Figure 7A . Expression levels of various GPI - APs in different clones of PIGG - KO HEK293 cells . Fluorescence intensities of NT5E in PIGG - KO clones # 4 , # 10 , # 17 , # 19 and wild type cells are 575 , 910 , 696 , 530 and 1548 while those of EphrinA5 in clones # 4 , # 17 , # 19 and wild type cells are 1431 , 2080 , 1868 and 3336 , respectively . Data information : Each FACS analysis was performed at least three times . B . Expression levels of GPI - APs in PIGG - KO clone # 19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells . Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886 , respectively . Data information : Each FACS analysis was performed at least three times .

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 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

Figure 7A. Expression levels of various GPI-APs in different clones of PIGG-KO HEK293 cells. Fluorescence intensities of NT5E in PIGG-KO clones #4, #10, #17, #19 and wild type cells are 575, 910, 696, 530 and 1548 while those of EphrinA5 in clones #4, #17, #19 and wild type cells are 1431, 2080, 1868 and 3336, respectively. Data information: Each FACS analysis was performed at least three times.B. Expression levels of GPI-APs in PIGG-KO clone #19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells. Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886, respectively. Data information: Each FACS analysis was performed at least three times.


Figure 8A . Expression levels of different GPI - APs in PIGG - KO HEK293 cells transiently co - transfected with PIGG cDNA or empty vector . Vertical bars represent % of levels of GPI - APs expression in non - rescued PIGG - KO cells relative to those in PIGG cDNA rescued PIGG - KO cells . Analysis was repeated 3 to 6 times and SD bars were shown . B . HA tagged NTNG2 and FLAG tagged DAF were transiently co - transfected together with PIGG cDNA or empty vector to PIGG - KO cells and were analyzed by FACS . Fluorescence intensity levels of NTNG2 in PIGG - KO cells and in PIGG rescued cells are 639 and 3426 , while those of DAF in PIGG - KO cells and in PIGG rescued cells are 562 and 599 , respectively . Each FACS analysis was performed at least three times .

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 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

Figure 8A. Expression levels of different GPI-APs in PIGG-KO HEK293 cells transiently co-transfected with PIGG cDNA or empty vector. Vertical bars represent % of levels of GPI-APs expression in non-rescued PIGG-KO cells relative to those in PIGG cDNA rescued PIGG-KO cells. Analysis was repeated 3 to 6 times and SD bars were shown.B. HA tagged NTNG2 and FLAG tagged DAF were transiently co-transfected together with PIGG cDNA or empty vector to PIGG-KO cells and were analyzed by FACS. Fluorescence intensity levels of NTNG2 in PIGG-KO cells and in PIGG rescued cells are 639 and 3426, while those of DAF in PIGG-KO cells and in PIGG rescued cells are 562 and 599, respectively. Each FACS analysis was performed at least three times.


Figure 1 ( C ) Exemplary images of COS - 7 cells expressing PEX - mRFP - FKBP before ( upper panels ) and 30 minutes after ( middle panels ) rapalog addition in the presence of a translocating motor - KIF1C - MDC - FRB and a non - translocating motor - KIF3B - MDC - FRB . Yellow lines indicate COS7 cell outline . Lower panel represents the overlay of sequential binarized images color coded by time ( see also Appendix Figure S1 ) . Scale bar = 20 µm ( D ) Plots representing intensity time traces of R90 % for COS - 7 cells transfected with PEX - mRFP - FKBP and KIF1C - MDC - FRB ( n = 13 ) or KIF3B - MDC - FRB ( n = 10 ) . R90 % is the radius for each time point that includes 90 % of the total fluorescent intensity . Error bars indicate SEM . E ) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS - 7 cells transfected with kinesin constructs summarized in ( A ) . The number of analyzed cells is summarized in Appendix Table S1 . Kinesins were considered translocators , if they displaced peroxisomes ≥ 5 µm ( marked with a blue dashed line ) in 30 minutes after rapalog addition . Error bars indicate SEM . ( F ) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90 % ( summarized in Appendix figure S2 , Appendix Table S1 ) . Error bars indicate SEM .

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

Figure 1(C) Exemplary images of COS-7 cells expressing PEX-mRFP-FKBP before (upper panels) and 30 minutes after (middle panels) rapalog addition in the presence of a translocating motor - KIF1C-MDC-FRB and a non-translocating motor - KIF3B-MDC-FRB. Yellow lines indicate COS7 cell outline. Lower panel represents the overlay of sequential binarized images color coded by time (see also Appendix Figure S1). Scale bar = 20 µm(D) Plots representing intensity time traces of R90% for COS-7 cells transfected with PEX-mRFP-FKBP and KIF1C-MDC-FRB (n =13) or KIF3B-MDC-FRB (n =10). R90% is the radius for each time point that includes 90% of the total fluorescent intensity. Error bars indicate SEM.E) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS-7 cells transfected with kinesin constructs summarized in (A). The number of analyzed cells is summarized in Appendix Table S1. Kinesins were considered translocators, if they displaced peroxisomes ≥ 5 µm (marked with a blue dashed line) in 30 minutes after rapalog addition. Error bars indicate SEM.(F) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90% (summarized in Appendix figure S2, Appendix Table S1). Error bars indicate SEM.


Figure 2 ( A ) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX - mRFP - FKBP , KIF1C - MDC - FRB , KIF21A - MDC - FRB or KIF5B - MDC - FRB . The morphology of transfected cell is visualized using a BFP fill . Axons are marked with a blue line . Arrows mark peroxisome targeting to dendrites ( gray ) and axon ( blue ) 30 min after rapalog addition . Scale bar = 20 µm . ( B ) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C - MDC - FRB ( n = 6 neurons ) , KIF21A - MDC - FRB ( n = 4 ) and KIF5B - MDC - FRB ( n = 8 ) . Traces were normalized to the average intensity before rapalog addition , and to the background . C ) Quantitative representation of percentage of DIV14 neurons transfected as in ( A ) with constructs showed in Figure 1A , in which peroxisomes after addition of rapalog redistributed to either axons ( " a " , blue bars ) or to axons and at least 2 dendrites ( " a + d " , red bars ) . Kinesins were considered " dendrite targeting " if dendrites were targeted in ≥ 50 % of cells and are highlighted in red . Kinesin constructs are transfected as indicated ; the number of analyzed cells is summarized in Appendix Table S1 . Error bars indicate SEM ( D ) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in ( A ) . Scale bars : 20 µm ( E ) Percentage of DIV2 neurons transfected with KIF1C - MDC - FRB ( n = 9 neurons ) , KIF21A - MDC - FRB ( n = 14 ) and KIF5B - MDC - FRB ( n = 14 ) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and ( F ) Percentage of neurites of DIV2 neurons transfected as in ( E ) targeted with peroxisomes after addition of rapalog . Error bars indicate SEM ; Scale bars : 20 µm .

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

Figure 2(A) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX-mRFP-FKBP, KIF1C-MDC-FRB, KIF21A-MDC-FRB or KIF5B-MDC-FRB. The morphology of transfected cell is visualized using a BFP fill. Axons are marked with a blue line. Arrows mark peroxisome targeting to dendrites (gray) and axon (blue) 30 min after rapalog addition. Scale bar = 20 µm.(B) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C-MDC-FRB (n = 6 neurons), KIF21A-MDC-FRB (n = 4) and KIF5B-MDC-FRB (n = 8). Traces were normalized to the average intensity before rapalog addition, and to the background.C) Quantitative representation of percentage of DIV14 neurons transfected as in (A) with constructs showed in Figure 1A, in which peroxisomes after addition of rapalog redistributed to either axons ("a", blue bars) or to axons and at least 2 dendrites ("a+d", red bars). Kinesins were considered "dendrite targeting" if dendrites were targeted in ≥ 50% of cells and are highlighted in red. Kinesin constructs are transfected as indicated; the number of analyzed cells is summarized in Appendix Table S1. Error bars indicate SEM(D) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in (A). Scale bars: 20 µm(E) Percentage of DIV2 neurons transfected with KIF1C-MDC-FRB (n=9 neurons), KIF21A-MDC-FRB (n=14) and KIF5B-MDC-FRB (n=14) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and (F) Percentage of neurites of DIV2 neurons transfected as in (E) targeted with peroxisomes after addition of rapalog. Error bars indicate SEM; Scale bars: 20 µm.


Figure 3 ( A ) Representative confocal images of a hippocampal neuron ( DIV14 ) immunostained for MAP2 , β - III - tubulin and DCLK1 . Scale bar = 20 µm ( B ) Quantification of the average ratio of DCLK1 , MAP1A , MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons . Per condition 6 - 9 cells were analyzed . Error bars indicate SEM . ( C ) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β - gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β - gal ( red ) and DCLK1 ( green ) . ( D ) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh # 1 ( n = 16 neurons ) , DCLK1 sh # 2 ( n = 16 ) and DCLK1 sh # 3 ( n = 18 ) . Non - transfected cells were taken as a control ( n = 48 ) . N = 2 . Error bars indicate SEM ; * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) . ( E ) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER ( control ) or pSUPER encoding one of three DCLK1 shRNAs . Levels of actin were used as an equal loading control . ( F ) Quantification of protein levels of DCLK1 of samples represented in ( E ) . N = 3 , error bars indicate SEM , * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) . ( G ) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX - mRFP - FKBP , KIF1C ( MDC ) - FRB and the indicated shRNA before and 30 minutes after rapalog addition . BFP fill was co - transected to visualize the morphology of transfected neuron . Axons are marked with a blue line . Arrows mark dendrite ( gray ) and axon ( blue ) targeting of peroxisomes after rapalog addition . Scale bar = 20 µm ( H ) Quantitative representation of the percentage of DIV14 neurons transfected with PEX - RFP together with either KIF1C , KIF21A or KIF5B and an indicated shRNA . as in ( A ) in which after addition of rapalog peroxisomes redistributed to axons ( " a " , blue bars ) , to both axons and dendrites ( " a + d " , red bars ) or did not redistribute into any neuronal compartment ( " nt " - no targeting , gray bars ) . Per condition 19 - 44 neurons were analyzed ; N = 2 .

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 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 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 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 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 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 3(A) Representative confocal images of a hippocampal neuron (DIV14) immunostained for MAP2, β-III-tubulin and DCLK1. Scale bar = 20 µm(B) Quantification of the average ratio of DCLK1, MAP1A, MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons. Per condition 6-9 cells were analyzed. Error bars indicate SEM.(C) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β-gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β-gal (red) and DCLK1 (green).(D) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh#1 (n = 16 neurons), DCLK1 sh#2 (n=16) and DCLK1 sh#3 (n=18). Non-transfected cells were taken as a control (n = 48). N=2. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).(E) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER (control) or pSUPER encoding one of three DCLK1 shRNAs. Levels of actin were used as an equal loading control.(F) Quantification of protein levels of DCLK1 of samples represented in (E). N = 3, error bars indicate SEM, *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).(G) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX-mRFP-FKBP, KIF1C (MDC)-FRB and the indicated shRNA before and 30 minutes after rapalog addition. BFP fill was co-transected to visualize the morphology of transfected neuron. Axons are marked with a blue line. Arrows mark dendrite (gray) and axon (blue) targeting of peroxisomes after rapalog addition. Scale bar = 20 µm(H) Quantitative representation of the percentage of DIV14 neurons transfected with PEX-RFP together with either KIF1C, KIF21A or KIF5B and an indicated shRNA. as in (A) in which after addition of rapalog peroxisomes redistributed to axons ("a", blue bars), to both axons and dendrites ("a+d", red bars) or did not redistribute into any neuronal compartment ("nt"-no targeting, gray bars). Per condition 19-44 neurons were analyzed; N = 2.


Figure 4 ( A - G ) Representative frame ( A , D ) and a kymograph ( B , F ) of a simultaneous two - color movie of a hippocampal neuron expressing NPY - mCherry and either KIF1A - GFP ( A , B ) , BDNF - GFP ( D ) or TfR - GFP ( F ) . Scale bar = 20 µm ( A , D ) , 5 µm ( B , F ) . ( C ) Quantification of vesicle colocalization in cells described in ( A , B ) The quantifications indicate the % of KIF1Avesicles that are NPY positive - left bar quantified from images of fixed cells presented in ( A ) and right bar quantified from kymographs as presented in ( B ) ( only moving fraction ) . ( E ) Quantification of vesicle colocalization in cells described in ( D ) - left bar indicates the % of NPYvesicles that are BDNF positive , the right bar indicates the % of BDNFvesicles that are NPY positive . ( G ) Quantification of vesicle colocalization in cells described in ( F ) . The left bar indicates the % of NPYvesicles that are TfR positive , the right bar indicates the % of TfR that are NPY positive . ( H , I ) Quantification of run lengths ( H ) and velocities ( I ) of vesicles in dendrites of hippocampal neurons transfected at DIV10 - 14 for 4 days with either NPY - GFP ( n = 924 movements ) or TfR - GFP ( n = 286 ) or KIF1A - GFP ( n = 174 ) together with a morphology marker BFP . N = 2 . Error bars indicate SEM ; * * * p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) . ( K ) Representative kymographs of NPY - GFP - labeled dense core vesicle motility in primary dendrites of transfected neurons . DIV10 - 14 hippocampal neurons were cotransfected , for 4 days , with plasmids encoding NPY - GFP , BFP fill and a KIF21 shRNA mix ( n = 30 ) , KIF1shRNA mix ( n = 31 ) , KIF1A shRNA ( n = 19 ) , KIF1B shRNA ( n = 19 ) or KIF1C shRNA ( n = 11 ) . pSUPER was used as a control ( n = 15 ) . Scale bar = 5 µm . ( L ) Quantification of the average number of dense core vesicle entries into the primary dendrite in 100 s after photobleaching . N = 2 - 3 . Error bars indicate SEM ; * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) . ( M ) Representative kymographs of TfR - GFP - labeled recycling endosome motility in primary dendrites . DIV10 - 14 Hippocampal neurons were cotransfected with TfR - GFP together with a BFP fill and a KIF1shRNA mix ( n = 11 ) . pSUPER was used as a control ( n = 20 ) . Scale bar = 5 µm . ( N ) Quantification of the average number of recycling endosome entries into the primary dendrite in 50 s . N = 2 . Error bars indicate SEM . ns - not significant ( Mann - Whitney test ) . ( O ) Representative images of hippocampal neurons ( DIV4 ) cotransfected with an empty pSUPER ( control ) or pSUPER encoding an indicated shRNA together with a GFP fill fixed at DIV8 and immunostained for MAP2 and sodium channels ( AIS ) . Scale bar = 50 µm . ( P ) Sholl analysis of cells described in ( O ) Error bars indicate SEM ; * - p < 0 . 05 , * * - p < 0 . 01 , and * * * - p < 0 . 001 ( two - way ANOVA test followed by Bonferroni ' s post hoc test ) . ( R ) Analysis of the total dendritic length of cells described in ( O ) Error bars indicate SEM ; * - p < 0 . 05 , * * - p < 0 . 01 and * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) . Per condition 21 - 24 neurons ( n ) were analyzed ; N = 2 .

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 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 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 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 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 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

Figure 4(A-G) Representative frame (A, D) and a kymograph (B, F) of a simultaneous two-color movie of a hippocampal neuron expressing NPY-mCherry and either KIF1A-GFP (A, B), BDNF-GFP (D) or TfR-GFP (F). Scale bar = 20 µm (A, D), 5 µm (B, F). (C) Quantification of vesicle colocalization in cells described in (A, B) The quantifications indicate the % of KIF1Avesicles that are NPY positive - left bar quantified from images of fixed cells presented in (A) and right bar quantified from kymographs as presented in (B) (only moving fraction). (E) Quantification of vesicle colocalization in cells described in (D) - left bar indicates the % of NPYvesicles that are BDNF positive, the right bar indicates the % of BDNFvesicles that are NPY positive. (G) Quantification of vesicle colocalization in cells described in (F). The left bar indicates the % of NPYvesicles that are TfR positive, the right bar indicates the % of TfR that are NPY positive.(H, I) Quantification of run lengths (H) and velocities (I) of vesicles in dendrites of hippocampal neurons transfected at DIV10-14 for 4 days with either NPY-GFP (n = 924 movements) or TfR-GFP (n = 286) or KIF1A-GFP (n = 174) together with a morphology marker BFP. N = 2. Error bars indicate SEM; *** p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test ).(K) Representative kymographs of NPY-GFP-labeled dense core vesicle motility in primary dendrites of transfected neurons. DIV10-14 hippocampal neurons were cotransfected, for 4 days, with plasmids encoding NPY-GFP, BFP fill and a KIF21 shRNA mix (n = 30), KIF1shRNA mix (n = 31), KIF1A shRNA (n = 19), KIF1B shRNA (n = 19) or KIF1C shRNA (n = 11). pSUPER was used as a control (n = 15). Scale bar = 5 µm.(L) Quantification of the average number of dense core vesicle entries into the primary dendrite in 100 s after photobleaching. N = 2-3. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).(M) Representative kymographs of TfR-GFP-labeled recycling endosome motility in primary dendrites. DIV10-14 Hippocampal neurons were cotransfected with TfR-GFP together with a BFP fill and a KIF1shRNA mix (n = 11). pSUPER was used as a control (n = 20). Scale bar = 5 µm.(N) Quantification of the average number of recycling endosome entries into the primary dendrite in 50 s. N = 2. Error bars indicate SEM. ns - not significant (Mann-Whitney test).(O) Representative images of hippocampal neurons (DIV4) cotransfected with an empty pSUPER (control) or pSUPER encoding an indicated shRNA together with a GFP fill fixed at DIV8 and immunostained for MAP2 and sodium channels (AIS). Scale bar = 50 µm.(P) Sholl analysis of cells described in (O) Error bars indicate SEM; * - p < 0.05, ** - p < 0.01, and *** - p < 0.001 (two-way ANOVA test followed by Bonferroni's post hoc test). (R) Analysis of the total dendritic length of cells described in (O) Error bars indicate SEM; * - p < 0.05, ** - p < 0.01 and *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test). Per condition 21-24 neurons (n) were analyzed; N = 2.


Figure 5 ( B ) Representative kymographs of dense - core vesicle motility in proximal dendrites of transfected neurons . DIV 10 - 14 hippocampal neurons were co - transfected for 4 days with NPY - GFP and pSUPER ( control ) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live - cell imaging . Scale bar = 5 µm . ( C ) Quantification of the average number of dense - core vesicle entries into dendrites of neurons transfected as in ( B ) during 100 s of the live recording after photobleaching . Per condition 10 - 32 cells were analyzed . Error bars indicate SEM ; * * * - p < 0 . 001 ( Kruskal - Wallis test followed by post hoc Dunn ' s test ) . N = 2 . ( D , E ) DIV10 - 14 hippocampal neurons were co - transfected for 4 days with either pSUPER ( control , n = 20 neurons ) or DCLK1 shRNA together with TfR - GFP ( n = 16 ) . ( D ) Representative kymographs of recycling endosome motility in dendrites . Scale bar = 5 µm . ( E ) Quantification of the average number of recycling endosome entries into dendrites during 50 s of the live recording after photobleaching . Error bars indicate SEM ; ns - not significant ( Mann - Whitney test ) . N = 2 .

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 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

Figure 5(B) Representative kymographs of dense-core vesicle motility in proximal dendrites of transfected neurons. DIV 10-14 hippocampal neurons were co-transfected for 4 days with NPY-GFP and pSUPER (control) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live-cell imaging. Scale bar = 5 µm.(C) Quantification of the average number of dense-core vesicle entries into dendrites of neurons transfected as in (B) during 100 s of the live recording after photobleaching. Per condition 10-32 cells were analyzed. Error bars indicate SEM; *** - p < 0.001 (Kruskal-Wallis test followed by post hoc Dunn's test). N = 2.(D, E) DIV10-14 hippocampal neurons were co-transfected for 4 days with either pSUPER (control, n = 20 neurons) or DCLK1 shRNA together with TfR-GFP (n = 16).(D) Representative kymographs of recycling endosome motility in dendrites. Scale bar = 5 µm. (E) Quantification of the average number of recycling endosome entries into dendrites during 50 s of the live recording after photobleaching. Error bars indicate SEM; ns - not significant (Mann-Whitney test). N = 2.


Figure 6 ( A ) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1 , DIV7 , DIV14 and DIV21 hippocampal neurons . ( B ) Representative image of a hippocampal neuron ( DIV14 ) immunostained for DCLK1 and β - III - tubulin . Scale bar = 20 µm . ( C , D ) Representative image of a COS - 7 cell transfected with DCLK1 - GFP ( C ) or DCLK1 ( ΔKD ) - GFP ( D ) and stained for detyrosinated , tyrosinated or α - tubulin . ( E ) Representative image of a COS - 7 cell transfected with DCLK1 - GFP and TagRFP - EB3 and recorded during live - cell imaging . Representative GFP was drawn ( over a region marked with white boxes ) showing that DCLK1 - GFP colocalizes with EB3 - TagRFP - T . Scale bars = 20 um .

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 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 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 6(A) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1, DIV7, DIV14 and DIV21 hippocampal neurons.(B) Representative image of a hippocampal neuron (DIV14) immunostained for DCLK1 and β-III-tubulin. Scale bar = 20 µm.(C, D) Representative image of a COS-7 cell transfected with DCLK1-GFP (C) or DCLK1(ΔKD)-GFP (D) and stained for detyrosinated, tyrosinated or α-tubulin.(E) Representative image of a COS-7 cell transfected with DCLK1-GFP and TagRFP-EB3 and recorded during live-cell imaging. Representative GFP was drawn (over a region marked with white boxes) showing that DCLK1-GFP colocalizes with EB3-TagRFP-T. Scale bars = 20 um.


Figure 7 ( A ) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0 . Samples were analyzed by western blot with indicated antibodies . ( B , E ) Representative images of a hippocampal cell transfected at DIV10 with RFP ( to visualize transfected cells ) , pSUPER ( control ) or DCLK1 shRNA , fixed at DIV14 and stained for EB3 . Black lines indicate the neuronal soma outline . Scale bars = 10 µm . ( C , D ) Quantification of the number of EB3 comets in the dendrite ( C ) and in the soma ( D ) . 25 - 26 neurons were analyzed per condition , N = 2 . Error bars indicate SEM , * * * - p < 0 . 001 , * * - p < 0 , 01 ( unpaired t - test ) . ( F - I ) Hippocampal neurons were co - transfected with MACF18 - GFP and pSUPER ( control ) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life - cell microscopy . ( F ) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation . Scale bar = 10 µm ( C ) . ( G , I ) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before ( G ) and after ( I ) photoablation . Eighteen neurons were analyzed per condition ; N = 2 . ( H ) Scheme of live - cell imaging after photoablation . ( J , K ) Biotin pull - downs ( PD ) from extracts of HEK293 transfected with biotin - tagged DCLK1 and ( J ) KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB , KIF5B - MDC - HA - FRB constructs and probed for HA / DCLK1 , ( K ) different GFP - tagged KIF1A truncation constructs and probed for GFP / DCLK1 . The ratio input / pellet is 2 % for all pull - down experiments . ( L ) GFP pull - down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB and KIF1C - MD - HA and probed for HA / GFP . LacZ - HA was used as a negative control .

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

Figure 7(A) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0. Samples were analyzed by western blot with indicated antibodies.(B, E) Representative images of a hippocampal cell transfected at DIV10 with RFP (to visualize transfected cells), pSUPER (control) or DCLK1 shRNA, fixed at DIV14 and stained for EB3. Black lines indicate the neuronal soma outline. Scale bars = 10 µm.(C, D) Quantification of the number of EB3 comets in the dendrite (C) and in the soma (D). 25-26 neurons were analyzed per condition, N=2. Error bars indicate SEM, *** - p < 0.001, ** - p<0,01 (unpaired t-test).(F-I) Hippocampal neurons were co-transfected with MACF18-GFP and pSUPER (control) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life-cell microscopy. (F) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation. Scale bar = 10 µm (C). (G, I) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before (G) and after (I) photoablation. Eighteen neurons were analyzed per condition; N=2. (H) Scheme of live-cell imaging after photoablation.(J, K) Biotin pull-downs (PD) from extracts of HEK293 transfected with biotin-tagged DCLK1 and (J) KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB, KIF5B-MDC-HA-FRB constructs and probed for HA/DCLK1, (K) different GFP-tagged KIF1A truncation constructs and probed for GFP/DCLK1. The ratio input/pellet is 2% for all pull-down experiments.(L) GFP pull-down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB and KIF1C-MD-HA and probed for HA/GFP. LacZ-HA was used as a negative control.


Figure 1B . IHLs were isolated from the liver of Tie2 - GFP mice intrasplenically injected with NaCl ( n = 3 ) or with 5x103 CT26 at the specified time points ( day 25 post - injection n = 6 , day 35 post - injection n = 5 ) . The number of GFP + TEMs was estimated by flow cytometry as 7AAD - / CD45 + / CD11b + / CD11c - / GFP + cells per liver ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . C . Representative images ( top panels ) and corresponding H & amp ; amp ; E micrographs ( bottom panels ) of the liver from control ( NaCl - injected , left panels ) or Tie2 - GFP mice 25 and 35 days ( center and right panels , respectively ) post - intrasplenic injection of 5x103 CT26 . Large metastatic foci in the liver of mice that received CT26 cells are indicated ( arrowheads ) . The dashed line in the H & amp ; amp ; E panels identifies the metastasis margin ; ✱ = CRC metastatic area ; scale bars = 100μm . D . Liver RNA was extracted from mice transplanted and injected as described in B ( NaCl n = 4 , day 25 n = 7 , day 35 n = 4 ) . The average GFP expression value of NaCl injected mice estimated by quantitative real - time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . E . Confocal immunofluorescence images of representative liver sections from Tie2 - GFP mice that were injected intrasplenically with either NaCl ( upper panels ) or 5x103 CT26 ( bottom panels , 25 days post - injection ) . Note that TEMs ( identified as GFP + , macrophage mannose receptor [ MMR ] + and F4 / 80 + cells ) gather in the proximity of CRC metastatic foci ( identified by the dashed lines ) ; ✱ = CRC metastatic area ; scale bars = 100μm .

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

Figure 1B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.C. Representative images (top panels) and corresponding H&amp;E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&amp;E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm.D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.


Figure 2A . Contrast - enhanced magnetic resonance imaging ( MRI ) of the liver from representative Tie2 - GFP mice ( red frame ) or Tie2 - IFNα ( blue frame ) that were intrasplenically injected with 5x103 CT26 . Red arrows identify CRC liver metastases of representative z - sections . Tumors were characterized as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences , respectively . Each panel refers to a single mouse analyzed at different time points ; n . a . = not assessed , refers to a mouse euthanized before the specified time point ; scale bars = 5mm . B . Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis . Mice were treated as described in A . Mock / Tie2 - GFP n = 11 , Tie2 - IFNα n = 9 ; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point ; p - values were calculated by Fisher ' s exact test . C . Tumor volume quantification measured by MRI analysis of the same mice described in B . Each symbol corresponds to the same mouse analyzed at different time points ; horizontal bars = mean values ; note that Mock / Tie2 - GFP mice were euthanized or died before day 54 ; p - values were calculated by Mann - Whitney test . D , E . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 ( D ) and Irf7 ( E ) within brain ( n = 4 , n = 2 ) , kidney ( n = 4 , n = 3 ) and metastatic liver ( n = 4 , n = 7 ) of Tie2 - GFP or Tie2 - IFNα mice euthanized 25 days post - intrasplenic injection of 5x103 CT26 . The basal expression of Oas1 and Irf7 estimated in brain of control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values depicted . Mean values are shown ; error bars = S . E . M . ; p - value was calculated by Mann - Whitney test . F . Eighty - four or 54 days post - NaCl or 5x103 CT26 intrasplenic injection , Sham / CTRL ( n = 5 ) or Tie2 - IFNα mice ( n = 7 ) were infected with LCMV ( Armstrong strain , 200 pfu intraperitoneally injected ) . Eight days post - infection white blood cells were isolated and analyzed for LCMV specific CD8 + T cell response . Left panel : percentage of total CD8 + T cells ; middle panel : percentage of CD8 + / NP118 + T cells ( NP118 = recombinant dimeric H - 2d / Ig fusion protein complexed with the immune - dominant H - 2d - restricted LCMV NP118 - 126 peptide ) ; right panel : percentage of CD8 + / IFNγ + T cells after in vitro stimulation with virus - specific H - 2d - restricted peptide ( NP118 - 126 ) ; data pooled from two independent experiments ; mean values are shown ; error bars = S . E . M . ; differences were not statistically significant ( by unpaired Student ' s t - Test ) . G . Kaplan - Meier survival curves of the indicated groups of mice described in A . Sham ( n = 3 ) , Mock ( wt ) / Tie2 - GFP ( n = 11 ) ; Tie2 - IFNα ( n = 9 ) ; data pooled from three independent experiments ; p = 0 . 001 by log - rank / Mantel - Cox test . The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal ( red frame : Mock / Tie2 - GFP , day 35 post - CT26 injection ) opposed to the lack of lesions in Tie2 - IFNα treated mice at later time points ( blue frame : day 450 post - CT26 injection ) .

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

Figure 2A. Contrast-enhanced magnetic resonance imaging (MRI) of the liver from representative Tie2-GFP mice (red frame) or Tie2-IFNα (blue frame) that were intrasplenically injected with 5x103 CT26. Red arrows identify CRC liver metastases of representative z-sections. Tumors were characterized as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Each panel refers to a single mouse analyzed at different time points; n.a.=not assessed, refers to a mouse euthanized before the specified time point; scale bars=5mm.B. Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis. Mice were treated as described in A. Mock/Tie2-GFP n=11, Tie2-IFNα n=9; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point; p-values were calculated by Fisher's exact test.C. Tumor volume quantification measured by MRI analysis of the same mice described in B. Each symbol corresponds to the same mouse analyzed at different time points; horizontal bars=mean values; note that Mock/Tie2-GFP mice were euthanized or died before day 54; p-values were calculated by Mann-Whitney test.D, E. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 (D) and Irf7 (E) within brain (n=4, n=2), kidney (n=4, n=3) and metastatic liver (n=4, n=7) of Tie2-GFP or Tie2-IFNα mice euthanized 25 days post-intrasplenic injection of 5x103 CT26. The basal expression of Oas1 and Irf7 estimated in brain of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values depicted. Mean values are shown; error bars=S.E.M.; p-value was calculated by Mann-Whitney test.F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student's t-Test).G. Kaplan-Meier survival curves of the indicated groups of mice described in A. Sham (n=3), Mock(wt)/Tie2-GFP (n=11); Tie2-IFNα (n=9); data pooled from three independent experiments; p=0.001 by log-rank/Mantel-Cox test. The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal (red frame: Mock/Tie2-GFP, day 35 post-CT26 injection) opposed to the lack of lesions in Tie2-IFNα treated mice at later time points (blue frame: day 450 post-CT26 injection).


Figure 3A . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2 - GFP or Tie2 - IFNαmice that were intrasplenically injected with either NaCl ( n = 4 and n = 4 , respectively ) or 5x105CT26 - RFP and euthanized 5 minutes ( n = 9 , n = 5 ) , 3 days ( n = 6 , n = 3 ) and 7 days ( n = 5 , n = 3 ) thereafter . The basal expression of Irf7 estimated in control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values observed at the following time points post - injection . Data pooled from four independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . The increase in Irf7 expression levels from the liver of Tie2 - GFPmice was statistically significant ( p = 0 . 004 by one - way Anova test , not reported on graph ) . B . Representative RFP immunostaining from the liver of Tie2 - GFP mice ( left panels ) or Tie2 - IFNα mice ( right panels ) at the indicated time points after 5x105 CT26 - RFP intrasplenic injection . Arrowheads highlight single or clustered RFP positive cells ; scale bars = 100μm . C . Immunostaining quantification of hepatic CT26 - RFP arrival ( 5 minutes post - injection : Tie2 - GFP n = 6 , Tie2 - IFNα n = 2 ) and expansion ( day 3 post - injection : Tie2 - GFP n = 6 , Tie2 - IFNα n = 3 ; day 7 post - injection : Tie2 - GFP n = 5 , Tie2 - IFNα n = 3 ) in the liver of Tie2 - GFP mice or Tie2 - IFNα mice that were injected with 5x105 CT26 - RFP as described in B . Data pooled from two independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . The increased percent of RFP + areas in the liver of Tie2 - GFP mice was statistically significant ( p = 0 . 0009 by one - way Anova test , not reported on graph ) .

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Figure 3A. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2-GFP or Tie2-IFNαmice that were intrasplenically injected with either NaCl (n=4 and n=4, respectively) or 5x105CT26-RFP and euthanized 5 minutes (n=9, n=5), 3 days (n=6, n=3) and 7 days (n=5, n=3) thereafter. The basal expression of Irf7 estimated in control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed at the following time points post-injection. Data pooled from four independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increase in Irf7 expression levels from the liver of Tie2-GFPmice was statistically significant (p=0.004 by one-way Anova test, not reported on graph).B. Representative RFP immunostaining from the liver of Tie2-GFP mice (left panels) or Tie2-IFNα mice (right panels) at the indicated time points after 5x105 CT26-RFP intrasplenic injection. Arrowheads highlight single or clustered RFP positive cells; scale bars=100μm.C. Immunostaining quantification of hepatic CT26-RFP arrival (5 minutes post-injection: Tie2-GFP n=6, Tie2-IFNα n=2) and expansion (day 3 post-injection: Tie2-GFP n=6, Tie2-IFNα n=3; day 7 post-injection: Tie2-GFP n=5, Tie2-IFNα n=3) in the liver of Tie2-GFP mice or Tie2-IFNα mice that were injected with 5x105 CT26-RFP as described in B. Data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increased percent of RFP+ areas in the liver of Tie2-GFP mice was statistically significant (p=0.0009 by one-way Anova test, not reported on graph).


Figure 4A . Representative contrast - enhanced MRI panels depicting the liver of Mock / Tie2 - GFP ( red frame ) or Tie2 - IFNα ( blue frame ) chimeric mice . Each frame shows the metastatic progression 14 and 21 days post - intrasplenic injection of 5x104 MC38 within the indicated chimeric group . BM donors and recipient mouse strains are indicated on the left of each frame ; red arrows or dashed red lines identify CRC liver metastases of representative z - sections . Tumors were identified as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences respectively ; scale bars = 5mm . B . Tumor volume quantification ( based on MRI analyses ) of lesions detected in the same livers described in A . Chimeric groups and number of mice analyzed are listed ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

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Figure 4A. Representative contrast-enhanced MRI panels depicting the liver of Mock/Tie2-GFP (red frame) or Tie2-IFNα (blue frame) chimeric mice. Each frame shows the metastatic progression 14 and 21 days post-intrasplenic injection of 5x104 MC38 within the indicated chimeric group. BM donors and recipient mouse strains are indicated on the left of each frame; red arrows or dashed red lines identify CRC liver metastases of representative z-sections. Tumors were identified as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences respectively; scale bars=5mm.B. Tumor volume quantification (based on MRI analyses) of lesions detected in the same livers described in A. Chimeric groups and number of mice analyzed are listed; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


Figure 5B . Tumor volume quantification measured by MRI analysis of Tie2 - GFP ( n = 6 ) or Tie2 - IFNα mice ( n = 9 ) at the indicated time points post - transplant , one additional Tie2 - IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs ' test and not further analyzed ; mean values are shown ; error bars = S . E . M . ; p = 0 . 051 , by Mann - Whitney test . C . Images ( top panels ) and corresponding contrast - enhanced MRI ( middle and bottom panels ) of the liver from representative Tie2 - GFP mice or Tie2 - IFNα mice that were intrahepatically injected with 5x103 CT26 . White dashed lines identify macroscopic lesions . Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z - stacks . Tumors detected by MRI analysis appeared as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences , respectively . Scale bars = 5mm . D . Percentage of mice with peritoneal carcinomatosis ( defined by the presence of multiple intra - peritoneal lesions ) determined by MRI analysis from the same mice described in B . Note that while intrahepatic number of lesions remained similar between time points , extrahepatic tumor spreading was significantly reduced in Tie2 - IFNα mice . P - values were calculated by Fisher ' s exact test . E . Confocal immunofluorescence images of representative liver sections from Tie2 - GFP mice at distal ( upper panels ) or proximal ( bottom panels ) areas to CRC liver metastasis , 30 days post HSPC transplant . Note that TEMs were identified as GFP + cells and by the concomitant expression of different levels of the myeloid cell marker CD11b + as highlighted in the inset ( merge panels , white arrows ) ; the dashed line identifies the metastasis margin ; ✱ = CRC metastatic area ; scale bars = 100μm ; insets depict the corresponding areas identified by dashed squares in the merge panels , magnified 1 . 5 - folds . F , G . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2 - GFP ( n = 4 ) or Tie2 - IFNα ( n = 4 ) mice euthanized 30 days post - transplant . The basal expression of Irf7 estimated in liver of control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values observed . Mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . H . Kaplan - Meier survival curves of the indicated groups of mice . Tie2 - GFP ( n = 3 ) ; Tie2 - IFNα ( n = 5 ) ; p = 0 . 007 by log - rank / Mantel - Cox test .

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Figure 5B. Tumor volume quantification measured by MRI analysis of Tie2-GFP (n=6) or Tie2-IFNα mice (n=9) at the indicated time points post-transplant, one additional Tie2-IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs' test and not further analyzed; mean values are shown; error bars=S.E.M.; p=0.051, by Mann-Whitney test.C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm.D. Percentage of mice with peritoneal carcinomatosis (defined by the presence of multiple intra-peritoneal lesions) determined by MRI analysis from the same mice described in B. Note that while intrahepatic number of lesions remained similar between time points, extrahepatic tumor spreading was significantly reduced in Tie2-IFNα mice. P-values were calculated by Fisher's exact test.E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice at distal (upper panels) or proximal (bottom panels) areas to CRC liver metastasis, 30 days post HSPC transplant. Note that TEMs were identified as GFP+ cells and by the concomitant expression of different levels of the myeloid cell marker CD11b+ as highlighted in the inset (merge panels, white arrows); the dashed line identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm; insets depict the corresponding areas identified by dashed squares in the merge panels, magnified 1.5-folds.F, G. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2-GFP (n=4) or Tie2-IFNα (n=4) mice euthanized 30 days post-transplant. The basal expression of Irf7 estimated in liver of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed. Mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.


Figure 6A . IHL and TEM characterization in the liver of patients described in Appendix Fig S6A . Liver samples from surgical resections ( n = 9 ) were weighted and IHLs were isolated , counted and normalized over tissue weight ( left panel ) . Flow cytometry analysis revealed that TEMs , identified as 7AAD - / CD45 + / CD11b + / CD14 + / Tie2 + cells , are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions ( right panel ) . Control = hepatic hemangioma ; Distal = distal to CRC liver metastasis ( > 1 cm from the lesion ) ; Proximal = proximal to CRC liver metastasis ( < 1 cm from the lesion ) ; p - values were calculated by Wilcoxon matched pairs Test . B - C . Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal ( > 1 cm ; left panels ) or proximal ( < 1 cm ; right panels ) sites from the CRC liver lesion . C . Higher magnification of the panels identified by a rectangle in panel B . Negative control ( bottom panels ) is obtained by omitting primary anti - TIE2 antibodies . TIE2immunostaining , indicates that sites that are distal from the lesion contain only TIE2 + cells with an apparent endothelial morphology ( characterized by an elongated appearance , arrows ) while sites that are proximal to the lesion also contain additional TIE2 + cells with an apparent monocyte - like morphology ( characterized by a round appearance , arrowheads ) . The dashed lines identifies the metastasis margin ; ✱ = CRC metastatic area . Scale bars of upper panels = 50μm ; middle and bottom panels = 20 μm .

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Figure 6A. IHL and TEM characterization in the liver of patients described in Appendix Fig S6A. Liver samples from surgical resections (n=9) were weighted and IHLs were isolated, counted and normalized over tissue weight (left panel). Flow cytometry analysis revealed that TEMs, identified as 7AAD-/CD45+/CD11b+/CD14+/Tie2+ cells, are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions (right panel). Control=hepatic hemangioma; Distal=distal to CRC liver metastasis (>1 cm from the lesion); Proximal=proximal to CRC liver metastasis (<1 cm from the lesion); p-values were calculated by Wilcoxon matched pairs Test.B-C. Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal (>1 cm; left panels) or proximal (<1 cm; right panels) sites from the CRC liver lesion. C. Higher magnification of the panels identified by a rectangle in panel B. Negative control (bottom panels) is obtained by omitting primary anti-TIE2 antibodies. TIE2immunostaining, indicates that sites that are distal from the lesion contain only TIE2+ cells with an apparent endothelial morphology (characterized by an elongated appearance, arrows) while sites that are proximal to the lesion also contain additional TIE2+ cells with an apparent monocyte-like morphology (characterized by a round appearance, arrowheads). The dashed lines identifies the metastasis margin; ✱=CRC metastatic area. Scale bars of upper panels=50μm; middle and bottom panels=20 μm.


Figure 1 ( B ) Cells expressing fluorescently - tagged α2 - tubulin ( atb2 ) were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs recorded within the final 1 . 1 µm of the cell for each strain . Red bars signify the mean ( C ) Kymographs showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged microtubules ( MT ) in the presence ( top panels ) and absence ( bottom panels ) of Mcp1 . Banding on MT , caused by unequal incorporation of fluorescence , illustrates the force exerted on the MT by continued growth into the cell cortex . Dashed yellow line indicates the cell end ( D ) MT growth speed was calculated from kymographs from control ( n = 16 ) and ∆ mcp1 cells ( n = 11 ) , Klp5 / Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control ( n = 44 ) and ∆ mcp1 cells ( n = 32 ) ( E ) Average intensity of Klp5 / Klp6 at the plus ends of iMTs from multiple kymographs of control ( n = 19 ) or ∆ mcp1 cells ( n = 14 ) ( F ) Mixing experiment to compare fluorescently - tagged Klp5 / Klp6 levels between cells either expressing ( blue , closed arrowheads ) or deleted ( red , open arrowhead ) for Mcp1 distinguished by the absence of fluorescently - tagged nuclear envelope protein Cut11 ( left panel ) . Scale bar , 5µm . Box plot ( right panel ) shows quantitated fluorescence values for nuclear levels of Klp5 / Klp6 in control ( n = 44 ) and ∆ mcp1 cells ( n = 45 ) and at the MT plus end in control ( n = 64 ) and ∆ mcp1 cells ( n = 35 ) prior to

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Figure 1(B) Cells expressing fluorescently-tagged α2-tubulin (atb2) were imaged every 5 seconds and the dwell time of ~100 individual iMTs recorded within the final 1.1 µm of the cell for each strain. Red bars signify the mean(C) Kymographs showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged microtubules (MT) in the presence (top panels) and absence (bottom panels) of Mcp1. Banding on MT, caused by unequal incorporation of fluorescence, illustrates the force exerted on the MT by continued growth into the cell cortex. Dashed yellow line indicates the cell end(D) MT growth speed was calculated from kymographs from control (n=16) and ∆mcp1 cells (n=11), Klp5/Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control (n=44) and ∆mcp1 cells (n=32)(E) Average intensity of Klp5/Klp6 at the plus ends of iMTs from multiple kymographs of control (n=19) or ∆mcp1 cells (n=14)(F) Mixing experiment to compare fluorescently-tagged Klp5/Klp6 levels between cells either expressing (blue, closed arrowheads) or deleted (red, open arrowhead) for Mcp1 distinguished by the absence of fluorescently-tagged nuclear envelope protein Cut11 (left panel). Scale bar, 5µm. Box plot (right panel) shows quantitated fluorescence values for nuclear levels of Klp5/Klp6 in control (n=44) and ∆mcp1 cells (n=45) and at the MT plus end in control (n=64) and ∆mcp1 cells (n=35) prior to shrinkage


Figure 2 ( A ) Log phase cells expressing fluorescently - tagged MTs were imaged and the levels of either fluorescently - tagged Klp5 / Klp6 or Tea2 at the plus ends of growing MTs determined . Measurements ( klp5 - mNG klp6 - mNG , n = 305 ; tea2 - GFP , n = 359 ) were plotted against microtubule ( MT ) length and third order polynomial curves fitted to the data ( B ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Tea2 kinesin in control cells ( top panels ) or in the absence of Klp6 ( middle panels ) or Mcp1 ( bottom panels ) . Dashed yellow line indicates the cell end ( C ) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in ( B ) for control ( n = 16 ) , ∆ klp6 ( n = 13 ) or ∆ mcp1 ( n = 14 ) cells ( D ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Klp5 / Klp6 in control ( top panels , repeated from 1C ) or ∆ tea2 cells ( bottom panels ) . Dashed yellow line indicates the cell end ( E ) Cells expressing fluorescently - tagged tubulin were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs for each condition recorded within the final 1 . 1 µm of the cell . Red bars signify the

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Figure 2(A) Log phase cells expressing fluorescently-tagged MTs were imaged and the levels of either fluorescently-tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs determined. Measurements (klp5-mNG klp6-mNG, n=305; tea2-GFP, n=359) were plotted against microtubule (MT) length and third order polynomial curves fitted to the data(B) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end (C) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n=16), ∆klp6 (n=13) or ∆mcp1 (n=14) cells (D) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Klp5/Klp6 in control (top panels, repeated from 1C) or ∆tea2 cells (bottom panels). Dashed yellow line indicates the cell end(E) Cells expressing fluorescently-tagged tubulin were imaged every 5 seconds and the dwell time of ~100 individual iMTs for each condition recorded within the final 1.1 µm of the cell. Red bars signify the mean


Figure 3 ( A ) Kymograph ( top left panels ) showing fluorescently - tagged Tea2 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the position of the cell end . Plots ( bottom left panels ) show both the relative fluorescence intensity and position for Tea2 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar 2µm . Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 ( green ) and Tip1 ( magenta ) over time ( top right ) and the distance of these pixels from the cell end ( bottom right )

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Figure 3(A) Kymograph (top left panels) showing fluorescently-tagged Tea2 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the position of the cell end. Plots (bottom left panels) show both the relative fluorescence intensity and position for Tea2 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar 2µm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 (green) and Tip1 (magenta) over time (top right) and the distance of these pixels from the cell end (bottom right)


Figure 4 ( A ) Kymograph ( top left panels ) showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the cell end . Plots ( bottom left panels ) show both the fluorescence intensity and position of Klp5 / Klp6 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar , 2µm . Intensity of the maximal value pixel at the plus end ( top right panel ) and its position relative to the cell end ( bottom right panel ) for both Klp5 / Klp6 ( green ) and Tip1 ( magenta ) plotted relative to MT shrinkage for this kymograph

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Figure 4 (A) Kymograph (top left panels) showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2µm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph


Figure 1 ( A ) Time - dependent killing of a panel of bacterial isolates in the presence of meropenem ( MEM10 , 10 µg / ml ) . Cells were grown overnight and sub - cultured for 30 minutes at 37 ° C before being exposed to meropenem at 10 µg / mL and moved to a 37 ° C stationary incubator . Samples were taken at each indicated time point and spot - titered onto LB medium to quantify colony forming units ( CFU / mL ) throughout meropenem treatment . ( B ) images of cells corresponding to the time points in ( A ) were taken after labeling with membrane stain FM4 - 64 . E . cloacae , Enterobacter cloacae ; K . aerogenes , Klebsiella aerogenes ; K . pneumoniae , Klebsiella pneumoniae ; E . coli , Escherichia coli

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Figure 1(A) Time-dependent killing of a panel of bacterial isolates in the presence of meropenem (MEM10, 10 µg/ml). Cells were grown overnight and sub-cultured for 30 minutes at 37°C before being exposed to meropenem at 10 µg/mL and moved to a 37°C stationary incubator. Samples were taken at each indicated time point and spot-titered onto LB medium to quantify colony forming units (CFU/mL) throughout meropenem treatment. (B) images of cells corresponding to the time points in (A) were taken after labeling with membrane stain FM4-64. E. cloacae, Enterobacter cloacae; K. aerogenes, Klebsiella aerogenes; K. pneumoniae, Klebsiella pneumoniae; E. coli, Escherichia coli


Figure 2 ( D ) Regulatory modes of representative VxrB - controlled genes in response to PenG . Upper plot shows VxrB binding patterns ( assessed via ChIP - Seq ) , lower panel shows transcriptional response upon overexpression of VxrB D78E ( assessed via RNA - Seq ) . RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG ( 100 µg / ml for 3 hours ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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Figure 2(D) Regulatory modes of representative VxrB-controlled genes in response to PenG. Upper plot shows VxrB binding patterns (assessed via ChIP-Seq), lower panel shows transcriptional response upon overexpression of VxrB D78E (assessed via RNA-Seq). RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG (100 µg/ml for 3 hours). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


Figure 3Reduction of cell wall synthesis gene expression causes a tolerance defect . ( C ) Survival of a ∆ pbp1b ∆ pbp1a Piptg : pbp1a strain after 6 hours of PenG exposure . Pup indicates conditions of high IPTG concentrations ( 100 µM ) , Pdown low IPTG concentration ( 2 µM ) . Survival fraction is cfu / ml of PenG - treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration . ( D ) Time - dependent killing experiment in the presence of PenG ( 100 µg / ml , 10 x MIC ) . All data are means ( + / - standard error ) of 3 independent biological replicates .

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Figure 3Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.(D) Time-dependent killing experiment in the presence of PenG (100 µg/ml, 10 x MIC). All data are means (+/- standard error) of 3 independent biological replicates.


Figure 4 ( C ) Time - course of iron accumulation in PenG - treated WT and ∆ vxrAB cells as measured by ICP - MS . Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ P = 0 . 001 ; ∗ ∗ ∗ P < 0 . 0003 ( paired t - test ) .

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Figure 4(C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test).


Figure 5 ( B ) . To test PenG - mediated induction of Fur - regulated genes , S1 nuclease mapping assays were performed on the small RNA ryhB , the RyhB target sodB , hutA / B , coding for heme transport systems and vc2212 , which encodes a component of a ferric citrate uptake system ) . Cells were grown to mid - exponential phase followed by exposure to H2O2 or PenG for the indicated duration . Numbers represent expression levels normalized to untreated control ( averages from three independent replicates ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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Figure 5(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


Figure 6 ( B ) and ( C ) Iron uptake mutants restore ∆ vxrAB growth on 10 % sucrose . The indicated strains were grown to exponential phase ( OD600 ~ 0 . 5 ) in LB medium and spot - plated on 10 % Sucrose plates with or without additional iron sulfate . An example is shown in ( B ) , quantification of cfu is shown in ( C ) . Data information : Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ ∗ P < 0 . 0003 ; ∗ ∗ ∗ ∗ P < 0 . 0001 ( paired t - test ) .

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Figure 6(B) and (C) Iron uptake mutants restore ∆vxrAB growth on 10% sucrose. The indicated strains were grown to exponential phase (OD600~0.5) in LB medium and spot-plated on 10% Sucrose plates with or without additional iron sulfate. An example is shown in (B), quantification of cfu is shown in (C). Data information: Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗∗ P<0.0003; ∗∗∗∗ P<0.0001 (paired t-test).


Figure 7 ( B ) Excretion of V . cholerae . Fecal samples were collected every two hours and assessed for their viable cell content ( Colony Forming Units , CFU ) to monitor the level of V . cholerae excretion . Each symbol represents fecal samples from an individual mouse ( n = 4 ) . The number of bacterial CFU was normalized per milligram of feces ( CFU / mg ) . Error bars represent standard deviation . ∗ ∗ P < 0 . 005 ; ∗ ∗ ∗ P < 0 . 001 ( two - way ANOVA ) .

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Figure 7(B) Excretion of V. cholerae. Fecal samples were collected every two hours and assessed for their viable cell content (Colony Forming Units, CFU) to monitor the level of V. cholerae excretion. Each symbol represents fecal samples from an individual mouse (n=4). The number of bacterial CFU was normalized per milligram of feces (CFU/mg). Error bars represent standard deviation. ∗∗ P<0.005; ∗∗∗ P<0.001 (two-way ANOVA).


Figure 8 ( E ) Time - dependent killing of respiratory chain mutants ( ∆ nqrA , encoding NADH dehydrogenase and ∆ ubiA , encoding an early step of ubiquinone synthesis ) in the presence of PenG ( 100 µg / ml , 10xMIC ) . All strains were grown in LB 0 . 2 % glucose . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation . ( F ) Growth - phase dependent killing assay of ROS detoxification systems ( ∆ fur , ∆ sodB , and ∆ oxyR1 ∆ katG ∆ katB ) . At designated time - points , a 5 ml aliquot was withdrawn and exposed to 100 µg / ml PenG for 3 h . CFU / mL for each time point was assessed before ( solid lines ) and after addition of PenG ( dotted lines ) . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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Figure 8(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


Figure 1Possible correlation between AR - and UPR - associated gene expression was assessed in the global gene expression data available in the TCGA Prostate Adenocarcinoma cohort ( n = 190 ) ( http : / / www . cbioportal . org / public - portal / index . do ) . Tumors were stratified according to AR status into three groups , that is ARlow ( n = 60 ) , ARmedium ( n = 70 ) , and ARhigh ( n = 60 ) . The levels of UPR gene expression in the three groups were compared using Pearson ' s correlation analysis by the R software and presented as a heatmap . There were significant differences between the three groups ( Supplementary Table S2 ) . The expression profiles of some prominent UPR genes from the data in ( A ) , including ERN1 ( IRE1 ) , EDEM1 , ATF6 , and DNAJC3 ( P58IPK ) , are presented . P - values of the different genes are given .

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Figure 1Possible correlation between AR- and UPR-associated gene expression was assessed in the global gene expression data available in the TCGA Prostate Adenocarcinoma cohort (n = 190) (http://www.cbioportal.org/public-portal/index.do). Tumors were stratified according to AR status into three groups, that is ARlow (n = 60), ARmedium (n = 70), and ARhigh (n = 60). The levels of UPR gene expression in the three groups were compared using Pearson's correlation analysis by the R software and presented as a heatmap. There were significant differences between the three groups (Supplementary Table S2).The expression profiles of some prominent UPR genes from the data in (A), including ERN1 (IRE1), EDEM1, ATF6, and DNAJC3 (P58IPK), are presented. P-values of the different genes are given.


Figure 2A , B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR ( qPCR ) . Controls were treated with vehicle for 84 h and set to 100 . Data represent the mean of three independent experiments in triplicate , and bars represent SE . P - values ranged between 1 . 66 × 10 − 5 and 0 . 025 , and IRE1α expression in R1881 48 h was * P = 0 . 013 with respect to vehicle - treated cells using unpaired Student ' s t - test . C - E IRE1α , XBP - 1S , and XBP - 1U mRNA in CWR22xenografts grown in nude mice and collected at the indicated times after castration . The value at t = 0 was set to 1 . Columns represent the mean of at least three independent tumors for each time point , and bars represent SE . P - values ranged between 5 . 7 × 10 − 10 and 0 . 0002 . IRE1α expression at 48 h post - castration was 3 . 17 × 10 − 6 with respect to t = 0 using unpaired Student ' s t - test . F , G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis . Data presented are representative of three independent experiments .

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Figure 2A, B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR (qPCR). Controls were treated with vehicle for 84 h and set to 100. Data represent the mean of three independent experiments in triplicate, and bars represent SE. P-values ranged between 1.66 × 10−5 and 0.025, and IRE1α expression in R1881 48 h was *P = 0.013 with respect to vehicle-treated cells using unpaired Student's t-test.C-E IRE1α, XBP-1S, and XBP-1U mRNA in CWR22xenografts grown in nude mice and collected at the indicated times after castration. The value at t = 0 was set to 1. Columns represent the mean of at least three independent tumors for each time point, and bars represent SE. P-values ranged between 5.7  ×  10−10 and 0.0002. IRE1α expression at 48 h post-castration was 3.17 × 10−6 with respect to t = 0 using unpaired Student's t-test.F, G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis. Data presented are representative of three independent experiments.


Figure 3After starvation , LNCaP cells were transfected with control ( CTRL ) siRNA or AR siRNA . Cells were then treated with R1881 for the indicated times . Controls were treated with vehicle for 48 h . A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP cells . Expression in cells transfected with CTRL siRNA was set to 1 . Bars represent SE with * P = 0 . 001 indicating significant difference between AR siRNA - and control siRNA - transfected cells using paired Student ' s t - test . After starvation , LNCaP cells were transfected with control ( CTRL ) siRNA or AR siRNA . Cells were then treated with R1881 for the indicated times . Controls were treated with vehicle for 48 h . B - E Same as in ( A ) , but mRNA expression levels of the indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation . Expression in cells transfected with CTRL siRNA was set to 100 . Bars represent SE . P - values are shown indicating significant difference between AR siRNA - and control siRNA - transfected cells using unpaired Student ' s t - test . After starvation , LNCaP cells were transfected with control ( CTRL ) siRNA or AR siRNA . Cells were then treated with R1881 for the indicated times . Controls were treated with vehicle for 48 h . F Expression of the indicated proteins under conditions indicated on the top label was determined by Western blot analysis . Representative blots for three independent experiments are shown .

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Figure 3After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP cells. Expression in cells transfected with CTRL siRNA was set to 1. Bars represent SE with *P = 0.001 indicating significant difference between AR siRNA- and control siRNA-transfected cells using paired Student's t-test.After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.B-E Same as in (A), but mRNA expression levels of the indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation. Expression in cells transfected with CTRL siRNA was set to 100. Bars represent SE. P-values are shown indicating significant difference between AR siRNA- and control siRNA-transfected cells using unpaired Student's t-test.After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.F Expression of the indicated proteins under conditions indicated on the top label was determined by Western blot analysis. Representative blots for three independent experiments are shown.


Figure 4A , B LNCaP ( A ) or VCaP cells ( B ) were cultured and treated with vehicle ( C ) for 48 h or R1881 for 24 and 48 h . The cells were then fixed , and ChIP assay was performed as described in Materials and Methods using AR antibody . The data shown are representative of one experiment in duplicate . Error bars represent SE . * P < 0 . 01 for LNCaP and * P < 0 . 04 for VCaP indicate significant difference between C ( control ) and R1881 using unpaired Student ' s t - test .

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Figure 4A, B LNCaP (A) or VCaP cells (B) were cultured and treated with vehicle (C) for 48 h or R1881 for 24 and 48 h. The cells were then fixed, and ChIP assay was performed as described in Materials and Methods using AR antibody. The data shown are representative of one experiment in duplicate. Error bars represent SE. *P < 0.01 for LNCaP and *P < 0.04 for VCaP indicate significant difference between C (control) and R1881 using unpaired Student's t-test.