I ) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1 - GFP / Atg19 - mCherry / ypt7Δ versus prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells . The intensity of Ape1 - GFP was normalized over the average fluorescence intensity of Nuf2 - GFP measured in kinetochores as performed in [ 36 ] . Data information : a total of 54 prApe1 - GFP spots and 80 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19Δ / ypt7Δ cells , whereas 52 prApe1 - GFP spots and 98 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19 - mCherry / ypt7Δ control cells . Error bars indicate standard error of the mean ( SEM ) . * P = 0 . 03 ( Z 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 O O O O O O O

I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).


Figure 4 : Quantification of the Ape1 , Ams1 , Atg19 protein abundances in living cells . ( A ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , Ams1 - GFP / Atg19 - mCherry / ypt7Δ , Atg19 - GFP / Ape1 - mCherry / ypt7Δ S . cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots . Only co - localizing GFP and RFP spots were considered . BF represents bright - field images . ( B ) Bar plot showing the number of molecules of prApe1 , Ams1 and Atg19 found in Cvt vesicles in living cells . Data information : data are representative of two independent experiments in which a total of 217 prApe1 - GFP , 181 Ams1 - GFP and 233 Atg19 - GFP spots were analyzed . Error bars indicate standard deviation ( SD ) . * * * P < 0 . 001 ; * P = 0 . 0176 ( Z 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells. (A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images. (B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells. Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).


( A ) Fluorescence microscopy image of a resin section of prApe1 - GFP / Atg19 - mCherry / ypt7Δ cells , merge of green ( prApe1 - GFP ) and red ( Atg19 - mcherry ) channels . Tetraspeck beads are detected in the blue channel . ( B ) Electron tomography slice of the boxed area in ( A ) . The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50 % and 90 % localization accuracy , respectively . Insets show close - up views of a double ( top ) and single ( bottom ) membrane . Corresponding tomogram Movie EV2 . ( C ) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation . The average dimensions give rise to spheroids with an average ellipticity of 11 % ( shown in dark grey ) . Both prApe1 - GFP and prApe1 - GFP / Atg19 - mCherry datasets were included . The cases representing autophagosomes were excluded .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence microscopy image of a resin section of prApe1-GFP/Atg19-mCherry/ypt7Δ cells, merge of green (prApe1-GFP) and red (Atg19-mcherry) channels. Tetraspeck beads are detected in the blue channel. (B) Electron tomography slice of the boxed area in (A). The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50% and 90% localization accuracy, respectively. Insets show close-up views of a double (top) and single (bottom) membrane. Corresponding tomogram Movie EV2. (C) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation. The average dimensions give rise to spheroids with an average ellipticity of 11 % (shown in dark grey). Both prApe1-GFP and prApe1-GFP/Atg19-mCherry datasets were included. The cases representing autophagosomes were excluded.


( D ) Dense Cvt aggregate that is not surrounded by membrane . Corresponding tomogram Movie EV3 . ( E ) Cvt aggregate that is partially enwrapped by double membrane . Corresponding tomogram Movie EV4 . ( F ) Cvt vesicle fully enclosed by double membrane . Bottom : schematic representation . White arrowheads indicate the double membrane . Corresponding tomogram Movie EV5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dense Cvt aggregate that is not surrounded by membrane. Corresponding tomogram Movie EV3. (E) Cvt aggregate that is partially enwrapped by double membrane. Corresponding tomogram Movie EV4. (F) Cvt vesicle fully enclosed by double membrane. Bottom: schematic representation. White arrowheads indicate the double membrane. Corresponding tomogram Movie EV5.


Figure 6 : Model of the Cvt pathway . ( A ) prApe1 the major Cvt cargo forms homo - oligomers as dodecamers that ( B ) assemble into higher - order assemblies mediated by the N - terminal propeptide . ( C ) These assemblies are capable of binding Atg19 that forms a trimer in solution . ( D ) Ams1 , which forms a homo - oligomer as a tetramer , joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site . ( E ) prApe1 dodecamers , secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol . ( F ) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form ( G ) the Cvt vesicle . Cvt assembly stages in ( E ) , ( F ) and ( G ) are drawn respecting the determined average size of the vesicles , the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article . ( H ) After fusion with the vacuole , prApe1 is subjected to maturation while maintaining the dodecamer organization . ( I ) Ams1 does not undergo a maturation process in the vacuole .

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

Figure 6: Model of the Cvt pathway. (A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide. (C) These assemblies are capable of binding Atg19 that forms a trimer in solution. (D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site. (E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol. (F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article. (H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization. (I) Ams1 does not undergo a maturation process in the vacuole.


Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( A ) CTGF interacts with LRP4 but not with MuSK . Full - length CTGF ( CTGF - full - myc ) was immunoprecipitated with the ectodomain of LRP4 ( LRP4ect - Flag , lane 6 ) but not with the ectodomain of MuSK ( MuSKect - Flag , lane 3 ) . However , when CTGF - full - myc , LRP4ect , and MuSKect - Flag were expressed together , CTGF - full - myc was immunoprecipitated with MuSKect - Flag ( lane 5 ) . The CT domain of CTGF ( CTGF - CT - myc ) was sufficient to immunoprecipitate LRP4ect - Flag ( lane 7 ) . Predicted positions of full - length and shortened CTGF proteins are indicated by bars and / or asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (A) CTGF interacts with LRP4 but not with MuSK. Full-length CTGF (CTGF-full-myc) was immunoprecipitated with the ectodomain of LRP4 (LRP4ect-Flag, lane 6) but not with the ectodomain of MuSK (MuSKect-Flag, lane 3). However, when CTGF-full-myc, LRP4ect, and MuSKect-Flag were expressed together, CTGF-full-myc was immunoprecipitated with MuSKect-Flag (lane 5). The CT domain of CTGF (CTGF-CT-myc) was sufficient to immunoprecipitate LRP4ect-Flag (lane 7). Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.


Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( B ) Deletion of the CT domain of human CTGF ( CTGF - ΔCT _ ΔTSP1 and CTGF - ΔCT ) impairs the interaction between CTGF and LRP4ect - Flag . Predicted positions of full - length and shortened CTGF proteins are indicated by bars and / or asterisks .

Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (B) Deletion of the CT domain of human CTGF (CTGF-ΔCT_ΔTSP1 and CTGF-ΔCT) impairs the interaction between CTGF and LRP4ect-Flag. Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.


Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( C ) Anti - CTGF antibody immunoprecipitated human LRP4 deleted with the 1st , 2nd , and 4th β - propeller domains ( Δ1 , Δ2 , and Δ4 ) but not with the 3rd β - propeller domain ( ∆ 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (C) Anti-CTGF antibody immunoprecipitated human LRP4 deleted with the 1st, 2nd, and 4th β-propeller domains (Δ1, Δ2, and Δ4) but not with the 3rd β-propeller domain (∆3).


Cell - free plate - binding assays . A purified tagged - protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase ( AP ) was overlaid . Bound protein was quantified by measuring AP activity . ( D ) LRP4ect - Flag , but not MuSKect - Flag , bound to overlaid CTGF - full - mycAP and CTGF - CT - mycAP . In contrast , LRP4ect - Flag did not bind to CTGF - ΔCT - mycAP . Rspo2 - mycAP were used as Control - mycAP and control - myc , respectively . Mean and SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for the three panels in ( D ) p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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

Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (D) LRP4ect-Flag, but not MuSKect-Flag, bound to overlaid CTGF-full-mycAP and CTGF-CT-mycAP. In contrast, LRP4ect-Flag did not bind to CTGF-ΔCT-mycAP. Rspo2-mycAP were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (D) p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


Cell - free plate - binding assays . A purified tagged - protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase ( AP ) was overlaid . Bound protein was quantified by measuring AP activity . ( E , upper panel ) LRP4ect - Flag bound to overlaid MuSKect - mycAP in a dose - dependent manner of CTGF - full - myc , but not of CTGF - ΔCT - myc . ( E , lower panel ) LRP4ect - Flag bound to overlaid agrin - mycAP independent of CTGF - full - myc . Rspo2 - mycAP and Rspo2 - myc were used as Control - mycAP and control - myc , respectively . Mean and SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for the three panels in ( E ) . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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

Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (E, upper panel) LRP4ect-Flag bound to overlaid MuSKect-mycAP in a dose-dependent manner of CTGF-full-myc, but not of CTGF-ΔCT-myc. (E, lower panel) LRP4ect-Flag bound to overlaid agrin-mycAP independent of CTGF-full-myc. Rspo2-mycAP and Rspo2-myc were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (E). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


( A ) Representative immunoblotting of Lrp4 , MuSK , Transferrin Receptor ( TfR ) , CTGF , and β - actin in whole - cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM ( 1 ng / ml ) agrin and / or 250 pM ( 100 ng / ml ) CTGF for 24 h . CTGF increases Lrp4 on the plasma membrane in the presence of agrin .

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

(A) Representative immunoblotting of Lrp4, MuSK, Transferrin Receptor (TfR), CTGF, and β-actin in whole-cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM (1 ng/ml) agrin and/or 250 pM (100 ng/ml) CTGF for 24 h. CTGF increases Lrp4 on the plasma membrane in the presence of agrin.


( B ) C2C12 myotubes were treated with 1 pM agrin and / or 250 pM CTGF for 6 h . Lrp4 was immunoprecipitated ( IP ) with an anti - Lrp4 antibody , and phosphorylated Lrp4 was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody .

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

(B) C2C12 myotubes were treated with 1 pM agrin and/or 250 pM CTGF for 6 h. Lrp4 was immunoprecipitated (IP) with an anti-Lrp4 antibody, and phosphorylated Lrp4 was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody.


( C ) Representative immunoblotting of Lrp4 , MuSK , CTGF , and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18 . 5 of wild - type ( WT ) and Ctgf - / - mice .

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(C) Representative immunoblotting of Lrp4, MuSK, CTGF, and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18.5 of wild-type (WT) and Ctgf-/- mice.


( D ) Representative immunoblotting of Y755 - phosphorylated MuSK , total MuSK , and β - actin in the diaphragm at E18 . 5 of wild - type ( WT ) , Ctgf - / - , and Ctgf + / - mice .

(D) Representative immunoblotting of Y755-phosphorylated MuSK, total MuSK, and β-actin in the diaphragm at E18.5 of wild-type (WT), Ctgf-/-, and Ctgf+/- mice.


( A ) C2C12 myotubes were treated with indicated concentrations of agrin - mycAP , CTGF - mycAP , and / or CTGF - ∆ CT - mycAP for 3 h . Total MuSK was immunoprecipitated ( IP ) with an anti - MuSK antibody , and phosphorylated MuSK was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . CTGF - mycAP , but not CTGF - ∆ CT - mycAP , enhanced agrin - mediated MuSK phosphorylation .

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

(A) C2C12 myotubes were treated with indicated concentrations of agrin-mycAP, CTGF-mycAP, and/or CTGF-∆CT-mycAP for 3 h. Total MuSK was immunoprecipitated (IP) with an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. CTGF-mycAP, but not CTGF-∆CT-mycAP, enhanced agrin-mediated MuSK phosphorylation.


( B ) C2C12 myotubes were treated with 5 pM agrin - mycAP , 250 pM CTGF - mycAP , and / or 250 pM CTGF - ∆ CT - mycAP for 18 h . BSA was added to the control medium . AChRs and the plasma membrane were visualized with Alexa594 - conjugated α - bungarotoxin ( red ) and CellMask ( green ) , respectively . Scale bar = 20 µm . Arrowheads point to the AChR clusters with an axis length of 4 μm or more . CTGF enhances agrin - mediated AChR clustering .

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(B) C2C12 myotubes were treated with 5 pM agrin-mycAP, 250 pM CTGF-mycAP, and/or 250 pM CTGF-∆CT-mycAP for 18 h. BSA was added to the control medium. AChRs and the plasma membrane were visualized with Alexa594-conjugated α-bungarotoxin (red) and CellMask (green), respectively. Scale bar = 20 µm. Arrowheads point to the AChR clusters with an axis length of 4 μm or more. CTGF enhances agrin-mediated AChR clustering.


( C ) C2C12 myotubes were infected with lentivirus expressing shControl , shCtgf - 1 , or shCtgf - 2 . Doxycycline was added for 2 days to induce shRNA expression . BSA or agrin was added to the medium at 10 pM . Total MuSK was immunoprecipitated ( IP ) by an anti - MuSK antibody , and phosphorylated MuSK was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . Ctgf knockdown suppressed MuSK phosphorylation .

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

(C) C2C12 myotubes were infected with lentivirus expressing shControl, shCtgf-1, or shCtgf-2. Doxycycline was added for 2 days to induce shRNA expression. BSA or agrin was added to the medium at 10 pM. Total MuSK was immunoprecipitated (IP) by an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. Ctgf knockdown suppressed MuSK phosphorylation.


( D ) Ten pM agrin was added to the medium . AChRs were visualized with Alexa594 - conjugated α - bungarotoxin ( red signals ) in the infected myotube ( green GFP signals ) . Ctgf knockdown decreased the number and length of AChR clusters . Scale bar = 10 µm .

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(D) Ten pM agrin was added to the medium. AChRs were visualized with Alexa594-conjugated α-bungarotoxin (red signals) in the infected myotube (green GFP signals). Ctgf knockdown decreased the number and length of AChR clusters. Scale bar = 10 µm.


( A ) Quantitative RT - PCR of Chrne , Musk , Lrp4 , and Ctgf in C2C12 myotubes treated with 10 ng / ml BSA or recombinant human CTGF .

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(A) Quantitative RT-PCR of Chrne, Musk, Lrp4, and Ctgf in C2C12 myotubes treated with 10 ng/ml BSA or recombinant human CTGF.


( B ) Quantitative RT - PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng / ml agrin and / or 10 ng / ml neuregulin - 1 .

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(B) Quantitative RT-PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng/ml agrin and/or 10 ng/ml neuregulin-1.


( C ) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18 . 5 . Co - localization of AChR ( red ) and CTGF ( green ) is indicated in the merged images . Red arrow points to an AChR cluster . Scale bar = 20 μm .

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(C) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18.5. Co-localization of AChR (red) and CTGF (green) is indicated in the merged images. Red arrow points to an AChR cluster. Scale bar = 20 μm.


( D ) Quantitative RT - PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse . Mean and SD ( n = 3 mice ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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(D) Quantitative RT-PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse. Mean and SD (n = 3 mice) are indicated. p-value < 0.05 by two-way repeated measures ANOVA. p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


( A ) Representative confocal images of the left diaphragm at E18 . 5 labeled with α - bungarotoxin ( red ) and anti - peripherin antibody ( green ) to visualize AChRs and the motor axon , respectively . The endplates of Ctgf - / - diaphragm were smaller ( lower left panel ) than those of the wild - type diaphragm ( upper left panel ) . In the Ctgf - / - diaphragm , 10 . 0 ± 3 . 2 % ( mean and SD , n = 105 in 5 embryos ) of peripherin - positive axons passed through the AChR - positive areas . In contrast , in the wild - type diaphragm , 2 . 0 ± 1 . 4 % ( mean and SD , n = 102 in 5 embryos ) of peripherin - positive axons passed through the AChR - positive areas . Quantification was performed in a blinded manner . Scale bar = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-peripherin antibody (green) to visualize AChRs and the motor axon, respectively. The endplates of Ctgf-/- diaphragm were smaller (lower left panel) than those of the wild-type diaphragm (upper left panel). In the Ctgf-/- diaphragm, 10.0 ± 3.2% (mean and SD, n = 105 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. In contrast, in the wild-type diaphragm, 2.0 ± 1.4% (mean and SD, n = 102 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. Quantification was performed in a blinded manner. Scale bar = 5 µm.


( B ) Representative confocal images of the left diaphragm at E18 . 5 labeled with α - bungarotoxin ( red ) and anti - synaptophysin antibody ( green ) to visualize AChRs and the nerve terminal , respectively . Synaptophysin signals mostly overlapped with α - bungarotoxin signals in the wild - type diaphragm , whereas synaptophysin signals extended into the axon in the Ctgf - / - diaphragm . Scale bar = 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

(B) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-synaptophysin antibody (green) to visualize AChRs and the nerve terminal, respectively. Synaptophysin signals mostly overlapped with α-bungarotoxin signals in the wild-type diaphragm, whereas synaptophysin signals extended into the axon in the Ctgf-/- diaphragm. Scale bar = 5 µm.


( C - E ) Blinded morphometric analysis of AChR signals ( C ) , synaptophysin signals ( D ) , and the ratio of synaptophysin signals not colocalized with AChR signals ( E ) of confocal images shown in ( A ) and ( B ) . AChR areas ( C ) , but not synaptophysin - positive areas ( D ) , were decreased in Ctgf - / - mice at E18 . 5 . ( E ) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf - / - diaphragm . Mean and SD ( n = 40 - 50 NMJs in 6 left diaphragms ) are indicated . p - value < 0 . 05 by one - way ANOVA for ( C and E ) , but not for ( D ) . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Blinded morphometric analysis of AChR signals (C), synaptophysin signals (D), and the ratio of synaptophysin signals not colocalized with AChR signals (E) of confocal images shown in (A) and (B). AChR areas (C), but not synaptophysin-positive areas (D), were decreased in Ctgf-/- mice at E18.5. (E) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf-/- diaphragm. Mean and SD (n = 40-50 NMJs in 6 left diaphragms) are indicated. p-value < 0.05 by one-way ANOVA for (C and E), but not for (D). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


( F ) Representative electron micrographs of the NMJs in the diaphragm of wild - type and Ctgf - / - mice at E18 . 5 . In Ctgf - / - mice , synaptic vesicles are sparser and the number of mitochondria at the nerve terminals is reduced compared to those in wild - type mice . See Table 1 for morphometric analysis . Regions indicated by rectangles are enlarged in the insets . Arrowheads point to synaptic vesicles ( SV ) . Mit , mitochondria . Blinded morphometric measurements are shown in Table 1 . Scale bar = 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative electron micrographs of the NMJs in the diaphragm of wild-type and Ctgf-/- mice at E18.5. In Ctgf-/- mice, synaptic vesicles are sparser and the number of mitochondria at the nerve terminals is reduced compared to those in wild-type mice. See Table 1 for morphometric analysis. Regions indicated by rectangles are enlarged in the insets. Arrowheads point to synaptic vesicles (SV). Mit, mitochondria. Blinded morphometric measurements are shown in Table 1. Scale bar = 500 nm.


( B - C ) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils . Data information : For each panel , data represents biological triplicates plated in technical duplicates . Statistical analysis was completed using one - way ANOVA , multiple comparison test . * P < . 05 ; * * P < . 01 ; * * * P < . 001 ; * * * * P < . 0001 . Center values denote the mean , and error bars denote 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 O O O O O O O O O

(B-C) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.


( D - E - F ) The expression of cyclooxygenases cox1 , cox2a and cox2b is enriched in macrophages . ( G - H ) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs . ( I - O ) The prostaglandin receptors ptger1a , ptger1b , ptger2a , ptger3 , ptger4a are mostly expressed in HSPCs . ( P - Q ) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs . Data information : For each panel , data represents biological triplicates plated in technical duplicates . Statistical analysis was completed using one - way ANOVA , multiple comparison test . * P < . 05 ; * * P < . 01 ; * * * P < . 001 ; * * * * P < . 0001 . Center values denote the mean , and error bars denote 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E-F) The expression of cyclooxygenases cox1, cox2a and cox2b is enriched in macrophages. (G-H) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs. (I-O) The prostaglandin receptors ptger1a, ptger1b, ptger2a, ptger3, ptger4a are mostly expressed in HSPCs. (P-Q) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.


( A ) Schematic indicating the imaging area in the tail at 72hpf , as indicated by the black box ; fluorescence imaging of the CHT in double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2 . ( B ) Quantification of runx1 : mcherry positive - cells ( C ) Quantification of cd45 : CFP positive - cells in double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2 . Data information : Statistical analysis was completed using one - way ANOVA , multiple comparison test . * P < . 05 ; * * P < . 01 ; * * * P < . 001 * * * * P < . 0001 . Scale bar 200μm ( 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 O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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 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 O O O

(A) Schematic indicating the imaging area in the tail at 72hpf, as indicated by the black box; fluorescence imaging of the CHT in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. (B) Quantification of runx1:mcherry positive-cells (C) Quantification of cd45:CFP positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. Data information: Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001 ****P<.0001. Scale bar 200μm (A).


( A ) WISH for cmyb expression at 60hpf in wild type and slco2b1 - / - embryos . ( B ) Quantification of cmyb - expressing cells . Each n represents the number of cmyb - expressing cells for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . Data information : Centre values denote the mean , and error values denote s . e . m . The statistical analysis was completed using an unpaired two tailed t test . * * * P < . 001 Scale bar 500μm ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WISH for cmyb expression at 60hpf in wild type and slco2b1-/- embryos. (B) Quantification of cmyb-expressing cells. Each n represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 500μm (A)


( C ) Schematic indicating the imaging area in the tail at 60hpf , as indicated by the black box ; fluorescence imaging in the CHT of kdrl : mCherry ; cmyb : GFP embryos injected with control - and slco2b1 - Mos . ( D ) Quantification of HSPCs associated to ECs . Each n represents the number of yellow spots for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . Data information : Centre values denote the mean , and error values denote s . e . m . The statistical analysis was completed using an unpaired two tailed t test . * * * P < . 001 Scale bar 200μm ( C )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic indicating the imaging area in the tail at 60hpf, as indicated by the black box; fluorescence imaging in the CHT of kdrl:mCherry;cmyb:GFP embryos injected with control- and slco2b1-Mos. (D) Quantification of HSPCs associated to ECs. Each n represents the number of yellow spots for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 200μm (C)


( E ) Experimental outline and quantification of time - lapse live imaging in controls and slco2b1 - morphants cmyb : GFP + cells , using Imaris software . Data information : The statistical analysis was completed using an unpaired two tailed t test . * * * P < .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Experimental outline and quantification of time-lapse live imaging in controls and slco2b1-morphants cmyb:GFP+ cells, using Imaris software. Data information: The statistical analysis was completed using an unpaired two tailed t test. ***P < .001


( A ) Anti - GFP and pH3 immunostainings of either controls ( Ctrl - Mo ) or slco2b1 - morphants ( slco2b1 - MO ) cmyb : GFP embryos . ( B ) Quantification of the number of the pH3 + HSCs in controls or slco2b1 - morphants . Centre values denote the mean , and error values denote s . e . m , statistical analysis was completed using an unpaired two tailed t test . * * * P < . 001 . Data information : Scale bar 50μm ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-GFP and pH3 immunostainings of either controls (Ctrl-Mo) or slco2b1-morphants (slco2b1-MO) cmyb:GFP embryos. (B) Quantification of the number of the pH3+ HSCs in controls or slco2b1-morphants. Centre values denote the mean, and error values denote s.e.m, statistical analysis was completed using an unpaired two tailed t test. ***P < .001. Data information: Scale bar 50μm (A)


( D ) Quantification of PGE2 concentration in control and slco2b1 morphants . The statistical analysis was completed using an unpaired two tailed t test * P < . 01 . Centre values denote the mean , and error bars denote s . e . m .

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

(D) Quantification of PGE2 concentration in control and slco2b1 morphants. The statistical analysis was completed using an unpaired two tailed t test *P < .01. Centre values denote the mean, and error bars denote s.e.m.


( E ) Fluorescence imaging in the CHT of cmyb : GFP embryos injected with control - and slco2b1 - MOs and treated with PGE2 . ( F ) Quantification of GFP positive cells . Statistical analysis : one - way ANOVA , multiple comparison , * P < . 01 ; * * * * P < . 0001 . Centre values denote the mean , and error bars denote s . e . m . Data information : Scale bar 200μm ( 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 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

(E) Fluorescence imaging in the CHT of cmyb:GFP embryos injected with control- and slco2b1-MOs and treated with PGE2. (F) Quantification of GFP positive cells. Statistical analysis: one-way ANOVA, multiple comparison, *P < .01; ****P < .0001. Centre values denote the mean, and error bars denote s.e.m. Data information: Scale bar 200μm (E).


( A ) Fluorescence imaging of the CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos injected with control - or slco2b1 - morpholinos , after treatment with DMSO . ( B ) Fluorescence imaging of the CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos injected with control - or slco2b1 - morpholinos , after treatment with DMSO + MTZ . ( C ) Fluorescence imaging of the CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos injected with control - or slco2b1 - morpholinos , after treatment with DMSO + MTZ + PGH2 . ( D ) Quantification of runx1 : mcherry positive - cells . Each dot represents the number of red cells for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . ( E ) Quantification of cd45 : CFP positive - cells . Each dot represents the number of blue cells for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . Data information for ( D ) and ( E ) : Statistical analysis was completed using one - way ANOVA , multiple comparison test . * * * * P < . 0001 . Centre values denote the mean , and error bars denote s . e . m Scale bar 200μm ( A - C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO. (B) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ. (C) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ+PGH2. (D) Quantification of runx1:mcherry positive-cells. Each dot represents the number of red cells for each embryo (biological replicates). Each experiment has been repeated three independent times. (E) Quantification of cd45:CFP positive-cells. Each dot represents the number of blue cells for each embryo (biological replicates). Each experiment has been repeated three independent times.Data information for (D) and (E): Statistical analysis was completed using one-way ANOVA, multiple comparison test. ****P<.0001. Centre values denote the mean, and error bars denote s.e.m Scale bar 200μm (A-C).


( A ) WISH for cmyb at 60hpf in kdrl : Gal4 + embryos injected with control - and slco2b1 - morpholinos and / or co - injected with the Tol2 - UAS : slco2b1 vector and tol2 mRNA . The region indicated by the dashed bas been magnified and shows in ( B ) . ( C ) Quantification and statistical analysis were completed using one - way ANOVA , multiple comparison test . * * * * P < . 0001 . Centre values denote the mean , and error bars denote s . e . m . Each dot represents the number of cmyb - expressing cells for each embryo ( biological replicates ) . Each experiment has been repeat three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WISH for cmyb at 60hpf in kdrl:Gal4+ embryos injected with control- and slco2b1- morpholinos and/or co-injected with the Tol2-UAS:slco2b1 vector and tol2 mRNA. The region indicated by the dashed bas been magnified and shows in (B). (C) Quantification and statistical analysis were completed using one-way ANOVA, multiple comparison test. ****P< .0001. Centre values denote the mean, and error bars denote s.e.m. Each dot represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeat three independent times.


( A ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) and CD31 ( endothelial cell marker ) at E13 and E16 stages of mouse fetal liver . ( B ) Distance distribution between cKithigh cells and endothelial cells ( n = 79 from 3 E13 fetal livers ; n = 92 from 3 E16 fetal livers ) , binned into 20μm intervals . Statistical analysis was completed using t - test . * P < . 01 ; * * P < . 001 ; * * * P < . 0001 . Centre values denote the mean , and error values denote s . e . m All nuclei marked with DAPI . Scale bar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and CD31 (endothelial cell marker) at E13 and E16 stages of mouse fetal liver. (B) Distance distribution between cKithigh cells and endothelial cells (n=79 from 3 E13 fetal livers; n=92 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test. *P<.01; **P<.001; ***P<.0001. Centre values denote the mean, and error values denote s.e.m All nuclei marked with DAPI. Scale bar 20μm


( C ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) and F4 / 80 ( macrophage cell marker ) at E13 and E16 stages of mouse fetal liver . ( D ) Distance distribution between cKithigh cells and macrophages ( n = 54 from 3 E13 fetal livers ; n = 64 from 3 E16 fetal livers ) , binned into 5μm intervals . Statistical analysis was completed using t - test . All nuclei marked with DAPI . Scale bar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and F4/80 (macrophage cell marker) at E13 and E16 stages of mouse fetal liver. (D) Distance distribution between cKithigh cells and macrophages (n=54 from 3 E13 fetal livers; n=64 from 3 E16 fetal livers), binned into 5μm intervals. Statistical analysis was completed using t-test. All nuclei marked with DAPI. Scale bar 20μm


( E ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) and MBP ( neutrophil cell marker ) at E13 and E16 stages of mouse fetal liver . ( F ) Distance distribution between cKithigh cells and neutrophils ( n = 45 from 3 E13 fetal livers ; n = 53 from 3 E16 fetal livers ) , binned into 20μm intervals . Statistical analysis was completed using t - test * * * P < . 001 . All nuclei marked with DAPI . Scale bar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and MBP (neutrophil cell marker) at E13 and E16 stages of mouse fetal liver. (F) Distance distribution between cKithigh cells and neutrophils (n=45 from 3 E13 fetal livers; n=53 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test ***P<.001. All nuclei marked with DAPI. Scale bar 20μm


A . Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO ( Ctrl ) for 1 h before fixation and immunostained with Hoechst ( blue ) , anti - CHMP4B ( green ) , anti - GAL3 ( red ) and anti - LAMP1 ( white ) are presented . Cells treated with LLOMe show increased recruitment of ESCRT - III protein to lysosomes when compared to Ctrl cells . Number of foci per cell were quantified and are indicated as mean ± SD . Data from > 86 cells per condition from three independent experiments . CHMP4B , GAL3 , LAMP1 foci per cell ( Ctrl versus LLOMe treatment ) : p = 0 . 0398 , p = 0 . 0033 , p = 0 . 2400 ( Student " s t ‐ test ) , respect Data information : Scale bars : 10 μm and 1 μm ( inset )

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 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 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

A. Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO (Ctrl) for 1 h before fixation and immunostained with Hoechst (blue), anti-CHMP4B (green), anti-GAL3 (red) and anti-LAMP1 (white) are presented. Cells treated with LLOMe show increased recruitment of ESCRT-III protein to lysosomes when compared to Ctrl cells. Number of foci per cell were quantified and are indicated as mean ± SD. Data from >86 cells per condition from three independent experiments. CHMP4B, GAL3, LAMP1 foci per cell (Ctrl versus LLOMe treatment): p=0.0398, p=0.0033, p=0.2400 (Student"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)


B . HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 treated as in ( A ) and stained with CD63 antibody are shown . Number of foci per cell were quantified and are indicated as mean ± SD . Data is quantified from > 87 cells per condition from three independent experiments . CHMP4B , GAL3 , CD63 foci per cell ( Ctrl versus LLOMe treatment ) : p = 0 . 0111 , p = 0 . 0050 , p = 0 . 3793 ( Student " s t ‐ test ) , respect Data information : Scale bars : 10 μm and 1 μm ( inset )

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

B. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 treated as in (A) and stained with CD63 antibody are shown. Number of foci per cell were quantified and are indicated as mean ± SD. Data is quantified from >87 cells per condition from three independent experiments. CHMP4B, GAL3, CD63 foci per cell (Ctrl versus LLOMe treatment): p=0.0111, p=0.0050, p=0.3793 (Student"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)


To screen for ESCRT proteins that are involved in the endolysosomal repair process , cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence . TSG101 , a component of the ESCRT - I complex , and EAP30 , an ESCRT - II protein , are clearly recruited to the sites of damage compared to the DMSO control ( Ctrl ) . As shown , the ESCRT - III complex together with ALIX is recruited to damaged endolysosomes . In contrast , HRS , a component of the ESCRT - 0 complex , and HD - PTP show no recruitment to damaged endomembranes . Number of foci per cell was quantified from > 85 cells per condition from three independent experiments and are presented as mean ± SD . Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student ' s t ‐ test , the p - values for which are : HRS p = 0 . 9257 , GAL3 p = 0 . 0050 ; TSG101 p = 0 . 0243 , GAL3 p = 0 . 0489 ; EAP30 p = 0 . 0006 , GAL3 p = 0 . 0307 ; CHMP2A p = 0 . 0284 , GAL3 p = 0 . 0260 ; CHMP4B p = 0 . 0028 , GAL3 p = 0 . 0414 ; VPS4A p = 0 . 0309 , GAL3 p = 0 . 0461 ; ALIX p = 0 . 0249 , GAL3 p = 0 . 0471 ; HD - PTP p = 0 . 4898 , GAL3 p = 0 . 0105 . Data information : Scale bars : 5 μm and 1 μm ( inset ) .

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

To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT-I complex, and EAP30, an ESCRT-II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT-III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT-0 complex, and HD-PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the p-values for which are: HRS p=0.9257, GAL3 p=0.0050; TSG101 p=0.0243, GAL3 p=0.0489 ; EAP30 p=0.0006, GAL3 p=0.0307; CHMP2A p=0.0284, GAL3 p=0.0260; CHMP4B p=0.0028, GAL3 p=0.0414; VPS4A p=0.0309, GAL3 p=0.0461; ALIX p=0.0249, GAL3 p=0.0471; HD-PTP p=0.4898, GAL3 p=0.0105. Data information: Scale bars: 5 μm and 1 μm (inset).


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs against : ( A ) HR 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system . A . Depletion of HRS does not alter the dynamics of CHMP4B - eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against: (A) HR 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system. A. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs agains ( B ) TSG10 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system B . Downregulation of TSG101 , with two independent siRNAs causes a delay in CHMP4B - eGFP recruitment as compared to siCtrl , indicating an important role of the ESCRT - I complex in recruiting the downstream components Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (B) TSG10 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system B. Downregulation of TSG101, with two independent siRNAs causes a delay in CHMP4B-eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT-I complex in recruiting the downstream components Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs agains ( C ) CHMP2 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system C . CHMP2A depletion accumulates and stabilizes CHMP4B - eGFP positive foci Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (C) CHMP2 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system C. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP positive foci Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs against ( D ) ALI 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system D . siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

O O O O O O O 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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (D) ALI 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system D. siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs against ( E ) both TSG101 and ALIX 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system E . Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

O O O O O O O O O O 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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (E) both TSG101 and ALIX 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system E. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


A . HeLa cells stably expressing CHMP4B - eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND - 99 and fixed at different time points as indicated . After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced . After 30 min , lysosomes gain back functionality ( judging by the increased number of Lysotracker foci ) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes . Representative confocal images for each time point are shown . Scale bars : 5

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 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

A. HeLa cells stably expressing CHMP4B-eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND-99 and fixed at different time points as indicated. After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm


B . Quantification graph ( > 250 cells per condition from four independent experiments ) showing CHMP4B and Lysotracker positive foci per cell at different time points . Error bars correspond to 95 % confidence

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B. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker positive foci per cell at different time points. Error bars correspond to 95% confidence intervals


C . Quantification graph showing dynamics of Lysotracker recovery in control ( siCtrl ) and both TSG101 and ALIX depleted cells . HeLa cells stably expressing CHMP4B - eGFP were co - transfected with siRNAs against TSG101 and ALIX . 48 h post - transfection , cells were pre - treated for 20 min with 75 nM Lysotracker Deep - Red , which was used as a read - out . While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl , simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair . Data from > 40 cells per condition from four independent live - cell imaging experiments are shown . Graph is normalized to the area occupied by the cells . Error bars correspond to 95 % confidence intervals .

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C. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101 and ALIX depleted cells. HeLa cells stably expressing CHMP4B-eGFP were co-transfected with siRNAs against TSG101 and ALIX. 48 h post-transfection, cells were pre-treated for 20 min with 75 nM Lysotracker Deep-Red, which was used as a read-out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live-cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.


Representative movie montage of a live - cell imaging experiment illustrating the dynamics of CHMP4B - eGFP and mCherry - Galectin3 recruitment at different time points before and after LLOMe treatment . Using the perfusion system , 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 and images were acquired every 20 sec for a period of 20 min . As shown in the representative movie montage and the quantification graph , CHMP4B recruitment precedes Galectin3 , which is an established marker for detection of damaged endolysosomal membranes . The quantification graph is normalized to the area occupied by the cells . Data were obtained from > 40 cells per condition from three independent experiments . Error bars correspond to 95 % confidence intervals . Data information : Scale bars : 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 B-CONTROLLED_VAR 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 O O O

Representative movie montage of a live-cell imaging experiment illustrating the dynamics of CHMP4B-eGFP and mCherry-Galectin3 recruitment at different time points before and after LLOMe treatment. Using the perfusion system, 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 and images were acquired every 20 sec for a period of 20 min. As shown in the representative movie montage and the quantification graph, CHMP4B recruitment precedes Galectin3, which is an established marker for detection of damaged endolysosomal membranes. The quantification graph is normalized to the area occupied by the cells. Data were obtained from >40 cells per condition from three independent experiments. Error bars correspond to 95% confidence intervals. Data information: Scale bars: 5 μm.


A . HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 were incubated with 250 μM LLOMe , fixed at different time points as indicated and stained for ubiquitin . As presented , CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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A. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


B . HeLa cells stably expressing CHMP4B - eGFP were treated as in ( A ) and stained for p62 . CHMP4B is recruited before p62 on the damaged lysosomes . The number of LAMP1 - positive foci appears stable independently of LLOMe treatment Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


C . Experimental setup as in ( A and B ) where HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 were stained for LC3 . As shown , CHMP4B is recruited prior to LC3 Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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C. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3 Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


A . HeLa cells were transfected with different siRNAs as indicated . 48 h post - transfection cells were treated with 250 μM LLOMe for 3 , 6 , 10 hours harvested and stained for Annexin V . Cells were immediately processed for flow cytometry . Already after 3 h of treatment with LLOMe , cells co - transfected with TSG101 + ALIX siRNA showed an increase in apoptotic cell death , whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 cells per condition were analyzed . Error bars represent SEM . Statistical significance was determined using one - way ANOVA test . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 .

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A. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


B . Experimental set up as in ( A ) with additional propidium iodide ( PI ) staining of transfected HeLa cells Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 cells per condition were analyzed . Error bars represent SEM . Statistical significance was determined using one - way ANOVA test . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 .

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B. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


A . HeLa cells stably expressing CHMP4B - eGFP were transfected with the mCherry - Galectin3 plasmid . 24 h later , cells were infected with WT Coxiella burnetti . Live - cell imaging started 24 h post - infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella - containing vacuole ( DRAQ5 labeling ) becoming positive for CHMP4B - eGFP and mCherry - Galectin3 . Data information : Scale bars : 50

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A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm


B . Bacterial viability and replication assay upon TSG101 KD . HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h . Then cells were infected with mCherry - Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells . 72 h later , infected cells were fixed , DAPI stained and processed for quantitative image analysis . Between 30 and 40 fields representing more than 9 , 000 cells per condition of three independent experiments were analyzed . Error bars represent SD . Statistical significance was determined using one - way ANOVA test . * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 . 001 .

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B. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001.


b - d ) Comparative characterization of CHO cell bulk transfection cultures , selected for stable expression of the UnaG - or EGFP - encoding reporter constructs . b ) Flow cytometry revealed efficient induction of green fluorescence , after treatment of both UnaG and EGFP - expressing CHO bulk cultures with CoCl2 , while growth under hypoxia ( 1 % oxygen ) for 12 hours , selectively induced green fluorescence only in dUnaG expressing cells . Under normoxia ( 21 % oxygen ) only background fluorescence was observed . c ) Assessment of the mean fluorescence intensity ( MFI ) to determine the activity of the 5xHRE - CMV promoter after 12 and 24hrs . Fluorescence intensity increased between 12 and 24hrs .

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b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.


d ) Representative images of dUnaG - or dEGFP - expressing CHO cells grown either under normoxia ( 21 % oxygen ) , CoCl2 - treatment or hypoxia ( 1 % oxygen ) for 12 hours . Scale bars =

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d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm


e ) Fluorescence intensity in dUnaG - or dEGFP - expressing CHO cell bulks grown under increasingly hypoxic conditions of 10 % , 5 % and 1 % oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5 % .

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e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.


f ) Maximal fluorescence of the UnaG - based hypoxia reporter in CHO cells grown at 1 % oxygen for 24 hrs hours was only observed at serum concentrations of 5 % FCS or higher , reflecting the dependence of UnaG on its serum cofactor bilirubin .

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f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.


b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP . Expression under hypoxia was induced by incubation in 1 % oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia . Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly - destabilized UnaG - variants , while the doubly destabilized doUnaG displayed the most rapid switching behavior , but the least brightness . To reduce phototoxicity , illumination intensity was kept minimal . Data points represent one viewfield of 425 µm2 ± 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 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

b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.


c ) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG . Open bars represent control cultures grown under normoxia . Solid bars indicate culture for 4 , 8 , 12 , 16 , 20 or 24 hrs under hypoxia ( 1 % oxygen ) . To assess deactivation , cultures previously kept under hypoxia ( 1 % oxygen ) for 24hrs were shifted to normoxia ( 21 % oxygen ) for the indicated times . Error bars represent standard errors .

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c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.


d ) Representative maximum intensity projections ( MIPs ) from the life cell cultures depicted in b . Cells were grown for the indicated times at hypoxic ( 1 % oxygen ) conditions and subsequently switched for the indicated times to normoxia ( 21 % oxygen ) ( red arrows in b ) to illustrate the dynamics of the different UnaG - based hypoxia sensors . Increasing destabilization is associated with reduced brightness but , also reduced background and improved switching behavior . Scale bars = 50 µm .

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d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.


a ) UnaG - based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model . 500 Gli36 glioblastoma cells , constitutively expressing mCherry and stably transfected with the HRE - dUnaG sensor construct , were stereotactically transplanted into the cortex of a SCIDmouse . Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation . Tumor cells are distinguished from the surrounding cortex by mCherry expression . Blood vessels were contrasted by immunostaining against PECAM - 1 . Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density ( outlined by white line in the composite panel ( bottom right ) . b ) Representative area from the tumor shown in a ) , which is located outside the viewfield in a ) positioned more closely to the tumor border . Also at the tumor border UnaG - expressing cells are preferentially observed at a distance to PECAM - 1 + vessels .

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a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCIDmouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels.


c ) Areas of dUnaG expression correlate well with HIF - 1α stabilization . Immunostaining for HIF - 1α ( cyan ) revealed the predominantly nuclear localization of stabilized HIF - 1α in cells that were also dUnaG - positive ( green ) . d ) Analysis of dUnaG expressing cells in c ) for mCherry fluorescence and HIF - 1α stabilization . dUnaG - positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF - 1α The threshold was set in either case to channel 70 of 256 intensity channels . Using this classification > 60 % of UnaG positive cells displayed only background level mCherry fluorescence . On the other hand , > 98 % of the UnaG expressing cells also expressed HIF - 1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo .

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c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo.


e ) The dUnaG - based senor was efficiently applied in intravital microscopy . 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG - sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy . We noted two distinct areas of intense UnaG fluorescence ( red lines ) . Shown is a MIP of a 60 µm z stack . The vasculature was contrasted by intravenous injection of non - functionalized Qdots . f ) Depth color coding of the MIP shown in e ) revealed the spatial relation between the vasculature , ( contrasted by Qdots ) , areas of increased dUnaG expression ( bordered by green lines ) and tumor cells expressing residual levels of mCherry . The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner ( red asterisk ) and the smaller vessels with a more axial orientation ( white arrows ) . Scale bars =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm


b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) in CHO cells stably transfected with the dUnOHR sensor . Hypoxia was induced by incubation in 1 % oxygen for 18 hrs , then the culture was switched to normoxia for 24 hrs , followed by another 14 hrs hypoxia and finally normoxia again . As expected UnaG fluorescence is efficiently induced under hypoxia , while mOrange fluorescence only appears after the switch to normoxia only . The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE - mCMV promoter activity . This behavior is repeated in subsequent hypoxia / normoxia cycles . The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period . Plotted is the average of the mean ± SEM . c ) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b . ) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange . Scale bars = 100 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.


a ) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct . The perimeter of the developing tumor ( white line in the merged picture , lower right panel ) was delineated by Hoechst 33342 staining . As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density . Vascular endothelium was identified by PECAM - 1 staining . 30 µm cryosection . Scale bar = 500µm . b ) Details of the tumor shown in a ) . Sparse , distinct cells are characterized by intense green and orange fluorescence ( shown as orange ) and are located preferentially to the edge of the hypoxic phalanx ( white arrows ) . A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence ( asterisks ) , indicative of previously hypoxic environment and subsequent reoxygenation . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm.


c ) Recently reoxygenated cells ( arrows ) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber ( double arrows ) . Scale bar = 100 µm .

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c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm.


d ) Previously hypoxic cells , frequently undergo apoptosis after reoxygenation . We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas . Scale bar = 50µm .

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d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.


e ) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis . Scale bar = 50µm .

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e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.


A - B . Correlation of MCU and MCUb expression levels with breast cancer clinical stages . Median - centered log2 mRNA expression levels of MCU and MCUb were collected from the TCGA breast cancer dataset ( http : / / tcga - data . nci . nih . gov / docs / publications / brca _ 2012 / ) . Data were plotted and analysed against tumor size ( T1 - T4 ) ( A ) and regional lymph node infiltration ( N0 - N3 ) ( B ) , according to the AJCC Cancer Staging Manual ( 7th edition ) . Linear regression analysis with different stages was implemented . Parameters of linear regression are shown . Number of samples for each stage are shown in parentheses .

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A-B. Correlation of MCU and MCUb expression levels with breast cancer clinical stages. Median-centered log2 mRNA expression levels of MCU and MCUb were collected from the TCGA breast cancer dataset (http://tcga-data.nci.nih.gov/docs/publications/brca_2012/). Data were plotted and analysed against tumor size (T1-T4) (A) and regional lymph node infiltration (N0-N3) (B), according to the AJCC Cancer Staging Manual (7th edition). Linear regression analysis with different stages was implemented. Parameters of linear regression are shown. Number of samples for each stage are shown in parentheses.


C - E . MCU silencing reduces Ca2 + mit uptake in TNBC cells . Cells were transfected with siMCU or siControl . After 48h , [ Ca2 + ] mit uptake upon ATP ( C , E ) or Histamine ( D ) stimulation was measured ( n = 10 ) . P - values : * * * p = 0 . 0008 , * * * p < 0 . 0001 , * * * p = 0 . 0001 , respectively .

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C-E. MCU silencing reduces Ca2+mit uptake in TNBC cells. Cells were transfected with siMCU or siControl. After 48h, [Ca2+]mit uptake upon ATP (C, E) or Histamine (D) stimulation was measured (n=10). P-values: ***p= 0.0008, ***p<0.0001, ***p= 0.0001, respectively.


F - H . MCU silencing impairs TNBC cell migration . Cells were transfected with siMCU or siControl . The day after transfection , a linear scratch was obtained on the cell monolayer through a vertically held P200 tip ( time - point 0h ) . Cell migration into the scratched area was monitored 48 hours later . The covered area was measured and expressed as a percentage relative to 0 - hour time - point ( n = 12 ) . P - value : * * * p < 0 . 0001 .

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F-H. MCU silencing impairs TNBC cell migration. Cells were transfected with siMCU or siControl. The day after transfection, a linear scratch was obtained on the cell monolayer through a vertically held P200 tip (time- point 0h). Cell migration into the scratched area was monitored 48 hours later. The covered area was measured and expressed as a percentage relative to 0-hour time-point (n=12). P-value: ***p<0.0001.