells overexpressing Atg1 ( B ) . Co ‐ expression of Atg1 and spaghetti ‐ squash ( A20A21 ) exhibited low level of phospho ‐ MRLC staining ( D ) . Atg1 ‐ induced high level of phospho ‐ MRLC staining was not suppressed by expression of caspase inhibitor p35 ( E ) or by depletion of Atg12 ( Atg12RNAi ) ( F ) . Bar , 20 μm .

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(A-F) Atg1‐induced myosin II activation depends on the kinase activity of Atg1. Third‐instar wing imaginal discs from ptc‐GAL4 UAS‐GFP controls or flies expressing indicated transgenes were stained with phospho‐MRLC (blue) and TRITC‐labelled phalloidin (red). Low level of phospho‐MRLC staining was observed in controls (A) and cells overexpressing kinase‐deficient Atg1‐KR (C), whereas a robust increase in phospho‐MRLC was found in cells overexpressing Atg1 (B). Co‐expression of Atg1 and spaghetti‐squash (A20A21) exhibited low level of phospho‐MRLC staining (D). Atg1‐induced high level of phospho‐MRLC staining was not suppressed by expression of caspase inhibitor p35 (E) or by depletion of Atg12 (Atg12RNAi) (F). Bar, 20 μm.


( H ) Sqa , but not Atg1 , directly phosphorylated Sqh in vitro . Flag - Atg1 , Flag - Atg1 ‐ KR , HA - Sqa and HA - Sqa ‐ KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [ γ ‐ 32P ] ATP and bacterially expressed recombinant wild ‐ type Sqh or SqhA20A21 . As shown on the autoradiogram ( top panel ) , wild ‐ type but not the kinase ‐ deficient Atg1 ( Atg1 ‐ KR ) and Sqa ( Sqa ‐ KA ) was autophosphorylated . No phosphorylation was seen with SqhA20A21 . The equal input of His ‐ fusion proteins is shown on the Coomassie staining . Anti ‐ Flag and anti ‐ HA immunoblottings ( IBs ) were used as controls to quantify the amount of proteins precipitated .

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(H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag-Atg1, Flag-Atg1‐KR, HA-Sqa and HA-Sqa‐KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ‐32P]ATP and bacterially expressed recombinant wild‐type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild‐type but not the kinase‐deficient Atg1 (Atg1‐KR) and Sqa (Sqa‐KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His‐fusion proteins is shown on the Coomassie staining. Anti‐Flag and anti‐HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated.


( A - K ) Genetic interactions between Atg1 , Sqa , and spaghetti ‐ squash ( Sqh ) . Compared with the ptc ‐ GAL4 controls ( A ) , expression of Atg1 or Sqa by ptc ‐ GAL4 resulted in missing anterior cross ‐ vein phenotypes ( B , C ) . However , RNAi ‐ mediated downregulation of Atg1 ( Atg1RNAi ) or Sqa ( SqaRNAi ) did not cause wing vein defects ( D , E ) . Depletion of Atg1 and Sqa suppressed Atg1 and Sqa ‐ induced wing vein defects , respectively ( F , I ) . Atg1 ‐ induced wing defects were modulated by depletion of Sqa ( G ) or by co ‐ expression of SqhA20A21 ( H ) . Nevertheless , Sqa ‐ induced wing vein defects were suppressed by co ‐ expression of SqhA20A21 ( J ) but not Atg1RNAi ( K ) .

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(A-K) Genetic interactions between Atg1, Sqa, and spaghetti‐squash (Sqh). Compared with the ptc‐GAL4 controls (A), expression of Atg1 or Sqa by ptc‐GAL4 resulted in missing anterior cross‐vein phenotypes (B, C). However, RNAi‐mediated downregulation of Atg1 (Atg1RNAi) or Sqa (SqaRNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa‐induced wing vein defects, respectively (F, I). Atg1‐induced wing defects were modulated by depletion of Sqa (G) or by co‐expression of SqhA20A21 (H). Nevertheless, Sqa‐induced wing vein defects were suppressed by co‐expression of SqhA20A21 (J) but not Atg1RNAi (K).


( M ) 293T cells were transfected with HA ‐ tagged Sqa WT ( wild ‐ type ) or KA , together with Flag ‐ tagged Atg1 WT or KR . The cells were lysed 48 h after transfection and immunoprecipitated ( IP ) with anti ‐ Flag antibodies . The immunoprecipitated proteins and the total cell lysates ( TCL ) were analysed by immunoblotting ( IB ) with antibodies as indicated .

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(M) 293T cells were transfected with HA‐tagged Sqa WT (wild‐type) or KA, together with Flag‐tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti‐Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated.


( N ) 293T cells transfected with Flag - Atg1 ‐ KR and various HA ‐ tagged Sqa contracts were subjected to immunoprecipitations with anti ‐ HA antibody . The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated .

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(N) 293T cells transfected with Flag-Atg1‐KR and various HA‐tagged Sqa contracts were subjected to immunoprecipitations with anti‐HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated.


( O ) Atg1 directly phosphorylated Sqa in vitro . Flag ‐ tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa ‐ K1 , Sqa ‐ K2 , and Sqa ‐ C in an in vitro kinase assay . The lower panels represent equal input of His ‐ fusion proteins and Atg1 immunoprecipitates .

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(O) Atg1 directly phosphorylated Sqa in vitro. Flag‐tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa‐K1, Sqa‐K2, and Sqa‐C in an in vitro kinase assay. The lower panels represent equal input of His‐fusion proteins and Atg1 immunoprecipitates.


( A ) Characterization of Atg1 ‐ dependent phosphorylation sites on Sqa . 293T cells transfected with Flag ‐ tagged Atg1 or Atg1 ‐ KR were subjected to immunoprecipitation with anti ‐ Flag antibodies , followed by in vitro kinase assays with bacterially expressed Sqa ‐ K2 , Sqa ‐ K2 ‐ T194A , Sqa ‐ K2 ‐ T239A , and Sqa ‐ K2 ‐ T279A as substrates . Atg1 but not Atg1 ‐ KR was autophosphorylated ( top panel ) . Relative phosphorylation levels of substrates were quantified . Data are represented as mean ± s . e . of triplicates . ( B - D ) Phosphorylation of Sqa at Thr ‐ 279 is critical for the kinase activity of Sqa .

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(A) Characterization of Atg1‐dependent phosphorylation sites on Sqa. 293T cells transfected with Flag‐tagged Atg1 or Atg1‐KR were subjected to immunoprecipitation with anti‐Flag antibodies, followed by in vitro kinase assays with bacterially expressed Sqa‐K2, Sqa‐K2‐T194A, Sqa‐K2‐T239A, and Sqa‐K2‐T279A as substrates. Atg1 but not Atg1‐KR was autophosphorylated (top panel). Relative phosphorylation levels of substrates were quantified. Data are represented as mean±s.e. of triplicates. (B-D) Phosphorylation of Sqa at Thr‐279 is critical for the kinase activity of Sqa.


( B ) HA ‐ tagged Sqa or Sqa ‐ T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti ‐ squash ( Sqh ) WT and A20A21 , in an in vitro kinase assay .

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(B) HA‐tagged Sqa or Sqa‐T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti‐squash (Sqh) WT and A20A21, in an in vitro kinase assay.


( C ) Clonal expression of Sqa but not Sqa ‐ T279A ( GFP ‐ positive cells ) in the larval wing imaginal discs resulted in a marked increase in phospho ‐ MRLC staining ( blue ) and actin reorganization ( red ) . Bar , 20 μm .

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(C) Clonal expression of Sqa but not Sqa‐T279A (GFP‐positive cells) in the larval wing imaginal discs resulted in a marked increase in phospho‐MRLC staining (blue) and actin reorganization (red). Bar, 20 μm.


( D ) The larval fat body of denoted genotypes were dissected , lysed , and subjected to western blot analysis using anti ‐ phospho ‐ MRLC and anti ‐ Sqh antibodies . For quantification , the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh

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(D) The larval fat body of denoted genotypes were dissected, lysed, and subjected to western blot analysis using anti‐phospho‐MRLC and anti‐Sqh antibodies. For quantification, the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh levels


( A , B ) Activation of myosin II on nutrient deprivation . The larval fat body of denoted genotypes under fed or starved conditions were dissected , lysed , and subjected to western blot analysis using antibodies specific for phospho ‐ myosin regulatory light chain ( MRLC ) and total MRLC . The Rheb , SqaRNAi , Sqa ‐ T279A , spaghetti ‐ squash ( SqhA20A21 ) , and Atg7RNAi transgenes were expressed under the control of hs - GAL4 driver ( B ) . For quantification , the relative phosphorylation levels of MRLC were quantified as in Figure 3D . Data are represented as mean ± s . e . of triplicates .

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(A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho‐myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa‐T279A, spaghetti‐squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs-GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates.


( C - J ) Starvation ‐ induced autophagosome formation was compromised by inhibition of myosin II activation . Compared with the fed condition ( C ) , starvation induced a robust formation of GFP - Atg8a puncta in larval fat ‐ body cells ( D ) . Clonal expression of Atg12RNAi ( E ) , Sqa ‐ T279A ( F ) , SqaRNAi ( G ) , or SqhA20A21 ( H ) markedly suppressed GFP - Atg8a puncta formation during starvation . The autophagic defects caused by Sqa ‐ T279A and SqaRNAi were rescued by co ‐ expression of the constitutively active SqhE20E21 ( I ) and SqhD20D21 ( J ) , respectively . Bar , 20 μm .

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(C-J) Starvation‐induced autophagosome formation was compromised by inhibition of myosin II activation. Compared with the fed condition (C), starvation induced a robust formation of GFP-Atg8a puncta in larval fat‐body cells (D). Clonal expression of Atg12RNAi (E), Sqa‐T279A (F), SqaRNAi (G), or SqhA20A21 (H) markedly suppressed GFP-Atg8a puncta formation during starvation. The autophagic defects caused by Sqa‐T279A and SqaRNAi were rescued by co‐expression of the constitutively active SqhE20E21 (I) and SqhD20D21 (J), respectively. Bar, 20 μm.


( K ) The average number of GFP - Atg8a marked autophagosomes per cell is shown ( data are represented as mean ± s . e . of 20 fat ‐ body samples imaged per genotype ; * * * P0 . 001 ) .

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(K) The average number of GFP-Atg8a marked autophagosomes per cell is shown (data are represented as mean±s.e. of 20 fat‐body samples imaged per genotype; ***P0.001).


( L ) Inhibition of myosin II activity increased sensitivity to starvation . Flies carrying transgenes under the control of fat body ‐ specific Cg - GAL4 driver were used for further analysis . Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions . Data are mean ± s . e . from triplicate experiments ( n = 100 flies / genotype / treatment ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . See Supplementary data for genotypes .

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(L) Inhibition of myosin II activity increased sensitivity to starvation. Flies carrying transgenes under the control of fat body‐specific Cg-GAL4 driver were used for further analysis. Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions. Data are mean±s.e. from triplicate experiments (n=100 flies/genotype/treatment). *P0.05, **P0.01, ***P0.001. See Supplementary data for genotypes.


( A ) Ulk1 interacted with ZIPK . 293T cells transfected with HA ‐ tagged ZIPK together with Flag ‐ tagged Ulk1 or Ulk1 ‐ KI were subjected to immunoprecipitations with anti ‐ Flag antibody . The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated .

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(A) Ulk1 interacted with ZIPK. 293T cells transfected with HA‐tagged ZIPK together with Flag‐tagged Ulk1 or Ulk1‐KI were subjected to immunoprecipitations with anti‐Flag antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated.


( B ) HA ‐ tagged kinase ‐ inactive ZIPK ‐ KA ( K42A ) was expressed in 293T cells with Flag ‐ tagged Ulk1 or Ulk1 ‐ KI . Cell lysates were incubated with or without alkaline phosphatase ( CIP ) and analysed by immunoblotting with antibodies as indicated .

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(B) HA‐tagged kinase‐inactive ZIPK‐KA (K42A) was expressed in 293T cells with Flag‐tagged Ulk1 or Ulk1‐KI. Cell lysates were incubated with or without alkaline phosphatase (CIP) and analysed by immunoblotting with antibodies as indicated.


( C ) Myosin II was activated upon nutrient deprivation in MCF7 cells . MCF7 cells were cultured in serum containing DMEM medium ( nutrient ‐ rich condition ) or in Earle ' s balanced salt solution ( EBSS ; starved condition ) for 2 h . The myosin II activity was significantly downregulated after EBSS ‐ starved cells were replenished with fresh DMEM medium containing 10 % FBS for 2 h . The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls . Each value represents mean ± s . e . of triplicates .

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(C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates.


( D ) MCF7 cells stably infected with lentivirus expressing control ( shLuc ) , Ulk1 or ZIPK shRNA were cultured in nutrient ‐ rich DMEM medium ( F ) or EBSS ( S ) for 2 h . Effects of Ulk1 and ZIPK knockdown on starvation ‐ induced myosin II activation were analysed by immunoblotting with antibodies as indicated . Data are mean ± s . e . of triplicates .

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(D) MCF7 cells stably infected with lentivirus expressing control (shLuc), Ulk1 or ZIPK shRNA were cultured in nutrient‐rich DMEM medium (F) or EBSS (S) for 2 h. Effects of Ulk1 and ZIPK knockdown on starvation‐induced myosin II activation were analysed by immunoblotting with antibodies as indicated. Data are mean±s.e. of triplicates.


( A ) MCF7 / GFP - LC3 cells stably infected with lentivirus expressing control ( shLuc ) , ZIPK , or non ‐ muscle myosin heavy chain ‐ IIA ( NMHC ‐ IIA ) shRNA were cultured in nutrient ‐ rich medium ( DMEM ) or starvation medium ( Earle ' s balanced salt solution ; EBSS ) in the presence or absence of lysosomal inhibitor bafilomycin A1 ( BafA1 ) for 2 h . Depletion of ZIPK and NMHC ‐ IIA markedly inhibited starvation ‐ induced GFP - LC3 puncta formation . Quantification of the number of GFP - LC3 dots per cell ( lower panel ) was shown ( data are represented as mean ± s . e . of 100 cells , * * * P0 . 001 ) .

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(A) MCF7/GFP-LC3 cells stably infected with lentivirus expressing control (shLuc), ZIPK, or non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) shRNA were cultured in nutrient‐rich medium (DMEM) or starvation medium (Earle's balanced salt solution; EBSS) in the presence or absence of lysosomal inhibitor bafilomycin A1 (BafA1) for 2 h. Depletion of ZIPK and NMHC‐IIA markedly inhibited starvation‐induced GFP-LC3 puncta formation. Quantification of the number of GFP-LC3 dots per cell (lower panel) was shown (data are represented as mean±s.e. of 100 cells, ***P0.001).


( B ) Cells as in ( A ) were cultured in nutrient ‐ rich medium ( F ) or EBSS ( S ) with or without BafA1 for 2 h . Effects of ZIPK and NMHC ‐ IIA knockdown on starvation ‐ induced GFP - LC3 conversion were assessed by immunoblotting with anti ‐ LC3 , anti ‐ ZIPK , anti ‐ NMHC ‐ IIA , and anti ‐ tubulin antibodies . The relative ratio of LC3II / LC3I is shown at the right panel . Data are mean ± s . e . of triplicates .

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(B) Cells as in (A) were cultured in nutrient‐rich medium (F) or EBSS (S) with or without BafA1 for 2 h. Effects of ZIPK and NMHC‐IIA knockdown on starvation‐induced GFP-LC3 conversion were assessed by immunoblotting with anti‐LC3, anti‐ZIPK, anti‐NMHC‐IIA, and anti‐tubulin antibodies. The relative ratio of LC3II/LC3I is shown at the right panel. Data are mean±s.e. of triplicates.


( A ) The fat body of spaghetti ‐ squash ( sqhAX3 ) ; sqh - GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis . Sqh - GFP and phospho ‐ myosin regulatory light chain ( MRLC ) were enriched in cell - cell junction under nutrient ‐ rich ( fed ) condition . Under starvation conditions , Sqh - GFP and phospho ‐ MRLC were localized to both cell - cell junction and perinuclear region . Actin was stained with TRITC ‐ labelled phalloidin ( red ) and nucleus was stained with DAPI ( blue ) . Bar , 20 μm .

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(A) The fat body of spaghetti‐squash (sqhAX3); sqh-GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh-GFP and phospho‐myosin regulatory light chain (MRLC) were enriched in cell-cell junction under nutrient‐rich (fed) condition. Under starvation conditions, Sqh-GFP and phospho‐MRLC were localized to both cell-cell junction and perinuclear region. Actin was stained with TRITC‐labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm.


( B ) Starvation induced redistribution of myosin II in MCF7 / GFP - LC3 cells . Both phospho ‐ MRLC and MRLC were localized in peripheral region of cells in nutrient ‐ rich medium ( DMEM ) , whereas they redistributed to the perinuclear region and co ‐ localized with GFP - LC3 under starvation conditions ( Earle ' s balanced salt solution ; EBSS ) . Bar , 10 μm .

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(B) Starvation induced redistribution of myosin II in MCF7/GFP-LC3 cells. Both phospho‐MRLC and MRLC were localized in peripheral region of cells in nutrient‐rich medium (DMEM), whereas they redistributed to the perinuclear region and co‐localized with GFP-LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm.


( C ) MCF7 / GFP - LC3 cells cultured in nutrient ‐ rich ( DMEM ) or starved ( EBSS ) conditions were homogenized and subjected to centrifugation , and the resulting post ‐ nuclear supernatant ( PNS ) was fractionated by high ‐ speed centrifugation into membrane pellet and cytosol . Proteins were resolved by SDS - PAGE and immunoblotted with anti ‐ TGN46 antibody as a control for membrane ‐ association proteins , anti ‐ caspase ‐ 3 as a control for cytosolic proteins . The levels of phospho ‐ MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS ( n = 3 ) . Each value represents mean ± s . e . of three experiments . * P0 . 05 , * * P0 . 01 .

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(C) MCF7/GFP-LC3 cells cultured in nutrient‐rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by high‐speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS-PAGE and immunoblotted with anti‐TGN46 antibody as a control for membrane‐association proteins, anti‐caspase‐3 as a control for cytosolic proteins. The levels of phospho‐MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. *P0.05, **P0.01.


( A ) Immunofluorescence analysis of GFP - mAtg9 and phospho ‐ myosin regulatory light chain ( MRLC ) in MCF7 / GFP - mAtg9 cells . GFP - mAtg9 was enriched in the trans ‐ Golgi network ( TGN ) labelled with anti ‐ TGN46 antibody and phospho ‐ MRLC localized to cell peripheral in nutrient ‐ rich condition ( DMEM ) . GFP - mAtg9 redistributed and co ‐ localized with phospho ‐ MRLC in starvation condition ( Earle ' s balanced salt solution ; EBSS ) .

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(A) Immunofluorescence analysis of GFP-mAtg9 and phospho‐myosin regulatory light chain (MRLC) in MCF7/GFP-mAtg9 cells. GFP-mAtg9 was enriched in the trans‐Golgi network (TGN) labelled with anti‐TGN46 antibody and phospho‐MRLC localized to cell peripheral in nutrient‐rich condition (DMEM). GFP-mAtg9 redistributed and co‐localized with phospho‐MRLC in starvation condition (Earle's balanced salt solution; EBSS).


( B ) mAtg9 interacted with non ‐ muscle myosin heavy chain ‐ IIA ( NMHC ‐ IIA ) under starvation conditions . 293T cells ( right panel ) or 293T cells stably infected with lentivirus expressing control ( shLuc ) , UNC ‐ 51 ‐ like kinases ( Ulk1 ) or zipper ‐ interacting protein kinase ( ZIPK ) shRNA ( left panel ) were transfected with V5 ‐ tagged mAtg9 and GFP - tagged NMHC ‐ IIA . At 48 h after transfection , cells were incubated in either serum ‐ containing medium ( DMEM ) or EBSS for 2 h with or without the treatment of 50 μM ML ‐ 7 and blebbistatin ( Blebb ) . Cell lysates were immunoprecipitated with anti ‐ V5 antibody and immunoblotted with indicated antibodies .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mAtg9 interacted with non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) under starvation conditions. 293T cells (right panel) or 293T cells stably infected with lentivirus expressing control (shLuc), UNC‐51‐like kinases (Ulk1) or zipper‐interacting protein kinase (ZIPK) shRNA (left panel) were transfected with V5‐tagged mAtg9 and GFP-tagged NMHC‐IIA. At 48 h after transfection, cells were incubated in either serum‐containing medium (DMEM) or EBSS for 2 h with or without the treatment of 50 μM ML‐7 and blebbistatin (Blebb). Cell lysates were immunoprecipitated with anti‐V5 antibody and immunoblotted with indicated antibodies.


( C ) MCF7 / GFP - mAtg9 cells were stably infected with lentivirus expressing control ( shLuc ) , ZIPK , or NMHC ‐ IIA shRNA . Under starved conditions , mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells ( shLuc ) . Starvation ‐ induced GFP - mAtg9 redistribution was blocked in ZIPK and NMHC ‐ IIA knockdown cells . The images were analysed by quantifying mAtg9 localization in cells for either a TGN ‐ enriched or dispersed pattern . Data are represented as mean ± s . e . of 70 cells . * P0 . 05 , * * P0 . 01 . Bar , 10 μm .

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(C) MCF7/GFP-mAtg9 cells were stably infected with lentivirus expressing control (shLuc), ZIPK, or NMHC‐IIA shRNA. Under starved conditions, mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells (shLuc). Starvation‐induced GFP-mAtg9 redistribution was blocked in ZIPK and NMHC‐IIA knockdown cells. The images were analysed by quantifying mAtg9 localization in cells for either a TGN‐enriched or dispersed pattern. Data are represented as mean±s.e. of 70 cells. *P0.05, **P0.01. Bar, 10 μm.


( A ) Four fetuses ( named NW - 1 , NW - 2 , NW - 3 and NW - 4 ) with different eye morphology were retrieved at E44 by caesarean

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(A) Four fetuses (named NW-1, NW-2, NW-3 and NW-4) with different eye morphology were retrieved at E44 by caesarean section


( B ) DraI digestion of PCR products from the multiple organs of the four fetuses ( Upper ) . NC , negative control with no genomic DNA loaded . Mut , MITFL247S / L247S genomic DNA loaded . WT , LW genomic DNA loaded . Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis ( Bottom ) . Mut , MITFL247S / L247S genomic DNA loaded . WT , LW genomic DNA

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(B) DraI digestion of PCR products from the multiple organs of the four fetuses (Upper). NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (Bottom). Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded


( A ) Representative microscopic appearances of the retina and RPE cells ( arrowhead ) of a chimeric , a WT and a Mut fetus at the same gestational age by H & E staining . WT , Bama WT fetus . Mut , Bama MITFL247S / L247S fetus . NW - 2 , the chimeric fetus . Scale bars , 100

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(A) Representative microscopic appearances of the retina and RPE cells (arrowhead) of a chimeric, a WT and a Mut fetus at the same gestational age by H&E staining. WT, Bama WT fetus. Mut, Bama MITFL247S/L247S fetus. NW-2, the chimeric fetus. Scale bars, 100 μm


( B ) Representative immunofluorescence images showed the expression of multiple RPE markers Pax6 , MITF and Bestrophin in the chimeric fetuses . Blue , Hochst3342 . WT , Bama WT fetus as positive control . NW - 2 , the chimeric fetus . MITFL247S / L247S fetus as negative control . Scale bars , 50

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(B) Representative immunofluorescence images showed the expression of multiple RPE markers Pax6, MITF and Bestrophin in the chimeric fetuses. Blue, Hochst3342. WT, Bama WT fetus as positive control. NW-2, the chimeric fetus. MITFL247S/L247S fetus as negative control. Scale bars, 50 μm


( A ) Representative light microscopic images show that the in vitro cultured RPE cells exhibited a hexagonal shape . D3 , D5 and D11 , cultured for 3 days , 5 days and 11 days , respectively . Scale bars , 100

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(A) Representative light microscopic images show that the in vitro cultured RPE cells exhibited a hexagonal shape. D3, D5 and D11, cultured for 3 days, 5 days and 11 days, respectively. Scale bars, 100 μm


Representative immunofluorescence images showed the expression of RPE - specific markers , including RPE65 ( B in the cultured RPE cells . Scale bars , 50

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Representative immunofluorescence images showed the expression of RPE-specific markers, including RPE65 (B in the cultured RPE cells. Scale bars, 50 μm


Representative immunofluorescence images showed the expression of RPE - specific markers , includin ZO - 1 ( C ) and Bestrophin ( D ) , respectively , in the cultured RPE cells . Scale bars , 50

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Representative immunofluorescence images showed the expression of RPE-specific markers, includin ZO-1 (C) and Bestrophin (D), respectively, in the cultured RPE cells. Scale bars, 50 μm


( E ) Semi - quantitative RT - PCR analysis of RPE functional markers in cultured porcine RPE cells . GAPDH was used to confirm the cDNA quality of all the

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(E) Semi-quantitative RT-PCR analysis of RPE functional markers in cultured porcine RPE cells. GAPDH was used to confirm the cDNA quality of all the samples


( F ) Western blotting identified three RPE markers in porcine RPE cells . GAPDH served as a housekeeping gene

(F) Western blotting identified three RPE markers in porcine RPE cells. GAPDH served as a housekeeping gene control


( G ) ELISA identified secreted VEGF and PEDF in chimeric and WT porcine RPE cells . Media were collected at 48 hours after the cells were seeded onto 12 - well cell culture plates to be used for analyses . The results are representative of three independent experiments and were evaluated using a two - tailed t - test . No significant differences were identified . Data are presented as the mean ± standard error mean ( SEM ) . WT , RPE cells cultured from the NW - 8 fetus . CH , RPE cells cultured from chimeric NW - 7

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(G) ELISA identified secreted VEGF and PEDF in chimeric and WT porcine RPE cells. Media were collected at 48 hours after the cells were seeded onto 12-well cell culture plates to be used for analyses. The results are representative of three independent experiments and were evaluated using a two-tailed t-test. No significant differences were identified. Data are presented as the mean ± standard error mean (SEM). WT, RPE cells cultured from the NW-8 fetus. CH, RPE cells cultured from chimeric NW-7 fetus


( A ) The full - term chimera ( NW - 16 ) piglet was born and showed normal development , size and morphology of the

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(A) The full-term chimera (NW-16) piglet was born and showed normal development, size and morphology of the eyes


( B ) Digestion of the MITF PCR products in multiple organs of the full - term chimeric pig . NC , negative control with no genomic DNA loaded . Mut , MITFL247S / L247S genomic DNA loaded . WT , Bama GFP - labeled genomic DNA loaded . In the chimera piglet , GFP - specific primers were used to further confirm the chimeric contribution to multiple organs in the piglet . GAPDH was used to confirm the DNA quality of all the samples . NC , negative control with no genomic DNA loaded . Mut , Bama MITFL247S / L247S genomic DNA loaded . WT , Bama GFP - labeled genomic DNA

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(B) Digestion of the MITF PCR products in multiple organs of the full-term chimeric pig. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded. In the chimera piglet, GFP-specific primers were used to further confirm the chimeric contribution to multiple organs in the piglet. GAPDH was used to confirm the DNA quality of all the samples. NC, negative control with no genomic DNA loaded. Mut, Bama MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded


( C ) Representative microscopic appearances of the corneal epithelial cells of the chimeric piglet staining by H & E . Scale bars , 100

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(C) Representative microscopic appearances of the corneal epithelial cells of the chimeric piglet staining by H&E. Scale bars, 100 μm


( D ) Representative microscopic appearances of the retina cells of the chimeric piglet staining by H & E . Scale bars , 100

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(D) Representative microscopic appearances of the retina cells of the chimeric piglet staining by H&E. Scale bars, 100 μm


( A ) Representative GFP - labeled blastomeres that contributed to the chimeric porcine RPE cells are shown with immunofluorescence staining . Scale bars , 25

(A) Representative GFP-labeled blastomeres that contributed to the chimeric porcine RPE cells are shown with immunofluorescence staining. Scale bars, 25 μm


( B ) Representative GFP - labeled blastomeres that contributed to the chimeric porcine corneal epithelial cells . Almost all RPE cells and corneal epithelial cells of the chimera stained with anti - GFP antibody . Scale bars , 75

O O O O O O O O O O O O O O O O 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) Representative GFP-labeled blastomeres that contributed to the chimeric porcine corneal epithelial cells. Almost all RPE cells and corneal epithelial cells of the chimera stained with anti-GFP antibody. Scale bars, 75 μm


( C ) Representative immunofluorescence images showed the expression of an RPE marker ( BEST ) in GFP - labeled cells . Scale bars , 50

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

(C) Representative immunofluorescence images showed the expression of an RPE marker (BEST) in GFP-labeled cells. Scale bars, 50 μm