The left panel depicts a cryo - EM based atomic model of monomeric bovine F - ATP synthase ( pdb 5ara ) ( excluding subunits e , f , g , DAPIT , 6 . 8PL and A6L ) with OSCP ( cyan ) and subunit b ( yellow ) highlighted on the structure of F - ATP synthase in the lower part of the panel . The region H112 - E115 is shown as van der Waals spheres ( inflated for better visibility ) . The backbone of the stretch T130 - K135 is shown in red van der Waals spheres . The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk ( OSCP and subunits b , d , F6 ) displaying a large number of conformations in this region . Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112 - HRGE - 115 for the deprotonated ( HIS ) and the protonated form ( HIS + ) . Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112 - E115 is found in a large number of MD conformations . The larger conformational variability of the deprotonated form can also be appreciated . The right panel reports a sequence comparison of OSCP across 16 out of 197 species ( HRGE is boxed in red ; Sauria sequences are in the thin black box ) . Conservation , quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq , excluding Sauria and most divergent sequences , are described in the documentation of the program JalView [ 67 ] .

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

The left panel depicts a cryo-EM based atomic model of monomeric bovine F-ATP synthase (pdb 5ara) (excluding subunits e, f, g, DAPIT, 6.8PL and A6L) with OSCP (cyan) and subunit b (yellow) highlighted on the structure of F-ATP synthase in the lower part of the panel. The region H112-E115 is shown as van der Waals spheres (inflated for better visibility). The backbone of the stretch T130-K135 is shown in red van der Waals spheres. The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk (OSCP and subunits b, d, F6) displaying a large number of conformations in this region. Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112-HRGE-115 for the deprotonated (HIS) and the protonated form (HIS+). Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112-E115 is found in a large number of MD conformations. The larger conformational variability of the deprotonated form can also be appreciated. The right panel reports a sequence comparison of OSCP across 16 out of 197 species (HRGE is boxed in red; Sauria sequences are in the thin black box). Conservation, quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq, excluding Sauria and most divergent sequences, are described in the documentation of the program JalView [67].


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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


Circular representation of DNA methylation levels for mothers ( outer circle ) and children ( inner circle ) . The height of each bar indicates the methylation change between the smoking and non ‐ smoking group ( dark hue : hypermethylation , light hue : hypomethylation ) .

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Circular representation of DNA methylation levels for mothers (outer circle) and children (inner circle). The height of each bar indicates the methylation change between the smoking and non‐smoking group (dark hue: hypermethylation, light hue: hypomethylation).


( a ) WT and dnm1Δ cells expressing Vph1 - RFP and Idp1 - GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0 . 08 . The cells were then sampled daily and analysed by fluorescence microscopy ( × 100 objective ) during stationary phase mitophagy in lactate medium . Scale bar , 5 μm .

(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm.


( b ) WT , dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods , and samples ( 10 OD600 units ) were taken at each time point . Protein extracts were prepared and equal protein amounts ( 20 μg ) were subjected to SDS - polyacrylamide gel electrophoresis and immunoblotting with anti - GFP antibody .

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(b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody.


( c ) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b , using four independent experiments for each strain . Error bars indicate s . d . analysis of variance , P0 . 001 ; N = 4 .

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(c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.


( c ) Density plot of the reproducibility of biological replicates .

(c) Density plot of the reproducibility of biological replicates.


( a ) Ratios from proteins with quantifications from all samples were log2 transformed and z - score normalized . Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k - means . The proteins in each k - means cluster were tested for enrichment of GO CC ( indicated in red ) and GO BP ( indicated in blue ) terms .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized. Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k-means. The proteins in each k-means cluster were tested for enrichment of GO CC (indicated in red) and GO BP (indicated in blue) terms.


( b ) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins .

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(b) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins.


( d ) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins .

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(d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.


A comparison of the SILAC - derived abundance as a function of time , for several proteins that either show differential behaviour between WT and the mutants ( Idh2 , Idp1 and Aco2 ) and proteins that behave similarly between all the genetic backgrounds tested ( Hsp78 ) . Shown are mean protein ratios ( two replicates for mutants , three replicates for WT ) , error bars indicating peptide - based relative s . d .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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 comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.


WT ( a ) , dnm1Δ ( b ) and atg1Δ cells ( c ) and atg32Δ cells ( d ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 were incubated in SL medium for 7 days , and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti - GFP antibodies as described in the Methods section . Positive control used in c and d was from dnm1Δ cells expressing Idp1 - GFP from a plasmid at 4 - day incubation ( Fig . 1b , lane 4 ) . Release of free GFP in WT cells is reproducibly variable between these reporters , and varies between 0 ( Idh2 ) and 60 % ( Aco2 ) . ( e ) Densitometric quantification of the release of free GFP ( as % of total signal ) in four independent experiments , comparing the results between WT and dnm1Δ cells . Bars denote s . d . ( n = 4 ) .

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

WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).


( a ) Cells expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 as well as a plasmid - borne ( pDJB12 ) , mitochondrially targeted RFP ( mtRFP ) were incubated in SL - leucine medium for 3 days and imaged daily . Aco2 and Idp1 show clear mitophagic targeting ( white arrows point at specific examples ) by day 3 , whereas Idh2 and Hsp78 show weak or no mitophagy . In addition , Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker , whereas Idp1 - GFP and Aco2 - GFP show a near - complete co - localization with mtRFP . Scale bar , 5 μm . ( b ) Quantification of the frequency of mitophagy from a ; the percentage of cells showing mitophagic profiles in the red ( magenta bars ; plasmid - borne mtRFP ) or green ( turquoise bars ; integrated C - terminal GFP chimerae ) was calculated for each GFP chimera . Idp - GFP , red N = 76 , green N = 82 ; Aco2 - GFP , red N = 79 , green N = 82 ; Idh2 - GFP , red N = 72 , green N = 72 ; Hsp78 - GFP , red N = 70 , green N = 70 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.


WT and dnm1Δ cells expressing integrated Hsp78 - GFP and plasmid - borne mtRFP were grown on lactate medium for 3 days . Cells were imaged daily and the images were analysed using ImageJ as described in Methods . Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities , respectively . Diagonal concentrations of dots indicate spatial correlation between the signals . The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3 - day time point in the dnm1Δ mutant , relative to the WT cells . Scale bar , 5 μm .

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WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.


