( C ) Workflow of neurite outgrowth . ( D ) Representative pictures of single neurites at time - point 0h ( DIV3 ) 24h ( DIV4 ) . Neurons were infected 4h after plating with lentiviruses encoding GFP ( to visualize neurons ) together with shRNA expression cassettes ( sh1 sh2 ) targeting either NrCAM or carrying a scrambled ( scr ) control construct . of neurites were taken at three days in vitro ( DIV3 ) 24h later at DIV4 . In order to study effect of ADAM10 on neurite outgrowth , neurons were treated with ADAM10 inhibitor GI254023x , or vehicle ( control ) , at DIV3 , after taking first pictures with an epifluorescent microscope . differences in neurite were calculated as absolute values ( neurite at 24h minus neurite of 0h ) for neurites passing through middle channels of chambers . Only neurites that had already entered main channel at 0h had not yet left those channels at 0h were considered . red arrows indicate start end of respective measurements . scale bar indicates 40 µm . ( E ) One - way ANOVA with post hoc Dunnett ' s . Given are mean + / - standard error of them mean ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , n - numbers for each condition are shown in graph in 3E ) .

O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).


( A ) Wt neurons were kept in culture until DIV5 , then were treated with acitretin ( 4 µM ) or vehicle control for 48h , as described earlier ( Tippmann et al , 2009 ) . ( B ) Densitometric quantifications of Western Blots are shown . Given are mean + / - standard error of mean ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - n = 9 - 20 ) . mean of solvent treated were set to 1 .

O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE 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-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Wt neurons were kept in culture until DIV5, then the cells were treated with acitretin (4 µM) or vehicle control for 48h, as described earlier (Tippmann et al, 2009). (B) Densitometric quantifications of the Western Blots are shown. Given are mean +/- the standard error of the mean (*** p < 0.001; **** p < 0. 0001, two-sided students t-test n = 9-20). The mean levels of solvent treated cells were set to 1.


( C ) Detection of sNrCAM human serum albumin ( hSA ) in AD CSF that had been treated with acitretin ( 30 mg / d ) or placebo for 30 days ( n = 9 ) ( Endres et al , 2014 ) . ( D ) Densitometric of Western Blots in ( C ) . sNrCAM / hSA ratios were calculated , then treatment to baseline ratios ( t / b ) for every were calculated . Two - sided student ' s t - ( n = 9 ) . Given are mean + / - standard error of them mean . Representative Western Blots are shown .

O O O O O O B-ORGANISM B-TISSUE B-GENEPROD O O O O B-DISEASE B-TISSUE O O O O O B-SMALL_MOLECULE O O O 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-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O

(C) Detection of sNrCAM and human serum albumin (hSA) in AD patients CSF that had been treated with acitretin (30 mg/d) or placebo for 30 days (n = 9) (Endres et al, 2014). (D) Densitometric quantification of the Western Blots in (C). sNrCAM/hSA ratios were calculated, and then the treatment to baseline ratios (t/b) for every patient were calculated. Two-sided student's t-test (n = 9). Given are mean +/- the standard error of them mean. Representative Western Blots are shown.


( A ) To ensure proper synapse formation , neurons were kept in culture for ten days prior to treatment ( Wan et al , 2012 ) . Then , were treated with NMDA ( 50 µM ) , NMDA ( 50 µM ) GI254023x ( 5 µM ) , GI254023x ( 5 µM ) alone or vehicle for 30 minutes . Total mADAM10 remained unchanged by treatment , which is in with NMDA altering mainly intracellular localization of ADAM10 by driving to synaptic membranes ( Marcello et al , 2007 ) . Densitometric quantifications of Western Blots are shown . One - way ANOVA with post hoc Dunnett ' s ( * * * p < 0 . 001 , n = 7 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . was reprobed with different indicated antibodies .

O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies.


( B ) Wt neurons were kept in culture until DIV10 , then were treated with NMDA ( 50 µM ) , or vehicle for 30 minutes . surface were labeled with biotin enriched by streptavidin pull - down . biotinylated were detected by immunoblotting . Total lysates were analyzed to compare mNrCAMs surface / lysate . Densitometric quantifications of Western Blots are shown . Two - sided students t - ( * * p < 0 . 01 , n = 6 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . Representative Western Blots are shown .

O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O

(B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


Volcano plot of proteomic of CSF from AD treated with acitretin or placebo for 30 days . Every circle represents one . log10 transformed t - p - values ( again treatment / baseline for every ) are plotted against log2 transformed label - free ratios of acitretin placebo CSF . ADAM10 substrates are indicated in bold writing . In total , more than 50 ADAM10 substrates were identified , but only significantly altered substrates as well as NrCAM APP are indicated .

O O O B-EXP_ASSAY O B-TISSUE O B-DISEASE O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-SMALL_MOLECULE O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O

Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.


HEK293 were transiently transfected with hNrCAM , FLAG - tagged hTSPAN15 , either plasmids or empty vector . Detection of sAPPα sNrCAM in conditioned media ADAM10 in lysate . Densitometric of Western Blots are shown ( * * p < 0 . 01 ) , ( * * * p < 0 . 001 ) , ( * * * * p < 0 . 0001 ) , two - sided students t - , or one - way ANOVA with post hoc Dunnett ' s ( n = 6 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . Representative Western Blots are shown .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O

HEK293 cells were transiently transfected with hNrCAM, FLAG-tagged hTSPAN15, either plasmids or the empty vector. Detection of sAPPα and sNrCAM in the conditioned media and ADAM10 in the cell lysate. Densitometric quantification of the Western Blots are shown (** p < 0.01), (*** p < 0.001), (**** p < 0.0001), two-sided students t-test, or one-way ANOVA with post hoc Dunnett's test (n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


( A ) Time series of TIRF microscopyimages showing GFP - dynein ( green in merge ) localising to plus ends of dynamic Alexa568 - microtubules ( Alexa568 - MT , magenta in merge ) . concentrations were 10 nM GFP - dynein , 20 nM human dynactin , 20 nM EB1 17 . 5 µM Alexa568 - tubulin ( 5 % labelling ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O

(A) Time series of TIRF microscopyimages showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).


( B ) dual colour kymographs showing GFP - dynein ( green in merge ) localising to plus ends of dynamic Alexa568 - microtubules ( Alexa568 - MT , magenta in merge ) . concentrations were 10 nM GFP - dynein , 20 nM human dynactin , 20 nM EB1 17 . 5 µM Alexa568 - tubulin ( 5 % labelling ) .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O

(B) individual and dual colour kymographs showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).


