( c , d ) Bif - 1 + / + and Bif - 1 − / − MEFs were pre - treated with 10 mM 3 - methyladenine ( 3 - MA ) , 0 . 2 μM wortmannin ( WM ) or control DMSO for 30 min followed by culture in EBSS for the indicated times . The percentage of dead cells was determined by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) . Immunoblot analyses were performed to detect LC3 modification and caspase - 3 activation .

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

(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Immunoblot analyses were performed to detect LC3 modification and caspase-3 activation.


( e ) Atg5 + / + and Atg5 − / − MEFs were cultured in EBSS in the presence of 50 μM of z - VAD - fmk or control DMSO for 0 , 6 or 12 h . The percentage of dead cells was determined by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) .

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 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

(e) Atg5+/+ and Atg5−/− MEFs were cultured in EBSS in the presence of 50 μM of z-VAD-fmk or control DMSO for 0, 6 or 12 h. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).


( f ) Total cell lysates ( TCL ) were prepared from Atg5 + / + and Atg5 − / − MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses . Uncropped images of the immunoblots in a , b , d and f are shown in the Supplementary Information , Fig . S5 .

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 O O O O O O O O O O O O O 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) Total cell lysates (TCL) were prepared from Atg5+/+ and Atg5−/− MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses. Uncropped images of the immunoblots in a, b, d and f are shown in the Supplementary Information, Fig. S5.


( a ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in complete medium ( CM ) or EBSS for 3 h and analysed by transmission electron microscopy . Arrows indicate autophagosomes . The number of autophagosomes per cross - sectioned cell was counted ( mean ± s . d . ; n = 42 ) .

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

(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in complete medium (CM) or EBSS for 3 h and analysed by transmission electron microscopy. Arrows indicate autophagosomes. The number of autophagosomes per cross-sectioned cell was counted (mean ± s.d.; n = 42).


( b ) Bif - 1 + / + and Bif - 1 − / − MEFs were transfected with GFP - LC3 , cultured in LC3 or complete medium for 3 h , then analysed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was counted ( mean ± s . d . ; n = 35 ) .

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

(b) Bif-1+/+ and Bif-1−/− MEFs were transfected with GFP-LC3, cultured in LC3 or complete medium for 3 h, then analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 35).


( c ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti - LC3 , anti - α - tubulin and anti - β - actin antibodies .

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

(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti-LC3, anti-α-tubulin and anti-β-actin antibodies.


( d ) Bif - 1 − / − MEFs stably transfected with a Bif - 1 expression plasmid or control empty vector were cultured in EBSS for 0 , 3 or 6 h and subjected to immunoblot analysis . The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3 - II out of total LC3 ( LC3 - I + LC3 - II ) and are represented in bar graphs .

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

(d) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were cultured in EBSS for 0, 3 or 6 h and subjected to immunoblot analysis. The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3-II out of total LC3 (LC3-I + LC3-II) and are represented in bar graphs.


( e ) Stable Bif - 1 shRNA - expressing clones or control HeLa cells transfected with GFP - LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was quantified ( mean ± s . d . ; n = 66 ) .

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 B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O

(e) Stable Bif-1 shRNA-expressing clones or control HeLa cells transfected with GFP-LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 66).


( f ) Bif - 1 knockdown HeLa cells were transfected with GFP - LC3 in combination with Bif - 1 shRNA - resistant Bif - 1 - HcRed , Bif - 1ΔBAR - HcRed or empty HcRed vector for 20 h . The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP - LC3 dots per HcRed - positive cell was determined using a fluorescence microscope ( mean ± s . d . ; n = 35 ) .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-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) Bif-1 knockdown HeLa cells were transfected with GFP-LC3 in combination with Bif-1 shRNA-resistant Bif-1-HcRed, Bif-1ΔBAR-HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 35).


( a ) COS7 cells were transfected with Bif - 1 - HcRed together with GFP - Atg5 or GFP - LC3 . Twenty hours after transfection , the cells were cultured in EBSS ( middle and bottom panels ) or complete medium ( top panel ) for 2 h and subjected to confocal microscopy analysis . Magnified images are shown as insets . Representative Bif - 1 - Atg5 or Bif - 1 - LC - 3 double - positive foci are indicated by arrows .

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

(a) COS7 cells were transfected with Bif-1-HcRed together with GFP-Atg5 or GFP-LC3. Twenty hours after transfection, the cells were cultured in EBSS (middle and bottom panels) or complete medium (top panel) for 2 h and subjected to confocal microscopy analysis. Magnified images are shown as insets. Representative Bif-1-Atg5 or Bif-1-LC-3 double-positive foci are indicated by arrows.


( b ) COS7 cells transfected with Bif - 1 - GFP were cultured in EBSS for 2 h and the localization of Bif - 1 - GFP was examined by immunogold electron microscopy using anti - GFP antibodies . Arrows indicate representative gold particles that are detected on autophagosomal membranes . The scale bars represent 10 μm in the main images and 1 μm in the insets in a , and 1 μm in b .

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 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

(b) COS7 cells transfected with Bif-1-GFP were cultured in EBSS for 2 h and the localization of Bif-1-GFP was examined by immunogold electron microscopy using anti-GFP antibodies. Arrows indicate representative gold particles that are detected on autophagosomal membranes. The scale bars represent 10 μm in the main images and 1 μm in the insets in a, and 1 μm in b.


( a ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti - Bif - 1 monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies .

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

(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies.


( b ) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti - Flag monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Myc or anti - Flag polyclonal antibodies .

O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies.


( c ) 293T cells were cotransfected with the indicated plasmids for 22 h , starved in EBSS for 2 h and subjected to immunoprecipitation with anti - Flag monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Myc or anti - Flag polyclonal antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were cotransfected with the indicated plasmids for 22 h, starved in EBSS for 2 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies.


( d ) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h , starved in 2 h for 2 h and subjected to immunoprecipitation with anti - Bif - 1 monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Bif - 1 and Beclin 1 polyclonal antibodies . The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti - UVRAG polyclonal antibody . Uncropped images of the immunoblots are shown in the Supplementary Information , Fig . S5 .

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

(d) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h, starved in 2 h for 2 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Bif-1 and Beclin 1 polyclonal antibodies. The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti-UVRAG polyclonal antibody. Uncropped images of the immunoblots are shown in the Supplementary Information, Fig. S5.


( a ) HeLa cells were transfected with 0 . 2 μg GFP - LC3 and 0 , 0 . 2 or 0 . 6 μg of Bif - 1SH3 - Myc expression plasmids for 24 h . The total amount of plasmid DNA was adjusted at 0 . 8 μg per transfection with the pcDNA3 vector . The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP - LC3 dots per GFP - positive cell was determined using a fluorescence microscope ( mean ± s . d . ; n = 40 ) .

O O O 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 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

(a) HeLa cells were transfected with 0.2 μg GFP-LC3 and 0, 0.2 or 0.6 μg of Bif-1SH3-Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP-LC3 dots per GFP-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 40).


( b ) HeLa cells were transfected with GFP - LC3 in combination with either Bif - 1SH3 - HcRed , Endophilin A1SH3 - HcRed or empty HcRed vector for 20 h . The cells were then cultured in 2 h or complete medium for 2 h and the number of GFP - LC3 dots per HcRed - positive cell was counted using a fluorescence microscope ( mean ± s . d . ; n = 46 ) . Statistical significance in a and b was determined by Student ' s t - test and the asterisks indicate P 0 . 0001 .

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 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 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

(b) HeLa cells were transfected with GFP-LC3 in combination with either Bif-1SH3-HcRed, Endophilin A1SH3-HcRed or empty HcRed vector for 20 h. The cells were then cultured in 2 h or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was counted using a fluorescence microscope (mean ± s.d.; n = 46). Statistical significance in a and b was determined by Student's t-test and the asterisks indicate P 0.0001.


( c ) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti - Flag monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Myc and anti - Flag polyclonal antibodies . Uncropped images of the immunoblots in c are shown in the Supplementary Information , Fig . S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc and anti-Flag polyclonal antibodies. Uncropped images of the immunoblots in c are shown in the Supplementary Information, Fig. S5.


( a ) Bif - 1 + / + and Bif - 1 − / − MEFs were transfected with Flag - PI ( 3 ) KC3 , then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI ( 3 ) KC3 kinase assay . The expression of Flag - PI ( 3 ) KC3 was confirmed by immunoblot analysis ( left panel ) . A representative autoradiograph is shown ( middle ) . The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation ( right ) . The data are presented as means ± s . d . of five independent experiments .

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

(a) Bif-1+/+ and Bif-1−/− MEFs were transfected with Flag-PI(3)KC3, then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI(3)KC3 kinase assay. The expression of Flag-PI(3)KC3 was confirmed by immunoblot analysis (left panel). A representative autoradiograph is shown (middle). The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation (right). The data are presented as means ± s.d. of five independent experiments.


( b ) Bif - 1 + / + and Bif - 1 − / − MEFs were transfected with p40 ( phox ) PX - EGFP , cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope . The number of p40 ( phox ) PX - EGFP - positive vesicles per cell was counted ( mean ± s . d . ; n = 35 ) . Statistical significance in a and b was determined by Student ' s t - test .

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

(b) Bif-1+/+ and Bif-1−/− MEFs were transfected with p40(phox) PX-EGFP, cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope. The number of p40(phox) PX-EGFP-positive vesicles per cell was counted (mean ± s.d.; n = 35). Statistical significance in a and b was determined by Student's t-test.


( c ) Stable Bif - 1 shRNA clones or control HeLa cells were transfected with Flag - PI ( 3 ) KC3 alone or in combination with Flag - tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI ( 3 ) KC3 lipid kinase assay . The results were quantified and are shown as relative activities ( mean ± s . d . ; n = 3 ) .

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

(c) Stable Bif-1 shRNA clones or control HeLa cells were transfected with Flag-PI(3)KC3 alone or in combination with Flag-tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI(3)KC3 lipid kinase assay. The results were quantified and are shown as relative activities (mean ± s.d.; n = 3).


( d ) 293T cells were cotransfected with Flag - PI ( 3 ) KC3 and either wild - type Bif - 1 , Bif - 1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI ( 3 ) KC3 kinase assay ( mean ± s . d . ; n = 3 ) .

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

(d) 293T cells were cotransfected with Flag-PI(3)KC3 and either wild-type Bif-1, Bif-1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI(3)KC3 kinase assay (mean ± s.d.; n = 3).


( a ) Bif - 1 − / − mice exhibit moderate splenomegaly . The weight of spleens extirpated from Bif - 1 + / + ( n = 27 ) and Bif - 1 − / − ( n = 34 ) mice ( 6 - 18 - month old ) was measured . Statistical significance was determined by Wilcoxon rank test .

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

(a) Bif-1−/− mice exhibit moderate splenomegaly. The weight of spleens extirpated from Bif-1+/+ (n = 27) and Bif-1−/− (n = 34) mice (6-18-month old) was measured. Statistical significance was determined by Wilcoxon rank test.


( b ) Hematoxylin and eosin staining of tumour tissues . Tumours were observed in 3 of 21 ( 14 . 3 % ) and 26 of 29 ( 89 . 7 % ) autopsied Bif - 1 + / + and Bif - 1 − / − mice , respectively . The scale bars represent 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(b) Hematoxylin and eosin staining of tumour tissues. Tumours were observed in 3 of 21 (14.3%) and 26 of 29 (89.7%) autopsied Bif-1+/+ and Bif-1−/− mice, respectively. The scale bars represent 50 μm.


A - C AID expression was analyzed in colon and pancreatic human cell lines ( A ) qRT - PCR analysis of AID expression in LoVo and SW480 colon cell lines . n = 5 ( LoVo ) ; 3 ( SW480 ) . * * P - value : LoVo : 0 . 0017 ; SW480 : 0 . 0079 . ( B ) qRT - PCR analysis of AID expression in AsPC and PaTU - 8988S pancreatic cell lines ( n = 2 ) . * P - value : AsPC : 0 . 0369 ; PaTU - 8988S : 0 . 0119 .

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

A-C AID expression was analyzed in colon and pancreatic human cell lines (A) qRT-PCR analysis of AID expression in LoVo and SW480 colon cell lines. n = 5 (LoVo); 3 (SW480). **P-value: LoVo: 0.0017; SW480: 0.0079. (B) qRT-PCR analysis of AID expression in AsPC and PaTU-8988S pancreatic cell lines (n = 2). *P-value: AsPC: 0.0369; PaTU-8988S: 0.0119.


A - C AID expression was analyzed in pancreas explants from C57BL / 6 mice . Samples were treated as indicated with 50 ng / ml TNF - α . ( C ) qRT - PCR analysis of AID expression in pancreatic explants from wild - type mice ( n = 3 ) . * P = 0 . 0242 .

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 I-CONTROLLED_VAR I-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 O O O O O

A-C AID expression was analyzed in pancreas explants from C57BL/6 mice. Samples were treated as indicated with 50 ng/ml TNF-α. (C) qRT-PCR analysis of AID expression in pancreatic explants from wild-type mice (n = 3). *P = 0.0242.


D AID − / − or AID + / − mice were treated with 3 % DSS for 10 cycles , and colonic sections were analyzed by histology inspection after H / E staining . Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments . n = 28 ( AID − / − males ) ; 35 ( females ) ; 23 ( AID + / − males ) ; 25 ( females ) . P - value : male : 0 . 8 ; female : 0 . 246 .

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 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 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

D AID−/− or AID+/− mice were treated with 3% DSS for 10 cycles, and colonic sections were analyzed by histology inspection after H/E staining. Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments. n = 28 (AID−/− males); 35 (females); 23 (AID+/− males); 25 (females). P-value: male: 0.8; female: 0.246.


GFP immunofluorescence in colonic and pancreatic tissue from R26AID + / KIVillinCRE + / TG and R26AID + / KIp48CRE + / KI mice . Scale bar : 50 μm .

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GFP immunofluorescence in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. Scale bar: 50 μm.


qRT - PCR analysis of AID expression in colonic and pancreatic tissue from R26AID + / KIVillinCRE + / TG and R26AID + / KIp48CRE + / KI mice . n = 5 ( R26AID + / + VillinCRE + / TG ) ; 4 ( R26AID + / KIVillinCRE + / TG ) ; 2 ( R26AID + / + p48CRE + / KI ) ; 2 ( R26AID + / KIp48CRE + / KI ) . LPS + IL4 - stimulated B cells are shown as a positive control ( n = 2 ) . Bars show mean values ± SEM normalized to LPS + IL4 - treated B cells .

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qRT-PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells.


Kaplan - Meier survival curves for R26AID + / KIVillinCRE + / TG ( left ) ( n = 47 ( R26AID + / + VillinCRE + / TG ) ; 38 ( R26AID + / KIVillinCRE + / TG ) ) and R26AID + / KI p48CRE + / KI mice ( right ) ( n = 39 ( R26AID + / + p48CRE + / KI ) ; 23 ( R26AID + / KIp48CRE + / KI ) ) .

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Kaplan-Meier survival curves for R26AID+/KIVillinCRE+/TG (left) (n = 47 (R26AID+/+ VillinCRE+/TG); 38 (R26AID+/KIVillinCRE+/TG)) and R26AID+/KI p48CRE +/KI mice (right) (n = 39 (R26AID+/+p48CRE+/KI); 23 (R26AID+/KIp48CRE+/KI)).


A , B Analysis of AID mutagenic activity in Elastase1 ( A ) and Elastase2a ( B ) by next - generation sequencing . Pancreatic DNA was isolated from pools of R26AID + / KIp48CRE + / KI and control R26AID + / + p48CRE + / KI 20 - week - old mice , and then PCR - amplified with specific primers and sequenced as previously described ( Perez - Duran et al , 2012 ) . Graphs show cytosine mutation frequency overall ( total ) or at AGCT hotspots . n = 2 . * P - value : Elastase1 : 0 . 0382 ; Elastase2a : 0 . 009 .

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A, B Analysis of AID mutagenic activity in Elastase1 (A) and Elastase2a (B) by next-generation sequencing. Pancreatic DNA was isolated from pools of R26AID+/KIp48CRE+/KI and control R26AID+/+p48CRE+/KI 20-week-old mice, and then PCR-amplified with specific primers and sequenced as previously described (Perez-Duran et al, 2012). Graphs show cytosine mutation frequency overall (total) or at AGCT hotspots. n = 2. *P-value: Elastase1: 0.0382; Elastase2a: 0.009.


C HTM - mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days . Red lines show mean values . Results of two independent experiments are shown . * * * * P < 0 . 0001 . D Representative images of γH2AX staining in pancreas explants from R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) ( 40 × magnification ) . White arrow points a representative positive cell .

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C HTM-mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days. Red lines show mean values. Results of two independent experiments are shown. ****P < 0.0001.D Representative images of γH2AX staining in pancreas explants from R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom) (40× magnification). White arrow points a representative positive cell.


Representative images of Ki67 staining in pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) . Detail is shown on the right . Scale bar : 100 μm . Graph shows quantification of Ki67 - positive epithelial cells per mm2 of tissue ( n = 8 ) . * P = 0 . 0266 .

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Representative images of Ki67 staining in pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom). Detail is shown on the right. Scale bar: 100 μm. Graph shows quantification of Ki67-positive epithelial cells per mm2 of tissue (n = 8). *P = 0.0266.


Representative immunofluorescence staining of 20 - week - old R26AID + / KIp48CRE + / KI mice : blue , DAPI ; red , Ki67 ; green , CK8 . Scale bar : 50 μm . Detail is shown on the bottom .

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Representative immunofluorescence staining of 20-week-old R26AID+/KIp48CRE+/KI mice: blue, DAPI; red, Ki67; green, CK8. Scale bar: 50 μm. Detail is shown on the bottom.


Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Left : Graph shows mean fluorescence intensity . Each dot represents an individual mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 8 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 077 . Right : Representative FACS staining for RAE in explants from R26AID + / + p48CRE + / KI ( red ) and R26AID + / KIp48CRE + / KI mice ( black ) .

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Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Graph shows mean fluorescence intensity. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 8 (R26AID+/KIp48CRE+/KI). P = 0.077. Right: Representative FACS staining for RAE in explants from R26AID+/+p48CRE+/KI (red) and R26AID+/KIp48CRE+/KI mice (black).


Analysis of RAE expression by ddPCR in aged ( 75 - week - old ) mice . Data are presented as the percentage of positive drops normalized to the mean control value . Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 ( R26AID + / + p48CRE + / KI ) ; 9 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 012 .

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Analysis of RAE expression by ddPCR in aged (75-week-old) mice. Data are presented as the percentage of positive drops normalized to the mean control value. Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 (R26AID+/+p48CRE+/KI); 9 (R26AID+/KIp48CRE+/KI). *P = 0.012.


Analysis of killing activity . Primary explants of pancreatic cells from R26AID + / + p48CRE + / KI or R26AID + / KIp48CRE + / KI mice were cultured with primary NK cells , and killing activity was assessed as described in Materials and Methods ( n = 4 ) . P = 0 . 19 .

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Analysis of killing activity. Primary explants of pancreatic cells from R26AID+/+p48CRE+/KI or R26AID+/KIp48CRE+/KI mice were cultured with primary NK cells, and killing activity was assessed as described in Materials and Methods (n = 4). P = 0.19.


Hematoxylin - eosin ( HE ) staining of pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Left : Representative HE staining showing an immune infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 200 μm . Right : Quantification of number of foci per mm2 of tissue . n = 13 ( R26AID + / + p48CRE + / KI ) ; 21 ( R26AID + / KIp48CRE + / KI ) . * * P = 0 . 0098 .

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Hematoxylin-eosin (HE) staining of pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative HE staining showing an immune infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 200 μm. Right: Quantification of number of foci per mm2 of tissue. n = 13 (R26AID+/+p48CRE+/KI); 21 (R26AID+/KIp48CRE+/KI). **P = 0.0098.


CD3 immunohistochemistry of pancreas from 75 - week - old R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Left : Representative image of a CD3 infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 100 μm . Right : Quantification of the number of CD3 - positive cells per mm2 of tissue . n = 12 ( R26AID + / + p48CRE + / KI ) ; 13 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0149 .

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CD3 immunohistochemistry of pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative image of a CD3 infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 100 μm. Right: Quantification of the number of CD3-positive cells per mm2 of tissue. n = 12 (R26AID+/+p48CRE+/KI); 13 (R26AID+/KIp48CRE+/KI). *P = 0.0149.


Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20 - week - old R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Scale bar : 20 μm .

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Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Scale bar: 20 μm.


TNF - α expression . Total RNA was isolated from pancreas of aged ( 75 - week - old ) R26AID + / KIp48CRE + / KI and control mice , and TNF - α expression was quantified by qRT - PCR . Each dot represents an individual mouse . n = 8 ( R26AID + / + p48CRE + / KI ) ; 15 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0392 .

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TNF-α expression. Total RNA was isolated from pancreas of aged (75-week-old) R26AID+/KIp48CRE+/KI and control mice, and TNF-α expression was quantified by qRT-PCR. Each dot represents an individual mouse. n = 8 (R26AID+/+p48CRE+/KI); 15 (R26AID+/KIp48CRE+/KI). *P = 0.0392.


Cell death detection . Pancreas from 75 - week - old R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice were stained with anti - caspase - 3 . Left : Representative staining from an R26AID + / + p48CRE + / KI mouse ( top ) and an R26AID + / KIp48CRE + / KI mouse ( bottom ) . Scale bar : 50 μm . Right : Quantification of number of cells per mm2 of tissue . Each dot represents an individual mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 11 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 0545 .

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Cell death detection. Pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice were stained with anti-caspase-3. Left: Representative staining from an R26AID+/+p48CRE+/KI mouse (top) and an R26AID+/KIp48CRE+/KI mouse (bottom). Scale bar: 50 μm. Right: Quantification of number of cells per mm2 of tissue. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 11 (R26AID+/KIp48CRE+/KI). P = 0.0545.


( E ) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals : MN2a axons projecting to the MHE muscle , representing the wild - type ( wt ) innervation pattern ( magenta ) ; MN2a axons projecting to muscles other than the MHE termed abnormal innervation ( light grey ) ; abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects ( dark grey ) . Note , each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS - RFP or UAS - GFPRNAi . Each experiment was performed in triplicates , innervation rates were calculated from n = 56 for elav > RFP , n = 21 for elav > DfdRNAi , n = 10 for elav > hthRNAi and n = 7 for elav > mirrRNAi . In the case of Dfd knock - down , two different driver lines were used , the pan - neural elav - GAL4 and the motoneuron - specific OK6 - GAL4 drivers , respectively . Both result in similar phenotypes , highlighting that the elav - GAL4 driven effects are specific to MNs . p - values between two genetic conditions were calculated by a two - sided Fisher test . * * * : p < 0 . 005 .

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(E) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern (magenta); MN2a axons projecting to muscles other than the MHE termed abnormal innervation (light grey); abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects (dark grey). Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. Each experiment was performed in triplicates, innervation rates were calculated from n=56 for elav>RFP, n=21 for elav>DfdRNAi, n=10 for elav>hthRNAi and n=7 for elav>mirrRNAi. In the case of Dfd knock-down, two different driver lines were used, the pan-neural elav-GAL4 and the motoneuron-specific OK6-GAL4 drivers, respectively. Both result in similar phenotypes, highlighting that the elav-GAL4 driven effects are specific to MNs. p-values between two genetic conditions were calculated by a two-sided Fisher test. ***: p < 0.005.


( B ) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd ( purple ) , Myosin in muscles ( blue ) and FasII in axonal projections ( green ) in control ( elav > RFP ) animals and animals mis - expressing the Hox TFs Lab , Dfd and Scr by means of the elav - GAL4 driver . A zoom on the projections ( FasII staining ) of MN2a and MN3 to the MHE and MHD muscles of an early stage 17 Drosophila embryo are shown . Asterisks highlight the location of MN2a and MN3 , respectively . MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates the Dfd expressing MHE muscle ( as shown in the elav > RFP control ) , while MN3 is identified as the MN underneath MN2a , which normally innervates the MHD muscle ( as shown in the elav > RFP control ) . The panel on the right side represents a schematic drawing of the confocal image shown on the left side , summarizing the innervation of the anterior muscles ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control and mis - expression conditions .

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 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-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 O O O O 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and animals mis-expressing the Hox TFs Lab, Dfd and Scr by means of the elav-GAL4 driver. A zoom on the projections (FasII staining) of MN2a and MN3 to the MHE and MHD muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


( A ) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles ( blue ) and FasII in axonal projections ( green ) in control ( elav > RFP ) animals and in animals with reduced neuronal DIPkappa ( elav > DIPkappaRNAi ) as well as in animals , which mis - express DIPgamma or dpr1 in neuronal cells ( elav > DIPgamma , elav > dpr1 ) . Single channels focusing on the projections ( FasII staining ) of MN2a and / or MN3 to the MHE , MHD or LR muscles of an early stage 17 Drosophila embryo are shown . Asterisks highlight the location of MN2a and MN3 , respectively . MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates the Dfd expressing MHE muscle ( as shown in the elav > RFP control ) , while MN3 is identified as the MN underneath MN2a , which normally innervates the MHD muscle ( as shown in the elav > RFP control ) . The panel on the right represents a schematic drawing of the confocal images shown on the left side , summarizing the innervation of the anterior muscles ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control and mis - expression conditions .

O 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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR 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 O 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced neuronal DIPkappa (elav>DIPkappaRNAi) as well as in animals, which mis-express DIPgamma or dpr1 in neuronal cells (elav>DIPgamma, elav>dpr1). Single channels focusing on the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right represents a schematic drawing of the confocal images shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


( A ) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd ( purple ) , Myosin in muscles ( blue ) and FasII in axonal projections ( green ) in control ( elav > RFP ) animals and in animals with reduced Dfd expression in neurons ( elav > DfdRNAi ) or in muscles ( Mef2 > DfdRNAi ) or both in both tissues ( elav ; Mef2 > DfdRNAi ) . The single channels for the projections ( FasII staining ) of MN2a and / or MN3 to the MHE , MHD or LR muscles of an early stage 17 Drosophila embryo are shown . Asterisks highlight the location of MN2a and MN3 , respectively . MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates the Dfd expressing MHE muscle ( as shown in the elav > RFP control ) , while MN3 is identified as the MN underneath MN2a , which normally innervates the MHD muscle ( as shown in the elav > RFP control ) . The panel on the right side represents a schematic drawing of the confocal image shown on the left side , summarizing the innervation of the anterior muscles ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control and perturbation conditions .

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(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced Dfd expression in neurons (elav>DfdRNAi) or in muscles (Mef2>DfdRNAi) or both in both tissues (elav;Mef2>DfdRNAi). The single channels for the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and perturbation conditions.


( A ) Immuno - precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4 ° C overnight . The control antibody ( ab ) is a rabbit anti - yeast Mad2 .

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(A) Immuno-precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4°C overnight. The control antibody (ab) is a rabbit anti-yeast Mad2.


( B ) Single confocal section of a population of S2 cells . Examples of interphase cells are boxed in blue . Mitotic cells positive for the anti - phosphoHistone3 ( pH3 ) are boxed in red .

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(B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red.


( C ) Single confocal section of a S2 cell . Anti - Incenp , anti - Cid mark the centromeric chromatin and the inner KT , respectively .

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(C) Single confocal section of a S2 cell. Anti-Incenp, anti-Cid mark the centromeric chromatin and the inner KT, respectively.


( D - E ) Maximal projections of S2 cells imaged by STED microscopy . 7 z - sections for a total of 1 . 5 um ( D ) and 4 z - sections for a total of 0 . 5 um ( E ) are shown . Anti - CenpC and anti - Spc105R mark the proximal and the distal region of different portions of the outer KT , respectively .

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(D-E) Maximal projections of S2 cells imaged by STED microscopy. 7 z-sections for a total of 1.5 um (D) and 4 z-sections for a total of 0.5 um (E) are shown. Anti-CenpC and anti-Spc105R mark the proximal and the distal region of different portions of the outer KT, respectively.


( F - G ) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29 , nuclear pores ( WGA ) , and CyclinB ( F ) , or Snap29 , Spc105R , and the nuclear lamina ( LaminA ) ( G ) . Note the transient perinuclear enrichment of Snap29 in early prophase . WGA stains also cytoplasmic speckles in hemocyte - derived S2 cells ( Rizki Rizki , 1983 ) . High magnification of the insets in C - G shows localization of Snap29 relative to the indicated markers .

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(F-G) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29, nuclear pores (WGA), and CyclinB (F), or Snap29, Spc105R, and the nuclear lamina (LaminA) (G). Note the transient perinuclear enrichment of Snap29 in early prophase. WGA stains also cytoplasmic speckles in hemocyte-derived S2 cells (Rizki Rizki, 1983). High magnification of the insets in C-G shows localization of Snap29 relative to the indicated markers.


( H - J ) Immuno - gold localization of Snap29 in cryo - sections of S2 cells in pro - metaphase . Pseudo - coloring in red identifies chromosomes ( Ch ) , in yellow membrane organelles and in green MTs . The white arrows in H - I indicate examples of signal in the nucleoplasm that is not associated to membranes , but rather , to electron - dense material . MVB : multivesicular body ; NE : nuclear envelope ; PM : plasma membrane .

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(H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.


( A ) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs . A cartoon of full length Snap29 is shown above the control panel .

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(A) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs. A cartoon of full length Snap29 is shown above the control panel.


( B ) Quantification of the Snap29 signal as in panel A , considering > 30 KTs per sample . Relative mRNA expression of the downregulated genes versus control , measured by Q - PCR , is shown above the graph . Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs .

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(B) Quantification of the Snap29 signal as in panel A, considering > 30 KTs per sample. Relative mRNA expression of the downregulated genes versus control, measured by Q-PCR, is shown above the graph. Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs.


( C ) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over - expressed . Cartoons of the expressed Snap29 mutant forms are shown above the panels . Anti - pH3 labels mitotic cells and CenpC marks KTs . ( D ) Quantification of Snap29 as in panel C , considering > 28 KTs per sample . Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells .

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(C) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over-expressed. Cartoons of the expressed Snap29 mutant forms are shown above the panels. Anti-pH3 labels mitotic cells and CenpC marks KTs. (D) Quantification of Snap29 as in panel C, considering > 28 KTs per sample. Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells.


( A ) Selected frames of time - lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29 . Note that depleted cell does not display a recognizable metaphase plate , or fail to segregate all chromosomes correctly , or form tripolar spindles or micronuclei ( arrowheads ; arrowheads in control point to cells dividing normally ) .

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(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally).


( B ) Western blotting of extract from control cells and cells depleted of Snap29 ( dsSnap29 ) relative to the experiment in a .

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(B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a.


( C ) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells , based on time - lapse imaging of an average of > 28 individual cells per sample . P value is determined by two - tailed t - test considering all defects together .

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(C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together.


( D ) Selected frames of time - lapse imaging of control U2OS cells and U2OS cells treated for the indicated time . Note that the depleted cell at 48 - 66h display altered metaphase plate and chromosome segregation ( arrowheads ) .

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(D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads).


( E ) Western blot analysis of extracts relative to the experiments in panel d .

(E) Western blot analysis of extracts relative to the experiments in panel d.


( F ) Quantification of the time from prophase to entry in anaphase based on analysis of > 20 individual cells / sample by time - lapse microscopy . P values are determined by Kruskal - Wallis test with Dunn ' s multiple comparison analysis . OE : Over - expression .

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(F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn's multiple comparison analysis. OE: Over-expression.


( A ) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4 ° C and immunostained to reveal the presence of unattached kinetochores . CENPT labels all KTs , while MAD1 identifies unattached ones . Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate . ( B ) Quantification of the number of KTs based on attachment , position and MAD1 positivity . Each point per category represents one experiment in which 10 random KTs / cell of 20 cells / sample were counted . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison test . Note that in control cells most of the KT are attached , MAD1 - negative and aligned to the metaphase plate , while in SNAP29KD cells half of the KTs that are unattached , MAD1 - positive and not aligned to the metaphase plate .

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(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate.


( C ) Single confocal sections of HeLa cells . CREST and CENPT were used to label the centromere region and associated KTs , respectively . Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown . ( D ) Average of the inter KT distance based on in each of which 10 chromosomes / cell and 10 cells / sample were counted . P values are obtained by Kruskal - Wallis test with Dunn ' s multiple comparison analysis . Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs .

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(C) Single confocal sections of HeLa cells. CREST and CENPT were used to label the centromere region and associated KTs, respectively. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown. (D) Average of the inter KT distance based on in each of which 10 chromosomes/cell and 10 cells/sample were counted. P values are obtained by Kruskal-Wallis test with Dunn's multiple comparison analysis. Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs.


( E ) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA . ( F ) Averaged spindle pole length based on > 20 cells with bipolar spindles / sample . The P value is obtained by two - tailed paired t - test .

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(E) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA. (F) Averaged spindle pole length based on >20 cells with bipolar spindles/sample. The P value is obtained by two-tailed paired t-test.


( A - C , E ) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs .

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(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.


( D ) Immunoblotting of HeLa protein extracts , treated as indicated .

(D) Immunoblotting of HeLa protein extracts, treated as indicated.


( F ) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A - C , e relative to CREST . 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining . P values are obtained by 2 - way ANOVA with Sidak ' s multiple comparison analysis relative to localization of HEC1 , which is not lost on SNAP29KD cells .

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(F) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A-C, e relative to CREST. 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Sidak's multiple comparison analysis relative to localization of HEC1, which is not lost on SNAP29KD cells.


( A - C ) KNL1 localization analysis in control , SNAP29 KD and SNAP29 KD expressing full length human SNAP29 , CFP tagged , full length Drosophila Snap29 ( CFP - Snap29 ) , or the mutant forms schematized above the panels . Max projections of single cells in prophase are shown . ( D ) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST . 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells .

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(A-C) KNL1 localization analysis in control, SNAP29 KD and SNAP29 KD expressing full length human SNAP29, CFP tagged, full length Drosophila Snap29 (CFP-Snap29), or the mutant forms schematized above the panels. Max projections of single cells in prophase are shown. (D) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST. 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells.


( E - F ) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs . In cells expressing SNAP29 Q1 Q2 , SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 ( arrows ) .

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(E-F) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs. In cells expressing SNAP29 Q1 Q2, SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 (arrows).


( A - B ) Confocal sections of wild - type and Snap29 mutant eye - antennal imaginal discs ( eye disc portion to the left ) of L3 larvae . Discs were labeled with phalloidin and DAPI to visualize the subcortical f - actin cytoskeleton and nuclei , respectively . Arrowheads in B point to actin - rich unpolarized cells in apical , medial and basal sections of the same disc .

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(A-B) Confocal sections of wild-type and Snap29 mutant eye-antennal imaginal discs (eye disc portion to the left) of L3 larvae. Discs were labeled with phalloidin and DAPI to visualize the subcortical f-actin cytoskeleton and nuclei, respectively. Arrowheads in B point to actin-rich unpolarized cells in apical, medial and basal sections of the same disc.


( C ) High magnification of a single confocal cross - section of wild - type and Snap29 mutant eye disc tissues . Two facing folds of the tissue are separated by the apical lumen . aPKC and Dlg mark the apical and lateral plasma membrane domains , respectively . In Snap29 mutant tissue the apical - basal polarity is disrupted .

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(C) High magnification of a single confocal cross-section of wild-type and Snap29 mutant eye disc tissues. Two facing folds of the tissue are separated by the apical lumen. aPKC and Dlg mark the apical and lateral plasma membrane domains, respectively. In Snap29 mutant tissue the apical-basal polarity is disrupted.


( D ) Single confocal cross - sections of wild - type and Snap29 eye - antennal discs , expressing Puckered - LacZ ( Puc - lacZ ) , a reporter of JNK signaling activation .

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(D) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs, expressing Puckered-LacZ (Puc-lacZ), a reporter of JNK signaling activation.


( E - H ) Single confocal cross - sections of wild - type and Snap29 eye - antennal discs ( E - F ) , and of otherwise wild - type and Snap29 eye - antennal discs expressing the bacterial apoptosis inhibitor p35 ( G - H ) . Anti - activated - Caspase 3 ( Casp3 ) marks apoptotic cells .

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(E-H) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs (E-F), and of otherwise wild-type and Snap29 eye-antennal discs expressing the bacterial apoptosis inhibitor p35 (G-H). Anti-activated-Caspase 3 (Casp3) marks apoptotic cells.


( a ) vBcl - 2 binding proteins . After transfection , 293T cells were used for GST - pulldown and the purified proteins were detected by silver staining .

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(a) vBcl-2 binding proteins. After transfection, 293T cells were used for GST-pulldown and the purified proteins were detected by silver staining.


( c ) UVRAG binds Beclin1 through its CCD . After transfection with Beclin1 - V5 , together with full - length or fragments of Flag - UVRAG , 293T whole cell lysates ( WCLs ) were used for immunoprecipitation ( IP ) with anti - Flag followed by immunoblotting ( IB ) with anti - V5 .

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(c) UVRAG binds Beclin1 through its CCD. After transfection with Beclin1-V5, together with full-length or fragments of Flag-UVRAG, 293T whole cell lysates (WCLs) were used for immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-V5.


( d ) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI ( 3 ) KC3 . 293T cell lysates were used for immunoprecipitation with preimmune sera or anti - Beclin1 , followed by immunoblotting with anti - UVRAG or anti - Beclin1 , or for immunoprecipitation with preimmune sera or anti - UVRAG , followed by immunoblotting with anti - PI ( 3 ) KC3 .

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(d) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI(3)KC3. 293T cell lysates were used for immunoprecipitation with preimmune sera or anti-Beclin1, followed by immunoblotting with anti-UVRAG or anti-Beclin1, or for immunoprecipitation with preimmune sera or anti-UVRAG, followed by immunoblotting with anti-PI(3)KC3.


( e ) UVRAGCCD is required for Beclin1 interaction . After transfection with Beclin1 - V5 ( lanes 2 and 4 ) together with Flag - UVRAGΔCCD ( lanes 1 and 2 ) or Flag - UVRAGΔC2 ( lanes 3 and 4 ) , WCLs were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 .

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

(e) UVRAGCCD is required for Beclin1 interaction. After transfection with Beclin1-V5 (lanes 2 and 4) together with Flag-UVRAGΔCCD (lanes 1 and 2) or Flag-UVRAGΔC2 (lanes 3 and 4), WCLs were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5.


( f ) Beclin1 binds UVRAG through its CCD . After transfection with Flag - UVRAG together with full - length or fragments of Beclin1 - V5 , WCLs were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag .

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

(f) Beclin1 binds UVRAG through its CCD. After transfection with Flag-UVRAG together with full-length or fragments of Beclin1-V5, WCLs were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag.


( g ) An increase in UVRAG stability induced by Beclin1 . After transfection with Flag - UVRAG and empty vector or Flag - UVRAG and Beclin1 - V5 , WCLs were treated with cycloheximide ( CHX , 20 μg ml − 1 ) for the indicated time , followed by immunoblotting with anti - Flag .

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

(g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag-UVRAG and empty vector or Flag-UVRAG and Beclin1-V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag.


( h ) UVRAG directly interacts with Beclin1 . MBP - UVRAGCCD ( lane 1 ) , His6 - Beclin1CCD ( lane 2 ) or mixtures of both proteins in a concentration dependent manner ( lanes 3 - 8 ) , were subjected to native gel electrophoresis . The numbers indicate the concentration ( nmol ) of protein . Arrows indicate the purified MBP - UVRAGCCD , His6 - Beclin1CCD and complex between these two .

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

(h) UVRAG directly interacts with Beclin1. MBP-UVRAGCCD (lane 1), His6-Beclin1CCD (lane 2) or mixtures of both proteins in a concentration dependent manner (lanes 3-8), were subjected to native gel electrophoresis. The numbers indicate the concentration (nmol) of protein. Arrows indicate the purified MBP-UVRAGCCD, His6-Beclin1CCD and complex between these two.


( i ) Beclin1CCD and UVRAGCCD associate into a α - helical heterodimer . After removing the N - terminal His6 and MBP tags , the Beclin1CCD and the preformed Beclin1CCD - UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis . The raw data for this experiment are presented in the Supplementary Information , Fig . S5 .

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 O O O O O

(i) Beclin1CCD and UVRAGCCD associate into a α-helical heterodimer. After removing the N-terminal His6 and MBP tags, the Beclin1CCD and the preformed Beclin1CCD-UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


( a ) The Bcl - 2 binding domain of Beclin1 is sufficient to bind vBcl - 2 . At 48 h posttransfection with wild - type and mutant Beclin1 - V5 , together with HA - vBcl - 2 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - HA .

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

(a) The Bcl-2 binding domain of Beclin1 is sufficient to bind vBcl-2. At 48 h posttransfection with wild-type and mutant Beclin1-V5, together with HA-vBcl-2, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-HA.


( b ) The loss of Beclin1 interaction of vBcl - 2AAA mutant . At 48 h posttransfection with wild - type HA - vBcl - 2 or vBcl - 2AAA mutant , together with Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 , followed by immunoblotting with anti - HA .

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

(b) The loss of Beclin1 interaction of vBcl-2AAA mutant. At 48 h posttransfection with wild-type HA-vBcl-2 or vBcl-2AAA mutant, together with Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5, followed by immunoblotting with anti-HA.


( c ) vBcl - 2 interaction with wild - type UVRAG in the presence of Beclin1 . At 48 h posttransfection with Flag - UVRAG together with Beclin1 - V5 and HA - vBcl - 2 or vBcl - 2AAA mutant , WCLs of 293T cells were used for immunoprecipitation with anti - HA followed by immunoblotting with anti - Flag .

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

(c) vBcl-2 interaction with wild-type UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAG together with Beclin1-V5 and HA-vBcl-2 or vBcl-2AAA mutant, WCLs of 293T cells were used for immunoprecipitation with anti-HA followed by immunoblotting with anti-Flag.


( d ) vBcl - 2 interaction with PI ( 3 ) KC3 in the presence of Beclin1 . At 48 h posttransfection with HA - vBcl - 2 together with Flag - PI ( 3 ) KC3 and Beclin1 - V5 , WCLs of 293T cells were used for immunprecipitation with anti - HA followed by immunoblotting with anti - Flag .

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

(d) vBcl-2 interaction with PI(3)KC3 in the presence of Beclin1. At 48 h posttransfection with HA-vBcl-2 together with Flag-PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunprecipitation with anti-HA followed by immunoblotting with anti-Flag.


( e ) PI ( 3 ) KC3 N - terminal C2 domain binds Beclin1 . At 48 h posttransfection with Beclin1 - V5 together with full - length or fragments of Flag - PI ( 3 ) KC3 , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 . CAT , catalytic domain .

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

(e) PI(3)KC3 N-terminal C2 domain binds Beclin1. At 48 h posttransfection with Beclin1-V5 together with full-length or fragments of Flag-PI(3)KC3, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. CAT, catalytic domain.


( f ) Beclin1CCD and Beclin1ECD are sufficient for binding PI ( 3 ) KC3 . At 48 h posttransfection with Flag - PI ( 3 ) KC3 together with full - length or fragments of Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag . CT , C terminus ( aa 337 - 448 ) .

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

(f) Beclin1CCD and Beclin1ECD are sufficient for binding PI(3)KC3. At 48 h posttransfection with Flag-PI(3)KC3 together with full-length or fragments of Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. CT, C terminus (aa 337-448).


( g ) PI ( 3 ) KC3 interaction with UVRAG in the presence of Beclin1 . At 48 h posttransfection with Flag - UVRAGΔCCD or UVRAGCCD together with HA - PI ( 3 ) KC3 and Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - HA .

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

(g) PI(3)KC3 interaction with UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAGΔCCD or UVRAGCCD together with HA- PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA.


( a ) UVRAG expression and LC3 process in HCT116 cells . The same protein amounts of 293T , NIH3T3 , HCT116 and MCF7 cells were used for immunoblotting with anti - UVRAG and anti - tubulin . WCLs of 293T cells transfected with Flag - tagged UVRAG were included as controls . WCLs of HCT116 . vector and HCT116 . UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti - LC3 and anti - tubulin . The raw data for this experiment is presented in the Supplementary information , Fig . S5 .

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 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 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

(a) UVRAG expression and LC3 process in HCT116 cells. The same protein amounts of 293T, NIH3T3, HCT116 and MCF7 cells were used for immunoblotting with anti-UVRAG and anti-tubulin. WCLs of 293T cells transfected with Flag-tagged UVRAG were included as controls. WCLs of HCT116.vector and HCT116.UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti-LC3 and anti-tubulin. The raw data for this experiment is presented in the Supplementary information, Fig. S5.


( b ) Electron microscopy of autophagy in HCT116 cells . HCT116 . vector and HCT116 . UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy . Scale bars ( from left to right panels ) represent 2 nm , 0 . 5 nm , 0 . 2 nm and 0 . 2 nm . The arrows indicate autophagosome or autolysosome .

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(b) Electron microscopy of autophagy in HCT116 cells. HCT116.vector and HCT116.UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy. Scale bars (from left to right panels) represent 2 nm, 0.5 nm, 0.2 nm and 0.2 nm. The arrows indicate autophagosome or autolysosome.


( c ) UVRAG induces autophagosome formation in HCT116 cells . After transfection with GFP - LC3 vector , HCT116 . vector and HCT116 . UVRAG cells were maintained under normal conditions or starved for 3 h . GFP - LC3 was detected using an inverted fluorescence microscope . Arrows indicate autophagosomes . The scale bars represent 10 μm .

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(c) UVRAG induces autophagosome formation in HCT116 cells. After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions or starved for 3 h. GFP-LC3 was detected using an inverted fluorescence microscope. Arrows indicate autophagosomes. The scale bars represent 10 μm.


( d ) After transfection with GFP - LC3 vector , HCT116 . vector and HCT116 . UVRAG cells were maintained under normal conditions , starved for 3 h in the absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Autophagome was quantified as the mean ± s . d . of combined results from three independent experiments .

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

(d) After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Autophagome was quantified as the mean ± s.d. of combined results from three independent experiments.


( a ) HCT116 . vector and HCT116 . UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis . HCT116 . vector , HCT116 . UVRAG , and HCT116 . UVRAG ΔCCD cells were treated with or without wortmannin ( 1 μM ) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity . Data represents mean ± s . d . of three experiments .

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

(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments.


( b ) UVRAG induces autophagosome formation in NIH3T3 cells . After transfection with GFP - LC3 vector , NIH3T3 . vector and NIH3T3 . UVRAG cells were maintained under normal conditions , starved for 3 h in the absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Data represents mean ± s . d . of three experiments .

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

(b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP-LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.


( a ) Autophagic activity of UVRAG mutants . HCT116 cells expressing wild - type or mutant UVRAG were transfected with GFP - LC3 expression vector and incubated under normal or starvation conditions for 3 h . Autophagy was quantified as described in Fig . 3 .

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(a) Autophagic activity of UVRAG mutants. HCT116 cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 expression vector and incubated under normal or starvation conditions for 3 h. Autophagy was quantified as described in Fig. 3.


( b ) Effect of wild - type and mutant UVRAG on Beclin1 - mediated autophagy . MCF7 . vector , MCF7 . Beclin1 and MCF7 . Beclin1ΔECD cells were cotransfected with wild - type UVRAG or its mutant expression vector together with GFP - LC3 expression vector . At 16 - 18 h posttransfection , cells were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as described in Fig . 3 . The scale bars represent 20 μm .

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 B-CONTROLLED_VAR 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

(b) Effect of wild-type and mutant UVRAG on Beclin1-mediated autophagy. MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells were cotransfected with wild-type UVRAG or its mutant expression vector together with GFP-LC3 expression vector. At 16-18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as described in Fig. 3. The scale bars represent 20 μm.


( c , d ) Interdependence between UVRAG and Beclin1 for autophagosome induction . HCT116 . vector and HCT116 . UVRAG cells were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these cells were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . The scale bars represent 20 μm . In all figures , data represents mean ± s . d . of three experiments . The raw data for c and d are presented in the Supplementary Information , Fig S5 .

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

(c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction. HCT116.vector and HCT116.UVRAG cells were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


( c , d ) Interdependence between UVRAG and Beclin1 for autophagosome induction . MCF7 . vector , MCF7 . Beclin1 and MCF7 . Beclin1ΔECD cells ( d ) were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these cells were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . The scale bars represent 20 μm . In all figures , data represents mean ± s . d . of three experiments . The raw data for c and d are presented in the Supplementary Information , Fig S5 .

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

(c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction.MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells (d) were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


( a ) Increased Beclin1 - PI ( 3 ) KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔCCD cells were used for immunoprecipitation with anti - PI ( 3 ) KC3 followed by immunoblotting with anti - Beclin1 . WCLs were used for immunoblotting with anti - Beclin1 and anti - PI ( 3 ) KC3 . HCT116 . UVRAG cells expressing Flag - tagged UVRAG were incubated under nutrient - rich conditions ( lanes 1 and 2 ) , starvation for 4 h ( lane 3 ) , or 2 μM rapamycin treatment for 2 h ( lane 4 ) . WCLs were then used for immunoprecipitation with anti - Beclin1 ( lanes 2 , 3 and 4 ) or preimmune antibody ( lane 1 ) followed by immunoblotting with anti - Flag . The raw data for this experiment are presented in the Supplementary Information , Fig . S5 .

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

(a) Increased Beclin1-PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3)KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3)KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


( b ) Increased PI ( 3 ) KC3 activity by UVRAG . At 48 h posttransfection with Flag - tagged PI ( 3 ) KC3 with Beclin1 ( lane 1 ) , with Beclin1 + UVRAG ( lane 2 ) or with Beclin1 + UVRAGΔCCD vector ( lane 3 ) , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by an assay for PI ( 3 ) KC3 lipid kinase activity , as described in the Methods . At 16 h posttransfection with p40 ( phox ) PX - EGFP fusion , HCT116 . vector and HCT116 . UVRAG cells were detected using an inverted fluorescence microscope . p40 ( phox ) PX - EGFP - positive vesicles were quantified as the mean ± s . d . of combined results from three independent experiments . The scale bars represent 10 μm .

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

(b) Increased PI(3)KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3)KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3)KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX-EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX-EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm.


( c ) Suppression of autophagy by γHV68 vBcl - 2 . At 18 h posttransfection with GFP - LC3 vector , NIH3T3 . vector , NIH3T3 . vBcl - 2 and NIH3T3 . vBcl - 2AAA , cells were incubated under normal or starvation conditions for 4 h . Subsequently , GFP - LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s . d . of combined results from three independent experiments .

O O O O O 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 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

(c) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP-LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.


( a ) Proliferation rates . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD and HCT116 . UVRAGCCD cells ( 1 × 105 per well ) were seeded into 6 - well microtitre plates . Cell numbers were counted on the days indicated . The results represent means of three independent assays .

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

(a) Proliferation rates. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 105 per well) were seeded into 6-well microtitre plates. Cell numbers were counted on the days indicated. The results represent means of three independent assays.


( b ) Anchorage independence . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD and HCT116 . UVRAGCCD cells ( 1 × 104 ) were incubated in soft agar as described in the Methods . After ten days , colonies were photographed and counted . The scale bars represent 50 μm .

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

(b) Anchorage independence. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 104) were incubated in soft agar as described in the Methods. After ten days, colonies were photographed and counted. The scale bars represent 50 μm.