Figure 1 . DENND2B binds ITSN Purified GST - SH3 - containing proteins were incubated with lysates of HEK - 293T cells expressing Flag - DENND2B . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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Figure 1. DENND2B binds ITSN  Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Purified GST - SH3 domains of ITSN were incubated with lysates of HEK - 293T cells expressing Flag - DENND2B . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Lysates from HEK - 293T cells expressing Flag - ITSN - s and GFP or GFP - DENND2B were incubated with protein A beads or protein A beads coupled to anti - GFP . Bound proteins were detected by Western blot .

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Lysates from HEK-293T cells expressing Flag-ITSN-s and GFP or GFP-DENND2B were incubated with protein A beads or protein A beads coupled to anti-GFP. Bound proteins were detected by Western blot.


D , E HeLa cells expressing the indicated constructs . Flag - ITSN - s was visualized with anti - Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively . Scale bar : 5 µm . Boxed regions are magnified on the bottom . White arrows and red arrows point to colocalizing and non - colocalizing structures , respectively .

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D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.


Figure 2 . DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK - 293T cells were treated with DMSO or okadaic acid ( OA ) , lysed and analyzed by Western blot .

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Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot.


B HEK - 293T cells expressing Flag - DENND2B were treated with DMSO or 250 nM OA , and cell lysates were incubated with GST - proteins . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry . Low abundance peptides not included . Refer to Table EV1 for a complete peptide list .

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C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.


E HEK - 293T cells expressing Flag - DENND2B were treated with DMSO , 250 nM OA or with increasing concentrations of PKD inhibitor CID755673 . Cell lysates were analyzed by Western blot .

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E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.


F HEK - 293T cells expressing Flag - DENND2B or control cells were treated with 250 nM OA or DMSO , incubated with protein G beads coupled to anti - Flag and analyzed by Western blot .

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F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.


G Lysates from HEK - 293T cells expressing Flag - DENND2B and PKD - dominant negative ( DN ) were incubated with protein G beads or protein G beads coupled to anti - Flag . Bound proteins were detected by Western blot .

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G Lysates from HEK-293T cells expressing Flag-DENND2B and PKD-dominant negative (DN) were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.


H HEK - 293T cells co - expressing Flag - DENND2B with pcDNA3 or PKD - constitutively active ( CA ) , were incubated with protein G beads or protein G beads coupled to anti - Flag and bound proteins were detected by Western blot .

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H HEK-293T cells co-expressing Flag-DENND2B with pcDNA3 or PKD-constitutively active (CA), were incubated with protein G beads or protein G beads coupled to anti-Flag and bound proteins were detected by Western blot.


I Quantification of H where PKD - CA treatment is normalized to pcDNA3 control . Mean + / - SD . Welch ' s t - test * p = 0 . 016 , n = 6 for both treatment groups from 3 independent experiments .

I Quantification of H where PKD-CA treatment is normalized to pcDNA3 control. Mean +/- SD. Welch's t-test *p=0.016, n=6 for both treatment groups from 3 independent experiments.


Figure 3 . 14 - 3 - 3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST - 14 - 3 - 3 WT or K50E were incubated with lysates of HEK - 293T cells expressing GFP - DENND2B WT or S30A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


B HEK - 293T cells expressing Flag - DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST - 14 - 3 - 3 . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


C Quantification of B . where bound DENND2B is normalized to the SM . Mean + / - SD . Welch ' s t - test * * p = 0 . 006 n = 8 for both treatment groups from 5 independent

C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experiments


D HEK - 293T cells expressing Flag - DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST - 14 - 3 - 3 . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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D HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


E Quantification of D . where bound DENND2B is normalized to the SM . Mean + / - SD . Welch ' s t - test * p = 0 . 040 . n = 5 for both treatment groups from 3 independent

E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experiments


F GST - SH3A was incubated with lysates of HEK - 293T cells expressing GFP - DENND2B WT or S30A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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F GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


G Lysates of HEK - 293T cells expressing Flag - DENND2B were pre - incubated with increasing concentrations of purified 14 - 3 - 3 WT and subsequently incubated with GST - SH3A ( top panel ) or GST - Grb2 ( bottom panel ) . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Figure 4 . DENND2B promotes EGFR recycling A , B HeLa cells expressing the indicated constructs . Boxed regions are magnified on the bottom . Scale bars : 5 µm in top panel and 1 µm bottom panel . White arrows and arrowheads point to colocalization on vesicles and plasma membrane , respectively .

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Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.


C Transduced MCF10A cells were quantified by real - time PCR . Mean + / - SD , ANOVA with Dunnett post - hoc test * * * p = 0 . 000 for both comparisons , n = 3 from 3 independent experiments .

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C Transduced MCF10A cells were quantified by real-time PCR. Mean +/- SD, ANOVA with Dunnett post-hoc test ***p=0.000 for both comparisons, n=3 from 3 independent experiments.


D Transduced HEK - 293T cells expressing Flag - DENND2B were analyzed by Western blot .

D Transduced HEK-293T cells expressing Flag-DENND2B were analyzed by Western blot.


E Transduced MCF10A cells , were biotinylated and cell lysates were incubated with streptavidin beads . Bound proteins were detected by Western blot .

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E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.


F Quantification of E . where surface EGFR is normalized to total EGFR . Mean + / - SD , Anova with Dunnett post - hoc test * p = 0 . 013 and * * * p = 0 . 000 . n = 6 for all treatment groups from 4 independent

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F Quantification of E. where surface EGFR is normalized to total EGFR. Mean +/- SD, Anova with Dunnett post-hoc test *p=0.013 and ***p=0.000. n=6 for all treatment groups from 4 independent experiments


G Quantification of E where total EGFR is normalized to Hsc70 . Mean + / - SD n = 3 for all treatment groups from 3 independent

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G Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experiments


I MCF10A cells expressing GFP - DENND2B were labelled for surface EGFR on ice , incubated at 16 ° C to allow internalization , acid washed to remove remaining labelled - EGFR and processed for immunofluorescence to visualize the internal pool of EGFR ( post - endocytosis , top panel ) or incubated at 37 ° C to allow recycling , acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR ( post - recycling , bottom panel ) .

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I MCF10A cells expressing GFP-DENND2B were labelled for surface EGFR on ice, incubated at 16°C to allow internalization, acid washed to remove remaining labelled-EGFR and processed for immunofluorescence to visualize the internal pool of EGFR (post-endocytosis, top panel) or incubated at 37°C to allow recycling, acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR (post-recycling, bottom panel).


J Quantification of I top panel , internal EGFR post - endocytosis . Mean fluorescence per cell + / - SD , n = 13 for GFP - DENND2B and n = 21 for control cells pooled from 2 independent experiments .

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J Quantification of I top panel, internal EGFR post-endocytosis. Mean fluorescence per cell +/- SD, n=13 for GFP-DENND2B and n=21 for control cells pooled from 2 independent experiments.


K Quantification of I bottom panel , internal EGFR post - recycling . Mean fluorescence per cell + / - SD , n = 10 for GFP - DENND2B and n = 26 for control cells pooled from 2 independent experiments .

K Quantification of I bottom panel, internal EGFR post-recycling. Mean fluorescence per cell +/- SD, n=10 for GFP-DENND2B and n=26 for control cells pooled from 2 independent experiments.


Figure 5 . EGF treatment dissociates DENND2B from ITSN A HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for the indicated times and cell lysates were analyzed by Western blot .

Figure 5. EGF treatment dissociates DENND2B from ITSN A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot.


B Quantification of A where P - PKD is normalized to Hsc70 . Mean + / - SD . ANOVA with Dunnett post - test * p = 0 . 015 and * * p = 0 . 003 n = 3 from 3 independent experiments .

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B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments.


C HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti - Flag . Bound proteins were detected by Western blot .

C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.


D Quantification of B where P - PKD substrate is normalized to Flag - DENND2B . Mean + / - SD Welch ' s t - test * p = 0 . 036 , n = 4 from 3 independent experiments .

D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments.


E HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for 5 min and cell lysates were incubated with GST - SH3A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


F Quantification of E where bound Flag - DENND2B is normalized to SM . Mean + / - SD , Welch ' s t - test * p = 0 . 012 , n = 6 from 3 independent experiments .

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F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments.


Wild - type mice have small defined crypts in the small intestine with little nuclearβ - catenin at the bottom of the crypt . The small intestine of AhCreERAPCfl / fl , AhCreERCatnblox ( ex3 ) / lox ( ex3 ) ( day 5 ) or AhCreERGsk3alphafl / fl betafl / fl ( day 6 ) show the crypt - progenitor cell ( CPC ) phenotype with increased crypt size ( red bar ) and nuclearβ - catenin ( arrows ) along the crypt - villus axis . Scale bar , 100 μm .

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Wild-type mice have small defined crypts in the small intestine with little nuclearβ-catenin at the bottom of the crypt. The small intestine of AhCreERAPCfl/fl, AhCreERCatnblox(ex3)/lox(ex3) (day 5) or AhCreERGsk3alphafl/fl betafl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclearβ-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm.


A heterozygous activation of β - catenin ( AhCreER Catnblox ( ex3 ) / + ) shows no increase in crypt size or nuclear β - catenin at days 5 - 10 . At day 15 post - induction , accumulation of nuclear β - catenin ( arrows ) becomes evident with a dramatic CPC phenotype at about day 25 . Scale bar , 100 μm .

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A heterozygous activation of β-catenin (AhCreER Catnblox(ex3)/+) shows no increase in crypt size or nuclear β-catenin at days 5-10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm.


Culture of small intestinal crypts of WT and VilCreER Apcfl / fl ( or AhCreER Apcfl / fl ) mice with / without R - spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc - deficient spheres . Representative photos were taken at day 5 in culture . Black scale bar , 50 μm .

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Culture of small intestinal crypts of WT and VilCreER Apcfl/fl (or AhCreER Apcfl/fl) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc-deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm.


At day 5 post - induction , only crypts from AhCreERCatnblox ( ex3 ) / lox ( ex3 ) but not AhCreERCatnblox ( ex3 ) / + survive in culture without the addition of R - spo1 . At day 10 post - induction , we observed a mixed phenotype of more organoid - like structures in AhCreERCatnblox ( ex3 ) / + compared to spheres from Catnblox ( ex3 ) / lox ( ex3 ) crypts in the first week of culture . Black scale bar , 50 μm .

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At day 5 post-induction, only crypts from AhCreERCatnblox(ex3)/lox(ex3) but not AhCreERCatnblox(ex3)/+ survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCreERCatnblox(ex3)/+ compared to spheres from Catnblox(ex3)/lox(ex3)crypts in the first week of culture. Black scale bar, 50 μm.


Quantification of organoids / spheres at day 10 post - induction ( N = 2 or 3 mice per genotype , mean of 6 technical replicates per mouse ) .

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Quantification of organoids/spheres at day 10 post-induction (N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).


Table shows that only homozygous activation of β - catenin ( Lgr5CreER Catnblox ( ex3 ) / lox ( ex3 ) ) or loss of Apc ( Lgr5CreER Apcfl / fl ) in Lgr5 - positive stem cells results in colonic lesions in cohorts , sampled when signs of sickness were apparent . There were no lesions observed when only one allele of β - catenin ( Lgr5CreER Catnblox ( ex3 ) / + ) was mutated ( chi - squared test , P < 0 . 001 ) .

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Table shows that only homozygous activation of β-catenin (Lgr5CreER Catnblox(ex3)/lox(ex3)) or loss of Apc (Lgr5CreER Apcfl/fl) in Lgr5-positive stem cells results in colonic lesions in cohorts, sampled when signs of sickness were apparent. There were no lesions observed when only one allele of β-catenin (Lgr5CreER Catnblox(ex3)/+) was mutated (chi-squared test, P < 0.001).


Colonic lesions were scored from H & amp ; amp ; E by identification of disorganised epithelium . Staining for β - catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions . Scale bar , 100 μm .

Colonic lesions were scored from H&amp;E by identification of disorganised epithelium. Staining for β-catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions. Scale bar, 100 μm.


Staining of small intestinal and colonic crypts for E - cadherin . Scale bar , 100 μm .

Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm.


Western blot of purified crypts from the small intestine and colon ( N = 4 mice ) .

Western blot of purified crypts from the small intestine and colon (N = 4 mice).


qRT - PCR of whole tissue from small intestine and colon . Expression of mRNA ( 2 ( − dCt ) ) calculated relative to GAPDH ( N = 3 mice ) . Statistics : one - sided Mann - Whitney U - test .