( C ) Merged triple colour single channel kymographs showing microtubule end tracking of GFP - dynein ( green in merge ) Alexa647 - EB3 ( red in merge ) on dynamic Alexa568 - microtubules ( blue in merge ) . Concentrations were 10 nM GFP - dynein , 10 nM human dynactin , 10 nM Alexa647 - EB3 , 17 . 5 µM tubulin .

O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O

(C) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP-dynein (green in merge) and Alexa647-EB3 (red in merge) on dynamic Alexa568-microtubules (blue in merge). Concentrations were 10 nM GFP-dynein, 10 nM human dynactin, 10 nM Alexa647-EB3, and 17.5 µM tubulin.


( D , E ) Dual single colour kymographs of GFP - dynein ( green ) on dynamic Alexa568 - microtubules ( magenta ) in absence of either dynactin ( D ) . Concentrations of present as in A .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of either dynactin (D) . Concentrations of the proteins present as in A.


( D , E ) Dual single colour kymographs of GFP - dynein ( green ) on dynamic Alexa568 - microtubules ( magenta ) in absence of EB ( E ) . Concentrations of present as in A .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of EB proteins (E). Concentrations of the proteins present as in A.


( F ) Averaged profiles of GFP - dynein at growing microtubule plus ends in presence of both human dynactin EB1 ( blue squares , as in A ) , in absence of dynactin ( red circles , as in D ) , or in absence of an EB ( black triangles , as in E ) . Mean values from at least two separate experiments per condition ( with a total of at least 30 kymographs ) are shown ; error bars are s . e .

O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of both human dynactin and EB1 (blue squares, as in A), in the absence of dynactin (red circles, as in D), or in the absence of an EB protein (black triangles, as in E). Mean values from at least two separate experiments per condition (with a total of at least 30 kymographs) are shown; error bars are s. e.


( G ) Kymographs of GFP - dyneinmicrotubule end tracking in absence of ATP ( in contrast to other experiments that contain ATP ) . Other conditions as in A . For merged kymographs , microtubule plus minus ends are labelled by ( + ) ( − ) . Experiments were performed at 30 ° C .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

(G) Kymographs of GFP-dyneinmicrotubule end tracking in the absence of ATP (in contrast to all other experiments that contain ATP). Other conditions as in A. For merged kymographs, microtubule plus and minus ends are labelled by (+) and (−). Experiments were performed at 30°C.


( A ) Merged triple colour single channel TIRF microscopy kymographs of GFP - dynein showing end tracking , processive motility diffusive static binding in presence of DDB components EB1 . concentrations are 10 nM GFP - dynein ( green in merge ) , 20 nM human dynactin , 20 nM EB1 , 200 nM Alexa647 - BicD2 - N ( magenta in merge ) , 17 . 5 µM Alexa568 - tubulin ( blue in merge ) . Alexa647 - BicD2 - N often co - localises with processively moving occasionally with statically bound GFP - dynein , but not with plus end - tracking GFP - dynein .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O

(A) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP-dynein showing end tracking, processive motility and diffusive and static binding in the presence of all DDB components and EB1. Protein concentrations are 10 nM GFP-dynein (green in merge), 20 nM human dynactin, 20 nM EB1, 200 nM Alexa647-BicD2-N (magenta in merge), and 17.5 µM Alexa568-tubulin (blue in merge). Alexa647-BicD2-N often co-localises with processively moving and occasionally with statically bound GFP-dynein, but not with plus end-tracking GFP-dynein.


( B ) Averaged profiles of GFP - dynein ( green squares ) Alexa647 - BicD2 - N ( magenta circles ) at growing microtubule plus ends . Mean values from three separate experiments per condition ( with a total of at least 75 kymographs ) are shown ; error bars are s . e .

O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

(B) Averaged fluorescence intensity profiles of GFP-dynein (green squares) and Alexa647-BicD2-N (magenta circles) at growing microtubule plus ends. Mean values from three separate experiments per condition (with a total of at least 75 kymographs) are shown; error bars are s. e.


( C , D ) Dual single colour kymographs of an Atto565 - microtubule ( magenta ) in presence of GFP - dynein ( green ) , human dynactin EB1 , either ( C ) in absence or ( D ) presence of 5 µM BicD2 - N ; other conditions as in A . increased BicD2 - N in D compared to A strongly reduces microtubule plus end tracking of GFP - dynein .

O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O

(C, D) Dual and single colour kymographs of an Atto565-microtubule (magenta) in the presence of GFP-dynein (green), human dynactin and EB1, and either (C) in the absence or (D) presence of 5 µM BicD2-N; other conditions as in A. The increased BicD2-N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP-dynein.


( E ) Averaged profiles of GFP - dynein at growing microtubule plus ends for condition without BicD2 - N ( red circles , as in C ) or with 5 µM BicD2 - N ( black squares , as in D ) . Mean values from three separate experiments per condition ( with a total of at least 106 kymographs ) are shown ; error bars are s . e . Microtubule plus minus ends in merged kymographs are labelled by ( + ) ( − ) . Experiments were performed at 30 ° C .

O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O 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-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(E) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the condition without BicD2-N (red circles, as in C) or with 5 µM BicD2-N (black squares, as in D). Mean values from three separate experiments per condition (with a total of at least 106 kymographs) are shown; error bars are s. e. Microtubule plus and minus ends in merged kymographs are labelled by (+) and (−). Experiments were performed at 30°C.


( A ) Example dual colour kymograph a schematic showing how of initiation of processive runs was measured . concentrations were 10 nM GFP - dynein , 20 nM pig dynactin , 5 µM BicD2 - N , 20 nM EB1 17 . 5 µM Atto647 - tubulin .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O

(A) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured. The protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 5 µM BicD2-N, 20 nM EB1 and 17.5 µM Atto647-tubulin.


( B ) Histograms of spatial initiation probabilities of processive runs within first 7 . 2 µm from growing microtubule plus ends in absence ( black bars ) , or presence of 10 nM EB1 ( red bars ) . 199 221 initiation positions were determined for two conditions ( from three experiments each ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(B) Histograms of the spatial initiation probabilities of processive runs within the first 7.2 µm from growing microtubule plus ends in the absence (black bars), or presence of 10 nM EB1 (red bars). 199 and 221 initiation positions were determined for the two conditions (from three experiments each).


( C ) Averaged profiles of GFP - dynein at growing microtubule plus ends for conditions as in B . Mean values from three separate experiments per condition ( with a total of at least 69 kymographs ) are shown ; error bars are s . e . Experiments were performed at 30 ° C .

O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the conditions as in B. Mean values from three separate experiments per condition (with a total of at least 69 kymographs) are shown; error bars are s. e. Experiments were performed at 30°C.


( D ) Histogram of spatial initiation probabilities of DDB on static GMPCPP - microtubules . 294 initiation positions were detemined ( from three experiments ) . Concentrations of DDB components as in B .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O

(D) Histogram of spatial initiation probabilities of DDB on static GMPCPP-microtubules. 294 initiation positions were detemined (from three experiments). Concentrations of DDB components as in B.


A , B ) Dual single colour kymographs showing under certain conditions p150 or dynactin - dependent microtubule plus end tracking of dynein in absence of EB : ( A ) 10 nM GFP - dynein ( green ) tracking growing end of a Atto565 - microtubule ( magenta ) in presence of pig dynactin at an elevated of 80 nM . Experiments in ( A ) ( B ) were performed in standard buffer ( BRB20 supplemented with 50 mM KCl , for details see Methods ) .

O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (A) 10 nM GFP-dynein (green) tracking the growing end of a Atto565-microtubule (magenta) in the presence of pig dynactin at an elevated concentration of 80 nM. Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).


( A , B ) Dual single colour kymographs showing under certain conditions p150 or dynactin - dependent microtubule plus end tracking of dynein in absence of EB : ( B ) 10 nM GFP - dynein ( green ) tracking plus end of an Atto565 - microtubule ( blue ) in presence of 10 nM Alexa647 - p150 ( a fragment containing first 517 of p150 ; called p150 - N in text ) ( red ) . Experiments in ( A ) ( B ) were performed in standard buffer ( BRB20 supplemented with 50 mM KCl , for details see Methods ) .

O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (B) 10 nM GFP-dynein (green) tracking the plus end of an Atto565-microtubule (blue) in the presence of 10 nM Alexa647-p150 (a fragment containing the first 517 amino acids of p150; called p150-N in the text) (red). Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).


( C , D ) Dual single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength : ( C ) In standard buffer , 10 nM Alexa647 - p150 ( green ) accumulates at plus end on GMPCPP - stabilised segment of a growing Atto565 - microtubule ( magenta ) .

O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O

(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (C) In standard assay buffer, 10 nM Alexa647-p150 (green) accumulates at the plus end and on the GMPCPP-stabilised segment of a growing Atto565-microtubule (magenta).


( C , D ) Dual single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength : ( D ) At higher ionic strength ( BRB80 supplemented with 60 mM KCl , see Methods ) , Alexa647 - p150 ( green ) binds only weakly to microtubule without a detectable plus end preference . concentrations as in C . Atto565 - tubulin was always 17 . 5 µM tubulin . was 30 ° C .

O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O

(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (D) At higher ionic strength (BRB80 supplemented with 60 mM KCl, see Methods), Alexa647-p150 (green) binds only weakly to the microtubule without a detectable plus end preference. Protein concentrations as in C. The Atto565-tubulin concentration was always 17.5 µM tubulin. The temperature was 30°C.


( A - C ) Kymographs showing GFP - dynein ( green ) on dynamic Atto647N - microtubules ( magenta ) in presence of regulators at different Lis1 concentrations . concentrations were 10 nM GFP - dynein , 20 nM pig dynactin , 20 nM EB1 , 5 µM BicD2 - N , ( A ) either no Lis1 , ( B ) 1 μM mCherry - Lis1 , or ( C ) 5 μM mCherry - Lis1 . Together with BicD2 , Lis1 increases number of processive dynein runs high concentrations of Lis1 restore plus - end localisation of dynein in presence of BicD2 - N . Microtubule orientation as indicated .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR O O O O

(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated.


( D ) Averaged profiles of GFP - dynein at growing microtubule plus ends in presence of EB1 , dynactin 5 µM BicD2 - N ( black squares , as in A ) , in presence of 1 µM mCherry - Lis1 ( blue circles , as in B ) 5 µM mCherry - Lis1 ( red triangles , as in C ) . Mean values from three separate experiments per condition ( with a total of at least 49 kymographs ) are shown ; error bars are s . e .

O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Averaged intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of EB1, dynactin and 5 µM BicD2-N (black squares, as in A), in the presence of 1 µM mCherry-Lis1 (blue circles, as in B) and 5 µM mCherry-Lis1 (red triangles, as in C). Mean values from three separate experiments per condition (with a total of at least 49 kymographs) are shown; error bars are s. e.


( E ) average number of processive DDB runs per µm microtubule for three different Lis1 conditions ( as in A , B , C ) . Error bars are s . d . * p = 0 . 001 , * * p = 0 . 003 , * * * p = 0 . 001 ( unpaired t - ) . 218 , 969 , 646 processive DDB events were analysed for three conditions , respectively ( from two experiments each ) .

O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O

(E) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions (as in A, B and, C). Error bars are s. d. *p = 0.001, **p = 0.003, ***p = 0.001 (unpaired t-test). 218, 969, and 646 processive DDB events were analysed for the three conditions, respectively (from two experiments each).


( F ) Bar graphs showing mean velocity mean run of processive DDB events for three Lis1 conditions ( as in A , B C ) . Error bars are s . e . m . Mean velocities were 0 . 37 ± 0 . 02 µm / s ( A ) , 0 . 38 ± 0 . 03 µm / s ( B ) , 0 . 35 ± 0 . 03 µm / s ( C ) . Mean run lengths were 3 . 1 ± 0 . 4 µm ( A ) , 3 . 2 ± 0 . 5 µm ( B ) , 2 . 9 ± 0 . 3 µm ( C ) . For each condition over 300 complexes were analysed from three different data sets .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions (as in A, B and C). Error bars are the s.e.m. Mean velocities were 0.37 ± 0.02 µm/s (A), 0.38 ± 0.03 µm/s (B), and 0.35 ± 0.03 µm/s (C). Mean run lengths were 3.1 ± 0.4 µm (A), 3.2 ± 0.5 µm (B), and 2.9 ± 0.3 µm (C). For each condition over 300 complexes were analysed from three different data sets.


( G ) Histogram of spatial initiation probabilities of DDB runs in presence of EB1 Lis1 . 381 initiation positions were determined ( from two experiments ) . Experiments were performed at 30 ° C .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(G) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1. 381 initiation positions were determined (from two experiments). Experiments were performed at 30°C.


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , WDR74 HAUS5 promoters for IgG DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, WDR74 and HAUS5 promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , COX14 , HLA - E PSMB8 promoters for IgG DVL3 in MCF7 ( NTC , shA DVL3 , shB DVL3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O

(D) ChIP-qPCRs at RFX5-GBAT2, COX14, HLA-E and PSMB8 promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( E ) RT - qPCR - of expression changes of RFX5 , GBAT2 , WDR74 , HCG15 , POU2F3 , SNORD3B , APOC1P1 , HLA - A , TAP1 , PSMD8 , PDG1 JUNB genes in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) . ( F ) RT - qPCR - of expression changes of RFX5 , GBAT2 , PCAT6 , ZNF672 , SSC5D , APOC1P1 , HLA - A , HLA - F TAP1 genes in MCF7 ( NTC , shA DVL3 , shB DVL3 ) . D

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O

(E) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells. D


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , KDM5B , APOC1P1 , PSMB8 promoters for IgG H3K4me3 in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , PCAT6 , APOC1P1 HAUS5 promoters for IgG H3K4me3 in MCF7 ( NTC , shA DVL3 , shB DVL3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O

(D) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , PSMB8 promoters for IgG KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , H4K8ac peak ( fourth row ) ChIP - Seq data in MDA - MB - 468 for PRDM10 , TRIM26 , BTN2A1 , PNRC2 , MR1 genes , visualized by IGV . green rectangular bars are locations of statistically - qualified peak of H3K27ac or H4K8ac in MDA - MB - 468 .

O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O

(D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.


( E ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , H4K8ac peak ( fourth row ) ChIP - Seq data in MCF7 for WDR74 , YWHAQ , CDNF / HSPA14 , BTN2A1 , MR1 genes , visualized by IGV . turquoise purple rectangular bars are locations of statistically - qualified peak of H3K27ac or H4K8ac in MCF7 , respectively .

O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

(E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.


( D ) RT - qPCR - of expression changes of DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) . ( E ) RT - qPCR - of expression changes of KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) . ( F ) RT - qPCR - of expression changes of DVL3 in MCF7 ( NTC , shA DVL3 , shB DVL3 ) . ( G ) RT - qPCR - of expression changes of KMT2D in MCF7 ( NTC , shA DVL3 , shB DVL3 ) . ( H

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O

(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H


( H ) Immunofluorescence of KMT2D localization in MDA - MB - 468 . were probed with KMT2D antibody ( red ) . nucleus was stained with DAPI ( blue ) actin filaments ( green ) were stained with Phalloidin . Merge of KMT2D ( red ) ( blue ) is shown as KMT2D / DAPI , merge of KMT2D ( red ) actin ( green ) is shown as KMT2D / ACTIN , , merge of KMT2D ( red ) , ( blue ) actin ( green ) is shown as MERGE for NTC , shA DVL3 shB DVL3 . Scale bar :

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O

(H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm


( C ) IVIS showing representative tumors generated by injecting luciferase - expressing MDA - MB - 468 ( NTC , shA DVL3 or shB DVL3 ) in mammary fat pad of SCID mice . Color scale ( luminescent signal ) : blue , least intense signal ; red , most intense signal . ( D ) of luminescence changes in tumors generated by injecting luciferase - expressing MDA - MB - 468 ( NTC , shA DVL3 or shB DVL3 ) in mammary fat pad of SCID mice , Average luminescence was used to measure volume of ; n ≥ 9 . (

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O B-DISEASE B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O B-DISEASE B-ORGANISM O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9. (


( G ) IVIS showing representative tumors generated by injecting luciferase - expressing MCF7 ( NTC , shA DVL3 ) in mammary fat pad of SCID mice . Color scale ( luminescent signal ) : blue , least intense signal ; red , most intense signal . ( H ) of luminescence changes in tumors generated by injecting luciferase - expressing MCF7 ( NTC , shA DVL3 ) in mammary fat pad of SCID mice . Average luminescence was used to measure volume of . n = 7 .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O B-DISEASE B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O B-DISEASE B-ORGANISM O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.


( A ) Immunofluorescence of HA - tagged DVL3 localization in MDA - MB - 468 . were probed with an HA antibody ( red ) . nucleus was stained with DAPI ( blue ) actin filaments ( green ) were stained with Phalloidin . Merge of actin ( green ) ( blue ) is shown as ACTIN / DAPI , merge of DVL3 ( red ) ( blue ) is shown as HA / DAPI , merge of DVL3 ( red ) actin ( green ) is shown as HA / ACTIN , , merge of DVL3 ( red ) , ( blue ) actin ( green ) is shown as MERGE for empty vector ( EV ) , HA - DVL3 - WT HA - DVL3 - NESm transient transfected . Scale bar : 10µm .

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(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.


( B ) RT - qPCR - of expression changes of DVL3 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) . ( C ) RT - qPCR - of expression changes of KMT2D in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) . ( D ) RT - qPCR - of expression changes of ARAP1 , RFX5 , GBAT2 , WDR74 , SNORD3D , HAUS5 , TAP1 , HLA - A , PDG1 , , MR1 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) . ( E

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O

(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (D) RT-qPCR-based analysis of expression changes of ARAP1 , RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (E


( A ) Representative clones screened for CD24 expression under regulation of tetracycline . were exposed to 1 µg / ml tetracycline for 48 h . 20 µg from each sample were subjected to Western blot for CD24 analyzed with anti - CD24 SWA11 mAb . was reprobed with anti - tubulin antibody to assess uniformity of sample loading .

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(A) Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 µg/ml tetracycline for 48 h. 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading.


( D ) Western blot of EXO - CD24 exosomes . Tetracycline ( Tet ) was used to induce CD24 expression in from which exosomes are derived . HSP70 was used as an exosomal marker .

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(D) Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker.


( C ) Western blot of EXO - mCD24 exosomes . HSP70 was used as an exosomal marker .

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(C) Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker.


( B ) respiratory rate ( C ) Blood oxygen ( SpO2 ) before treatment ( Day 1 , measured before administration of first dose ) after treatment ( Day 7 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for Blood oxygen ( SpO2 ) respiratory rate . these are presented graphically via box plots before after treatment .

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(B) respiratory rate and (C) Blood oxygen (SpO2) and before treatment (Day 1, measured before administration of first dose) and after treatment (Day 7). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for Blood oxygen (SpO2) and respiratory rate. these are presented graphically via box plots before and after treatment.


Systemic CRP . Values are normalized against baseline value ( Day 0 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for CRP . these are presented graphically via box plots before after treatment ( n = 35 )

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Systemic CRP levels. Values are normalized against baseline value (Day 0). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for CRP levels. these are presented graphically via box plots before and after treatment (n=35 patients)


( B ) Systemic cytokine . Cytokine were measured during ( Day 3 / 4 ) or after ( Day 7 / 8 ) treatment with EXO - CD24 using Quantibody Multiplex ELISA Array . Values are normalized against baseline value ( Day 0 ) . Samples from 11 were analysed . Results from 8 are shown ( results for 3 are not shown because one or more baseline results were below detection level ) . Box plot of Systemic cytokine by days after treatment ( minimum , maximum , median , first quartile , third quartile ) .

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(B) Systemic cytokine levels. Cytokine levels were measured during (Day 3/4) or after (Day 7/8) treatment with EXO-CD24 using Quantibody Multiplex ELISA Array. Values are normalized against baseline value (Day 0). Samples from 11 patients were analysed. Results from 8 patients are shown (results for 3 patients are not shown because one or more baseline results were below detection level). Box plot of Systemic cytokine levels by days after treatment (minimum, maximum, median, first quartile, third quartile).


( C ) During course of EXO - CD24 treatment follow - up , cytokine were measured for of ( n = 24 ) .

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(C) During the course of EXO-CD24 treatment and follow-up, cytokine levels were measured for the rest of the patients (n=24 patients).


( D ) Monitoring systemic IL - 6 cytokine level following EXO - CD24 treatment . Samples from 17 , representing 4 experimental groups , were analysed using BD ™ Cytometric Bead Array ( CBA ) .

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(D) Monitoring systemic IL-6 cytokine level following EXO-CD24 treatment. Samples from 17 patients, representing the 4 experimental groups, were analysed using the BD™ Cytometric Bead Array (CBA).


A . GFP - Snc1 - PEM accumulates in atg1Δ , atg8Δ , atg11Δ mutant . Lysates were prepared from wild type ( WT ) , ypt1 - 1 ( for comparison ) , atg1Δ , atg8Δ , atg11Δ mutant transformed with a 2μ plasmid expressing GFP - Snc1 - PEM from TPI promoter . level of GFP - Snc1 - PEM was determined using immunoblot with anti - GFP antibodies . bands were quantified increase in level in mutant versus WT is shown under blot adjusted to G6PDH loading control .

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A. GFP-Snc1-PEM protein accumulates in atg1Δ, atg8Δ, and atg11Δ mutant cells. Lysates were prepared from wild type (WT), ypt1-1 (for comparison), atg1Δ, atg8Δ, and atg11Δ mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control.


B . GFP - Snc1 - PEM accumulates in aberrant ER structures in atg1Δ , atg8Δ , atg11Δ mutant . ER - marker Sec61 was tagged with mCherry in strains from panel A , were examined by live - microscopy . Shown from left to right : DIC , GFP , mCherry , merge , % with intracellular GFP - Snc1 - PEM ( number of with internal GFP - Snc1 - PEM / number of visualized ) , % in which intra - Snc1 - PEM co - localizes with Sec61 .

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B. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1Δ, atg8Δ, and atg11Δ mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61.


C . UPR is induced in atg1Δ , atg8Δ , atg11Δ mutant . from panel A were transformed with a second plasmid that expresses β - gal from a UPR promoter . UPR was determined expressed as % of WT response .

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C. UPR is induced in atg1Δ, atg8Δ, and atg11Δ mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response.


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM level (

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM level ( D ) , intra - accumulation of GFP - Snc1 - PEM , ( E ) ,

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E),


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM level ( D ) , intra - accumulation of GFP - Snc1 - PEM , ( E ) , induction of UPR response ( F ) . Wild type ( WT ) , atg19Δ , atg11Δ , atg32Δ , atg36Δ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for panels A - C , respectively . E . Shown from left to right : DIC , GFP , % with intracellular Snc1 - PEM structures . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19Δ, atg11Δ, atg32Δ, and atg36Δ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . While level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ atg2 ∆ , but not atg18 ∆ , mutant , it does not accumulate in aberrant structures does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , atg2 ∆ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . tested : level of GFP - Snc1 - PEM ( A ) ,

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A),


A - C . While level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ atg2 ∆ , but not atg18 ∆ , mutant , it does not accumulate in aberrant structures does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , atg2 ∆ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . tested : level of GFP - Snc1 - PEM ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B )

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B)


A - C . While level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ atg2 ∆ , but not atg18 ∆ , mutant , it does not accumulate in aberrant structures does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , atg2 ∆ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . tested : level of GFP - Snc1 - PEM ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B ) , induction of UPR response ( C , atg1 ∆ is shown as a positive control ) .

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B), and induction of the UPR response (C,atg1∆ is shown as a positive control).


D - G . Atg9 is epistatic to Atg11 in macro - ER - phagy . ATG9 was deleted in wild type atg11 ∆ mutant effects of overexpression of GFP - Snc1 - PEM were determined in single double mutants as described in Fig 1 legend . D . Deletion of ATG9 in wild type ( WT ) or atg11 ∆ mutant results in an increase of GFP - Snc1 - PEM level similar to increase in atg11 ∆ mutant .

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D-G. Atg9 is epistatic to Atg11 in macro-ER-phagy. ATG9 was deleted in wild type and atg11∆ mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in Fig 1 legend. D. Deletion of ATG9 in wild type (WT) or atg11∆ mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in atg11∆ mutant cells.


E . Deletion of ATG9 in wild type or atg11 ∆ mutant results in an increase of intracellular GFP - Snc1 - PEM . However , only ~ 20 % of atg9 ∆ single - , atg9 ∆ atg11 ∆ double - mutant accumulate GFP - Snc1 - PEM in aberrant structures , as compared with ~ 75 % of atg11 ∆ mutant . Shown from top to bottom : DIC , GFP , % with aberrant intracellular GFP - Snc1 - PEM structures , of GFP - Snc1 - PEM inside / PM ( 30 were analyzed for each ) .

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E. Deletion of ATG9 in wild type or atg11∆ mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the atg9∆ single-, and atg9∆ atg11∆ double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of atg11∆ mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain).


F . UPR is induced in atg11 ∆ , but not in atg9 ∆ single - atg9 ∆ atg11 ∆ double - mutant overexpressing GFP - Snc1 - PEM .

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F. UPR is induced in atg11∆, but not in atg9∆ single- and atg9∆ atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM.


G . UPR can be induced in atg9 ∆ single - , atg9 atg11 ∆ double - mutant overexpressing GFP - Snc1 - PEM by tunicamycin . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

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G. UPR can be induced in atg9∆ single-, and atg9 atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . ATG9 was deleted in ypt1 - 1 , WT sec12ts mutant effect of overexpression of GFP - Snc1 - PEM was determined in single double mutant . Experiments were performed as described for Fig 1A - 1C , respectively . A . Snc1 - PEM is increased ~ 3 fold in atg9 ∆ sec12ts as compared to ~ 20 fold in ypt1 - 1 mutant . Importantly , atg9 ∆ is epstatic to ypt1 - 1 , but not to sec12ts , mutation . Left , immuno - blot , increase of level in mutant versus WT is shown under blot ; right , a bar graph summarizing quantified

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A-C.ATG9 was deleted in ypt1-1, WT and sec12ts mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed as described for Fig 1A-1C, respectively. A. Snc1-PEM is increased ~3 fold in atg9∆ and sec12ts as compared to ~20 fold in ypt1-1 mutant cells. Importantly, atg9∆ is epstatic to the ypt1-1, but not to the sec12ts, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data


. Whereas deletion of ATG9 in ypt1 - 1 mutant results in three fold lower accumulation of GFP - Snc1 - PEM in aberrant structures ( 85 % to 27 % ) , its deletion in sec12ts mutant results in two fold increased accumulation ( ~ 40 % to ~ 80 % ) . Shown from top to bottom : DIC , GFP , % with intracellular Snc1 - PEM structures .

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. Whereas deletion of ATG9 in ypt1-1 mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in sec12ts mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.


C . Deletion of ATG9 in ypt1 - 1 mutant overexpressing Snc1 - PEM results in ~ 3 . 5 lower UPR ( p - value < 0 . 0005 ) , but a slightly increased UPR in sec12ts mutant ( p - value = 0 . 05 ) .

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C. Deletion of ATG9 in ypt1-1 mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in sec12ts mutant cells (p-value = 0.05).


D . Atg9 is present on aberrant ER structures that accumulate in ypt1 - 1 mutant . WT ypt1 - 1 mutant expressing Atg9 - mCherry from chromosome GFP - Snc1 - PEM from a 2μ plasmid were analyzed by live - microscopy . Whereas in WT ( top ) , GFP - Snc1 - PEM localizes to Atg9 - mCherry to intracellular , two co - localize in 100 % of ypt1 - 1 mutant ( bottom ) that accumulate intracellular GFP - Snc1 - PEM structures ( ~ 80 % ) . Shown from left to right : DIC , GFP , mCherry , merge , % with intracellular Snc1 - PEM , % with co - localization ( number of with observed phenotype / total number of analyzed ) .

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D. Atg9 is present on aberrant ER structures that accumulate in ypt1-1 mutant cells. WT and ypt1-1 mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the ypt1-1 mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed).


E . GFP - Snc1 - PEM is delivered to vacuole for degradation in sec12ts mutant . Accumulation of GFP - Snc1 - PEM in vacuoles of sec12ts mutant , with without deletion of vacuolar protease Pep4 , was determined using FM4 - 64 dye , which labels vacuolar . Deletion of PEP4 in sec12ts mutant results in an increase of in which GFP - Snc1 - PEM accumulates inside vacuole ( from 8 % to 100 % ) . Shown from left to right : DIC , GFP , FM4 - 64 ( vacuolar ) , merge , % with aberrant GFP - Snc1 - PEM structures , % with GFP - Snc1 - PEM outside vacuole , % with GFP - Snc1 - PEM inside vacuole . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

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E. GFP-Snc1-PEM is delivered to the vacuole for degradation in sec12ts mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of sec12ts mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . ypt1 - 1 mutation is epistatic to vps21 ∆ in ER - phagy . yDsRed - Snc1 - PEM was overexpressed in WT , vps21 ∆ , ypt1 - 1 ypt1 - 1vps21 ∆ double mutant that also expressed autophagosomal marker yEGFP - Atg8 . were analyzed by live - microscopy . Shown from left to right : DIC , DsRed , GFP , merge , % Atg8 dots , number of Atg8 dots per , % in which Atg8 dots co - localize with Snc1 - PEM . About 50 % of WT 85 % of vps21 ∆ mutant contain ~ 1 dot of Atg8 representing AP . Importantly , in ~ 70 % of vps21 ∆ mutant Snc1 - PEM co - localizes with APs , as compared to ~ 4 % in WT . In contrast , ~ 90 % ypt1 - 1 ypt1 - 1vps21 ∆ mutant contain three APs per , Snc1 - PEM does not co - localize with them . Arrows point to co - localization ; arrowheads point to either Atg8 dots or GFP - Snc1 - PEM that do not co - localize .

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A. The ypt1-1 mutation is epistatic to vps21∆ in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, vps21∆, ypt1-1 and ypt1-1vps21∆ double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of vps21∆ mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the vps21∆ mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% ypt1-1 and ypt1-1vps21∆ mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize.


following effects of overexpression of GFP - Snc1 - PEM in WT ypt1 - 1 mutant , without with ire1Δ or hac1Δ , were analyzed as described for Fig 1A - 1C , respectively : level ( B ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( C ) , UPR induction ( D ) . B . Deletion of either Ire1 or Hac1 does not affect level of Snc1 - PEM accumulation in WT or ypt1 - 1 mutant .

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The following effects of overexpression of GFP-Snc1-PEM in WT and ypt1-1 mutant cells, without and with ire1Δ or hac1Δ, were analyzed as described for Fig 1A-1C, respectively: protein level (B), accumulation of GFP-Snc1-PEM in aberrant structures (C), and UPR induction (D). B. Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or ypt1-1 mutant cells.


C . Deletion of either Ire1 or Hac1 does not affect of WT ypt1 - 1 mutant that accumulate aberrant intra - Snc1 - PEM . Shown from top to bottom : DIC , GFP , % with intracellular Snc1 - PEM structures .

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C. Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1-1 mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.


D . Deletion of either Ire1 or Hac1 obliterate UPR in both WT ypt1 - 1 mutant . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

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D. Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1-1 mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . Overexpressed Snq2 - GFP accumulates in ER induces UPR in ypt1 - 1 mutant . Snq2 - yEGFP was overexpressed in WT ypt1 - 1 mutant following effects were analyzed as described for Fig 1A - 1C , respectively : increase in level of Snq2 - yEGFP ( A )

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A)


A - C . Overexpressed Snq2 - GFP accumulates in ER induces UPR in ypt1 - 1 mutant . Snq2 - yEGFP was overexpressed in WT ypt1 - 1 mutant following effects were analyzed as described for Fig 1A - 1C , respectively : increase in level of Snq2 - yEGFP ( A ) , accumulation of Snq2 - yEGFP in aberrant ER structures ( B ) ,

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B),


A - C . Overexpressed Snq2 - GFP accumulates in ER induces UPR in ypt1 - 1 mutant . Snq2 - yEGFP was overexpressed in WT ypt1 - 1 mutant following effects were analyzed as described for Fig 1A - 1C , respectively : increase in level of Snq2 - yEGFP ( A ) , accumulation of Snq2 - yEGFP in aberrant ER structures ( B ) , induction of UPR response ( C , GFP - Snc1 - PEM is shown for comparison ) .

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B), and induction of the UPR response (C, GFP-Snc1-PEM is shown for comparison).


D - F . Synergistic effect of co - overexpression of Snq2 - GFP with DsRed - Snc1 - PEM in atg11 ∆ mutant . WT ( left ) atg11 ∆ mutant ( right ) were transformed with plasmids for overexpression of Snq2 , Snc1 - PEM or both Snq2 Snc1 - PEM . D . Immunoblot . Shown from top to bottom : Snq2 - yEGFP ( using anti - GFP antibodies ) , Ds - Red - Snc1 - PEM ( using anti - Snc1 antibodies ) , G6PDH ( loading control ) , a bar graph showing fold increase of Snq2 - yEGFP ( green ) Ds - Red - Snc1 - PEM ( red ) in atg11 ∆ mutant over WT .

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D-F. Synergistic effect of co-overexpression of Snq2-GFP with DsRed-Snc1-PEM in atg11∆ mutant cells. WT (left) and atg11∆ mutant cells (right) were transformed with plasmids for overexpression of Snq2, Snc1-PEM or both Snq2 and Snc1-PEM. D. Immunoblot analysis and quantification. Shown from top to bottom: Snq2-yEGFP (using anti-GFP antibodies), Ds-Red-Snc1-PEM (using anti-Snc1 antibodies), G6PDH (loading control), and a bar graph showing fold increase of Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) in atg11∆ mutant cells over WT.


E . Accumulation of macro - ER - phagy cargos in aberrant intracellular structures using live - microscopy . Shown from left to right : DIC , GFP , DsRed Merge . Shown from top to bottom : Snq2 , Snc1 - PEM , Snq2 + Snc1 - PEM , % with co - localization ( relevant only for co - overexpression ) ( % with ER - phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig ) . F . level of intracellular structures ( over PM ) in atg11 ∆ mutant . bar graph shows Snq2 - yEGFP ( green ) Ds - Red - Snc1 - PEM ( red ) ( 20 were analyzed for each ) . When co - overexpressed in atg11 ∆ mutant , level of either accumulating in aberrant structures is ~ 5 fold higher than when overexpressed individually . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

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E. Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig). F. Fluorescence level of intracellular structures (ratio over PM) in atg11∆ mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in atg11∆ mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . A diagram showing two groups of ER - resident : those that become macro - ER - phagy cargos ( in green ) , those that do not ( in blue or grey ) . Like overexpressed Snc1 - PEM or Snq2 , ER - resident Sec61 ( translocon subunit ) Hmg1 ( sterol biogenesis ) are transported to vacuole via macro - ER - phagy . In contrast , ER - to - Golgi exit regulators Sec12 Sec13 ER lumen chaperone Kar2 are not co - transported to lysosome through macro - ER - phagy . Four or six different strains were used for this : WT ( YPT1 ) , pep4 ∆ , pep4 prb1 ∆ , ypt1 - 1 , ypt1 - 1pep4 ∆ , ypt1 - 1pep4 ∆ prb1 ∆ . In each , one ER - resident was tagged at its C - terminus ( except for Kar2 ) . ( pep4 ∆ strains were not used in experiments because they do not show full defect ) . level of ER - resident was determined by immunoblot in that either do not ( B - D ) or do overexpress GFP - Snc1 - PEM ( F - H ) . B - C . Sec61 - 3xHA ( B ) Sec13 - 3x HA ( C ) expressing ( 2 independent un - transformed colonies ) were tested by immunoblot ( using anti - HA antibodies ) . Shown from top to bottom : genotype , HA - tagged , G6PDH ( loading control ) , of HA - tagged expressed as average fold of WT ( p - value = 0 . 025 for Sec61 ) .

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A. A diagram showing two groups of ER-resident proteins: those that become macro-ER-phagy cargos (in green), and those that do not (in blue or grey). Like overexpressed Snc1-PEM or Snq2, the ER-resident membrane proteins Sec61 (translocon subunit) and Hmg1 (sterol biogenesis) are transported to the vacuole via macro-ER-phagy. In contrast, the ER-to-Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co-transported to the lysosome through macro-ER-phagy. Four or six different strains were used for this analysis: WT (YPT1), pep4∆, and pep4 prb1∆, ypt1-1, ypt1-1pep4∆, and ypt1-1pep4∆ prb1∆. In each strain, one ER-resident protein was tagged at its C-terminus (except for Kar2). (The pep4∆ strains were not used in all experiments because they do not show the full defect). The level of ER-resident proteins was determined by immunoblot analysis in cells that either do not (B-D) or do overexpress GFP-Snc1-PEM (F-H). B-C. Sec61-3xHA (B) and Sec13-3x HA (C) expressing cells (2 independent un-transformed colonies) were tested by immunoblot analysis (using anti-HA antibodies). Shown from top to bottom: strain genotype, HA-tagged protein, G6PDH (loading control), quantification of HA-tagged protein expressed as average fold of WT (p-value = 0.025 for Sec61).


D . Summary of immunoblot analyses of ER - resident level in that do not overexpress GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots are shown in Fig 6B - 6C S6A - S6C Fig . In YPT1 , of Sec61 Hmg1 ( green ) are slightly increased ( 14 38 % , respectively ) in strains defective in vacuolar proteolysis ; of Sec12 , Sec13 Kar2 are not increased ( blue grey ) . of Sec61 Hmg1 ( green ) are increased by 2 - 2 . 5 fold in ypt1 - 1 mutant when compared to WT , regardless if they are defective in vacuolar proteolysis or not . In contrast , of Sec12 , Sec13 Kar2 are only slightly increased ( blue grey ) .

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D. Summary of immunoblot analyses of ER-resident proteins level in cells that do not overexpress GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6B-6C and S6A-S6C Fig. In YPT1 cells, the levels of Sec61 and Hmg1 (green) are slightly increased (14 and 38%, respectively) in strains defective in vacuolar proteolysis; the levels of Sec12, Sec13 and Kar2 are not increased (blue and grey). The levels of Sec61 and Hmg1 (green) are increased by 2-2.5 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey).


E . level of ER - resident Sec61 is similar whether GFP - Snc1 - PEM is overexpressed or not . Wild - type were transformed with a 2μ plasmid , either empty or for overexpression of GFP - Snc1 - GEM ( 2 independent transformants ) . Shown from top to bottom : plasmid , Sec61 , G6PDH ( loading control ) , GFP - Snc1 - PEM , of Sec61 expressed as average fold of WT with empty plasmid .

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E. The level of the ER-resident protein Sec61 is similar whether GFP-Snc1-PEM is overexpressed or not. Wild-type cells were transformed with a 2μ plasmid, either empty or for overexpression of GFP-Snc1-GEM (2 independent transformants). Shown from top to bottom: plasmid, Sec61, G6PDH (loading control), GFP-Snc1-PEM, and quantification of Sec61 expressed as average fold of WT with empty plasmid.


F - G . Sec61 - 3xHA ( F ) Sec13 - 3xHA ( G ) expressing were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM . lysates were tested by immunoblot ( using ant - HA anti - GFP antibodies ) . Shown from top to bottom : genotype , specific ER - resident tested , of ER - resident bands expressed as average fold of WT , GFP - Snc1 - PEM , of GFP - Snc1 - PEM bands expressed as average fold of WT , G6PDH ( loading control ) .

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F-G. Sec61-3xHA (F) and Sec13-3xHA (G) expressing cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM. Cell lysates were tested by immunoblot analysis (using ant-HA and anti-GFP antibodies). Shown from top to bottom: strain genotype, the specific ER-resident protein tested, quantification of the ER-resident protein bands expressed as average fold of WT, GFP-Snc1-PEM, quantification of the GFP-Snc1-PEM bands expressed as average fold of WT, and G6PDH (loading control).


H . Summary of immunoblot of ER - resident level in overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots are shown in Fig 6F 6G S6F - S6H Fig . In YPT1 , like Snc1 - PEM ( black ) , of Hmg1 Sec61 ( green ) are increased > 10 fold in strains defective in vacuolar proteolysis . In contrast , of Sec12 , Sec13 Kar2 are not changed ( blue grey ) . Like Snc1 - PEM ( black ) , of Hmg1 Sec61 ( green ) are increased ~ 15 fold in ypt1 - 1 mutant when compared to WT , regardless if they are defective in vacuolar proteolysis or not . In contrast , of Sec12 , Sec13 Kar2 are only slightly increased ( blue grey ) . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

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H. Summary of immunoblot analysis of ER-resident proteins level in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6F and 6G and S6F-S6H Fig. In YPT1 cells, like Snc1-PEM (black), the levels of Hmg1 and Sec61 (green) are increased >10 fold in strains defective in vacuolar proteolysis. In contrast, the levels of Sec12, Sec13 and Kar2 are not changed (blue and grey). Like Snc1-PEM (black), the protein levels of Hmg1 and Sec61 (green) are increased ~15 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey). +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . In defective in vacuolar proteolysis ( pep4 ∆ prb1 ∆ ) , Hmg1 co - localizes with 70 % of intra - Snc1 - PEM , but Sec13 does not ( at least 36 were analyzed for each ) . No co - localization was observed for resident ER overexpressed Snc1 - PEM in WT ( PEP4PRB1 ) . Hmg1 ( left ) Sec13 ( right ) were tagged with mCherry at their C - terminus in PEP4PRB1 ( WT ) pepb4 ∆ prb1 ∆ mutant . were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM analyzed by live - microscopy . Shown from top to bottom : DIC , GFP , mCherry , merge ( yellow ) , % with intra - Snc1 - PEM , % co - localization of ER with intra - Snc1 - PEM .

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A. In cells defective in vacuolar proteolysis (pep4∆ prb1∆), Hmg1 co-localizes with 70% of intra-cellular Snc1-PEM, but Sec13 does not (at least 36 cells were analyzed for each strain). No co-localization was observed for resident ER proteins and overexpressed Snc1-PEM in WT cells (PEP4PRB1). Hmg1 (left) and Sec13 (right) were tagged with mCherry at their C-terminus in PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells. Cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM and analyzed by live-cell microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge (yellow), % cells with intra-cellular Snc1-PEM, and % co-localization of the ER protein with intra-cellular Snc1-PEM.


B . ER - resident Hmg1 is delivered to vacuole with GFP - Snc1 - PEM . vacuole of PEP4PRB1 ( WT ) pepb4 ∆ prb1 ∆ mutant expressing Hmg1 - mCherry overexpressing GFP - Snc1 - PEM ( see Panel A , left ) was stained with CMAC - Arg ( blue ) , were analyzed by live - microscopy . In WT ( top ) , there is no intracellular Snc1 - PEM red - labeled ER is distinguished from blue vacuole . In contrast , in ~ 75 % pep4 ∆ prb1 ∆ mutant ( bottom ) , which are defective in vacuolar proteolysis , Hmg1 ( red ) co - localizes with Snc1 - PEM ( green ) in vacuole ( blue ) . Shown from left to right : DIC , GFP , mCherry , blue , merge ( shows merge of three colors ) , % with intracellular Snc1 - PEM , % in which Hmg1 with Snc1 - PEM co - localize , % co - localization of two with vacuolar dye ( 38 were analyzed for each ) .

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B. The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% pep4∆ prb1∆ mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain).