Expression of PID - 5 : : mTagRFP - T ( C ) in perinuclear granules in the germline . DEPS - 1 : : GFP mark P granules . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm .

Expression of PID-5::mTagRFP-T (C) in perinuclear granules in the germline. DEPS-1::GFP mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 4 : : mTagRFP - T ( D ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm .

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

Expression of PID-4::mTagRFP-T (D) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 5 : : mTagRFP - T ( E ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm .

Expression of PID-5::mTagRFP-T (E) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


F ) Box - plots representing the distance ( µm ) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition.


Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 4 : : mTagRFP - T ( G ) The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar = 25

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

Expression of PID-2::eGFP (xfSi145) together with PID-4::mTagRFP-T (G) The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µ


Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 5 : : mTagRFP - T ( H ) . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar = 25 µm .

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

Expression of PID-2::eGFP (xfSi145) together with PID-5::mTagRFP-T (H). The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µm.


A - E ) Expression of 3xFLAG : : GFP : : ZNFX - 1 and PGL - 1 : : mTagRFP - T in a wild type ( A ) , pid - 2 ( B ) , pid - 4 ; pid - 5 ( C ) , pid - 4 ( D ) , and pid - 5 ( E ) mutant backgrounds . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown . Arrow heads indicate individual condensates . Scale bar : 25 µm . F ) Quantification of the ratio Z / P granules in wild type , pid - 2 , pid - 4 / - 5 , pid - 4 and pid - 5 mutant backgrounds . The number of granules is indicated in brackets , next to the genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Expression of 3xFLAG::GFP::ZNFX-1 and PGL-1::mTagRFP-T in a wild type (A), pid-2 (B), pid-4;pid-5 (C), pid-4 (D), and pid-5 (E) mutant backgrounds. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown. Arrow heads indicate individual condensates. Scale bar: 25 µm. F) Quantification of the ratio Z / P granules in wild type, pid-2, pid-4/-5, pid-4 and pid-5 mutant backgrounds. The number of granules is indicated in brackets, next to the genotype.


Expression of PID - 4 : : mTagRFP - T ( G ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Scale bar : 25 µm .

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

Expression of PID-4::mTagRFP-T (G) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.


Expression of PID - 4 : : mTagRFP - T H ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Scale bar : 25 µm .

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

Expression of PID-4::mTagRFP-T H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.


I ) Box - plots representing the distance ( µm ) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition . The median is represented by a line . The interquartile range ( IQR ) , 25th to 75th percentile , is represented by the upper and lower lines , respectively , and whiskers represent the first quartile ( down to - 1 . 5 * IQR ) or the third quartile ( up to + 1 . 5 * IQR ) . P - values were calculated using a t - test ( two - tailed ) .

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

I) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using a t-test (two-tailed).


( A ) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA : active UC ( n = 26 ) , active CD ( n = 34 ) , control ( Con ) ( n = 26 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA: active UC (n=26), active CD (n=34), control (Con) (n=26).


( B ) Age - and sex - matched Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - mice ( n = 9 ) and Atx + / + ; Il10 - / - littermates ( n = 9 ) were examined for the development of spontaneous colitis . Body weight changes were monitored every other day , starting at the age of 37 days and ending at the age of 6 months . Results are means ± SD . Data were compared by Two - way ANOVA ( with treatment and times ) , followed by the multiple - comparison Bonferroni t test to assess differences between groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Age- and sex-matched Atx∆ΜΕ/∆ΜΕ;Il10-/- mice (n=9) and Atx+/+;Il10-/- littermates (n=9) were examined for the development of spontaneous colitis. Body weight changes were monitored every other day, starting at the age of 37 days and ending at the age of 6 months. Results are means ± SD. Data were compared by Two-way ANOVA (with treatment and times), followed by the multiple-comparison Bonferroni t test to assess differences between groups.


( C ) Gross appearance of the full - length mouse colon . Each line on a ruler represents a millimeter ( mm ) .

O 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) Gross appearance of the full-length mouse colon. Each line on a ruler represents a millimeter (mm).


( D and E ) H & E stained section of the mouse colon . Insets are enlarged in the lower panel . Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope ( n = 14 / group ) ( E ) .

O O O O O O O O O O O O O O O O O O O O O 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 and E) H&E stained section of the mouse colon. Insets are enlarged in the lower panel. Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope (n=14/group) (E).


( F ) Histological parameters were quantified with H & E - colon sections ( n = 10 / group ) ( D ) and shown as mean ± SEM .

O O O O O O O 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) Histological parameters were quantified with H&E-colon sections (n=10/group) (D) and shown as mean ± SEM.


( G ) Survival of Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - ( n = 17 ) and Atx + / + ; Il10 - / - littermates ( n = 18 ) was analyzed by the Kaplan - Meier method ( Log - rank P = 0 . 0046 ) .

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

(G) Survival of Atx∆ΜΕ/∆ΜΕ;Il10-/- (n=17) and Atx+/+;Il10-/- littermates (n=18) was analyzed by the Kaplan-Meier method (Log-rank P=0.0046).


( H ) Presented are H & E - stained sections of the small intestine in a ' Swiss - Roll ' form . Inset areas are enlarged in the right panel .

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

(H) Presented are H&E-stained sections of the small intestine in a 'Swiss-Roll' form. Inset areas are enlarged in the right panel.


( A ) Total cell lysates of peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were subjected to immunoblotting analysis to confirm Atx deletion .

(A) Total cell lysates of peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were subjected to immunoblotting analysis to confirm Atx deletion.


( B ) Peritoneal macrophages from the mice were subjected to the ESI - MS / MS method to quantify endogenous LPA . Individual molecular species of LPA and total LPA ( inset graph ) were compared . The data are shown as mean ± SEM ( n = 3 / group ) * P < 0 . 05 ( one - tailed unpaired t - test ) .

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

(B) Peritoneal macrophages from the mice were subjected to the ESI-MS/MS method to quantify endogenous LPA. Individual molecular species of LPA and total LPA (inset graph) were compared. The data are shown as mean ± SEM (n=3/group) *P < 0.05 (one-tailed unpaired t-test).


( C and D ) Thioglycolate - elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages ( C ) and DCs ( D ) . Scale bar indicates 1 μm .

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

(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.


Lipid rafts from peritoneal macrophages ( E , F ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . Protein was purified and concentrated with the 3K cut - off filter , followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin - 1 .

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

Lipid rafts from peritoneal macrophages (E, F) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin-1.


Lipid rafts from mouse macrophage Raw264 . 7 cells treated with Atx inhibitor ( Atx - i ) PF8380 ( 50 μM , 30 min ) or vehicle ( Veh . , DMSO 0 . 1 % ) ( G ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . Protein was purified and concentrated with the 3K cut - off filter , followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin - 1 .

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

Lipid rafts from mouse macrophage Raw264.7 cells treated with Atx inhibitor (Atx-i) PF8380 (50 μM, 30 min) or vehicle (Veh., DMSO 0.1%) (G) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin-1.


( H ) Peritoneal macrophages were stained with Alexa Fluor 594 - cholera toxin subunit B ( Red ) . The plasma membrane was examined with FV10i confocal scanning microscopy . Inset areas were magnified at the right panel . Each scale bar indicates 10 μm .

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

(H) Peritoneal macrophages were stained with Alexa Fluor 594-cholera toxin subunit B (Red). The plasma membrane was examined with FV10i confocal scanning microscopy. Inset areas were magnified at the right panel. Each scale bar indicates 10 μm.


Peritoneal macrophages ( A ) were stimulated with LPS ( 20 ng / mL , 20 min ) Cell lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody .

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

Peritoneal macrophages (A) were stimulated with LPS (20 ng/mL, 20 min) Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.


Raw264 . 7 cells ( B ) were stimulated with LPS ( 20 ng / mL , 20 min ) in the presence / absence of ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) . Cell lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody .

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

Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.


The physical interaction of TLR4 - CD14 was examined by FRET analysis . HEK293 cells were transfected with TLR4 - EYFP ( yellow ) and CD14 - ECFP ( blue ) ( C ) followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . Cells were washed with PBS and fixed , then visualized with filter sets for CFP and YFP . FRET images were visualized with the FRET filter set , expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins ( arbitrary linear units of fluorescence intensity ) . Presented are the representative images from at least three independent experiments . Scale bar indicates 5 μm .

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

The physical interaction of TLR4-CD14 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


The physical interaction of TLR4 - TLR4 was examined by FRET analysis . HEK293 cells were transfected with TLR4 - EYFP and TLR4 - ECFP ( D ) , followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . Cells were washed with PBS and fixed , then visualized with filter sets for CFP and YFP . FRET images were visualized with the FRET filter set , expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins ( arbitrary linear units of fluorescence intensity ) . Presented are the representative images from at least three independent experiments . Scale bar indicates 5 μm .

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

The physical interaction of TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


( A and B ) Peritoneal macrophages from Atx + / + ( A ) and Atx ∆ ΜΕ / ∆ ΜΕ ( B ) mice were stimulated with LPS ( 20 ng / mL , 20 min ) , followed by fixation in 4 % paraformaldehyde for 15 min at room temperature and staining with TLR4 - anti rabbit FITC ( Green ) , early endosomal marker EEA1 - anti mouse Dylight405 ( Blue ) , and Alexa Fluor 594 - CTXB ( Red ) . TLR4 internalization was examined with confocal microscopy . Presented are the representative from 3 independent experiments in which more than 95 % of the cells exhibited similar results . Scale bar , 10 μm .

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

(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated with LPS ( 20 ng / mL , 20 min ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments .

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

Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 , were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2, were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . regular Akt were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. regular Akt were used for a loading control. Presented are the representative image from at least three independent experiments.


Atx - heterozygous ( Atx - Het ) mice and wild type littermates ( C ) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . RasGAP levels were used for a loading control . Presented are the representative image from at least three independent experiments .

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

Atx-heterozygous (Atx-Het) mice and wild type littermates (C) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. RasGAP levels were used for a loading control. Presented are the representative image from at least three independent experiments.


( D ) Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . , β - Actin were used for a loading control . Presented are the representative image from at least three independent experiments .

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

(D) Raw264.7 cells were stimulated by LPS with the ATX inhibitor. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. , β-Actin were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with TLR2 ligand Peptidoglycan ( PGN , 30 ng / mL ) ( E ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TLR2 ligand Peptidoglycan (PGN, 30 ng/mL) (E) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with Pam3CSK4 ( 1 μg / mL ) ( F ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with Pam3CSK4 (1 μg/mL) (F) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with IL - 1β ( 100 ng / mL ) for the indicated time points . : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with IL-1β (100 ng/mL) for the indicated time points. : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with TNFα ( 25 ng / mL ) ( H ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TNFα (25 ng/mL) (H) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


Peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates ( A ) were stimulated with LPS ( 50 ng / mL , 8 h ) The culture medium was collected for ELISA to measure the level of secreted cytokines . All assays were performed in triplicate , and data are shown as mean ± SEM .

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

Peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates (A) were stimulated with LPS (50 ng/mL, 8 h) The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.


Raw264 . 7 cells ( B ) were stimulated with LPS ( 50 ng / mL , 8 h ) in the presence / absence of the ATX inhibitor ( 50 μM , 30 min ) . The culture medium was collected for ELISA to measure the level of secreted cytokines . All assays were performed in triplicate , and data are shown as mean ± SEM .

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

Raw264.7 cells (B) were stimulated with LPS (50 ng/mL, 8 h) in the presence/absence of the ATX inhibitor (50 μM, 30 min). The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.


Peritoneal macrophages were stimulated with LPS or vehicle ( endotoxin free water ) , after which intracellular cytokine production was measured through FACS analysis . With the scatter dot plot , the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry . The overlay plot represents the cytometry data .

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

Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.


( F ) To induce sepsis via LPS injection ( Shirey et al . , 2013 ; Voss et al . , 2016 ) , age ( 9 - 10 weeks ) - and sex - matched Atx ∆ ΜΕ / ∆ ΜΕ mice ( n = 9 ) and Atx + / + littermates ( n = 21 ) were injected with LPS ( i . p . 25 mg / kg ) . Survival was monitored for up to 16 h , and analyzed by the Kaplan - Meier method ( Log - rank P = 0 . 0004 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) To induce sepsis via LPS injection (Shirey et al., 2013; Voss et al., 2016), age (9-10 weeks)- and sex-matched Atx∆ΜΕ/∆ΜΕ mice (n = 9) and Atx+/+ littermates (n = 21) were injected with LPS (i.p. 25 mg/kg). Survival was monitored for up to 16 h, and analyzed by the Kaplan-Meier method (Log-rank P=0.0004).


( G ) Blood samples were collected from Atx ∆ ΜΕ / ∆ ΜΕ ( n = 7 ) and Atx + / + ( n = 6 ) mice 10 h after LPS injection , after which the serum TNFα protein level was measured . The data are shown as mean ± SEM .

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

(G) Blood samples were collected from Atx∆ΜΕ/∆ΜΕ (n = 7) and Atx+/+ (n = 6) mice 10 h after LPS injection, after which the serum TNFα protein level was measured. The data are shown as mean ± SEM.


Confocal laser scanning micrographs of the macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates . Cells were incubated with IgG - opsonized latex beads in the absence / presence of LPS ( 20 ng / mL , 2 h ) and stained with Phalloidin ( F - actin ) and DAPI . Internalized beads were examined under a higher ( A )

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

Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A)


Confocal laser scanning micrographs of the macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates . Cells were incubated with IgG - opsonized latex beads in the absence / presence of LPS ( 20 ng / mL , 2 h ) and stained with Phalloidin ( F - actin ) and DAPI . Internalized beads were examined under a lower magnification ( B ) to quantify % phagocytosis by dividing latex bead - positive cell numbers by the total number of DAPI - positive cells ( n = 11 - 16 per group ) . The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM ( B ) . * * P < 0 . 01 ( Mann - Whitney U test ) .

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

Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test).


( C ) LPS - stimulated macrophages were co - incubated with IgG - opsonized latex beads for 40 min . Phagocytic cups were visualized by F - actin staining with Phalloidin - iFluor 647 . The arrow indicates the phagocytic cup formed in the plasma membrane . Scale bar is 10 μm .

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

(C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm.


After LPS ( 20 ng / mL , 4 h ) treatment , LPS - induced iNos , Arginase - 1 ( Arg - 1 ) , and Ym - 1 mRNA expression were evaluated by semi - quantitative PCR ( D ) .

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

After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D).


With LPS stimulation ( 20 ng / mL , 8 h ) , iNOS protein production was examined by immunoblotting analysis ( E ) .

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

With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).


( A ) in vitro kinase assays were performed using purified recombinant kinases ( ULK1 and ULK2 ) and substrate ( ATG16L1 ) in the presence of radiolabelled ATP . ULK and ATG16L1 inputs were examined by western blot ( WB ) and substrate phosphorylation was analyzed by autoradiography ( AR ) . Data information : Unless otherwise indicated experiments were performed three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times.


( B ) Full - length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay . ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography . Data information : Unless otherwise indicated experiments were performed three times .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. Data information: Unless otherwise indicated experiments were performed three times.


( D ) Full - length or mutated HA - ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay . Inputs were analysed by WB and target phosphorylation by AR . Data information : Unless otherwise indicated experiments were performed three times .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. Data information: Unless otherwise indicated experiments were performed three times.


( E ) HEK293A cells were transfected with wild - type or phospho - dead ATG16L1 in the presence of wild - type or kinase - dead ULK1 . Phosphorylation of ATG16L1 ( S278 or S287 ) and inputs were examined by WB . Data information : Unless otherwise indicated experiments were performed three times .

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

(E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated experiments were performed three times.


( A ) Wild - type , ULK1 / 2 double knockout ( dKO ) , or IKKα KO mouse embryonic fibroblasts ( MEFs ) were incubated with either complete medium , amino acid - deficient DMEM , or HBSS for 1 hour . Samples were immunoblotted using the indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( B ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs were infected with log phase Salmonella for 2 hours ; bacteria - containing media was then removed and cells were incubated with gentamycin ( 50 µg / mL ) - containing DMEM for 2 hours . Samples were immunoblotted using the indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( C ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs cells were infected with Salmonella for 1 hours . Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B . Representative images are shown ( scale bars , 10 μm and 3 μm ) . Quantification was generated from 8 fields of view from a representative experiment . The experiments were repeated twice . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( D ) Wild - type , ULK1 / 2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour . Xenophagy rates were examined through Colony Forming Unit ( CFU ) assays . Quantification of infection rates by immunofluorescence is demonstrated in the right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( A ) HEK293A cells were transfected with either flag - tagged WT ATG16L1 or T300A ATG16L1 . ULK1 was co - transfected in increasing amounts where indicated . Cleavage of ATG16L1 was analyzed by WB of whole cell lysates . Levels of ATG16L1 cleavage were quantified from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( B ) HEK293A cells were transfected with either tagged wild - type , T300A , or S278 / T300A ATG16L1 in the presence or absence of ULK1 . Cleavage of ATG16L1 was analyzed by WB . Levels of ATG16L1 cleavage were measured from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with either tagged wild-type, T300A, or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analyzed by WB. Levels of ATG16L1 cleavage were measured from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( C ) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan - caspase inhibitor Z - VAD - FMK ( 15 µM ) for 4 hours . Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( D ) Wild - type or T300A - expressing HEK293A were treated with Salmonella in the presence or absence of ULK1 / 2 inhibitor ( 16 μM ) for the indicated time points . Expression of ATG16L1 was analysed by WB . The experiments were performed twice .

(D) Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice.


( E ) ATG16L1 knock - out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour . Xenophagy rates were examined through CFU assays . Quantification of infection rates by immunofluorescence is demonstrated in the right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(E) ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( A ) Wild - type MEF cells were infected with Salmonella for 25 minutes . Phospho - ATG16L1 , total ATG16L1 , and LPS were stained and analysed by immunofluorescence . Representative immunofluorescent images are shown ( scale bars , 10 μm and 1 μm ) . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm). Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( B ) Wild - type and ULK1 / 2 dKO were infected with Salmonella for 25 minutes . Immunofluorescence was performed using antibodies against LPS and ATG16L1 . Representative immunofluorescent images are shown on the left panel ( scale bars , 10 μm and 2 μm ) . Quantification of ATG16L1 - positive bacteria from 7 fields of view from a representative experiment is shown in the right panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( C ) ATG16L1 knock - out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . Bacteria were stained using anti - LPS antibodies to analyze localization in addition to ATG16L1 . Representative immunofluorescent images of ATG16L1 and LPS are shown ( scale bars , 5μm and 1 μm ) . Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( D ) ATG16L1 knock - out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . Bacteria were stained using anti - LPS antibodies to analyze localization in addition to the autophagy marker LC3B . Representative immunofluorescent images of LC3B and LPS are shown ( scale bars , 5μm and 1 μm ) . Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( D ) Number of putative enhancer regions per cell line . Bar depicts the mean . Diamonds represent individual numbers in each cell line and are color - coded according to cancer

O O O O O O O O O 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) Number of putative enhancer regions per cell line. Bar depicts the mean. Diamonds represent individual numbers in each cell line and are color-coded according to cancer type


( E ) Heat maps of DNase I signal for enhancer regions ( rows ) . For intergenic enhancers ( left ) the signal is aligned at the upstream ( relative to + strand ) enhancer edge , shown from 4 kbp upstream to 5 kbp downstream , and ordered by increasing length of enhancer regions . For intragenic enhancers ( right ) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp . Exemplary data from LoVo colorectal cancer cells . ( F ) Heat maps as in ( E ) showing composite ChIP - seq peak coverage from 326 TFs

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

(E) Heat maps of DNase I signal for enhancer regions (rows). For intergenic enhancers (left) the signal is aligned at the upstream (relative to + strand) enhancer edge, shown from 4 kbp upstream to 5 kbp downstream, and ordered by increasing length of enhancer regions. For intragenic enhancers (right) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp. Exemplary data from LoVo colorectal cancer cells. (F) Heat maps as in (E) showing composite ChIP-seq peak coverage from 326 TFs


( B ) Bidirectional promoters show higher mRNA synthesis . Boxplots showing TT - seq signal for mRNAs from unidirectional ( n = 39 , 376 ) and bidirectional ( n = 83 , 363 ) promoters . Bidirectional and unidirectional promoters were defined for each cell line separately and then plotted together . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown . P - value by two - sided Mann - Whitney U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bidirectional promoters show higher mRNA synthesis. Boxplots showing TT-seq signal for mRNAs from unidirectional (n=39,376) and bidirectional (n=83,363) promoters. Bidirectional and unidirectional promoters were defined for each cell line separately and then plotted together. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( A ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the TAL1 locus ( hg38 ; chr1 : 47 , 194 , 268 - 47 , 242 , 880 ; ( Kent et al . , 2002 ) ) in the leukemia cell lines Jurkat and K562 . For better visualization , TT - seq coverage is cut at 200 ( purple lines ) . eRNAs are shown between the plus and minus strand . Red line indicates the short 12 bp insertion in Jurkat cells , introducing novel binding sites for the transcription factor

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the TAL1 locus (hg38; chr1:47,194,268-47,242,880; (Kent et al., 2002)) in the leukemia cell lines Jurkat and K562. For better visualization, TT-seq coverage is cut at 200 (purple lines). eRNAs are shown between the plus and minus strand. Red line indicates the short 12 bp insertion in Jurkat cells, introducing novel binding sites for the transcription factor MYB


( B ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the 2 . 8 Mbp gene desert surrounding MYC ( hg38 ; chr8 : 127 , 042 , 406 - 129 , 779 , 109 ; ( Kent et al . , 2002 ) ) in fourteen cancer cell lines . eRNAs represent the union from all cell lines . Genome - wide association studies ( GWAS ) risk loci contain GWAS single nucleotide polymorphisms ( SNPs ) for prostate cancer ( PrCa ) , acute lymphocytic leukemia ( ALL ) , breast cancer ( BrCa ) , colorectal cancer ( CRC ) , and glioblastoma ( GBM ) including SNPs in linkage disequilibrium ( r2 ≥ 0 . 8 ) . TT - seq coverage is displayed between 8 - 250 to allow for better visualization of the eRNA signal surrounding MYC . Dashed rectangle is shown at full scale on the right . Note : TT - seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements ( e . g . translocations ) affecting the shown region in the used cancer cell line clones .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.


( A ) Med1 - defined enhancers ranked by Med1 ChIP - seq signal ( Yan et al . , 2013 ) in LoVo colorectal cancer cells . Super - enhancers ( black rectangles ) were determined as enhancer regions lying above the inflection point of the

O O O O O O O O O O O O O O O O O O O O O O O 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) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve


( B ) Increased eRNA synthesis from super - enhancers . Boxplots comparing eRNA synthesis ( TT - seq signal ) at Med1 - defined typical enhancers ( n = 2 , 111 ) and super - enhancers ( n = 130 ) in LoVo cells ( analysis was restricted to intergenic enhancers Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown . P - value by two - sided Mann - Whitney U - test .

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

(B) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( C ) Super - enhancers are associated with the production of long eRNAs . Boxplots showing length of TT - seq - defined eRNAs originating from Med1 - defined super - enhancers ( n = 552 ) or other enhancer regions ( n = 6 , 934 ) . Median , hinges , whiskers and notches are shown as in ( B ) . P - value by two - sided Mann - Whitney U - test .

(C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test.


( D ) Barplot showing the number of TT - seq enhancer regions per Med1 - defined super - enhancer region in LoVo cells . Super - enhancers spanning a protein - coding TSS ( n = 32 ) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT - seq enhancer calling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling.


( E ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the PHLDA1 locus ( hg38 ; chr12 : 75 , 612 , 350 - 76 , 042 , 552 ; in the colorectal cancer cell line HCT 116 . TT - seq coverage is cut at 250 ( purple lines ) to allow for better visualization . eRNAs are shown between the plus and minus strand . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) comprise ChIP - seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region . Binding regions for selected TFs ( TCF7l2 , YY1 , SP1 ) are shown below together with p300 and RNA polymerase II ( Pol II ) . TT - seq enhancer regions are shown at the bottom together with a H3K27ac - defined super - enhancer annotation , which was downloaded from SEdb 2019 ) . At date of publication no Med1 ChIP - seq data was available for HCT 116 .

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

(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.


( A ) Unsupervised clustering of all pairwise Spearman correlations of normalized TT - seq signal for mRNAs ( left ) and eRNAs ( right ) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Unsupervised clustering of all pairwise Spearman correlations of normalized TT-seq signal for mRNAs (left) and eRNAs (right) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type, respectively.


( B ) Heatmaps showing mRNAs ( left ) and eRNAs ( right ) as being synthesized ( red , normalized TT - seq signal FPK ≥ 15 ) or not ( blue , normalized TT - seq signal FPK < 15 ) for all fourteen cancer cell lines ( columns ) . Heatmaps show only transcripts ( rows ) with normalized FPK ≥ 30 in at least one of the cell lines ( mRNAs , n = 14 , 040 ; eRNAs n = 48 , 105 ) . Heatmaps are ordered by increasing cell type specificity , as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmaps showing mRNAs (left) and eRNAs (right) as being synthesized (red, normalized TT-seq signal FPK ≥ 15) or not (blue, normalized TT-seq signal FPK < 15) for all fourteen cancer cell lines (columns). Heatmaps show only transcripts (rows) with normalized FPK ≥ 30 in at least one of the cell lines (mRNAs, n=14,040; eRNAs n=48,105). Heatmaps are ordered by increasing cell type specificity, as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in.


( D ) Boxplots showing normalized TT - seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed ( from left to right : n = 890 ; 500 ; 494 ; 214 ; 238 ; 239 ; 378 ; 62 ; 153 ; 879 ; 586 ; 173 ; 601 ; 232 ) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed . All pairwise comparisons are significantly different ( P - values < 1e - 9 , two - sided Mann - Whitney U - test ) . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots showing normalized TT-seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed (from left to right: n=890; 500; 494; 214; 238; 239; 378; 62; 153; 879; 586; 173; 601; 232) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed. All pairwise comparisons are significantly different (P-values < 1e-9, two-sided Mann-Whitney U-test). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown.


UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the BMP4 locus ( hg38 ; chr14 : 53 , 935 , 697 - 54 , 264 , 430 ; in LoVo colorectal cancer cells . TT - seq coverage is cut at 500 ( purple lines ) to allow for better visualization . eRNAs and enhancers are shown above and below the minus strand , respectively . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) include ChIP - seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region . Significant promoter capture Hi - C ( CHi - C ) interactions are shown in

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

UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; in LoVo colorectal cancer cells. TT-seq coverage is cut at 500 (purple lines) to allow for better visualization. eRNAs and enhancers are shown above and below the minus strand, respectively. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region. Significant promoter capture Hi-C (CHi-C) interactions are shown in purple


Representative microscopic views of Ndrg4 + / + and Ndrg4 - / - murine intestinal sections ( n = 4 , 12 months of age ) reveal an even distribution of alkaline phosphatase along the enterocyte brush border , and a similar number and distribution of Paneth cells ( Lysozyme ) in the small intestine ; and neuroendocrine ( Chromogranin A ) , Goblet ( PAS + ) , and proliferating ( Ki67 ) cells in the colon . Scale bars , 50 µm .

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

Representative microscopic views of Ndrg4+/+ and Ndrg4-/- murine intestinal sections (n=4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (Chromogranin A), Goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm.


Representative microscopic views of the myenteric plexus of Ndrg4 + / + and Ndrg4 - / - murine colonic sections labeled with either TuJ1 and S100 ( B , n = 3 ) or HuC / D ( C , n = 6 ) did not reveal structural or organizational differences in the ganglionic network of Ndrg4 + / + and Ndrg4 - / - mice . Scale bars , 100 µm . Quantification of the enteric neuronal cell number ( HuC / D ) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4 + / + and Ndrg4 - / - mice ( n = 6 ) .

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

Representative microscopic views of the myenteric plexus of Ndrg4+/+ and Ndrg4-/- murine colonic sections labeled with either TuJ1 and S100 (B, n=3) or HuC/D (C, n=6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4+/+ and Ndrg4-/- mice. Scale bars, 100 µm. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4+/+ and Ndrg4-/- mice (n=6).


In contrast , Ndrg4fl / fl and Ndrg4fl / fl - VillinCre mice have a similar bodyweight ( I ) and develop an equal number of adenomas ( J ) with an equivalent diameter ( K , L ) . N = 13 vs . 12 in I , n = 13 vs . 11 in J and n = 10 vs . 8 in K .

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

In contrast, Ndrg4fl/fl and Ndrg4fl/fl-VillinCre mice have a similar bodyweight (I) and develop an equal number of adenomas (J) with an equivalent diameter (K,L). N=13 vs. 12 in I, n=13 vs. 11 in J and n=10 vs. 8 in K.


Compared to Ndrg4 + / + ENS - derived medium , addition of medium derived from Ndrg4 - / - ENS cultures enhances the relative growth of intestinal epithelial organoids ( E , IEOs ) and accelerates the formation of new crypt buds per IEO in the course of five days ( F ) .

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

Compared to Ndrg4+/+ ENS-derived medium, addition of medium derived from Ndrg4-/- ENS cultures enhances the relative growth of intestinal epithelial organoids (E, IEOs) and accelerates the formation of new crypt buds per IEO in the course of five days (F).


Box plot showing the quantitative protein expression results of nanoLC - MS / MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4 - / - compared to the Ndrg4 + / + ENS cell secretome ( n = 4 ; NSAF , normalized spectral abundance factor ) . Data are analyzed with R version 3 . 5 . 2 and the ibb R package . Each dot within the box plot represents the NSAF of an individual sample ; the inside band reflects the median , and the bottom and top of the box the first and third quartile , respectively . The whiskers reflect the minimum and maximal values within 1 . 5 × the interquartile range . NSAF values were compared using the Mann - Whitney U test .

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

Box plot showing the quantitative protein expression results of nanoLC-MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4-/- compared to the Ndrg4+/+ ENS cell secretome (n=4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann-Whitney U test.


Representative immunohistochemistry ( C ) and more detailed immunofluorescence ( D , E ) labeling indicate that Nid1 ( C , D ) and Fbln2 ( C , E ) are expressed within the myenteric plexus and by primary ENS - cells ( ( C ) , black arrowheads , scale bars , 50 µm ; ( D , E ) , n = 3 , scale bars , 10 µm ) .

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

Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS-cells ((C), black arrowheads, scale bars, 50 µm; (D, E), n=3, scale bars, 10 µm).


Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia ( Black arrowheads ) and interconnecting nerve fibers , and in a limited level throughout the epithelium ( n = 3 ) . Scale bar , 50 µm .

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

Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia (Black arrowheads) and interconnecting nerve fibers, and in a limited level throughout the epithelium (n=3). Scale bar, 50 µm.


Addition of NID1 & FBLN2 to the Matrigel ® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition .

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

Addition of NID1&FBLN2 to the Matrigel® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition.


Box plot reflecting the quantitative nanoLC - MS / MS analysis results shows the up - regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome ( n = 17 ) compared to the normal colon tissue secretome ( n = 17 ) . Data are analyzed with R version 3 . 5 . 2 and the ibb R package . Within the box plots , each dot represents the log10 transformed raw count of an individual sample ; the inside band is the median value ; the bottom and top of the box the first and third quartile , respectively ; and the whiskers reflect the minimum and maximal values within 1 . 5 × the interquartile range .

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

Box plot reflecting the quantitative nanoLC-MS/MS analysis results shows the up-regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome (n=17) compared to the normal colon tissue secretome (n=17). Data are analyzed with R version 3.5.2 and the ibb R package. Within the box plots, each dot represents the log10 transformed raw count of an individual sample; the inside band is the median value; the bottom and top of the box the first and third quartile, respectively; and the whiskers reflect the minimum and maximal values within 1.5× the interquartile range.


Representative images of human colonic cancer epithelium ( n = 3 ) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium . Scale bar , 50 μm .

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

Representative images of human colonic cancer epithelium (n=3) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium. Scale bar, 50 μm.


( A , B ) t - SNE visualization of 20 , 009 human retinal cells colored by ( A ) annotation of 18 transcriptionally distinct clusters ( C0 - C17 ) and ( B ) their distribution in 3 donor retina samples .

O O O O O O O O O O O O O O O O O O O O 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) t-SNE visualization of 20,009 human retinal cells colored by (A) annotation of 18 transcriptionally distinct clusters (C0-C17) and (B) their distribution in 3 donor retina samples.


C ) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters . The size of each circle depicts the percentage of cells expressing the marker within the cluster . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥10 nTrans (number of transcripts).


D ) t - SNE plots showing expression of a set of selected marker genes for major retinal classes .

D) t-SNE plots showing expression of a set of selected marker genes for major retinal classes.


E ) Correlation matrix for the identified 18 clusters . The upper triangle depicts the z - value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters .

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

E) Correlation matrix for the identified 18 clusters. The upper triangle depicts the z-value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters.


F ) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters . The rows correspond to top 10 genes most selectively upregulated in individual clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) and the columns show individual cells ordered by cluster ( C0 - C17 ) .

F) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered by cluster (C0-C17).


A ) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters . Brown colour indicates ≥ 100 nTrans ( number of transcripts ) .

O O O O O O O O O O O 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) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters. Brown colour indicates ≥100 nTrans (number of transcripts).


B ) Representation of the three donor retina samples in the six rod photoreceptor clusters .

B) Representation of the three donor retina samples in the six rod photoreceptor clusters.


C ) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters .

C) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters.


D ) Distribution of rod photoreceptor populations with high MALAT1 expression ( MALAT1 - hi ) or low MALAT1 expression ( MALAT1 - lo ) in three donor retina samples .

D) Distribution of rod photoreceptor populations with high MALAT1 expression (MALAT1-hi) or low MALAT1 expression (MALAT1-lo) in three donor retina samples.


E ) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer ( ONL ) . INL : inner nuclear layer ; OPL : outer plexiform layer . Scale bar = 20µm .

E) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer (ONL). INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm.


A , B ) ( A ) t - SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters ( CCA0 - CCA12 ) and ( B ) their distribution in 3 donor retina samples .

O O O O O O O O O O O O O O O O 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) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples.


C ) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters . The size of each circle depicts the percentage of cells expressing the marker within the cluster . Brown colour indicates ≥ 50 nTrans ( number of transcripts ) .

C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts).


D ) t - SNE plots showing expression of MALAT1 .

D) t-SNE plots showing expression of MALAT1.


E ) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1 - hi ( CCA1 , CCA10 ) and MALAT1 - lo ( CCA0 ) subpopulations . x - axis depicts normalized transcript levels .

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

E) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels.


F ) t - SNE plots showing expression of major ribosomal genes .

F) t-SNE plots showing expression of major ribosomal genes.


G ) Heatmap of differentially expressed genes between the two MALAT1 - hi clusters CCA1 and CCA10 . The rows correspond to top 10 genes most selectively upregulated in individual clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) and the columns show individual cells ordered in CCA1 and CCA10 .

G) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.


Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post - mortem ( 7 and 13 hours , Retina 7 ) , showing decreases in MALAT1 - hi rod subpopulations in the outer nuclear layer ( ONL ) at later time point . INL : inner nuclear layer ; OPL : outer plexiform layer . Scale bar = 20µm . White arrows indicated MALAT1 - hi rod photoreceptors .

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

Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.


A ) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) .

A) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts).


C ) Scatter plots showing expression of OPN1LW / OPN1MW or OPN1SW in individual cone photoreceptors for population C10 . The colour depicts expression level for OPN1LW / OPN1MW in individual cells .

O O O O O O O O O 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) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells.


D ) Heatmap of top 20 differentially expressed genes between L / M cones and S cones . The colour depicts normalised gene expression ( z - score capped at 2 . 5 ) .

D) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5).


E ) Expression pattern of glial markers in Muller glia ( C9 ) and retinal astrocytes ( C16 ) . x - axis depicts normalized transcript levels .

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

E) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.


C ) t - SNE analysis of the human Müller glia cell line MIO - M1 with the retinal cell types identified in this human neural retina atlas .

O O O O O 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) t-SNE analysis of the human Müller glia cell line MIO-M1 with the retinal cell types identified in this human neural retina atlas.


A ) GST pulldown used to analyse TBC1D14 interactors . Recombinant GST or GST - TBC1D14 was incubated with or without HEK 293A lysate . Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS - PAGE , with visible bands being excised from the gel and analysed by mass spectrometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST pulldown used to analyse TBC1D14 interactors. Recombinant GST or GST-TBC1D14 was incubated with or without HEK 293A lysate. Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS-PAGE, with visible bands being excised from the gel and analysed by mass spectrometry.


B ) Lysates of HEK293A cells expressing either GFP or GFP - TBC1D14 were subjected to immunoprecipitation ( IP ) with GFP - Trap , and then immunoblotted for GFP , TRAPPC12 and TRAPPC4 .

B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4.


C ) Lysates of cells expressing GFP or GFP - TRAPPC3 were subjected to IP with GFP - Trap , and then immunoblotted for TRAPPC4 , GFP and TBC1D14 .

O O O O O O 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) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14.


D ) Cells expressing GFP - TBC1D14 , generating a tubulated endosomal compartment , were fed Alexa647 labelled Transferrin ( white , blue in merge ) for 15 minutes in full medium , fixed , stained for endogenous TRAPPC4 ( red ) and analysed by confocal microscopy . White arrowheads indicate transfected cells , yellow arrowheads depict regions of colocalisation in inset . Scale bars = 10 μm .

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

D) Cells expressing GFP-TBC1D14, generating a tubulated endosomal compartment, were fed Alexa647 labelled Transferrin (white, blue in merge) for 15 minutes in full medium, fixed, stained for endogenous TRAPPC4 (red) and analysed by confocal microscopy. White arrowheads indicate transfected cells, yellow arrowheads depict regions of colocalisation in inset. Scale bars = 10 μm.


E ) Cells treated as in D were stained for endogenous TRAPPC12 ( red ) and analysed by confocal microscopy . Scale bars = 10

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

E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μm


F ) Cells in complete medium were fixed , stained for endogenous TBC1D14 ( green ) , RAB1B ( red ) and TRAPPC4 ( white , blue in merge ) and analysed by confocal microscopy . Inset shows protein localisation in juxtanuclear area . Yellow arrowheads show regions of colocalisation Scale bars = 10 μm . Data shown are representative from 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells in complete medium were fixed, stained for endogenous TBC1D14 (green), RAB1B (red) and TRAPPC4 (white, blue in merge) and analysed by confocal microscopy. Inset shows protein localisation in juxtanuclear area. Yellow arrowheads show regions of colocalisation Scale bars = 10 μm. Data shown are representative from 3 independent experiments.


A ) HEK293A cells transfected with GFP ( upper panels ) or GFP - TBC1D14 ( lower panels ) ( green ) were labelled with anti - RAB11A ( red ) and anti - RAB1B ( white , blue in merge ) antibodies . Arrows depict transfected cells . Inset : GFP - TBC1D14 transfected cell showing triple co - localisation between GFP - TBC1D14 , RAB11A and RAB1B ( white arrows ) and double co - localisation between GFP - TBC1D14 and RAB11A ( yellow arrows ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells transfected with GFP (upper panels) or GFP-TBC1D14 (lower panels) (green) were labelled with anti-RAB11A (red) and anti-RAB1B (white, blue in merge) antibodies. Arrows depict transfected cells. Inset: GFP-TBC1D14 transfected cell showing triple co-localisation between GFP-TBC1D14, RAB11A and RAB1B (white arrows) and double co-localisation between GFP-TBC1D14 and RAB11A (yellow arrows).


B ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB11A ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation .

B) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB11A (white, blue in merge). White arrowheads indicate regions of triple co-localisation.


C ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB1B ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation , yellow arrowheads regions of double co - localisation between GFP - TBC1D14 and TRAPPC4 . All scale bars = 20 μm .

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

C) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB1B (white, blue in merge). White arrowheads indicate regions of triple co-localisation, yellow arrowheads regions of double co-localisation between GFP-TBC1D14 and TRAPPC4. All scale bars = 20 μm.


B ) HEK 293A cell lysates ( from approximately 8 × 106 cells per construct ) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP , TRAPPC12 and TRAPPC4 . Data are representative of 3 independent experiments .

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

B) HEK 293A cell lysates (from approximately 8 × 106 cells per construct) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP, TRAPPC12 and TRAPPC4. Data are representative of 3 independent experiments.


A ) Cells expressing the indicated GFP - fusions ( green ) were loaded with Alexa647 - Transferrin ( red ; arrowheads indicate transfected cells ) for 15 minutes , fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy . Scale bars = 10 μm . Arrows in inset indicate the juxtanuclear

A) Cells expressing the indicated GFP-fusions (green) were loaded with Alexa647-Transferrin (red; arrowheads indicate transfected cells) for 15 minutes, fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy. Scale bars = 10 μm. Arrows in inset indicate the juxtanuclear ERC


B ) Cells transfected as in ( A ) were loaded with Alexa - 647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods . At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry . Results are expressed as a percentage of Tfn fluorescence at t = 0 and are the mean of 3 independent experiments , ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells transfected as in (A) were loaded with Alexa-647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods. At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry. Results are expressed as a percentage of Tfn fluorescence at t=0 and are the mean of 3 independent experiments, ± s.e.m.


C ) Cells transfected as in ( A ) were stained for the cis - Golgi marker GM130 . Arrowheads depict transfected cells . Scale bars = 10 μm . Bar chart is quantification of fragmented Golgi complex ( > 100 cells per condition from three independent experiments , ± s . e . m . * * p < 0 . 01 , one way ANOVA with Sidak ' s post - test ) .

C) Cells transfected as in (A) were stained for the cis-Golgi marker GM130. Arrowheads depict transfected cells. Scale bars = 10 μm. Bar chart is quantification of fragmented Golgi complex (> 100 cells per condition from three independent experiments, ± s.e.m. ** p < 0.01, one way ANOVA with Sidak's post-test).