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qRT-PCR of whole tissue from small intestine and colon. Expression of mRNA (2(−dCt)) calculated relative to GAPDH (N = 3 mice). Statistics: one-sided Mann-Whitney U-test.


Proximity ligation assay for E - cadherin and β - catenin . For each mouse ( N = 6 ) , at least 10 crypts per tissue were analysed to calculate the mean . Statistics : one - sided Mann - Whitney U - test ; white scale bar , 50 μm .

Proximity ligation assay for E-cadherin and β-catenin. For each mouse (N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann-Whitney U-test; white scale bar, 50 μm.


Immunoprecipitation ( IP ) of E - cadherin from VilCreER Catnblox ( ex3 ) / lox ( ex3 ) mice , 24 h ( day 1 , top ) and 48 h ( day 2 , bottom ) after induction . The ratio of wild - type ( WT , top lane ) and mutant exon 3 β - catenin ( Δex3 , bottom lane ) bound to E - cadherin ( IP : Ecad ) was calculated for each tissue . Graphs show average of experiments ( N = 3 mice for each genotype and time point ) ; error bars represent s . e . m .

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Immunoprecipitation (IP) of E-cadherin from VilCreER Catnblox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.


IP of E - cadherin from VilCreER Catnblox ( ex3 ) / + mice at day 4 and day 8 . Graphs show average of experiments ( N = 3 mice for each genotype and time point ) ; error bars represent s . e . m .

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IP of E-cadherin from VilCreER Catnblox(ex3)/+ mice at day 4 and day 8. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.


AhCreERCatnblox ( ex3 ) / + compared to AhCreER Catnblox ( ex3 ) / + Cdh1fl / + at day 10 post - induction . Note the presence of colonic lesions in the colon of AhCreER Catnblox ( ex3 ) / + Cdh1fl / + mice ( arrows ) . Scale bar , 100 μm ; red bar indicates proliferative zone ( BrdU ) .

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AhCreERCatnblox(ex3)/+ compared to AhCreER Catnblox(ex3)/+ Cdh1fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCreER Catnblox(ex3)/+ Cdh1fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU).


Scoring of BrdU - positive cells per half - crypt in the small intestine of wild - type , AhCreERCdh1fl / + , AhCreERCatnblox ( ex3 ) / + and AhCreERCatnblox ( ex3 ) / + Cdh1fl / + mice at day 10 post - induction . N ≥ 3 , at least 25 crypts per mouse were scored . There was significantly higher proliferation in the AhCreERCatnblox ( ex3 ) / + Cdh1fl / + mice ( P = 0 . 028 , one - sided Mann - Whitney U - test ) .

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Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCreERCdh1fl/+, AhCreERCatnblox(ex3)/+ and AhCreERCatnblox(ex3)/+Cdh1fl/+mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCreERCatnblox(ex3)/+Cdh1fl/+mice (P = 0.028, one-sided Mann-Whitney U-test).


In vitro growth of crypts ( small intestine ) from mice as indicated at day 5 post - induction without R - spo1 . Quantification of sphere - forming efficiency of AhCreERCatnblox ( ex3 ) / + AhCreERCatnblox ( ex3 ) / + Cdh1fl / + , day 5 post - induction . N = 3 mice per genotype , mean of 6 technical replicates per mouse , P = 0 . 04 one - sided Mann - Whitney U - test .

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In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCreERCatnblox(ex3)/+AhCreERCatnblox(ex3)/+Cdh1fl/+, day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann-Whitney U-test.


Survival of Lgr5CreERCatnblox ( ex3 ) / + and Lgr5CreERCatnblox ( ex3 ) / + Cdh1fl / + shows significant acceleration ( P = 0 . 000123 , log - rank test ) after E - cadherin reduction . About 85 % ( 6 / 7 mice ) had colonic lesions , as identified with β - cateninIHC in contrast to Lgr5CreERCatnblox ( ex3 ) / + mice .

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Survival of Lgr5CreERCatnblox(ex3)/+ and Lgr5CreERCatnblox(ex3)/+Cdh1fl/+ shows significant acceleration (P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-cateninIHC in contrast to Lgr5CreERCatnblox(ex3)/+mice.


Proximity ligation assay for E - cadherin : β - catenin on a tissue microarray of SPT patients . PLA dots / cells in normal and tumour tissue were counted . Each dot in the boxplot represents the average for a single patient . Staining for β - catenin confirms accumulation of nuclear β - catenin in tumour tissue . Representative pictures are shown . N = 18 , statistics : Mann - Whitney U - test , P = 0 . 0009051 . White scale bar , 50 μm .

Proximity ligation assay for E-cadherin:β-catenin on a tissue microarray of SPT patients. PLA dots/cells in normal and tumour tissue were counted. Each dot in the boxplot represents the average for a single patient. Staining for β-catenin confirms accumulation of nuclear β-catenin in tumour tissue. Representative pictures are shown. N = 18, statistics: Mann-Whitney U-test, P = 0.0009051. White scale bar, 50 μm.


Correlation of several Wnt target genes with the expression of E - cadherin ( CDH1 ) was analysed in 269 HCC patients ( TCGA provisional ) , and linear regression line ( blue ) and confidence region ( shaded ) were added .

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Correlation of several Wnt target genes with the expression of E-cadherin (CDH1) was analysed in 269 HCC patients (TCGA provisional), and linear regression line (blue) and confidence region (shaded) were added.


( A ) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues ( T ) and adjacent nontumor tissues ( N ) . The graph represents CRIP1 protein expression normalized to GAPDH levels ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , paired ( A , Student ' s t -

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(A) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues (T) and adjacent nontumor tissues (N). The graph represents CRIP1 protein expression normalized to GAPDH levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired (A, Student's t-test


( B ) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues . Scale bars : 20 μm . Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , paired B ) Student ' s t -

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(B) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues. Scale bars: 20 μm. Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired B) Student's t-test


( C ) CRIP1 mRNA expression in the TCGA - LIHC dataset . ( D ) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , unpaired Student ' s t -

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(C) CRIP1 mRNA expression in the TCGA-LIHC dataset. (D) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired Student's t-test


The protein ( F ) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes . ( G ) Relative CRIP1 protein level compared to normal LO2 hepatocytes . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , unpaired G ) Student ' s t -

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The protein (F) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes. (G) Relative CRIP1 protein level compared to normal LO2 hepatocytes. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired G) Student's t-test


( H - I ) Kaplan - Meier survival analyses of OS ( H ) and PFS ( I ) based on CRIP1 expression levels in 108 HCC patients . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using log - rank test ( H - I ) .

(H-I) Kaplan-Meier survival analyses of OS (H) and PFS (I) based on CRIP1 expression levels in 108 HCC patients. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using log-rank test (H-I).


( B ) Tumor sphere formation assay in CRIP1 - overexpressing Huh7 and Hep3B cells . The data are shown as the relative fold change spheres compared with the vector group ( right ) . Scale bar : 50 μm . Data information : Data represent the mean ± SD at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test ( B ,

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(B) Tumor sphere formation assay in CRIP1-overexpressing Huh7 and Hep3B cells. The data are shown as the relative fold change spheres compared with the vector group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (B,


( C ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - overexpressing Huh7 and Hep3B cells .

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(C) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 and Hep3B cells.


( H ) Tumor sphere formation assay in CRIP1 - knockdown MHCC - 97H and HepG2 cells . The data are shown as the relative fold change spheres compared with the siCtrl group ( right ) . Scale bar : 50 μm . Data information : Data represent the mean ± SD at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test H ,

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(H) Tumor sphere formation assay in CRIP1-knockdown MHCC-97H and HepG2 cells. The data are shown as the relative fold change spheres compared with the siCtrl group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test H,


( I ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - knockdown MHCC - 97H and HepG2 cells .

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(I) Western blot analysis of HCC cancer stem cell markers in CRIP1-knockdown MHCC-97H and HepG2 cells.


( G ) Western blot analysis of cancer stem cell markers in the indicated groups .

(G) Western blot analysis of cancer stem cell markers in the indicated groups.


( A ) Western blot analysis of BBOX1 protein levels in CRIP1 - overexpressing Huh7 and Hep3B cells . ( B ) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC - 97H and HepG2 cells .

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(A) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells. (B) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC-97H and HepG2 cells.


( D ) Western blot analysis of BBOX1 in CRIP1 - overexpressing Huh7 cells treated with CHX ( 100 μg / ml ) for the indicated durations ( lfte ) . The graph shows quantification of relative BBOX1 levels ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 and * * P < 0 . 01 . Differences were tested using an unpaired 2 - tailed Student ' s t - test D ) .

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(D) Western blot analysis of BBOX1 in CRIP1-overexpressing Huh7 cells treated with CHX (100 μg/ml) for the indicated durations (lfte). The graph shows quantification of relative BBOX1 levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01. Differences were tested using an unpaired 2-tailed Student's t-test D).


( E ) Western blot analysis of BBOX1 protein levels in CRIP1 - overexpressing Huh7 and Hep3B cells treated with MG132 ( 10 μM ) for 10 h . D ) .

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(E) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells treated with MG132 (10 μM) for 10 h. D).


( F ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 and Hep3B cells .

(F) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells.


( G ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs .

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(G) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs.


( H ) Co - immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1 , STUB1 , and BBOX1 in Huh7 and Hep3B cells .

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(H) Co-immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1, STUB1, and BBOX1 in Huh7 and Hep3B cells.


( I ) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1 .

(I) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1.


( J ) Co - immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1 - Myc and CRIP1 siRNAs .

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(J) Co-immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1-Myc and CRIP1 siRNAs.


( B ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant .

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(B) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.


( C ) Western blot analysis of BBOX1 - WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1 .

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(C) Western blot analysis of BBOX1-WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1.


tumor sphere formation ability and quantification of spheres ( E ) , in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant . Scale bar : 50 μm . Data information : Data represent the mean ± SD of at least three independent experiments . * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t -

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tumor sphere formation ability and quantification of spheres (E), in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test


protein levels of HCC cancer stem cell markers ( G ) in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant .

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protein levels of HCC cancer stem cell markers (G) in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.


Representative images of tumor sphere formation and quantification of spheres ( C ) in CRIP1 - overexpressing Huh7 cells with or without iCRT3 ( 50 μM ) for 24 h . Scale bar : 50 μm . Data information : Data represent the mean ± SD of at least three independent experiments . * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t -

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Representative images of tumor sphere formation and quantification of spheres (C) in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P <0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test


( E ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - overexpressing Huh7 cells with or without iCRT3 ( 50 μM ) for 24 h .

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(E) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h.


( F ) Coimmunoprecipitation and immunoblot analyses of β - catenin acetylation in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) for 24 h .

(F) Coimmunoprecipitation and immunoblot analyses of β-catenin acetylation in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24 h.


( G ) Western blot analysis of cytoplasmic and nuclear fractions of β - catenin in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) for 24h .

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(G) Western blot analysis of cytoplasmic and nuclear fractions of β-catenin in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24h.


( I ) Co - immunoprecipitation and immunoblot analyses of β - catenin ubiquitination in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) and EX527 ( 20 μM ) for 24 h .

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(I) Co-immunoprecipitation and immunoblot analyses of β-catenin ubiquitination in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) and EX527 (20 μM) for 24 h.


( A ) IHC analysis of CRIP1 , BBOX1 , β - catenin , CD133 , OCT4 , and NANOG using TAM ( left ) . Scale bars : 20 μm . Correlation analysis of CRIP1 expression with BBOX1 , β - catenin , CD133 , OCT4 , and NANOG expression in HCC tissues ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . . * * P < 0 . 01 and * * * P < 0 . 001 . Chi - square test was performed to determine the correlation ( A ) .

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(A) IHC analysis of CRIP1, BBOX1, β-catenin, CD133, OCT4, and NANOG using TAM (left). Scale bars: 20 μm. Correlation analysis of CRIP1 expression with BBOX1, β-catenin, CD133, OCT4, and NANOG expression in HCC tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments.. **P <0.01 and ***P < 0.001. Chi-square test was performed to determine the correlation (A).


Heatmaps of AIB1 - YAP co - activated , YAP activated , AIB1 - YAP corepressed , and YAP repressed genes shown in panel ( A ) . Red and blue indicate high and low levels , respectively . RNA - sequencing was performed in triplicate and statistics in Panel ( A - B ) were calculated by EdgeR with Benjamini - Hochberg multiple test correction in R .

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Heatmaps of AIB1-YAP co-activated, YAP activated, AIB1-YAP corepressed, and YAP repressed genes shown in panel (A). Red and blue indicate high and low levels, respectively. RNA-sequencing was performed in triplicate and statistics in Panel (A-B) were calculated by EdgeR with Benjamini-Hochberg multiple test correction in R.


Select genes are regulated by YAP and AIB1 , others are YAP regulated independently of AIB1 . Endogenous gene expression changes assessed by RT - qPCR .

Select genes are regulated by YAP and AIB1, others are YAP regulated independently of AIB1. Endogenous gene expression changes assessed by RT-qPCR.


Significant genomic co - occupancy between AIB1 and TEAD in normal breast and breast cancer cells . Overlap of AIB1 and TEAD4 ChIP - seq peaks in MCF10A and MCF7 cell lines . Select peaks from MCF10A ChIP - seq shown . ChIP - sequencing performed in triplicate .

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Significant genomic co-occupancy between AIB1 and TEAD in normal breast and breast cancer cells. Overlap of AIB1 and TEAD4 ChIP-seq peaks in MCF10A and MCF7 cell lines. Select peaks from MCF10A ChIP-seq shown. ChIP-sequencing performed in triplicate.


TEAD binding motifs are enriched within AIB1 occupied sites across the genome . HOMER de novo motif analysis of AIB1 ChIP - seq peaks in MCF10A and MCF7 cells .

TEAD binding motifs are enriched within AIB1 occupied sites across the genome. HOMER de novo motif analysis of AIB1 ChIP-seq peaks in MCF10A and MCF7 cells.


AIB1 modulates TEAD transcriptional activity in a YAP dependent manner . HEK293T cells were transfected with CTGF - promoter reporter and expression vectors of indicated proteins .

AIB1 modulates TEAD transcriptional activity in a YAP dependent manner. HEK293T cells were transfected with CTGF-promoter reporter and expression vectors of indicated proteins.


AIB1 is engaged with TEAD on co - activated target promoters in normal cells . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A cells . Levels of precipitated chromatin assessed by qPCR .

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AIB1 is engaged with TEAD on co-activated target promoters in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.


AIB1 is engaged with TEAD on co - activated target promoters in early stage breast cancer cells . Sequential ChIP - reChIP performed with indicated antibodies in MCFDCIS cells . Levels of precipitated chromatin assessed by qPCR .

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AIB1 is engaged with TEAD on co-activated target promoters in early stage breast cancer cells. Sequential ChIP-reChIP performed with indicated antibodies in MCFDCIS cells. Levels of precipitated chromatin assessed by qPCR.


ANCO1 is recruited to YAP - TEAD by AIB1 . Positive Proximity Ligation Assay ( PLA ) signal ( white foci , falsely colored 594nm signal ) shown between ANCO1 - TEAD , ANCO1 - YAP , AIB1 - TEAD , and AIB1 - YAP , in control cells are lost in AIB1 knockdown . Representative images of Proximity Ligation Assay ( PLA ) between indicated proteins in MCF10A shGFP ( control ) and shAIB1 cell lines using antibodies ( species ) as indicated ; Scale bars shown indicate 25μm .

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ANCO1 is recruited to YAP-TEAD by AIB1. Positive Proximity Ligation Assay (PLA) signal (white foci, falsely colored 594nm signal) shown between ANCO1-TEAD, ANCO1-YAP, AIB1-TEAD, and AIB1-YAP, in control cells are lost in AIB1 knockdown. Representative images of Proximity Ligation Assay (PLA) between indicated proteins in MCF10A shGFP (control) and shAIB1 cell lines using antibodies (species) as indicated; Scale bars shown indicate 25μm.


YAP overexpression represses , AIB1 knockdown dysregulates , and ANCO1 knockdown amplifies genes at the 1q21 . 3 locus in MCF10A and MCFDCIS cells . Gene set enrichment analysis of RNA - seq data and cDNA array data generated from MCF10A cell lines as indicated .

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YAP overexpression represses, AIB1 knockdown dysregulates, and ANCO1 knockdown amplifies genes at the 1q21.3 locus in MCF10A and MCFDCIS cells. Gene set enrichment analysis of RNA-seq data and cDNA array data generated from MCF10A cell lines as indicated.


YAP represses 1q21 . 3 localized genes . Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP / S94A ; levels were assessed by RT - qPCR .

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YAP represses 1q21.3 localized genes. Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP/S94A; levels were assessed by RT-qPCR.


ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells ; levels were assessed by RT - qPCR .

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ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells; levels were assessed by RT-qPCR.


YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown . Levels of endogenous 1q21 . 3 genes were assessed in YAP or S94A - expressing , control or shANCO1 MCF10A cells by RT - qPCR .

YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown. Levels of endogenous 1q21.3 genes were assessed in YAP or S94A-expressing, control or shANCO1 MCF10A cells by RT-qPCR.


TEAD is engaged at annotated enhancer regions within 1q21 . 3 . Analysis of TEAD4 ChIP - seq in MDA - MB - 231 ( GSE66081 ) , MCF7 , and A549 Cells ( GSE32465 ) at 1q21 . 3 with enhancers scored by GeneHancer . Enhancer regions of interest indicated between dotted lines .

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TEAD is engaged at annotated enhancer regions within 1q21.3. Analysis of TEAD4 ChIP-seq in MDA-MB-231(GSE66081), MCF7, and A549 Cells (GSE32465) at 1q21.3 with enhancers scored by GeneHancer. Enhancer regions of interest indicated between dotted lines.


TEAD , YAP , AIB1 , and ANCO1 are engaged at annotated enhancer regions within 1q21 . 3 . Chromatin Immunoprecipitation ( ChIP ) performed with indicated antibodies in MCF10A cells .

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TEAD, YAP, AIB1, and ANCO1 are engaged at annotated enhancer regions within 1q21.3. Chromatin Immunoprecipitation (ChIP) performed with indicated antibodies in MCF10A cells.


AIB1 is engaged with TEAD on superenhancer sites at 1q21 . 3 in normal cells . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A cells . Levels of precipitated chromatin assessed by qPCR .

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AIB1 is engaged with TEAD on superenhancer sites at 1q21.3 in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.


ANCO1 is recruited to superenhancers at 1q21 . 3 by AIB1 and is significantly reduced at 1q21 . 3 following AIB1 knockdown . ChIP performed with ANCO1 antibodies in control or shAIB1 cells . Levels of precipitated chromatin assessed by qPCR .

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ANCO1 is recruited to superenhancers at 1q21.3 by AIB1 and is significantly reduced at 1q21.3 following AIB1 knockdown. ChIP performed with ANCO1 antibodies in control or shAIB1 cells. Levels of precipitated chromatin assessed by qPCR.


YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown , but further enhanced by ANCO1 knockdown . Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days . 10x and 40x magnifications shown , Scale bar = 200μm .

YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown, but further enhanced by ANCO1 knockdown. Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days. 10x and 40x magnifications shown, Scale bar = 200μm.


Quantification of aberrant spheres per field displayed in panel ( A ) , n = number of spheres .

Quantification of aberrant spheres per field displayed in panel (A), n= number of spheres.


Quantification of size ( arbitrary units ) of aberrant spheres per field displayed in panel ( A ) , relative to control YAP S94A shGFP spheres . n = number of spheres .

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Quantification of size (arbitrary units) of aberrant spheres per field displayed in panel (A), relative to control YAP S94A shGFP spheres. n= number of spheres.


MCFDCIS xenograft tumors have increased expression of AIB1 - YAP , EMT , and 1q21 . 3 genes during the invasive transition . Schematic of experiment shown . Gene set enrichment analysis of RNA - seq data generated from MCFDCIS xenografts at indicated time points .

MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.


Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions . Representative images of H & E , p63 , and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions . Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions . 20x magnification ; Scale bar = 100μm . Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel ( E ) .

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Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions. Representative images of H&E, p63, and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions. Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions. 20x magnification; Scale bar = 100μm. Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel (E).


ANCO1 levels decrease in breast cancer . ANCO1 mRNA expression from normal tissue ( GTEX ) and Tumor ( TCGA ) for breast samples .

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ANCO1 levels decrease in breast cancer. ANCO1 mRNA expression from normal tissue (GTEX) and Tumor (TCGA) for breast samples.


Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions . DCIS lesions can be stratified to either ANCO1 negative ( center panel ) , ANCO1 positive ( right panel ) . Scale bar = 50μm .

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Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions. DCIS lesions can be stratified to either ANCO1 negative (center panel), ANCO1 positive (right panel). Scale bar = 50μm.


High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients . KM plot showing progression free survival in patients with stratified ANCO1 expression . n = 360 patients .

High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients. KM plot showing progression free survival in patients with stratified ANCO1 expression. n= 360 patients.


Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21 . 3 genes , without altered AIB1 - YAP co - activated gene expression . Dot plots showing TCGA Pan - Cancer data accessed via Xenabroswer . Patients were stratified to high or low expression of ANCO1 by median expression .

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Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21.3 genes, without altered AIB1-YAP co-activated gene expression. Dot plots showing TCGA Pan-Cancer data accessed via Xenabroswer. Patients were stratified to high or low expression of ANCO1 by median expression.


( C ) Flag - tagged Raptor was transfected with HA - tagged ArfGAP1 or HA - tagged RFP1 in wildtype ( WT ) or RagA / B knockout ( KO ) HEK293A cells for 24 hours . Cell lysates were immunoprecipitated ( IP ) with anti - Flag beads . IP or WCL samples were probed for HA - tagged ArfGAP1 or HA - tagged RFP1 , Flag - tagged Raptor , RagA , RagB , and Vinculin the loading control .

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(C) Flag-tagged Raptor was transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 in wildtype (WT) or RagA/B knockout (KO) HEK293A cells for 24 hours. Cell lysates were immunoprecipitated (IP) with anti-Flag beads. IP or WCL samples were probed for HA-tagged ArfGAP1 or HA-tagged RFP1, Flag-tagged Raptor, RagA, RagB, and Vinculin the loading control.


( G ) RagA / B KO HEK293A cells were transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) for 72 hours . Cell lysates were immunoprecipitated ( IP ) with anti - HA beads . IP or WCL samples were probed for HA and mTORC1 components mTOR , Raptor and mLST8 . Vinculin was probed as a control .

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(G) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) for 72 hours. Cell lysates were immunoprecipitated (IP) with anti-HA beads. IP or WCL samples were probed for HA and mTORC1 components mTOR, Raptor and mLST8. Vinculin was probed as a control.


( D ) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 ( shArfGAP1 ) or GFP ( shGFP ) control . Cells were cultured under normal condition ( NC ; lanes 1 and 7 ) , or starved of amino acids for 1 hour ( - AA 1h ; lanes 2 and 8 ) , followed by either maintaining in amino acid starvation media ( - AA ; lanes 3 and 9 ) , or addition of 1 mM of Leu ( + Leu ; lanes 4 and 10 ) , Gln ( + Gln ; lanes 5 and 11 ) , or Asn ( + Asn ; lanes 6 and 12 ) for 2 hours . mTORC1 activity was analyzed similar to ( B ) . Protein levels of ArfGAP1 were confirmed by immunoblotting . S6K1 and Actin were probed as controls .

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(D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls.


( E ) RagA / B KO HEK293A cells were co - transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) with Myc - tagged S6K1 . Cells were starved of amino acids ( - AA ) for 2 hours and then 4mM of Gln or Asn were added for 2 hours . mTORC1 was analyzed similar to ( B ) . IP or WCL samples were probed for HA , Myc , and phosphorylated S6K1 at Thr 389 . Actin serve as a loading control .

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(E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control.


( B ) Fluorescence Lifetime Imaging Microscopy ( FLIM ) and Förster resonance energy transfer ( FRET ) analysis were performed in wildtype ( WT , left ) or RagA / B KO ( middle ) HAP1 cells expressing mCherry - tagged ArfGAP1 and endogenously GFP - tagged Raptor . Cells either had amino acids ( + AA ) or were starved of amino acids ( - AA ) . At least 20 cells were analyzed in each group . Box plots shows the percentage of pixels with ≥ 30 % FRET efficiency of co - transfected cells . RFP1 was expressed and analyzed as a negative control . The whiskers of the boxplot represent the minimum and maximum values , while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median . Right - Expression level of RagA / B was confirmed Western blot analysis . Actin was used as a control .

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(B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.


( A ) Mouse embryonic fibroblast ( MEF ) cells were transfected with HA - tagged ArfGAP1 . Immunofluorescence staining was assessed for HA - tagged ArfGAP1 ( blue ) , mTOR ( green ) , and LAMP2 ( red , lysosomal marker ) . Left - A representative example of the immunofluorescence staining ( scale bar , 10 μm ) . The top left image shows the staining of HA - tagged ArfGAP1 , with a white outline highlighting the region that is positive for HA - tagged ArfGAP1 . This outline was overlaid with the mTOR channel ( top right ) and the LAMP2 channel ( bottom left ) . The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm ( Rizk et al . , 2014 ; bottom right ) . The co - localization of mTOR and LAMP2 was analyzed for the region within the outline ( HA - ArfGAP1 positive ) and the region outside of the outline ( HA - ArfGAP1 - negative ) , respectively . Right - In each microscopy field , the image was processed as described above , and the co - localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm ( Rizk et al . , 2014 ) . The quantification was performed for the subgroups of cells that are positively or negatively stained for HA - tagged ArfGAP1 , respectively , and compared using Student ' s t - test . Columns represent Mean ± SEM ( N = 3 ) .

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(A) Mouse embryonic fibroblast (MEF) cells were transfected with HA-tagged ArfGAP1. Immunofluorescence staining was assessed for HA-tagged ArfGAP1 (blue), mTOR (green), and LAMP2 (red, lysosomal marker). Left- A representative example of the immunofluorescence staining (scale bar, 10 μm). The top left image shows the staining of HA-tagged ArfGAP1, with a white outline highlighting the region that is positive for HA-tagged ArfGAP1. This outline was overlaid with the mTOR channel (top right) and the LAMP2 channel (bottom left). The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm (Rizk et al., 2014; bottom right). The co-localization of mTOR and LAMP2 was analyzed for the region within the outline (HA-ArfGAP1 positive) and the region outside of the outline (HA-ArfGAP1-negative), respectively. Right- In each microscopy field, the image was processed as described above, and the co-localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm (Rizk et al., 2014). The quantification was performed for the subgroups of cells that are positively or negatively stained for HA-tagged ArfGAP1, respectively, and compared using Student's t-test. Columns represent Mean ± SEM (N = 3).


( D ) MIA Paca - 2 cells stably expressing FLAG - tagged or HA - tagged TMEM192 cells were cultured under normal condition ( NC ) or starved of amino acids ( - AA ) , and then stimulated with 2 mM glutamine and asparagine ( + Gln / Asn ) for 2 hours . HA - tagged TMEM192 was immunoprecipitated ( IP ) with HA beads and IP and whole cell lysates ( WCL ) were probed for ArfGAP1 , HA , RagA , or p18 ( LAMTOR1 ) . Flag , pS6K1 ( mTORC1 activity ) , S6K1 , and Vinculin were probed for as controls .

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(D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls.


( D ) Top - Input of GST , GST - tagged ArfGAP1 , and GST - tagged ArfGAP1 L207D / V279D that was us in the in vitro binding assays . Bottom - Flag - tagged mTOR , Flag - tagged Raptor , and Flag - tagged mLST8 were overexpressed in HEK293A cells . Cell lysates were immunoprecipitated ( IP ) with anti - Flag beads and incubated with either GST ( control ) , GST - tagged ArfGAP1 , or GST - ArfGAP1 L207D / V279D . IP or WCL samples were probed for Flag or GST .

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(D) Top - Input of GST, GST-tagged ArfGAP1, and GST-tagged ArfGAP1 L207D/V279D that was us in the in vitro binding assays. Bottom - Flag-tagged mTOR, Flag-tagged Raptor, and Flag-tagged mLST8 were overexpressed in HEK293A cells. Cell lysates were immunoprecipitated (IP) with anti-Flag beads and incubated with either GST (control), GST-tagged ArfGAP1, or GST-ArfGAP1 L207D/V279D. IP or WCL samples were probed for Flag or GST.


( A ) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression . Overall survival and disease - free survival were compared between these two groups , as shown in Kaplan - Meier curves . Median survivals ( in months ) , log - rank P values , and hazard ratios ( HR ) with 95 % confidence intervals are indicated .

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(A) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression. Overall survival and disease-free survival were compared between these two groups, as shown in Kaplan-Meier curves. Median survivals (in months), log-rank P values, and hazard ratios (HR) with 95% confidence intervals are indicated.


( D ) MIA Paca - 2 cells stably expressing short hairpin RNAs ( shRNAs ) against ArfGAP1 or GFP ( control ) were plated and treated with mTOR inhibitor Torin 1 ( 250 nM ) or vehicle control for 48 hours prior to cell counting . Columns represent Mean ± SEM ( N = 6 ) . Student ' s t - test shGFP Vehicle vs . shGFP Torin 1 ( P = 2 . 83 E - 07 ) , shGFP Vehicle vs . shArfGAP1 Vehicle ( * P = 0 . 025 ) , shGFP Torin 1 vs , shArfGAP1 Torin 1 ( P = 0 . 139 ) , and shArfGAP1 Vehicle vs . shArfGAP1 Torin 1 ( P = 1 . 22 E - 07 ) . " ns " stands for " not significant " .

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(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".


( B ) Immunoblot showing CENP - AEA protein level at the indicated time in RPE - 1 cells .

(B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells.


( C ) Representative immunofluorescence images showing CENP - A reloading at CENP - B - marked centromeres in DLD - 1 cells . White dashed circles contour nuclei . Scale bar , 5 µm .

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(C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm.


( D ) Quantification of the percentage of cells showing centromeric CENP - A 24 hr or 48 hr after IAA WO . Each dot represents one experiment ( ~ 30 - 50 cells per condition per experiment ) , error bars represent standard deviation ( SD ) of 5 independent experiments .

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(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.


( E ) Quantification of centromeric CENP - A levels normalized to non - treated level . Each dot represents one experiment , error bars represent SD . Unpaired t test : * * * p = 0 . 0005 .

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(E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005.


( F ) Stills of live cell imaging to follow de novo CENP - AEA reloading in RPE - 1 cells harboring endogenously tagged CENP - Bmcherry . Images were taken every 15 min . White dashed circles contour nuclei prior / after mitosis and cells during mitosis based on bright field images .

(F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images.


( G ) Dot plot showing the timing of CENP - AEA reloading after anaphase onset in the indicated cell lines . Each dot represents one cell , error bars represent standard deviation .

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(G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation.