Figure 3A . Localization of the Golgi apparatus ( ManII - GFP ) and the centrosome ( 𝛾 - tubulin ) in E15 . 5 mCherry - electroporated radial glial cell . The Golgi apparatus is localized basally , away from the centrosome . Scale bar = 5µm . B . Average distance between the apical - most part of the Golgi apparatus and the apical surface in aRG cells . N = 224 cells from 3 independent brains . E . Live imaging of GFP - RAB6A in aRG cells at E15 . 5 allows tracking of individual RAB6A + vesicles in situ , from the basal Golgi apparatus towards the apical surface . Scale bar = 5µm . Distance = 5µm , time = 30 seconds . F . RAB6A + vesicle directionality in apical processes of aRG cells over one - minute movies . G . Relative time spent by RAB6A + vesicles in apical , basal or static phases . H . Velocity of apically and basally moving RAB6A + vesicles . Data information : ( F , G , H ) N = 388 vesicles from 30 cells . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median . I Live imaging of GFP - RAB6A in control , dynarrestin - treated and CC1 - p150 - expressing aRG cells at E15 . 5 . Scale bars = 5µm . Distance = 5µm , time = 30 seconds . J . Number of RAB6A + vesicles in the apical process of DMSO and dynarrestin - treated mouse aRG cells . K . Relative time spent by RAB6A + vesicles in apical movement phase , in DMSO and dynarrestin - treated mouse aRG cells . L . Number of RAB6A + vesicles in the apical process of mCherry and CC1 - p150 - expressing aRG cells . M . Relative time spent by RAB6A + vesicles in apical movement phase , in mCherry and CC1 - p150 - expressing aRG cells . DMSO treatment slightly affected RAB6A dynamics , as compared to mCherry control . Data information : ( J , K , L , M ) 216 vesicles from N = 11 cells for DMSO , 145 vesicles from N = 25 cells for dynarrestin , 173 vesicles from N = 17 cells for mCherry control , 71 vesicles from N = 15 cells for CC1 - p150 . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Localization of the Golgi apparatus (ManII-GFP) and the centrosome (𝛾-tubulin) in E15.5 mCherry-electroporated radial glial cell. The Golgi apparatus is localized basally, away from the centrosome. Scale bar = 5µm. B. Average distance between the apical-most part of the Golgi apparatus and the apical surface in aRG cells. N=224 cells from 3 independent brains.E. Live imaging of GFP-RAB6A in aRG cells at E15.5 allows tracking of individual RAB6A+ vesicles in situ, from the basal Golgi apparatus towards the apical surface. Scale bar = 5µm. Distance = 5µm, time = 30 seconds. F. RAB6A+ vesicle directionality in apical processes of aRG cells over one-minute movies. G. Relative time spent by RAB6A+ vesicles in apical, basal or static phases. H. Velocity of apically and basally moving RAB6A+ vesicles. Data information: (F, G, H) N= 388 vesicles from 30 cells. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.I Live imaging of GFP-RAB6A in control, dynarrestin-treated and CC1-p150-expressing aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time = 30 seconds. J. Number of RAB6A+ vesicles in the apical process of DMSO and dynarrestin-treated mouse aRG cells. K. Relative time spent by RAB6A+ vesicles in apical movement phase, in DMSO and dynarrestin-treated mouse aRG cells. L. Number of RAB6A+ vesicles in the apical process of mCherry and CC1-p150-expressing aRG cells. M. Relative time spent by RAB6A+ vesicles in apical movement phase, in mCherry and CC1-p150-expressing aRG cells. DMSO treatment slightly affected RAB6A dynamics, as compared to mCherry control. Data information: (J, K, L, M) 216 vesicles from N=11 cells for DMSO, 145 vesicles from N=25 cells for dynarrestin, 173 vesicles from N=17 cells for mCherry control, 71 vesicles from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


Figure 4C . RUSH assay for CRB3 - GFP in control ( mcherry ) and dynactin - inhibited radial glial cells ( CC1 - p150 - dsRed ) , electroporated at E . 15 . 5 and imaged at E16 . 5 . Scale bar = 5µm . D . CRB3 localization at the apical surface upon release . E . Percentage of cells with 100 % of CRB3 signal at the apical surface upon release . Data information : ( D , E ) mCherry ( control ) : N = 3 brains per timepoint ( 361 cells total ) . CC1 - p150 : N = 3 brains per timepoint ( 2 brains for T0 ) ( 268 cells total ) . Fisher ' s exact test and Benjamini - Hochberg procedure , * * * p ≤ 0 . 001 . All error bars indicate SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C. RUSH assay for CRB3-GFP in control (mcherry) and dynactin-inhibited radial glial cells (CC1-p150-dsRed), electroporated at E.15.5 and imaged at E16.5. Scale bar = 5µm. D. CRB3 localization at the apical surface upon release. E. Percentage of cells with 100% of CRB3 signal at the apical surface upon release. Data information: (D, E) mCherry (control): N= 3 brains per timepoint (361 cells total). CC1-p150: N=3 brains per timepoint (2 brains for T0) (268 cells total). Fisher's exact test and Benjamini-Hochberg procedure, *** p ≤ 0.001. All error bars indicate SD.


Figure 5A . PAX6 and phospho - Histone 3 ( p - H3 ) staining in WT and LIS1 KO E12 . 5 brains . Cortices were subdivided into 5 bins of equal size along the radial axis . Scale bar = 25µm . B . Percentage of PH3 + / PAX6 + cells located above the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1192 cells from N = 6 brains . LIS1 KO : 589 cells from N = 3 brains . Data information : ( B , Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD . C . Phospho - Vimentin ( p - VIM ) staining in WT and LIS1 KO E12 . 5 brains . Scale bar = 25µm . D . Percentage p - VIM + cells dividing ectopically , away from the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1056 cells from N = 6 brains . LIS1 KO : 879 cells from N = 3 brains . Data information : D ) Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. PAX6 and phospho-Histone 3 (p-H3) staining in WT and LIS1 KO E12.5 brains. Cortices were subdivided into 5 bins of equal size along the radial axis. Scale bar = 25µm. B. Percentage of PH3+/PAX6+ cells located above the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1192 cells from N=6 brains. LIS1 KO: 589 cells from N=3 brains. Data information: (B, Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.C. Phospho-Vimentin (p-VIM) staining in WT and LIS1 KO E12.5 brains. Scale bar = 25µm. D. Percentage p-VIM+ cells dividing ectopically, away from the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1056 cells from N=6 brains. LIS1 KO: 879 cells from N=3 brains. Data information: D) Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


Figure 6A . CRB3 and PALS1 staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm . B . Quantification of CRB3 staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 13 brains ) , LIS1 KO ( E12 . 5 , N = 4 brains ) ) and RAB6A / B dKO ( E15 . 5 , N = 4 brains ) . Data information : ( B , Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD . C . N - Cadherin staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm . D . Quantification of N - Cadh staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 6 brains ) , LIS1 KO ( E12 . 5 , N = 3 brains ) and RAB6A / B dKO ( E15 . 5 , N = 3 brains ) . Data information : D ) Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. CRB3 and PALS1 staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.B. Quantification of CRB3 staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=13 brains), LIS1 KO (E12.5, N=4 brains)) and RAB6A/B dKO (E15.5, N=4 brains). Data information: (B, Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.C. N-Cadherin staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.D. Quantification of N-Cadh staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=6 brains), LIS1 KO (E12.5, N=3 brains) and RAB6A/B dKO (E15.5, N=3 brains). Data information: D) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


Figure 1 ( C ) Quantification of renewed ECs ( RFP - only labelled cells ) , left graph , and number of ISC and EB cells ( double RFP + GFP + cells ) , right graph , in posterior midguts after 7 , 14 , or 21 days of tracing in normal ( ' Variable ' : gut scored n = 9 , 8 , 4 ) and low demand ( ' Low ' : n = 6 , 4 , 7 ) culturing conditions . Asterisks denote significances from one - way ANOVA with Bonferroni ' s Multiple Comparison Test ( * * , P = 0 . 0016 ; * * * * , P < 0 . 0001 ) . Data information In the boxplots , the box extends from the 25th to 75th percentiles and whiskers show all points , min to max . The line in the middle of the box is plotted at the median ( D ) Quantification of mitosis ( phospho - histone H3 ( PH3 ) + cells ) in low demand midguts at the indicated time points . * * , P = 0 . 0015 ; n . s . not significant from one - way ANOVA test . Data information : In the boxplots , the box extends from the 25th to 75th percentiles and whiskers show all points , min to max . The line in the middle of the box is plotted at the median ( E ) Representative adult midgut from control esgReDDM at day 14 after temperature shift in variable demand . ECs renewed are marked positively by persistent RFP labelling ( red - only cells ) . ( F and G ) Few ECs had been renewed in ' Low ' midguts after 14 ( F ) and 21 ( G ) days of tracing . Data information : Scale bars , 50µm . ( H - J ) Age - synchronized posterior midguts of control ( esgReDDM > , H ) and p35 overexpression in stem and progenitor cells ( esgReDDM > p35 , p35 I and J ) at 7 days after temperature shift . Arrows in ( H , I ) point to newly differentiated ECs . In ( J ) , tumour mass is found in the anterior midgut . Data information : Scale bars , 50µm . ( K ) , Quantification of mitosis PH3 + cells in posterior midgut ( pmg ) ( control : gut scored n = 13 and esg > p35 , p35 : n = 22 ] ) . Student t - test ( * * * * = P < 0 . 0001 ) . Data information : In the boxplots , the box extends from the 25th to 75th percentiles and whiskers show all points , min to max . ( L ) , Diap1 is monitored by the GFP . 4 . 3 . ( green ) reporter ( Djiane et al . , 2013 ; Zhang et al . , 2008 ) . Diap1 - GFP is detected in a subset of adult midgut esg + cells that are negative for the mitotic marker PH3 ( red , inset ) and for EE marker Prospero ( Pros , grey in inset ) . Diap1 - GFP is not detected in mature EC ( large nuclei cells , DAPI in blue ) . Data information : Scale bars , 50µm . ( M - O ) , Diap1 - GFP ( green ) co - localized almost 100 % with the EB marker GBE - Su ( H ) - lacZ ( red , M and O ) . Arrow points to a Diap1 - GFP - negative GBE - Su ( H ) - lacZ - positive cell . Data information : Scale bars , 50µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) Quantification of renewed ECs (RFP-only labelled cells), left graph, and number of ISC and EB cells (double RFP+ GFP+ cells), right graph, in posterior midguts after 7, 14, or 21 days of tracing in normal ('Variable': gut scored n= 9, 8, 4) and low demand ('Low': n= 6, 4, 7) culturing conditions. Asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (**, P = 0.0016; ****, P < 0.0001). Data information In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median(D) Quantification of mitosis (phospho-histone H3 (PH3)+ cells) in low demand midguts at the indicated time points. **, P = 0.0015; n.s. not significant from one-way ANOVA test. Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median(E) Representative adult midgut from control esgReDDM at day 14 after temperature shift in variable demand. ECs renewed are marked positively by persistent RFP labelling (red-only cells). (F and G) Few ECs had been renewed in 'Low' midguts after 14 (F) and 21 (G) days of tracing. Data information: Scale bars, 50µm.(H-J) Age-synchronized posterior midguts of control (esgReDDM>, H) and p35 overexpression in stem and progenitor cells (esgReDDM>p35, p35 I and J) at 7 days after temperature shift. Arrows in (H, I) point to newly differentiated ECs. In (J), tumour mass is found in the anterior midgut. Data information: Scale bars, 50µm.(K), Quantification of mitosis PH3+ cells in posterior midgut (pmg) (control: gut scored n=13 and esg>p35, p35: n=22]). Student t-test (**** = P < 0.0001). Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max.(L), Diap1 is monitored by the GFP.4.3. (green) reporter (Djiane et al., 2013; Zhang et al., 2008). Diap1-GFP is detected in a subset of adult midgut esg+ cells that are negative for the mitotic marker PH3 (red, inset) and for EE marker Prospero (Pros, grey in inset). Diap1-GFP is not detected in mature EC (large nuclei cells, DAPI in blue). Data information: Scale bars, 50µm.(M-O), Diap1-GFP (green) co-localized almost 100% with the EB marker GBE-Su(H)-lacZ (red, M and O). Arrow points to a Diap1-GFP-negative GBE-Su(H)-lacZ-positive cell. Data information: Scale bars, 50µm.


Figure 2 ( B ) Lineage tracing using the initiator caspase sensor act - DBS - S - QF ( 14 days of tracing ) combined with QUAS - FLP and act > FRT > lacZ are visualized by ßGalactosidase ( ßGal , green ) . ISCs are labelled by anti - Dl ( red , insets ) . Red arrowhead points to the ISC . Gut is counterstained with DAPI ( blue ) and DAPI ( grey , in the inset ) . Data information : Scale bars , 100µm ( C ) Control GBE - Su ( H ) ReDDM > and ( D ) GBE - Su ( H ) ReDDM > p35 , p35 midguts after 7 days of tracing . Data information : Scale bars , 100µm ( E - E ' ' ) Adult midgut klu > mCD8 : : GFP , GBE - Su ( H ) - lacZ stained for ß - Gal ( red ) and GFP ( green ) . DAPI counterstaining in blue ( E ' and E ' ' ) . The arrows point to examples of klu - negative Su ( H ) - lacZ - positive cells . The green label ( asterisk , E ) is in the overlying muscle . Data information : Scale bars 50µm . ( F - J ) Adult midguts of ( F ) control kluReDDM > ( G ) kluReDDM > Debcl - RNAi , ( H ) kluReDDM > Drice - RNAi , ( I ) kluReDDM > Diap1 - RNAi , ( J ) kluReDDM > Diap1 midguts after 7 days of tracing . The arrows point to examples of PH3 + labelled ISCs . Data information : Scale bars , 100µm ( K ) Quantification of klu + cells in the indicated midguts at day 7 in low demand ( Low ) or standard ( Variable ) culturing conditions ( n = 7 , 5 , 9 , 7 ) . ata information : ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction . ( L ) Intestinal cell turnover measured by quantification of the number of newly renewed ECs ( RFP - only cells : gut scored n = 9 , 13 , 9 , 13 ) . Data information : Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction . ( M ) Quantification of apoptotic progenitor cells ( esg + ) measured as GFP + TUNEL + cells ( n = 9 , 9 , 9 , 9 ) . Data information : Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction . ( N ) Quantification of klu + cells and renewed ECs in kluReDDM > Diap1 posterior midguts ( n = 8 ) relative to ' Low ' control in K and L , respectively . Data information : ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Lineage tracing using the initiator caspase sensor act-DBS-S-QF (14 days of tracing) combined with QUAS-FLP and act>FRT>lacZ are visualized by ßGalactosidase (ßGal, green). ISCs are labelled by anti-Dl (red, insets). Red arrowhead points to the ISC. Gut is counterstained with DAPI (blue) and DAPI (grey, in the inset). Data information: Scale bars, 100µm(C) Control GBE-Su(H)ReDDM> and (D) GBE-Su(H)ReDDM>p35, p35 midguts after 7 days of tracing. Data information: Scale bars, 100µm(E-E'') Adult midgut klu>mCD8::GFP, GBE-Su(H)-lacZ stained for ß-Gal (red) and GFP (green). DAPI counterstaining in blue (E' and E''). The arrows point to examples of klu-negative Su(H)-lacZ-positive cells. The green label (asterisk, E) is in the overlying muscle. Data information: Scale bars 50µm.(F-J) Adult midguts of (F) control kluReDDM> (G) kluReDDM>Debcl-RNAi, (H) kluReDDM>Drice-RNAi, (I) kluReDDM>Diap1-RNAi, (J) kluReDDM>Diap1 midguts after 7 days of tracing. The arrows point to examples of PH3+ labelled ISCs. Data information: Scale bars, 100µm(K) Quantification of klu+ cells in the indicated midguts at day 7 in low demand (Low) or standard (Variable) culturing conditions (n=7, 5, 9, 7). ata information: ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.(L) Intestinal cell turnover measured by quantification of the number of newly renewed ECs (RFP-only cells: gut scored n=9, 13, 9, 13). Data information: Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.(M) Quantification of apoptotic progenitor cells (esg+) measured as GFP+ TUNEL+ cells (n= 9, 9, 9, 9). Data information: Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.(N) Quantification of klu+ cells and renewed ECs in kluReDDM>Diap1 posterior midguts (n=8) relative to 'Low' control in K and L, respectively. Data information: ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.


Figure 3 ( A - E ) Confocal images of representative midguts of the indicated genotypes using kluReDDM for tracing and age - and EB - specific transgene expression at day 7 after temperature shift . ISC mitosis was monitored by using PH3 labelling ( white ) in all guts . ( B - E ) Accumulation and loss of klu + cells by loss ( B and E ) and gain ( C and D ) of N and EGFR signalling . In ( C ) , the accumulation of EC - committed EBs ( klu + cells ) in midguts with constitutively activated EGFR ( kluReDDM > EGFRact ) is accompanied with a non - autonomous increase in ISC mitosis . Inset in ( C ) shows labelling with Dl ( blue ) , revealing symmetric ISC mitoses Data information : Scale bar , 100µm for all images . ( F ) Quantification of the number of klu + ( EBs ) in the indicated midguts ( n = 21 , 14 , 5 , 6 , 8 , 6 , 4 , 10 , 12 , 9 , 13 , 14 ) . Data information : Note that impaired cell renewal reduces the intestinal width . * * , P < 0 . 01 ; * * * * , P < 0 . 0001 , one - way ANOVA with Bonferroni correction . ( G ) Quantification of apoptotic esg + ( TUNEL + GFP + ) cells in the indicated midguts ( n = 28 , 4 , 7 , 7 , 16 , 8 , 8 , 7 , 10 , 7 , 20 , 24 ) . Data information : Note that impaired cell renewal reduces the intestinal width . * * , P < 0 . 01 ; * * * * , P < 0 . 0001 , one - way ANOVA with Bonferroni correction . Confocal images of representative midguts of the indicated genotypes Rescue of the loss of EBs induced by gain of Notch ( H - RNAi ) by expressing Debcl - RNAi . Data information : Scale bar , 100µm for all images . Confocal images of representative midguts of the indicated genotypes escue of the loss of EBs induced by loss of EGFR ( EGFRDN ) by expressing Debcl - RNAi . ( J and K ) Impact of gain of expression ( J ) and loss by RNAi expression ( K ) of lz using kluReDDM White arrowheads point to examples of mitosis labelled by PH3 + . ( L and M ) Impact of gain of expression ( L ) or loss by RNAi expression ( M ) of klu ( M ) using kluReDDM . Data information : Scale bar , 100µm for all images . ( N - R ) Images of representative midguts of esgReDDM at day 7 after temperature shift of the indicated genotypes . PH3 labelling ( white ) detected rare mitoses in the mutant genotypes . Data information : Scale bar , 100µm for all images .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-E) Confocal images of representative midguts of the indicated genotypes using kluReDDM for tracing and age- and EB-specific transgene expression at day 7 after temperature shift. ISC mitosis was monitored by using PH3 labelling (white) in all guts. (B-E) Accumulation and loss of klu+ cells by loss (B and E) and gain (C and D) of N and EGFR signalling. In (C), the accumulation of EC-committed EBs (klu+ cells) in midguts with constitutively activated EGFR (kluReDDM> EGFRact) is accompanied with a non-autonomous increase in ISC mitosis. Inset in (C) shows labelling with Dl (blue), revealing symmetric ISC mitoses Data information: Scale bar, 100µm for all images.(F) Quantification of the number of klu+ (EBs) in the indicated midguts (n= 21, 14, 5, 6, 8, 6, 4, 10, 12, 9, 13, 14). Data information: Note that impaired cell renewal reduces the intestinal width. **, P< 0.01; ****, P< 0.0001, one-way ANOVA with Bonferroni correction.(G) Quantification of apoptotic esg+ (TUNEL+ GFP+) cells in the indicated midguts (n= 28, 4, 7, 7, 16, 8, 8, 7, 10, 7, 20, 24). Data information: Note that impaired cell renewal reduces the intestinal width. **, P< 0.01; ****, P< 0.0001, one-way ANOVA with Bonferroni correction.Confocal images of representative midguts of the indicated genotypes Rescue of the loss of EBs induced by gain of Notch (H-RNAi) by expressing Debcl-RNAi. Data information: Scale bar, 100µm for all images.Confocal images of representative midguts of the indicated genotypes escue of the loss of EBs induced by loss of EGFR (EGFRDN) by expressing Debcl-RNAi. (J and K) Impact of gain of expression (J) and loss by RNAi expression (K) of lz using kluReDDM White arrowheads point to examples of mitosis labelled by PH3+. (L and M) Impact of gain of expression (L) or loss by RNAi expression (M) of klu (M) using kluReDDM. Data information: Scale bar, 100µm for all images.(N-R) Images of representative midguts of esgReDDM at day 7 after temperature shift of the indicated genotypes. PH3 labelling (white) detected rare mitoses in the mutant genotypes. Data information: Scale bar, 100µm for all images.


Figure 4 ( C ) All EBs show Diap1 - GFP expression but at varying levels , consistent with EB death fate being tuned by apoptotic input counteracting survival signals . Data information : ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) . ( D ) Quantification of the number of Diap1 - GFP + . Note that in p35 - expressing midguts reared in ' Low ' , GFP levels are not increased , consistent with the p35 protein suppressing apoptosis downstream of Diap1 . Data information ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) . ( E ) Quantification of the number of newly renewed ECs in the indicated genotypes after 7 days Increase in EB numbers and EC turnover in the EGFRact midgut reflects both suppression of EB deaths and the non - autonomous stimulation of ISC in this background . Data information : ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) . ( F ) Representative images acquired using the same laser intensity are shown for Diap1 - GFP in the indicated illustrative examples . Scale bar , 100µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C) All EBs show Diap1-GFP expression but at varying levels, consistent with EB death fate being tuned by apoptotic input counteracting survival signals. Data information: (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).(D) Quantification of the number of Diap1-GFP+. Note that in p35-expressing midguts reared in 'Low', GFP levels are not increased, consistent with the p35 protein suppressing apoptosis downstream of Diap1. Data information (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).(E) Quantification of the number of newly renewed ECs in the indicated genotypes after 7 days Increase in EB numbers and EC turnover in the EGFRact midgut reflects both suppression of EB deaths and the non-autonomous stimulation of ISC in this background. Data information: (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).(F) Representative images acquired using the same laser intensity are shown for Diap1-GFP in the indicated illustrative examples. Scale bar, 100µm.


Figure 1 . Full - length TLN ( but not APP ) accumulates in a distinct compartment in PS1 − / − hippocampal neurons . Double - immunofluorescence staining for endogenous TLN ( green , B36 . 1 ) and APP ( red , mAb C1 6 . 1 ) in wild - type ( top ) and PS1 − / − ( bottom ) hippocampal neurons ( 15 - d culture ) . The inset shows an overview of the neuron . In wild - type neurons TLN labeling is essentially localized to the somatodendritic plasmalemma in contrast to APP , which is found in intracellular compartments . In addition , PS1 deficiency causes TLN , but not APP , to cluster in large somatic accumulations . Bars , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.Full-length TLN (but not APP) accumulates in a distinct compartment in PS1−/−hippocampal neurons. Double-immunofluorescence staining for endogenous TLN (green, B36.1) and APP (red, mAb C1 6.1) in wild-type (top) and PS1−/− (bottom) hippocampal neurons (15-d culture). The inset shows an overview of the neuron. In wild-type neurons TLN labeling is essentially localized to the somatodendritic plasmalemma in contrast to APP, which is found in intracellular compartments. In addition, PS1 deficiency causes TLN, but not APP, to cluster in large somatic accumulations. Bars, 10 μm.


Figure 2 . ( A ) Western blot analysis of endogenous TLN and APP in wild - type and PS1 − / − hippocampal neurons . PS1 deficiency results in the accumulation of APP - CTF . No TLN - CTF ( estimated ± 7 kD ) was detected , neither was it detected after SFV - induced exogenous overexpression . Also , overexpression of a TLNΔE fragment did not generate a PS1 - dependent TLN intracellular domain . ( B ) Different constructs fused to the Gal4VP16 domain were used in a γ - secretase reporter assay . Control transfections ( pIPspAdApt empty vector and pIPspAdApt - APP without Gal4VP16 ) show no luciferase activity . APP - Gal4VP16 and NotchΔE - Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT , L658 , 458 , or compound 32 ( 125 nM ) . No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN ( APP / TLNTMR chimaera ) , indicating that this region is not cleaved by γ - secretase . ( C ) Left : γ - secretase inhibitors do not induce the formation of TLN accumulations . Wild - type hippocampal neurons were treated with γ - secretase inhibitors ( 125 nM , daily from d 7 - 14 post - plating ) , fixed , and immunostained for TLN and the percentage of neurons with TLN accumulations was scored . Culturing neurons for 25 d increases the size and number ( insets ) , but not the frequency of TLN accumulations ( mean ± SEM , n = 3 , 200 - 700 neurons per time point ) . Right : Western blot showing that only APP - CTF accumulates after chronic treatment with γ - secretase inhibitors . ( D ) Adenoviral reintroduction of human PS1 suppresses TLN accumulations . 30 - 35 % of PS1 − / − neurons display TLN accumulations , in contrast to < 5 % in PS1 + / + and + / − cultures as well as in cultures expressing human PS1 in a PS1 − / − background . However , suppression of TLN accumulations was obtained by adenoviral introduction of human PS1 . A clear dose - dependent inhibition was seen with increasing MOI of adenoviral ( AV ) human PS1 , but also the familial Alzheimer ' s disease - linked PS1G384A and dominant - negative PS1D257A mutants . Control adenoviral infections with eGFP did not have a significant effect . After 9 - 11 d post - infection ( 15 d post - plating ) , neurons displayed significant PS1 protein expression as demonstrated by double staining for TLN and human PS1 ( mAb 5 . 2 ) . Bars , 20 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(A) Western blot analysis of endogenous TLN and APP in wild-type and PS1−/− hippocampal neurons. PS1 deficiency results in the accumulation of APP-CTF. No TLN-CTF (estimated ± 7 kD) was detected, neither was it detected after SFV-induced exogenous overexpression. Also, overexpression of a TLNΔE fragment did not generate a PS1-dependent TLN intracellular domain.(B) Different constructs fused to the Gal4VP16 domain were used in a γ-secretase reporter assay. Control transfections (pIPspAdApt empty vector and pIPspAdApt-APP without Gal4VP16) show no luciferase activity. APP-Gal4VP16 and NotchΔE-Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT, L658,458, or compound 32 (125 nM). No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN (APP/TLNTMR chimaera), indicating that this region is not cleaved by γ-secretase.(C) Left: γ-secretase inhibitors do not induce the formation of TLN accumulations. Wild-type hippocampal neurons were treated with γ-secretase inhibitors (125 nM, daily from d 7-14 post-plating), fixed, and immunostained for TLN and the percentage of neurons with TLN accumulations was scored. Culturing neurons for 25 d increases the size and number (insets), but not the frequency of TLN accumulations (mean ± SEM, n = 3, 200-700 neurons per time point). Right: Western blot showing that only APP-CTF accumulates after chronic treatment with γ-secretase inhibitors.(D) Adenoviral reintroduction of human PS1 suppresses TLN accumulations. 30-35% of PS1−/− neurons display TLN accumulations, in contrast to <5% in PS1+/+ and +/− cultures as well as in cultures expressing human PS1 in a PS1−/− background. However, suppression of TLN accumulations was obtained by adenoviral introduction of human PS1. A clear dose-dependent inhibition was seen with increasing MOI of adenoviral (AV) human PS1, but also the familial Alzheimer's disease-linked PS1G384A and dominant-negative PS1D257A mutants. Control adenoviral infections with eGFP did not have a significant effect. After 9-11 d post-infection (15 d post-plating), neurons displayed significant PS1 protein expression as demonstrated by double staining for TLN and human PS1(mAb 5.2). Bars, 20 μm.


Figure 3 . Wild - type and PS1 − / − cortical neurons were transduced with SFV - TLN ( A ) or - APP ( B ) , pulse labeled for 15 min with [ 35S ] methionine , and chased for different time periods . ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS - PAGE and phosphorimaging . The accumulation of an EndoH - resistant band indicates progressive maturation during Golgi passage ; however , the ratio of EndoH - resistant to - sensitive TLN ( A , top ) and APP ( B , top ) revealed no difference ( mean ± SEM , n = 3 ) . Instead , the half - life of overexpressed TLN ( A , bottom ) , but not full - length APP ( B , bottom ) , is significantly prolonged in neurons − / − neurons ( mean ± SEM , n = 3 ) . ( C ) Wild - type and PS1 − / − hippocampal neurons ( 12 d ) were scraped , pelleted , and treated with 10 mU EndoH , followed by Western blot detection of APP , TLN , PS1 , nicastrin ( NCT ) , synaptophysin , and ductin . TLN levels are increased almost fourfold in PS1 − / − neurons . No EndoH - sensitive TLN was detected in PS1 − / − neurons , indicating a complete maturation . APP - CTF shows a dramatic accumulation ( 44 - fold ; mean ± SEM , n = 3 ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.Wild-type and PS1−/− cortical neurons were transduced with SFV-TLN (A) or -APP (B), pulse labeled for 15 min with [35S]methionine, and chased for different time periods. ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS-PAGE and phosphorimaging. The accumulation of an EndoH-resistant band indicates progressive maturation during Golgi passage; however, the ratio of EndoH-resistant to -sensitive TLN (A, top) and APP (B, top) revealed no difference (mean ± SEM, n = 3). Instead, the half-life of overexpressed TLN (A, bottom), but not full-length APP (B, bottom), is significantly prolonged in neurons−/−neurons (mean ± SEM, n = 3).(C) Wild-type and PS1−/− hippocampal neurons (12 d) were scraped, pelleted, and treated with 10 mU EndoH, followed by Western blot detection of APP, TLN, PS1, nicastrin (NCT), synaptophysin, and ductin. TLN levels are increased almost fourfold in PS1−/− neurons. No EndoH-sensitive TLN was detected in PS1−/− neurons, indicating a complete maturation. APP-CTF shows a dramatic accumulation (44-fold; mean ± SEM, n = 3).


Figure 4 . TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways . ( A - F ) Early compartments . No colocalization was observed between TLN ( green in all panels ) and nuclei ( A , TOPRO - 3 ) , ER - markers BIP ( B ) and calnexin ( C ) , ERGIC - 53 ( D ) , β - COP ( E ) or GM130 ( F ) , and labeling cis - Golgi . ( G - L ) Late compartments . TLN accumulations are not accessible for exogenous biotin ( G ) . Early and recycling endosomes ( H , EEA1 ) and transferrin receptor ( I , TFR ) , late endosomes ( J , LBPA ) , and lysosomes ( K , Lamp - 2 ; L , catD ) also stained negative for TLN . Vertical sections ( x - z , arrowheads in I and J ) clearly distinguish TLN accumulations from recycling ( I , TFR ) and late endosomes ( J , LBPA ) . Bars , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways. (A-F) Early compartments. No colocalization was observed between TLN (green in all panels) and nuclei (A, TOPRO-3), ER-markers BIP (B) and calnexin (C), ERGIC-53 (D), β-COP (E) or GM130 (F), and labeling cis-Golgi. (G-L) Late compartments. TLN accumulations are not accessible for exogenous biotin (G). Early and recycling endosomes (H, EEA1) and transferrin receptor (I, TFR), late endosomes (J, LBPA), and lysosomes (K, Lamp-2; L, catD) also stained negative for TLN. Vertical sections (x-z, arrowheads in I and J) clearly distinguish TLN accumulations from recycling (I, TFR) and late endosomes (J, LBPA). Bars, 10 μm.


Figure 5 . Correlative light immuno - EM analysis of TLN accumulations . ( A ) Insert : overview of the selected neuron as seen by immunocytochemistry . The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno - EM level ( star ) . ( B ) Higher magnification of the boxed area in A . ( C ) High magnification of the boxed structure seen in B . 10 - nm gold particles label TLN . Note the complex composition of the internal membranes of the TLN - positive compartments . ( D ) Example of a TLN ( 15 - nm gold ) - containing structure obtained by the conventional cryosectioning technique . Note some heterogeneity in the internal membranes between C and D . Lamp - 1 ( 10 - nm gold ) is absent from the TLN - containing compartment . Arrows point to invaginations of the cytoplasm . N , nucleus ; P , plasma membrane . Bars : A , 5 μm ; B , 2 μm ; C , 500 nm ; D , 200 nm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.Correlative light immuno-EM analysis of TLN accumulations. (A) Insert: overview of the selected neuron as seen by immunocytochemistry. The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno-EM level (star). (B) Higher magnification of the boxed area in A. (C) High magnification of the boxed structure seen in B. 10-nm gold particles label TLN. Note the complex composition of the internal membranes of the TLN-positive compartments. (D) Example of a TLN (15-nm gold)-containing structure obtained by the conventional cryosectioning technique. Note some heterogeneity in the internal membranes between C and D. Lamp-1 (10-nm gold) is absent from the TLN-containing compartment. Arrows point to invaginations of the cytoplasm. N, nucleus; P, plasma membrane. Bars: A, 5 μm; B, 2 μm; C, 500 nm; D, 200 nm.


Figure 6 . TLN accumulations are distinct from lysosomes . A TLN ( 15 - nm gold ) - containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp - 1 ( 10 - nm gold ) . The TLN - positive compartment is clearly distinct in size and from lysosomes ( L ) and lacks Lamp - 1 . P , plasma membrane . Bar , 200 nm .

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Figure 6.TLN accumulations are distinct from lysosomes. A TLN (15-nm gold)-containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp-1 (10-nm gold). The TLN-positive compartment is clearly distinct in size and from lysosomes (L) and lacks Lamp-1. P, plasma membrane. Bar, 200 nm.


Figure 7 . ( B and C ) Double immunostaining of wild - type and PS1 − / − hippocampal neurons for TLN ( green ) and autophagic vacuole - associated Apg12p ( B ) and LC3 ( C ) . To detect both sets of pAbs , an alternative method was developed using biotinylated anti - TLN ( see Materials and methods ) . Each experiment included controls for specificity ( not depicted ) . In wild - type neurons , both Apg12p and LC3 ( B and C , top row ) were broadly distributed in cell bodies and neurites , but did not colocalize with TLN . Colocalization was only observed in multiple TLN accumulations in PS1 − / − hippocampal neurons ( second row in B and C , respectively ) . For Apg12p , coimmunostaining was also noticed in small discrete structures in the proximal dendrites ( B , bottom row ) . Bars , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(B and C) Double immunostaining of wild-type and PS1−/− hippocampal neurons for TLN (green) and autophagic vacuole-associated Apg12p (B) and LC3 (C). To detect both sets of pAbs, an alternative method was developed using biotinylated anti-TLN (see Materials and methods). Each experiment included controls for specificity (not depicted). In wild-type neurons, both Apg12p and LC3 (B and C, top row) were broadly distributed in cell bodies and neurites, but did not colocalize with TLN. Colocalization was only observed in multiple TLN accumulations in PS1−/− hippocampal neurons (second row in B and C, respectively). For Apg12p, coimmunostaining was also noticed in small discrete structures in the proximal dendrites (B, bottom row). Bars, 10 μm.


Figure 8 . ( A ) Intracellular sorting and maturation of catD . Wild - type and PS1 − / − neurons were metabolically labeled and chased for the indicated time periods . Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46 - kD intermediate vs . 52 - kD precursor ( mean ± SEM , n = 3 ) . ( B - D ) TLN localizes to autophagic vacuoles of catD − / − hippocampal neurons . Lysotracker staining revealed sparsely distributed small - sized organelles in wild - type and PS1 − / − neurons in contrast to catD − / − neurons where high numbers of large Lysotracker - positive organelles were found ( B , arrowheads ) . Although TLN accumulations in PS1 − / − were fully negative for Lysotracker , some acidic organelles tended to closely associate ( C , arrowheads , middle ) . In catD − / − neurons , TLN was clearly detected in the large acidic organelles ( C , bottom ) . Bars , 20 μm . ( D ) At the immuno - EM level , the large acidic organelles seen in catD − / − neurons represent dense autophagic vacuole - like structures ( star ) that label positive for TLN ( 10 - nm gold ) . M , mitochondrion . Bars , 200 nm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8.(A) Intracellular sorting and maturation of catD. Wild-type and PS1−/− neurons were metabolically labeled and chased for the indicated time periods. Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46-kD intermediate vs. 52-kD precursor (mean ± SEM, n = 3).(B-D) TLN localizes to autophagic vacuoles of catD−/−hippocampal neurons. Lysotracker staining revealed sparsely distributed small-sized organelles in wild-type and PS1−/− neurons in contrast to catD−/−neurons where high numbers of large Lysotracker-positive organelles were found (B, arrowheads). Although TLN accumulations in PS1−/− were fully negative for Lysotracker, some acidic organelles tended to closely associate (C, arrowheads, middle). In catD−/− neurons, TLN was clearly detected in the large acidic organelles (C, bottom). Bars, 20 μm. (D) At the immuno-EM level, the large acidic organelles seen in catD−/−neurons represent dense autophagic vacuole-like structures (star) that label positive for TLN (10-nm gold). M, mitochondrion. Bars, 200 nm.


Figure 9 . TLN mediates phagocytic uptake of microbeads in hippocampal neurons . ( A ) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast ( DIC ) . Already after 4 h , many beads stain positive for TLN ( arrowheads , top ) . At 48 h ( bottom ) , most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining . * , cell body . Note that microbeads were not found associated with preexisting TLN accumulations ( asterisk in top panels , 4 h ) . ( B ) Microbeads ( 24 h ) accumulate TLN and actin , as shown by phalloidin - Alexa 568 . An overview of the neuron is given in each top left inset , next to two detailed areas ( white boxes ) . The differential interference contrast ( DIC ) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin ( arrowheads ) . TOPRO - 3 ( blue ) marks the nucleus ( see also Fig . S1 , available at http : / / www . jcb . org / cgi / content / full / jcb . 200406060 / DC1 ) . ( C ) Similar as in B , but immunostained for TLN and endogenous PIP2 . The overview demonstrates the overall recruitment of PIP2 to microbeads ( arrows ) . The parallel insets show a detailed area ( white box ) with PIP2 being colocalized with TLN on individual microbeads ( arrowheads ) . Note that PIP2 occasionally colocalizes with TLN at the plasma membrane ( asterisk ) . Bars ( A - C ) , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9.TLN mediates phagocytic uptake of microbeads in hippocampal neurons. (A) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast (DIC). Already after 4 h, many beads stain positive for TLN (arrowheads, top). At 48 h (bottom), most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining. *, cell body. Note that microbeads were not found associated with preexisting TLN accumulations (asterisk in top panels, 4 h). (B) Microbeads (24 h) accumulate TLN and actin, as shown by phalloidin-Alexa 568. An overview of the neuron is given in each top left inset, next to two detailed areas (white boxes). The differential interference contrast (DIC) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin (arrowheads). TOPRO-3 (blue) marks the nucleus (see also Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406060/DC1). (C) Similar as in B, but immunostained for TLN and endogenous PIP2. The overview demonstrates the overall recruitment of PIP2 to microbeads (arrows). The parallel insets show a detailed area (white box) with PIP2 being colocalized with TLN on individual microbeads (arrowheads). Note that PIP2 occasionally colocalizes with TLN at the plasma membrane (asterisk). Bars (A-C), 10 μm.


Figure 1Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( A ) PS1 , PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N ( PS1 ) , BI - HF5C ( PS2 ) and N1660 ( NCT ) , respectively . ( B ) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687 . ( C ) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation / immunoblotting using antibodies 3552 / 2D8 . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( D ) Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 . Pooled clones of HEK293 / sw cells stably transfected with PS1 L166P were used as reference . Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( E ) PS1 expression levels were analyzed in cell lysates of HEK293 / sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12 , respectively . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( F ) Conditioned media were analyzed by ELISA specific for Aβ40 , Aβ42 and Aβ43 . Data represent mean ± s . e . m . ( n = 6 ) . Absolute levels and Aβ ratios are shown . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( G ) Secreted Aβ was analyzed as in ( D ) . To verify individual Aβ species , Aβ standards were co - migrated . Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( H ) Total Aβ in conditioned media was analyzed by MALDI - TOF MS following immunoprecipitation with antibody 4G8 . Observed ( Aβ42 , 4513 . 6 ; Aβ43 , 4615 . 3 ) and predicted molecular masses ( Aβ42 , 4514 . 1 ; Aβ43 , 4615 . 2 ) were in good agreement .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(A) PS1, PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI-HF5C (PS2) and N1660 (NCT), respectively.(B) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687. (C) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (D) Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8. Pooled clones of HEK293/sw cells stably transfected with PS1 L166P were used as reference. Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (E) PS1 expression levels were analyzed in cell lysates of HEK293/sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12, respectively.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (F) Conditioned media were analyzed by ELISA specific for Aβ40, Aβ42 and Aβ43. Data represent mean ± s.e.m. (n=6). Absolute levels and Aβ ratios are shown.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(G) Secreted Aβ was analyzed as in (D). To verify individual Aβ species, Aβ standards were co-migrated. Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(H) Total Aβ in conditioned media was analyzed by MALDI-TOF MS following immunoprecipitation with antibody 4G8. Observed (Aβ42, 4513.6; Aβ43, 4615.3) and predicted molecular masses (Aβ42, 4514.1; Aβ43, 4615.2) were in good agreement.


Figure 2 ( A ) PS1 / 2 - / - MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A . Antibody 5E12 was used for the detection of the PS1 CTF . ( B ) Stably transduced PS1 / 2 - / - MEF cells of A were transiently transfected with APPsw - 6myc and conditioned media were analyzed for Aβ species by Tris - Bicine urea SDS - PAGE . Individual Aβ species were verified by co - migration with Aβ standards ( left panel ) and quantified ( right panel ) . Data represent mean ± s . e . m . of n = 3 independently performed transfections . Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis . ( C ) Secreted Aβ from cells in B were quantified by ELISA . Data represent mean ± s . e . m . of n = 5 ( PS1 WT ) or 6 ( PS1 L435F ) independently performed transfections .

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Figure 2(A) PS1/2-/- MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A. Antibody 5E12 was used for the detection of the PS1 CTF.(B) Stably transduced PS1/2-/- MEF cells of A were transiently transfected with APPsw-6myc and conditioned media were analyzed for Aβ species by Tris-Bicine urea SDS-PAGE. Individual Aβ species were verified by co-migration with Aβ standards (left panel) and quantified (right panel). Data represent mean ± s.e.m. of n=3 independently performed transfections. Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis.(C) Secreted Aβ from cells in B were quantified by ELISA. Data represent mean ± s.e.m. of n=5 (PS1 WT) or 6 (PS1 L435F) independently performed transfections.


Figure 3Immunohistochemical detection of Aβ40 ( left column ) , Aβ42 ( medium column ) and Aβ43 ( right column ) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation ( cases # 1 and # 2 , 1st and 2nd rows ) , another FAD case with different PS1 mutation ( case # 3 , 3rd row ) and one SAD case ( case # 4 , forth row ) . In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40 . This is in contrast to Aβ plaques of the control FAD and SAD cases , in which solely Aβ42 predominates and Aβ43 is sparse ; even levels of Aβ40 seem to be lower than in the PS1 L435F cases . Note that in both PS1 L435F cases several plaques are larger than those seen in cases # 3 and # 4 , representing cotton wool plaques . Scale bar = 500 µm . Magnification is identical in all pictures .

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Figure 3Immunohistochemical detection of Aβ40 (left column), Aβ42 (medium column) and Aβ43 (right column) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation (cases #1 and #2, 1st and 2nd rows), another FAD case with different PS1 mutation (case #3, 3rd row) and one SAD case (case #4, forth row). In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40. This is in contrast to Aβ plaques of the control FAD and SAD cases, in which solely Aβ42 predominates and Aβ43 is sparse; even levels of Aβ40 seem to be lower than in the PS1 L435F cases. Note that in both PS1 L435F cases several plaques are larger than those seen in cases #3 and #4, representing cotton wool plaques. Scale bar = 500 µm. Magnification is identical in all pictures.


Figure 1B , phase contrast images of the morphological changes in MDR cells , residual MDR cells and regrown cells for Du145TXR or MCF - 7ADR . Red circles mark magnified areas ( bottom ) . Scale bars , 50 μm ( low magnification images ) . C - D , cell viability of residual MDR cells ( RMCs ) and MDR cells ( MCs ) of ( C ) Du145TXR or ( D ) MCF - 7ADR cells using the same combination treatment for 24 hours on the indicated days . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments . F - G , drug tolerant rate of ( F ) Du145 / Du145TXR ( 20 nM taxol / 20 nM taxol plus 50 μM verapamil ) or ( G ) MCF - 7 / MCF - 7ADR ( 200 nM adriamycin / 200 nM adriamycin plus 50 μM verapamil ) cells treated with drugs on the indicated days . Drug tolerant rate defined by comparing cell viability followed treatment , RMCs VS . MCs or CCs VS . PeCs , > 1 representing drug tolerant . Induction Time indicates the time of entering DTP state ; Sustaining Time indicates the time of maintaining DTP state . Blue colors represent control groups ; Red colors represent MDR cancer cells . Mean with ± SD .

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Figure 1B, phase contrast images of the morphological changes in MDR cells, residual MDR cells and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images).C-D, cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 hours on the indicated days. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate defined by comparing cell viability followed treatment, RMCs VS. MCs or CCs VS. PeCs, > 1 representing drug tolerant. Induction Time indicates the time of entering DTP state; Sustaining Time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD.


Figure 2D , heat map based on scoring rank indicated consistently expressed genes identified by RNA - seq of MCs VS . MPCs in Du145TXR and MCF - 7ADR . F , immunoblotting of NPC1L1 in MCs , MPCs and RCs of Du145TXR or MCF - 7ADR . G , immunoblotting of NPC1L1 in Du145TXR or MCF - 7ADR cells transfected with siNPC1L1 or siScramble . H - I , colony formation assay and quantification of ( H ) Du145TXR or ( I ) MCF - 7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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Figure 2D, heat map based on scoring rank indicated consistently expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR and MCF-7ADR.F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR. G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.H-I, colony formation assay and quantification of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


Figure 3A - C , flow cytometric analysis of ROS levels in Du145TXR or MCF - 7ADR cells treated with indicated agents for 12 and 36 hours . ( A ) Representative images and quantification of ROS in ( B ) Du145TXR or ( C ) MCF - 7ADR cells are shown . One - way ANOVA was used to analyze statistical differences . Mean with ± SD Data information : Results are representative of three independent experiments . D - E , cell viability of ( D ) Du145TXR or ( E ) MCF - 7ADR treated with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments . J - K , cell viability of ( J ) Du145TXR or ( K ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments.D-E, cell viability of (D) Du145TXR or (E) MCF-7ADR treated with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.J-K, cell viability of (J) Du145TXR or (K) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments.


Figure 4C - D , endocytosis analysis of ( C ) Du145TXR or ( D ) MCF - 7ADR cells treated with indicated agents for 8 hours , followed by staining with 250 μg / mL FITC - DEX for 4 hours . Scale bars , 10 μm . Data information : Results are representative of three independent experiments . E - F , phase contrast images of the morphological changes in ( E ) Du145TXR and ( F ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours . Scale bars , 10 or 25 μm . The red arrows indicate vacuoles . Data information : Results are representative of three independent experiments . I - J , colony formation assay and quantification of ( I ) Du145TXR or ( J ) MCF - 7ADR cells treated with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C-D, endocytosis analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments.E-F, phase contrast images of the morphological changes in (E) Du145TXR and (F) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours. Scale bars, 10 or 25 μm. The red arrows indicate vacuoles. Data information: Results are representative of three independent experiments.I-J, colony formation assay and quantification of (I) Du145TXR or (J) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


Figure 5C , immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours . Data information : Results are representative of three independent experiments . F - G , relative luciferase activities of wild - type ( WT ) or mutant ( MUT ) NPC1L1 promoter ( - 205 ~ - 215 , # 1 ) reporter constructs in ( F ) Du145TXR or ( G ) MCF - 7ADR cells treated with indicated agents . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments . J - K , colony formation assay and quantification of ( J ) Du145TXR or ( K ) MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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Figure 5C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments.F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


Figure 6immunohistochemical staining of Ki67 in indicated groups . ( D ) Representative images are shown . Scale bar , 50 μm . , immunohistochemical staining of cleaved - caspase 3 ( CC3 ) in indicated groups . ( F ) Representative images are shown . Scale bar , 50 μm . ( I ) Corresponding images and isolated tumors are shown . The red dotted line represents cured tumors .

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Figure 6immunohistochemical staining of Ki67 in indicated groups. (D) Representative images are shown. Scale bar, 50 μm. , immunohistochemical staining of cleaved-caspase 3 (CC3) in indicated groups. (F) Representative images are shown. Scale bar, 50 μm.(I) Corresponding images and isolated tumors are shown. The red dotted line represents cured tumors.


Figure 1A ) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1 . Coimmunoprecipitation ( CoIP ) experiments using TAP - tagged Nrd1 ( either wt or ∆ CID ) as the bait . Representative gel of one out of two independent experiments . C ) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1 . CoIP experiments using Nrd1 - TAP as the bait in a SEN1 or sen1 ∆ NIM background . Representative gel of one out of two independent experiments . D ) CoIP experiments using HA - tagged Sen1 , either wt or ∆ NIM , as the bait . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA prior to immunoprecipitation . In these experiment , Sen1 could not be detected in the input extracts .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1. Coimmunoprecipitation (CoIP) experiments using TAP-tagged Nrd1 (either wt or ∆CID) as the bait. Representative gel of one out of two independent experiments.C) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1. CoIP experiments using Nrd1-TAP as the bait in a SEN1 or sen1∆NIM background. Representative gel of one out of two independent experiments.D) CoIP experiments using HA-tagged Sen1, either wt or ∆NIM, as the bait. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA prior to immunoprecipitation. In these experiment, Sen1 could not be detected in the input extracts.


Figure 3A ) Deletion of the N - terminal of Sen1 is lethal . A ∆ sen1 strain ( YDL2767 ) covered by an URA3 - containing plasmid ( pFL38 ) expressing wt Sen1 was transformed with a TRP1 - plasmid ( pFL39 ) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left . After over - night growth in non - selective medium , cells were plated on minimal medium ( CSM ) containing 5 - fluorootic acid ( 5 - FOA ) to select those that have lost the URA3 plasmid . The absence of growth in 5 - FOA implies that the SEN1 version expressed in the TRP1 - plasmid does not support viability . B ) Deletion of the N - terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo . Northern blot analyses of three well - characterized NNS - targets , the CUT NEL025c and the snoRNAs SNR13 and SNR33 , in a Sen1 - AID ( auxin - induced degron ) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1 . Sen1 - AID was depleted for 2h by the addition of 500 μM indole - 3 - acetic acid ( IAA ) to monitor the capacity of the plasmid - borne versions of SEN1 to induce transcription termination . The expected RNA species resulting from inefficient termination ( RT for readthrough species ) are indicated . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control . C ) Overexpression of sen1 ∆ Nter restores viability . Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter ( pGAL ) in the presence of galactose . D ) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N - terminal domain . Northern blot analysis of a typical NNS - target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.B) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


Figure 4RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . A ) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site ( PAS ) of mRNAs . Values on the y axis correspond to the median coverage . B ) Box - plot representation of the readthrough ( RT ) index as a readout of the termination efficiency at each mRNA . The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS , divided by the average signal from the TSS to the PAS of each mRNA . Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR ( where IQR is the interquartile distance or distance between the first and the third quartile ) . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . For each mRNA ( n = 5752 ) , the values correspond to the average of two independent biological replicates . Data populations were compared using a t - test ( bilateral distributions , unpaired data ) and p - values ≥ 0 . 05 are indicated as ns ( not significant ) . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . C - D ) IGV screenshots of two well - characterized protein - coding genes displaying substantial defects in premature termination in some mutants . The values correspond to the counts per million of counts ( CPM ) multiplied by 10 . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . E ) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site ( TTS ) as the reference point . F ) Analysis of termination defects in the different mutants at CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS , divided by the average signal over a window of 100 nt downstream from the TSS ( CUT100 ) . For each CUT ( n = 329 ) , the values correspond to the average of two independent biological replicates . P - values < 0 . 001 are indicated by three asterisks . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . G - H ) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS , divided by the average signal over the mature snoRNA . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. A) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site (PAS) of mRNAs. Values on the y axis correspond to the median coverage. B) Box-plot representation of the readthrough (RT) index as a readout of the termination efficiency at each mRNA. The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS, divided by the average signal from the TSS to the PAS of each mRNA. Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR (where IQR is the interquartile distance or distance between the first and the third quartile). The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. For each mRNA (n=5752), the values correspond to the average of two independent biological replicates. Data populations were compared using a t-test (bilateral distributions, unpaired data) and p-values≥ 0.05 are indicated as ns (not significant).RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. C-D) IGV screenshots of two well-characterized protein-coding genes displaying substantial defects in premature termination in some mutants. The values correspond to the counts per million of counts (CPM) multiplied by 10.RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. E) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site (TTS) as the reference point. F) Analysis of termination defects in the different mutants at CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS, divided by the average signal over a window of 100 nt downstream from the TSS (CUT100). For each CUT (n=329), the values correspond to the average of two independent biological replicates. P-values < 0.001 are indicated by three asterisks. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. G-H) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants.RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA.RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs.