( C , D ) U2OS cells were treated with 30 µM 3 , 4 - DC for 6 h , and then endogenous TFEB translocation was assessed by immunostaining ( C ) . TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei ( D ) . Data are means ± SD ( * * * = p < 0 . 001 ) . Representative images are shown in ( C ) . Scale bar equals 10 µm .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 O O O O O O O O O O O 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, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm.


( E , F ) U2OS cells were treated with 30 µM 3 , 4 - DC for 6 h or were left untreated . Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page .

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

(E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page.


MEF wild type and TSC2 knockout ( TSC2 KO ) cells were treated with 3 , 4 - DC for 6 h . The cells were fixed and immunofluorescence was performed to detect endogenous TFEB . The ratio of TFEB intensities between nuclei and cytoplasm was calculated in ( H ) . Data are means ± SD ( * * * = p < 0 . 001 versus Ctr ; # # # = p < 0 . 001 versus WT / 3 , 4 - DC ) . Representative images are shown in ( G ) . Scale bar equals 10 µm .

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

MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm.


MEF wild type and TSC2 knockout ( TSC2 KO ) cells were treated with 3 , 4 - DC for 6 h . ( I ) After the treatment , nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels .

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

MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels.


( J - L ) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3 , 4 - DC . Antibodies against LC3 , p62 , TSC2 , or GAPDH were administrated to detect protein levels , and phosphorylation of mTOR substrate p70 S6K at Threonine 389 ( T389 ) was measured with the phosphorylation specific antibody ( P - p70 ( T389 ) ) and anti - p70 antibody . The bands intensities of LC3 - II , p62 , and GAPDH were measured with Image J , and the ratios of LC3 - II / GAPDH and p62 / GAPDH were calculated in ( K ) and ( L ) . Data are means ± SEM of at least three independent experiments ( * * = p < 0 . 01 , * * * = p < 0 . 001 vs Ctr ; # = p < 0 . 05 , # # = p < 0 . 01 vs WT / 3 , 4 - DC )

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

(J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC)


( M , N ) U2OS cells treated with 3 , 4 - DC or Torin for 6 h were collected and processed for western blot . TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody ( P - TFEB ( S211 ) ) and total TFEB protein level was also detected with anti - TFEB . The bands intensities of P - TFEB and total TFEB were measured with Image J , and their ratio was calculated in ( N ) . Data are means ± SEM of at least three independent experiments ( * = p < 0 . 05 vs Ctr ) .

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

(M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).


U2OS - GFP - LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS - PAGE and

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

U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


U2OS - GFP - LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT cells treated with 3 , 4 - DC ) . Representative images are shown Scale bar equals 10 µm .

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

U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm.


U2OS wild type ( wt ) and TFEB knockout ( TFEB KO ) GFP - LC3 cells were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS - PAGE and

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

U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


U2OS wild type ( wt ) and TFEB knockout ( TFEB KO ) GFP - LC3 cells were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy ata are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT cells treated with 3 , 4 - DC ) . Representative images are shown cale bar equals 10 µm .

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U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy ata are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown cale bar equals 10 µm.


U2OS wild type ( WT ) , TFEB or TFE3 knockout , or double knockout ( TF DKO ) cells were treated with 30 µM 3 , 4 - DC for 16 h . Following , cells were collected and SDS - PAGE and immunoblots were performed as described before . TFEB , TFE3 , LC3 , p62 , and GAPDH protein levels were measured with specific antibodies ( G ) .

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

U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G).


U2OS wild type ( WT ) , TFEB or TFE3 knockout , or double knockout ( TF DKO ) cells were treated with 30 µM 3 , 4 - DC for 16 h . GFP - LC3 dots were quantified as indicator of autophagy ( H ) . Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 versus Ctr , respectively ) .

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

U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively).


( I ) U2OS WT , knockout for TFEB ( TFEB - / - ) or TFE3 ( TFE3 - / - ) , double knockout ( DKO ) were treated with 3 , 4 - DC for 24 h and stained with LysoTracker Red for 30 min . Thereafter , the red ( positive ) dots were measured . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) .

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

(I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).


( J ) Relative mRNA expression levels of key autophagy genes such as Lamp1 , Lc3b and p62 were detected in controls and 3 , 4 - DC treated U2OS cells . Data is depicted as a heatmap showing means of at least three independent experiments ( * * * = p < 0 . 001 versus WT / Ctr and # = p < 0 . 05 , # # = p < 0 . 01 , as compared to WT treated with 3 , 4 - DC ) .

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

(J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC).


( A - D ) Female C57 / BL6 animals were treated with 3 , 4 - DC for 24 h . Following , liver and heart tissue was excised and cells were subjected to subcellular fractionation . Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 ( A , C ) . GAPDH and H3 were used as controls for cytosolic and nuclear fractions , respectively . Band intensities of nuclear TFEB , TFE3 and H3 were measured and ratios of nuclear TFs versus H3 ( TFsnuc / H3 ) were calculated in ( B , D ) . Data are means ± SEM of at least three mice ( * * * = p < 0 . 001 versus Ctr )

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

(A-D) Female C57/BL6 animals were treated with 3,4-DC for 24 h. Following, liver and heart tissue was excised and cells were subjected to subcellular fractionation. Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 (A, C). GAPDH and H3 were used as controls for cytosolic and nuclear fractions, respectively. Band intensities of nuclear TFEB, TFE3 and H3 were measured and ratios of nuclear TFs versus H3 (TFsnuc/H3) were calculated in (B, D). Data are means ± SEM of at least three mice (*** = p < 0.001 versus Ctr)


GFP - LC3 expressing mice were i . p . injected with 3 , 4 - DC for 24 h . Leupeptin ( Leu ) was used to test autophagic flux in vivo and GFP - LC3 dots were measured in liver tissue . Data are means ± SEM of at least three mice ( * = p < 0 . 05 versus ctr without Leu ; # = p < 0 . 05 versus ctr with Leu ) .

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

GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).


C57 / BL6 mice were i . p . injected with 3 , 4 - DC or were starved for 24 hours . Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control . Band intensities of GAPDH and LC3 - II were measured and ratios LC3 - II versus GAPDH ( LC3 - II / GAPDH ) were calculated Data are means ± SEM of at least three mice ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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

C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).


GFP - LC3 expressing mice were i . p . injected with 3 , 4 - DC for 24 h . Leupeptin ( Leu ) was used to test autophagic flux in vivo and GFP - LC3 dots were measured in heart ( I , J ) tissue . Data are means ± SEM of at least three mice ( * = p < 0 . 05 versus ctr without Leu ; # = p < 0 . 05 versus ctr with Leu ) .

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

GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in heart (I, J) tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).


C57 / BL6 mice were i . p . injected with 3 , 4 - DC or were starved for 24 hours . Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control . Band intensities of GAPDH and LC3 - II were measured and ratios LC3 - II versus GAPDH ( LC3 - II / GAPDH ) were calculated Data are means ± SEM of at least three mice ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).


( A ) Twelve weeks - old wild - type and cardiac specific Atg7 knockout ( Atg7cKO ) mice were injected with corn oil ( vehicle control , Ctr ) or 3 , 4 - DC 24 h before surgery and subjected to 3 h prolonged ischemia . Representative images of left ventricular ( LV ) myocardial sections after alcian blue and triphenyltetrazolium chloride ( TTC ) staining are depicted . Scale bar equals 1 mm .

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

(A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm.


( B , C ) The size of the infarction area ( indicated by pale color ) per area at risk ( AAR ) ( B ) and the global AAR per LV ( C ) were measured ( mean value ± SEM , * * p < 0 . 01 , # p < 0 . 05 vs WT / Ctr ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C) The size of the infarction area (indicated by pale color) per area at risk (AAR) (B) and the global AAR per LV (C) were measured (mean value ±SEM, **p<0.01, #p<0.05 vs WT/Ctr).


A . Induction of autophagy in murine MCA205 fibrosarcomas . Cells were treated with 3 , 4 DC alone or in combination with chloroquine , and were harvested 6 hours later for immunoblot detection of LC3 lipidation .

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

A. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation.


( C - E ) . Cytofluorometric characterization of tumor infiltrating lymphocytes ( TIL ) , in particular FOXP3 + regulatory T cells ( Treg ) , CD8 + cytotoxic T lymphocytes and the ratio of CD8 + T cells over Treg .

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

(C-E). Cytofluorometric characterization of tumor infiltrating lymphocytes (TIL), in particular FOXP3+ regulatory T cells (Treg), CD8+ cytotoxic T lymphocytes and the ratio of CD8+ T cells over Treg.


Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl / 6 mice treated as

Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl/6 mice treated as indicated


Growth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD ( H - I ) and were evolving in immunocompetent C57Bl / 6 mice or immunodeficient nu / nu mice ( G ) , treated as

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Growth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD (H-I) and were evolving in immunocompetent C57Bl/6 mice or immunodeficient nu/nu mice (G), treated as indicated


( J - L ) Immunocompetent C57Bl / 6 mice were subcutaneously inoculated with TFEB / TFE3 double knockdown MCA205 cells or its scramble control cells ( K ) . When tumors became palpable , mice were treated as indicated Tumor growth curves from mice subjected to 3 , 4 - DC administration alone or in combination with MTX are shown ( J , L ) . Asterisks indicate significant effect of MTX with respect to untreated controls ( mean value ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) , while hash symbols refer to the comparison of the effects of MTX plus 3 , 4 - DC to MTX alone ( # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 ) ( n = 6 - 14 ) .

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(J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14).


B Expression levels of RBM15 and AS - RBM15 in different blood lineages by meta - analysis of RNA - seq data [ 38 ] : HSC ( hematopoietic stem cells ) , MPP ( multipotent progenitor cells ) , CMP ( common myeloid progenitor cells ) , CLP ( common lymphoid progenitor cells ) , GMP ( Granulocyte - macrophage progenitor cells ) , MEP ( megakaryocyte - erythrocyte progenitor cells ) , MK ( megakaryocyte ) , and EB ( erythroid body ) .

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B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).


C Relative expression levels of AS - RBM15 ( left ) and RBM15 ( right ) in MEG - 01 cells treated with PMA were measured by real - time PCR assays ( n = 4 , mean ± SD ) . SD : standard deviation .

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C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.


D Relative expression levels of AS - RBM15 and RBM15 in CD34 + cells grown in pro - MK medium were measured by real - time PCR . GAPDH was used as an internal control ( n = 3 , mean ± SD ) . Also see Fig EV 1 .

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D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.


A CFU - MK assays with CD34 + cells expressing AS - RBM15 . MK colonies staining for acetylcholinesterase activity were counted ( right ) . P value was calculated using Student ' s t test ( n = 3 , mean ± SD ) .

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A CFU-MK assays with CD34+ cells expressing AS-RBM15. MK colonies staining for acetylcholinesterase activity were counted (right). P value was calculated using Student's t test (n=3, mean ± SD).


B The number of CD41 + CD42 + mature MK cells was determined by FACS analysis . CD34 + cells expressing sh - AS - RBM15 were grown in pro - MK medium containing thrombopoietin and stem cell factor ( TPO / SCF ) for five days ( n = 4 , mean ± SD ) .

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B The number of CD41+CD42+ mature MK cells was determined by FACS analysis. CD34+ cells expressing sh-AS-RBM15 were grown in pro-MK medium containing thrombopoietin and stem cell factor (TPO/SCF) for five days (n=4, mean ± SD).


D FACS analysis of CD34 + cells grown in pro - MK medium and expressing AS - RBM15 full length or Δ5 or Δ3 . The CD41 + CD42 + cell percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) .

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D FACS analysis of CD34+ cells grown in pro-MK medium and expressing AS-RBM15 full length or Δ5 or Δ3. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).


E The percentage of CD41 + CD42 + cells in CD34 + cells expressing AS - RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro - MK medium for five days . P value was calculated by t test , p < 0 . 01 , ( n = 3 , mean ± SD ) .

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E The percentage of CD41+CD42+ cells in CD34+ cells expressing AS-RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro-MK medium for five days. P value was calculated by t test, p<0.01, (n=3, mean ± SD).


A RBM15 protein levels in leukemia cell lines ( MEG - 01 , K562 , HEL ) were assessed by Western blotting ( WB ) ( left ) ( n = 3 ) . The mRNA levels of RBM15 in cells overexpressing AS - RBM15 were measured by real - time PCR ( right , n = 3 , mean ± SD ) . GAPDH was used as an internal control for WB and PCR .

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A RBM15 protein levels in leukemia cell lines (MEG-01, K562, HEL) were assessed by Western blotting (WB) (left) (n=3). The mRNA levels of RBM15 in cells overexpressing AS-RBM15 were measured by real-time PCR (right, n=3, mean ± SD). GAPDH was used as an internal control for WB and PCR.


B Protein levels ( left ) and mRNA levels ( right ) of RBM15 in MEG - 01 cells expressing two different shRNAs against AS - RBM15 respectively were measured using WBs and real - time PCR assays , respectively ( n = 3 , mean ± SD ) .

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B Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing two different shRNAs against AS-RBM15 respectively were measured using WBs and real-time PCR assays, respectively (n=3, mean ± SD).


C Protein levels ( left ) and mRNA levels ( right ) of RBM15 in MEG - 01 cells expressing truncated AS - RBM15 RNAs ( the schematic as shown on Fig 2C ) were measured using WB and real - time PCR respectively ( n = 3 , mean ± SD ) .

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C Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing truncated AS-RBM15 RNAs (the schematic as shown on Fig 2C) were measured using WB and real-time PCR respectively (n=3, mean ± SD).


D The role of AS - RBM15 in protein translation was assessed by reporter assays . The reporter under control of the CMV promoter contains the 5 ' UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control . The reporter plasmid was cotransfected with plasmids expressing full - length , exon 1 and Δ5 AS - RBM15 respectively . The vector reporter without 5 ' UTR is a negative control . ( n = 3 , mean ± SD ) . P values were calculated by one - way ANOVA test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5'UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5'UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).


A Cytoplasmic extracts from MEG - 01 cell lines stably expressing AS - RBM15 , Exon 1 , or shRNA - 02 against AS - RBM15 were fractionated through sucrose gradients ( 15 % - 60 % ) ( n = 4 ) . Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm ( A254 ) . The arrow indicates increasing polyribosome weights . Low molecular weight , LMW ; high molecular weight , HMW .

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A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW.


B The distributions of RBM15 mRNA ( top ) , GAPDH mRNA ( middle ) , and AS - RBM15 ( bottom ) were measured by real - time PCR and represented as percentages of total RNA in the fractions ( n = 3 ) .

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B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).


C MS2 pull - down assays : AS - RBM15 full - length ( FL ) and exon 1 were fused to MS2RNA as baits . Plasmids expressing MS2 binding protein ( HA - tagged ) and fusion baits were co - transfected into 293T cells . HA antibody was used to immunoprecipitateHA - MS2 binding protein as shown by a WB on the bottom panel . RBM15 mRNA and MS2 fusions were assessed by real - time PCR ( n = 3 , mean ± SD ) . Fold enrichment was calculated as 2 to the power of [ ( CI . R - Cb . R ) - ( CI . MS2 - Cb . MS2 ) ] . I : input i . e . the RNA in solution before immunoprecipitation , R : RBM15 mRNA , b : bead - bound mRNA , MS2 : RNA levels of MS2 fusions , C : number of cycles in real - time PCR . One - way ANONA for p value : p < 0 . 05 .

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C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2RNA as baits. Plasmids expressing MS2 binding protein ( HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitateHA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05.


D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS - RBM15 - MS2 and MS2 ( n = 3 ) .

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D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3).


E RBM15 mRNA interacts with AS - RBM15 via RNA - RNA interaction . AS - RBM15 - MS2 - associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15 , AS - RBM15 and MS2 under denaturing ( heat ) and non - denaturing conditions ( n = 3 ) .

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E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3).


F RNase A protection assays for detecting endogenous association of AS - RBM15 and RBM15 mRNA ( n = 4 ) . RNA was extracted from MEG - 01 cells with phenol and digested with RNase A . cDNA was synthesized after RNase A treatment and detected using AS - RBM15 exon - specific primers as shown in the schematic diagram on top . RT : reverse transcriptase .

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F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase.


G MEG - 01 cells expressing AS - RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes . Cells were harvested for WB using antibodies against GAPDH and RBM15 ( n = 2 ) .

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G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).


H The RBM15 5 ' UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS - RBM15 ( n = 4 , mean ± SD ) . P values were calculated by one - way ANOVA test ( * * p < 0 . 01 and * * * p < 0 . 001 ) .

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H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).


The subcellular distribution of RBM15 and AS - RBM15 in CD34 + cells grown in TPO - containing medium for 5 days . Cytoplasmic and nuclear RNA levels of AS - RBM15 and RBM15 were normalized to GAPDH ( control for cytoplasmic fraction ) and MALAT1 ( control for nuclear fraction ) , respectively , using the ΔΔCt method ( n = 4 , mean ± SD ) .

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The subcellular distribution of RBM15 and AS-RBM15 in CD34+ cells grown in TPO-containing medium for 5 days. Cytoplasmic and nuclear RNA levels of AS-RBM15 and RBM15 were normalized to GAPDH (control for cytoplasmic fraction) and MALAT1 (control for nuclear fraction), respectively, using the ΔΔCt method (n=4, mean ± SD).


A RUNX1 expression levels in human CD34 + cells grown in pro - MK medium in a time course were measured by real - time PCR ( n = 4 , mean ± SD ) .

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A RUNX1 expression levels in human CD34+ cells grown in pro-MK medium in a time course were measured by real-time PCR (n=4, mean ± SD).


B CD34 + cells were grown in pro - MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis . The CD41 + CD42 + cell percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) .

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B CD34+ cells were grown in pro-MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).


C The mRNA expression levels of RUNX1 , RBM15 , and AS - RBM15 in RUNX1 knockdown MEG - 01 cells were measured by real - time PCR ( n = 4 , mean ± SD ) .

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C The mRNA expression levels of RUNX1, RBM15, and AS-RBM15 in RUNX1 knockdown MEG-01 cells were measured by real-time PCR (n=4, mean ± SD).


D Chromatin immunoprecipitation using anti - RUNX1 antibody . Immunoprecipitated DNA fragments were measured by real - time PCR using HEL cells expressing shRUNX1 and control ( n = 4 , means ± SD ) .

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D Chromatin immunoprecipitation using anti-RUNX1 antibody. Immunoprecipitated DNA fragments were measured by real-time PCR using HEL cells expressing shRUNX1 and control (n=4, means ± SD).


E CD34 + human cord blood cells were transduced with MIG retroviruses co - expressing A / E ( AML1 - ETO , aka RUNX1 - ETO ) and GFP . Around 5 days after transduction , the GFP + CD11b - cells were sorted for real - time PCR ( n = 4 ) . Also see Fig EV6 for RUNX1 - ETO binding profile on RBM15 locus .

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E CD34+ human cord blood cells were transduced with MIG retroviruses co-expressing A/E (AML1-ETO, aka RUNX1-ETO) and GFP. Around 5 days after transduction, the GFP+CD11b- cells were sorted for real-time PCR (n=4). Also see Fig EV6 for RUNX1-ETO binding profile on RBM15 locus.


A ) Representative specimen of colorectal ( CRC ) primary tumour stained with haematoxylin / eosin / saffron ( HES ) , or antibodies against E - cadherin or Vimentin . i ) The blue , orange and pink dotted lines highlight the normal mucosa , the submucosa and the muscularis propria respectively . Red dotted line highlights the neoplastic tissue . Black arrowheads indicate the direction of invasion . Boxed regions ii and iii show high magnification of normal colonic glands ( ii ) and the CRC invasive front ( iii ) . Scale bar : 2 mm and 500 μm .

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A) Representative specimen of colorectal (CRC) primary tumour stained with haematoxylin/eosin/saffron (HES), or antibodies against E-cadherin or Vimentin. i) The blue, orange and pink dotted lines highlight the normal mucosa, the submucosa and the muscularis propria respectively. Red dotted line highlights the neoplastic tissue. Black arrowheads indicate the direction of invasion. Boxed regions ii and iii show high magnification of normal colonic glands (ii) and the CRC invasive front (iii). Scale bar: 2 mm and 500 μm.


B ) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin . Boxed regions i , ii and iii are high magnifications of the luminal cavity of normal colonic gland ( i ) and colorectal carcinoma glands ( ii and iii ) . Aarrowheads point to the apical pole enriched in villin . Scale bars : 50 μm .

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B) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin. Boxed regions i, ii and iii are high magnifications of the luminal cavity of normal colonic gland (i) and colorectal carcinoma glands (ii and iii). Aarrowheads point to the apical pole enriched in villin. Scale bars: 50 μm.


( C ) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16

(C) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16 patients


A ) Representative confocal images of non - invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen ( Ezrin ) , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii , iii and iv are displayed at high magnification . Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars show off - centered nuclei . Scale bars : 20 μm .

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A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.


Graphs representing the percentage of adenocarcinoma explants displaying lumens ( B ) from 8 patients . Evaluation performed from DIC observation of live tissues , at least 50 explants were analysed per patients . The error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( ns : non significant ) .

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Graphs representing the percentage of adenocarcinoma explants displaying lumens (B) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).


Graphs representing the percentage of adenocarcinoma explants displaying protrusions ( C ) from 8 patients . Evaluation performed from DIC observation of live tissues , at least 50 explants were analysed per patients . The error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( ns : non significant ) .

Graphs representing the percentage of adenocarcinoma explants displaying protrusions (C) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).


D ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel monitored by DIC microscopy over 2 days Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars point to lumens . Scale bars : 50 μm .

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

D) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel monitored by DIC microscopy over 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.


E ) Images of an invading gland of a colorectal NOS adenocarcinoma , at the light microscopic level ( i , semithin section , 100x objective ) , and as seen by electron microscopy ( ii , 1000x magnification ; and iii , 4000x magnification ) . Note that the bulk of the invading neoplastic gland is in a state of glandular organization ( regions denoted by arrowheads in the left panel ) : epithelia ( E , middle panel ) are polarized to form a lumen ( L ) , and are attached to a basal membrane ( asterisk ) by hemidesmosomes ; the gland is surrounded by fibroblasts ( F ) . However , this organization is focally disrupted at the tubular invasion pole ( arrows in the left panel ) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of an invading gland of a colorectal NOS adenocarcinoma, at the light microscopic level (i, semithin section, 100x objective), and as seen by electron microscopy (ii, 1000x magnification; and iii, 4000x magnification). Note that the bulk of the invading neoplastic gland is in a state of glandular organization (regions denoted by arrowheads in the left panel): epithelia (E, middle panel) are polarized to form a lumen (L), and are attached to a basal membrane (asterisk) by hemidesmosomes; the gland is surrounded by fibroblasts (F). However, this organization is focally disrupted at the tubular invasion pole (arrows in the left panel) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing (right panel).


A ) Representative images of Caco - 2 cysts transfected with control siRNA ( siGlo ) or siRNAs targeting ROCK1 and ROCK2 , fixed and stained for prominin - 1 , F - actin ( Phalloidin ) and Nuclei ( Hoechst ) . Confocal Z - sections of cysts are displayed . Arrowheads show non - protruding cells . Arrows point to protruding cells . White stars mark nuclei that are off - centered relative to the cyst ' s monolayer . Scale bars : 20 μm .

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 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 O O O 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) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.


B ) Representative confocal images of Caco - 2 cysts treated with ROCK inhibitors Y27632 and H1152 , non - treated ( NT ) or transduced with ROCK1 and ROCK2 shRNAs ( shROCK1 + 2 ) . The cysts were fixed 2 days after invasion and stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i and ii are displayed at high magnification . White arrowheads point to non - protruding cells , green arrowheads point to the apical pole , arrows point to protruding cells and white stars show off - centered nuclei . Scale bar : 20 μm .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O 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 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 O O O O O O 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 confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.


C ) Bar graph representing the percentage of protrusive Caco - 2 cysts from three independent experiments . Caco - 2 cysts non - treated ( NT ) , incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2 . The cysts were fixed 2 days after invasion , over 100 cysts were counted for each condition . Error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * p < 0 . 001 , * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).


E ) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non - treated ( NT ) , in ROCK inhibited conditions ( Y27632 , H1152 ) or in cells transfected with siRNA against ROCK1 and ROCK2 ( siROCK1 + 2 ) . The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).


F ) High magnification of boxed region from ( D ) . The top ( i ) and the bottom ( ii ) side of the Boyden chamber membrane were stained for F - actin , E - cadherin , ZO - 1 , Ezrin and Hoechst . Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion . Scale bars : 40 μm .

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

F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.


A ) Representative images of organoids formed from patient derived xenografts ( PDXs ) that have been embedded in collagen - I gels during 3 days , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) and ( + H1152 ) . At end - point , organoids were stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . i and ii are high magnification of boxed regions of + Y27632 and + H1152 organoids respectively . Arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments . Over 50 organoids were counted per condition ( Means ± SEM ) . P values were calculated using paired t - test ( * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001).


C ) Representative images of human adenocarcinoma explants after 2 days in 3D collagen - I gel , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) and ( + H1152 ) and stained for the apical pole ( Ezrin ) , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions of NT , + Y27632 , and + H1152 explants are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . D Explants displaying ( D ) lumens from 10 patients were quantified and results presented as percentage of total explants . Over 50 explants were counted per condition ( duplicate ) and per patient ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , n . s : non significant ) .

O O 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


Explants displaying ( E ) protrusive cells from 10 patients were quantified and results presented as percentage of total explants . Over 50 explants were counted per condition ( duplicate ) and per patient ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , n . s : non significant ) .

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

Explants displaying (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


F ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars point to lumens . Scale bars : 50 μm .

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

F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.


A ) Caco - 2 cysts expressing an empty vector ( pLKO ) or shROCK1 ( # 02 ) or shROCK2 ( # 36 ) after 2 days of invasion were fixed and stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii and iii are shown at high magnifications . Arrowheads point to non - protruding cells . Arrow points to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) The number of protrusive Caco - 2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts ( Means ± SEM of at least 3 independent experiments , paired t - test , * * * p < 0 . 001 , * p < 0 . 05 n . s : non significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant).


C ) Caco - 2 cysts stably expressing GFP alone or GFP - ROCK2DN # 2 , were submitted to a 2 days period invasion assay , then fixed and stained for F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii and iii are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . White and black stars show nuclei that engage in protrusions . Scale bars : 20μm . D ) Bargraph representing the percentage of protrusive cysts transduced with GFP ( CTRL , with or without Y27632 ) or with ROCK2DN # 2 ( Means ± SEM of at least 3 independent experiments , paired t - test , * * p < 0 . 01 n . s : non significant ) .

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

C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm. D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant).


E ) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole ( arrows ) reminiscent of collective invasion . T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus - ROCK2 - DA and treated with doxycycline 1μg / ml . Scale bars : 20μm .

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

E) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm.


F ) Bar graph representing the percentage of protrusive T84 cysts in control ( WT ) , treated with ROCK inhibitor ( Y27632 ) or transduced with lentiviruses expressing GFP or GFP - ROCK2 - DA after induction by Doxycycline ( Means ± SEM of at least 3 independent experiments ) ( paired t - test , * * * p < 0 . 001 , * p < 0 . 05 ) .

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

F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05).


G ) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set ( n = 477 patients with survival annotation ) using Kaplan - Meier survival analysis . The patient cohort was divided into tumours with or without a down - regulation of ROCK2 relatively to non - tumoural tissues ( expression cut - off value corresponds to the 1st decile of ROCK2 expression in non - tumoural sample , i . e . 7 . 6 ) . Log - rank test p - values for survival distribution difference are indicated at 3 and 5 years .

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

G) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set (n=477 patients with survival annotation) using Kaplan-Meier survival analysis. The patient cohort was divided into tumours with or without a down-regulation of ROCK2 relatively to non-tumoural tissues (expression cut-off value corresponds to the 1st decile of ROCK2 expression in non-tumoural sample, i.e. 7.6). Log-rank test p-values for survival distribution difference are indicated at 3 and 5 years.


B ) DIC acquisition of fixed mosaic Caco - 2 cysts expressing GFP alone , GFP - ROCK2DN # 1 or GFP - ROCK2DN # 2 . Boxed regions i and ii are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . Scale bars : 20μm .

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 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

B) DIC acquisition of fixed mosaic Caco-2 cysts expressing GFP alone, GFP-ROCK2DN#1 or GFP-ROCK2DN#2. Boxed regions i and ii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. Scale bars: 20μm.


C ) Bar graph representing the percentage of mosaic Caco - 2 cysts transduced with GFP or GFP - ROCK2DN # , with or without Y27632 , harbouring a protruding morphology ( Means ± SEM of at least 3 independent experiments , unpaired t - test , * * * p < 0 . 001 , * p < 0 . 05 ) .

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 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

C) Bar graph representing the percentage of mosaic Caco-2 cysts transduced with GFP or GFP-ROCK2DN#, with or without Y27632, harbouring a protruding morphology (Means ± SEM of at least 3 independent experiments, unpaired t-test, ***p<0.001, *p<0.05).


D ) Quantification of the fate of the GFP positive cells , leader or follower , in mosaic cysts transduced with GFP - alone and treated with Y27632 ( GFP + Y27632 ) or GFP - ROCK2DN # 2 from 3 independent experiments ( Means ± SEM , unpaired t - test , * * * p < 0 . 001 ) .

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

D) Quantification of the fate of the GFP positive cells, leader or follower, in mosaic cysts transduced with GFP-alone and treated with Y27632 (GFP+Y27632) or GFP-ROCK2DN#2 from 3 independent experiments (Means ± SEM, unpaired t-test, ***p<0.001).


E ) Representative pull - down of RAC1 - GTP by PAK - Crib , in Caco - 2 cells treated with ROCK inhibitor Y27632 , H1152 or blebbistatin and lysed at 12 or 16 hours . Immunoblot analyses were performed with anti - Rac1 and anti - GAPDH antibody from three independent experiments .

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

E) Representative pull-down of RAC1-GTP by PAK-Crib, in Caco-2 cells treated with ROCK inhibitor Y27632, H1152 or blebbistatin and lysed at 12 or 16 hours. Immunoblot analyses were performed with anti-Rac1 and anti-GAPDH antibody from three independent experiments.


F ) DIC acquisition of fixed Caco - 2 cysts treated with ROCK inhibitors alone ( Y27632 or H1152 ) , or in combination with RAC1 inhibitor NSC23766 . Arrowheads point to non - protruding cells and arrow point to protruding cell . Scale bar 20 μm .

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

F) DIC acquisition of fixed Caco-2 cysts treated with ROCK inhibitors alone (Y27632 or H1152), or in combination with RAC1 inhibitor NSC23766. Arrowheads point to non-protruding cells and arrow point to protruding cell. Scale bar 20 μm.


G ) Bar graph representing the percentage of protruding Caco - 2 cysts in control conditions ( non - treated , NT ) or treated with NSC23766 , Y27632 or H1152 ( Means ± SEM of at least 3 independent experiments , unpaired t - test , * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p < 0 .

O O 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 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

G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05


A ) siRNA screen performed in Caco - 2 cysts treated with Y27632 : Caco - 2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days . Then , Caco - 2 cysts were treated with Y27632 and the serum gradient was induced . After 2 days , the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining . The bar graph represents the mean invasion rate . Each condition was duplicated . The red dashed line represent the median invasion rate , the red full line indicates the 3 fold decrease in invasion rate . Three hits were identified : # 2 : ARHGEF18 + RNEF , # 7 : SGEF + ARHGEF16 and # 24 : FARP1 + FARP2 ( Mean ± SD ) .

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

A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .


B ) Caco - 2 cysts stably expressing empty vector ( pGIPZ ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632 , allowed to invade for 2 days , fixed and stained for F - actin ( Phalloidin ) . Boxed regions i , ii , iii and iv are shown at high magnification . Arrowheads point to non - protruding cells , arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . C ) Bar graphs representing the percentage of protrusive cysts formed from Caco - 2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments ( Means ± SEM , paired t - test , ns : non significant , * * p < 0 . 01 ) .

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

B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01).


D ) Representative acquisition of Caco - 2 cells overexpressing GFP - FARP2 grown in 2D and stained with ZO - 1 , in non - treated ( NT ) and after 2h with Y27632 or H1152 . The dashed lines crossing the cell - cell junctions ( i , ii and iii ) are representative of the linescans measurements . Scale bars : 20μm . E ) Representative fluorescence intensity of ZO - 1 ( red ) staining and GFP - FARP2 ( green ) along the dashed line i , ii and iii represented corresponding to non - treated , Y27632 or H1152 treatments )

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

D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments)


F ) Bar graph representing the average ratio of GFP - FARP2 over ZO - 1 fluorescence intensities measured with linescans crossing cell - cell junctions . The quantification was performed on 71 cells expressing GFP - FARP2 - FL from 3 independent experiments . For each cell , between 10 and 15 lines ( 12 pixels wide ) were drawn across random region of cell - cell junctions positive for ZO - 1 staining ( Mean ± SEM Unpaired t - test * * p < 0 . 01 , * p < 0 . 05 ) .

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

F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).


A ) Caco - 2 cysts stably expressing GFP ( treated or not with Y27632 ) , or Cherry - FARP2 - full length ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH 730 / 733 ( FARP2 - del / Mut ) were allowed to invade for 2 days , fixed and stained for F - actin . Boxed regions i , ii , iii , iv and v are shown at high magnification . Arrowheads point to non - protruding cells , arrow points to protruding cells , white star shows nucleus that engages in protrusion . Scale bars : 20μm . B ) Bargraph representing the percentage of protrusive cysts expressing GFP ( treated or not with Y27632 ) or the mentioned GFP - FARP2 constructs from 3 independent experiments ( Means ± SEM , paired t - test , * * p < 0 , 01 , n . s : non significant ) .

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

A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant).


C ) Representative images of Caco - 2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin - IIA and allowed to invade for 2 days . After fixation , the cysts were stained for F - actin ( Phalloidin ) and nuclei ( DAPI ) . Representative Y27632 - induced « invasive » phenotype and Blebbistatin - induced « dendritic » phenotype are displayed . Arrows point to protruding cells , white star shows nucleus that engages in protrusion and arrowheads point to " dendritic " protrusions . D ) Bar graph representing the percentage of cysts with the " invasive " or " dendritic " phenotypes after treatment with Y27632 , Blebbistatin or siMyosin - IIA or siMyosin - IIB from at least 3 independent experiments ( Means ± SEM , unpaired t - test , * p < 0 , 05 , n . s : non significant ) .

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

C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions. D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant).


E ) Mature Caco - 2 cysts were transduced with GFP or Cherry tagged FARP2 - full length ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH / 730 / 733 ( FARP2 - del / Mut ) , and treated ( or not , NT ) with Blebbistatin . 2 days after invasion , the cysts were fixed and stained for F - actin . Boxed regions i , ii , iii and iv are shown at high magnification . Arrowheads point to non - protruding cells ; arrow points to protruding cells and white stars show nucleus that engage in protrusions . Scale bars : 20 μm . F ) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin , from at least 3 independent experiments ( Means ± SEM , t - test , * * p < 0 , 01 , n . s : non significant ) .

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

E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).


Purified CUL3WT and CUL3Δ403 - 459 analysed by SDS - PAGE and Coomassie blue staining , following expression in SF21 cells , and DAC - tag - affinity purification and tag cleavage ( see Materials and Methods ) . Bands migrating between 62 - and 98 - kDa protein markers are CUL3WT and CUL3Δ403 - 459 , respectively , while the band below 17 kDa is Rbx1 . The immunoblot for RBX1 is shown on the right . The band in CUL3WT denoted with * is co - purified insect cell CAND1 , which was later removed by SEC . All protein identities were confirmed by mass spectrometry .

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

Purified CUL3WT and CUL3Δ403-459 analysed by SDS-PAGE and Coomassie blue staining, following expression in SF21 cells, and DAC-tag-affinity purification and tag cleavage (see Materials and Methods). Bands migrating between 62- and 98-kDa protein markers are CUL3WT and CUL3Δ403-459, respectively, while the band below 17 kDa is Rbx1. The immunoblot for RBX1 is shown on the right. The band in CUL3WT denoted with * is co-purified insect cell CAND1, which was later removed by SEC. All protein identities were confirmed by mass spectrometry.


KLHL3 binding to CUL3WT and CUL3Δ403 - 459 is comparable as shown by pull - down assays with a KLHL3 resin generated from cross - linking anti - KLHL3 antibody to Protein G sepharose , and then binding saturating amounts of KLHL3 to the anti - KLHL3 resin ( denoted KLHL3R ) , the control resin ( ControlR ) is Protein G sepharose ( see Materials and Methods for further detail ) . CUL3WT or CUL3Δ403 - 459 was incubated with either KLHL3R or ControlR at 4 ° C for 1 h and then washed thrice with 1 × PBS , resin volumes were minimised , and SDS - Laemmli buffer was used to elute protein from resin prior to analysis by SDS - PAGE and staining with colloidal blue .

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

KLHL3 binding to CUL3WT and CUL3Δ403-459 is comparable as shown by pull-down assays with a KLHL3 resin generated from cross-linking anti-KLHL3 antibody to Protein G sepharose, and then binding saturating amounts of KLHL3 to the anti-KLHL3 resin (denoted KLHL3R), the control resin (ControlR) is Protein G sepharose (see Materials and Methods for further detail). CUL3WT or CUL3Δ403-459 was incubated with either KLHL3R or ControlR at 4°C for 1 h and then washed thrice with 1× PBS, resin volumes were minimised, and SDS-Laemmli buffer was used to elute protein from resin prior to analysis by SDS-PAGE and staining with colloidal blue.


UBE2D3 ˜ ubiquitin - release assays analysed by non - reducing SDS - PAGE and immunodetection . His - tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE , and charging was stopped by ATP depletion with apyrase . No CUL3 ( negative control , lane 1 ) , CUL3WT or CUL3Δ403 - 459 was then added to the charged E2 and the activity of the RING domain , RBX1 , monitored by the hydrolysis of ubiquitin from UBE2D3 , visualised by immunodetection with anti - His antibody ( upper panel , " ˜ " denotes thioester bond ) . The lower panel is an immunoblot for CUL3 , showing equivalent amount of both CUL3WT and CUL3Δ403 - 459 was added to the reaction mix ; over time , the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin . The right two lanes are control lanes incubated for 10 min with E2 ˜ Ubi but then treated with reducing agent prior to SDS - PAGE .

B-MEASURED_VAR O B-MEASURED_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 B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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-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 O O O O O O O O O O B-CONTROLLED_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

UBE2D3˜ubiquitin-release assays analysed by non-reducing SDS-PAGE and immunodetection. His-tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE, and charging was stopped by ATP depletion with apyrase. No CUL3 (negative control, lane 1), CUL3WT or CUL3Δ403-459 was then added to the charged E2 and the activity of the RING domain, RBX1, monitored by the hydrolysis of ubiquitin from UBE2D3, visualised by immunodetection with anti-His antibody (upper panel, "˜" denotes thioester bond). The lower panel is an immunoblot for CUL3, showing equivalent amount of both CUL3WT and CUL3Δ403-459 was added to the reaction mix; over time, the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin. The right two lanes are control lanes incubated for 10 min with E2˜Ubi but then treated with reducing agent prior to SDS-PAGE.


Structural model of CUL3WT based on the structure of CUL1 ( PDB : 1LDK ) , highlighting the residues deleted in the PHA2E mutant which are located close to the N - terminal domain ( NTD ) and C - terminal domain ( CTD ) border , using Phyre2 and Chimera ( see Materials and Methods ) . The NTD is coloured purple , the CTD cyan , and residues 403 - 459 orange .

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

Structural model of CUL3WT based on the structure of CUL1 (PDB:1LDK), highlighting the residues deleted in the PHA2E mutant which are located close to the N-terminal domain (NTD) and C-terminal domain (CTD) border, using Phyre2 and Chimera (see Materials and Methods). The NTD is coloured purple, the CTD cyan, and residues 403-459 orange.


Time course of in vitro neddylation reactions . Purified NAE , UBE2M and Nedd8 were mixed with either CUL3WT or CUL3Δ403 - 459 and incubated at 30 ° C for the time indicated . Due to the covalent attachment of Nedd8 ( 8 . 6 kDa ) , modified proteins have decreased mobility . The band shift observed for CUL3WT ( lane 1 versus lane 2 ) is representative of a single Nedd8 modification , while the multiple bands in the CUL3Δ403 - 459 reaction reflect the attachment of up to three Nedd8 molecules ( lane 5 versus lane 8 ) .

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

Time course of in vitro neddylation reactions. Purified NAE, UBE2M and Nedd8 were mixed with either CUL3WT or CUL3Δ403-459 and incubated at 30°C for the time indicated. Due to the covalent attachment of Nedd8 (8.6 kDa), modified proteins have decreased mobility. The band shift observed for CUL3WT (lane 1 versus lane 2) is representative of a single Nedd8 modification, while the multiple bands in the CUL3Δ403-459 reaction reflect the attachment of up to three Nedd8 molecules (lane 5 versus lane 8).


HEK - 293 cells over - expressing either FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 were immunoprecipitated with M2 Flag - binding sepharose , in the presence of a deneddylase inhibitor ( OPT ) . Extract , cells were lysed in the presence of OPT and clarified by centrifugation . IP : M2 , clarified supernatant samples were incubated for 1 h at 4 ° C with M2 ( anti - Flag ) sepharose , then washed with PBS and eluted with SDS - Laemmli buffer , prior to SDS - PAGE and immunodetection with anti - FLAG antibody . Bands are labelled as follows : 1 . CUL3WT , 2 . CUL3WT - N8 , 3 . CUL3Δ403 - 459 , 4 . CUL3Δ403 - 459 - N8 , 5 . CUL3Δ403 - 459 - 2 ( N8 ) .

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

HEK-293 cells over-expressing either FLAG-CUL3WT or FLAG-CUL3Δ403-459 were immunoprecipitated with M2 Flag-binding sepharose, in the presence of a deneddylase inhibitor (OPT). Extract, cells were lysed in the presence of OPT and clarified by centrifugation. IP:M2, clarified supernatant samples were incubated for 1 h at 4°C with M2 (anti-Flag) sepharose, then washed with PBS and eluted with SDS-Laemmli buffer, prior to SDS-PAGE and immunodetection with anti-FLAG antibody. Bands are labelled as follows: 1. CUL3WT, 2. CUL3WT-N8, 3. CUL3Δ403-459, 4. CUL3Δ403-459-N8, 5. CUL3Δ403-459-2(N8).


Neddylation in HEK - 293 cells over - expressing FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 was blocked by treatment with a Nedd8 - E1 - enzyme inhibitor , 3 μM MLN4924 ( Millennium Pharmaceuticals ) , in cells over - expressing FLAG - CUL3WT and FLAG - CUL3Δ403 - 459 respectively . Cell extracts were sampled over time in the presence of OPT to prevent de - neddylation and subjected to SDS - PAGE and immunodetection of the FLAG tag .

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

Neddylation in HEK-293 cells over-expressing FLAG-CUL3WT or FLAG-CUL3Δ403-459 was blocked by treatment with a Nedd8-E1-enzyme inhibitor, 3 μM MLN4924 (Millennium Pharmaceuticals), in cells over-expressing FLAG-CUL3WT and FLAG-CUL3Δ403-459 respectively. Cell extracts were sampled over time in the presence of OPT to prevent de-neddylation and subjected to SDS-PAGE and immunodetection of the FLAG tag.


FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 was immunoprecipitated with M2 - sepharose as described in ( F ) . + MLN4924 indicates cell culture media supplementation with 3 μM MLN4924 for 5 h prior to cell lysis to achieve complete deneddylation of CUL3 . Following SDS - PAGE , immunoblotting with COP9 Signalosome ( CSN ) or CAND1 antibodies determined the interaction between these Cullin regulators and FLAG - CUL3WT and FLAG - CUL3Δ403 - 459 , respectively .

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

FLAG-CUL3WT or FLAG-CUL3Δ403-459 was immunoprecipitated with M2-sepharose as described in (F). +MLN4924 indicates cell culture media supplementation with 3 μM MLN4924 for 5 h prior to cell lysis to achieve complete deneddylation of CUL3. Following SDS-PAGE, immunoblotting with COP9 Signalosome (CSN) or CAND1 antibodies determined the interaction between these Cullin regulators and FLAG-CUL3WT and FLAG-CUL3Δ403-459, respectively.


GST - CAND1 pull - down assays with CUL3 proteins . Purified GST or GST - CAND1 immobilised on glutathione - sepharose was mixed with either purified CUL3WT or CUL3Δ403 - 459 and incubated at 4 ° C for 1 h . The sepharose was then washed thrice and eluted from the resin by incubation with SDS - Laemmli buffer prior to SDS - PAGE and Coomassie staining . CUL3WT but not CUL3Δ403 - 459 interacts with CAND1 .

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

GST-CAND1 pull-down assays with CUL3 proteins. Purified GST or GST-CAND1 immobilised on glutathione-sepharose was mixed with either purified CUL3WT or CUL3Δ403-459 and incubated at 4°C for 1 h. The sepharose was then washed thrice and eluted from the resin by incubation with SDS-Laemmli buffer prior to SDS-PAGE and Coomassie staining. CUL3WT but not CUL3Δ403-459 interacts with CAND1.