( F ) Intravital microscopy quantification of the rolling and adhesion frequencies and rolling velocities of neutrophils recruited to venules irrigating inflamed ( TNFα - injected ) cremaster muscle . Data are means ± SEM . ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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(F) Intravital microscopy quantification of the rolling and adhesion frequencies and rolling velocities of neutrophils recruited to venules irrigating inflamed (TNFα-injected) cremaster muscle. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KO mice . These animals were fed a ND or MCD for 3 weeks . ( A ) Neutrophils and monocytes as a percentage of circulating leukocytes , measured in total blood .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (A) Neutrophils and monocytes as a percentage of circulating leukocytes, measured in total blood.


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KOmice . These animals were fed a ND or MCD for 3 weeks . ( B ) Representative H & amp ; amp ; E and oil red - stained liver sections after 3 weeks of treatment . Scale Bar : 50μm .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KOmice. These animals were fed a ND or MCD for 3 weeks. (B) Representative H&amp;E and oil red-stained liver sections after 3 weeks of treatment. Scale Bar: 50μm.


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KO mice . These animals were fed a ND or MCD for 3 weeks . ( C ) Liver triglyceride and ( D ) plasma transaminase activity ( ALT ) at the end of the diet period .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (C) Liver triglyceride and (D) plasma transaminase activity (ALT) at the end of the diet period.


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KO mice . These animals were fed a ND or MCD for 3 weeks . ( E ) Total RNA was extracted from livers , and chemokine and cytokine mRNA levels were determined by qRT - PCR . mRNA expression was normalized to the amount of Gapdh mRNA . Data are means ± SEM . ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (E) Total RNA was extracted from livers, and chemokine and cytokine mRNA levels were determined by qRT-PCR. mRNA expression was normalized to the amount of Gapdh mRNA. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


( A , B and C ) Plasma levels of 3 - hydroxybutyrate in WT and p38γ / δ - / - ( A ) , Lyzs - Cre and p38γ / δLyzs - KO mice ( B ) and Lyzs - Cre Ly6G Ab treated after MCD diet ( C ) .

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(A, B and C) Plasma levels of 3-hydroxybutyrate in WT and p38γ/δ-/- (A), Lyzs-Cre and p38γ/δLyzs-KO mice (B) and Lyzs-Cre Ly6G Ab treated after MCD diet (C).


B Antiproliferative activities of XMU - MP - 5 and crizotinib against EML4 - ALK Ba / F3 and parental Ba / F3 cells , respectively . Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay . Each concentration was performed in triplicate . Data were presented as Mean ± SEM from three independent experiments ( n = 3 ) .

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B Antiproliferative activities of XMU-MP-5 and crizotinib against EML4-ALK Ba/F3 and parental Ba/F3 cells, respectively. Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3) .


C XMU - MP - 5 downregulates ALK signaling pathways in EML4 - ALK Ba / F3 cells . Cells were treated with XMU - MP - 5 or crizotinib for 4 h , and then analyzed by immunobloting .

C XMU-MP-5 downregulates ALK signaling pathways in EML4-ALK Ba/F3 cells. Cells were treated with XMU-MP-5 or crizotinib for 4 h, and then analyzed by immunobloting.


C XMU - MP - 5 suppressed ALK signaling pathway in H3122 cells .

C XMU-MP-5 suppressed ALK signaling pathway in H3122 cells.


E Comparison of tumor volume of different treatment groups in 11 days . BALB / c nude mice bearing H3122 xenograft tumors were treated with vehicle , XMU - MP - 5 and crizotinib by tail vein injection once daily . Data were shown as Mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test . N . S . , P = 0 . 1063 ; * * P = 0 . 0097 .

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E Comparison of tumor volume of different treatment groups in 11 days. BALB/c nude mice bearing H3122 xenograft tumors were treated with vehicle, XMU-MP-5 and crizotinib by tail vein injection once daily. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test. N.S., P = 0.1063; **P = 0.0097.


G Representative images of tumors analyzed by H & E and IHC staining . Scale bars , 100 μm .

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G Representative images of tumors analyzed by H&E and IHC staining. Scale bars, 100 μm.


A Cell viability was measured by MTS assay . Each concentration was performed in triplicate . The exact IC50 value was presented as Mean ± SEM from three independent experiments ( n = 3 ) and shown in Appendix Table S6 . IC50 fold changes of wild type and mutant EML4 - ALK Ba / F3 cell lines to parental Ba / F3 cell line of XMU - MP - 5 and crizotinib were determined .

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A Cell viability was measured by MTS assay. Each concentration was performed in triplicate. The exact IC50 value was presented as Mean ± SEM from three independent experiments (n = 3) and shown in Appendix Table S6. IC50 fold changes of wild type and mutant EML4-ALK Ba/F3 cell lines to parental Ba/F3 cell line of XMU-MP-5 and crizotinib were determined.


B Heatmap of the anti - proliferative activity of ALK inhibitors across Ba / F3 cell lines with different ALK mutations .

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B Heatmap of the anti-proliferative activity of ALK inhibitors across Ba/F3 cell lines with different ALK mutations.


C , D In vivo activity of XMU - MP - 5 against L1196M xenograft mouse model . BALB / c nude mice bearing L1196M Ba / F3 xenograft tumors were treated with vehicle , 30 mg / kg or 60 mg / kg XMU - MP - 5 by tail vein injection once daily . Tumor volume ( C ) and tumor weight ( D ) were shown as mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test for ( C ) , N . S . , P = 0 . 1482 ; * P = 0 . 037 . Statistical comparasions were performed using a one - way ANOVA with Dunnett test for ( D ) , N . S . , P = 0 . 2162 ; * P = 0 . 0122 .

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C, D In vivo activity of XMU-MP-5 against L1196M xenograft mouse model. BALB/c nude mice bearing L1196M Ba/F3 xenograft tumors were treated with vehicle, 30 mg/kg or 60 mg/kg XMU-MP-5 by tail vein injection once daily. Tumor volume (C) and tumor weight (D) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (C), N.S., P = 0.1482; *P = 0.037. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (D), N.S., P = 0.2162; *P = 0.0122.


E , F Efficacy of XMU - MP - 5 in G1202R Ba / F3 xenograft mouse model . BALB / c nude mice bearing G1202R Ba / F3 cells xenograft tumors were administrated with vehicle or XMU - MP - 5 by tail vein injection twice daily . Lorlatinib was used as a positive control by oral administration . Tumor volume ( E ) and tumor weight ( F ) were shown as mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test for ( E ) , Vehicle vs XMU - MP - 5 30 mg / kg , * P = 0 . 0433 ; Vehicle vs XMU - MP - 5 30 mg / kg BID , * * P = 0 . 0093 ; Vehicle vs Lorlatinib , * * P = 0 . 0029 . Statistical comparasions were performed using a one - way ANOVA with Dunnett test for ( F ) . Vehicle vs XMU - MP - 5 30 mg / kg , * * * P = 0 . 0008 ; Vehicle vs XMU - MP - 5 30 mg / kg BID , * * * P = 0 . 0003 ; Vehicle vs Lorlatinib , * * * P = 0 . 0002 .

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E, F Efficacy of XMU-MP-5 in G1202R Ba/F3 xenograft mouse model. BALB/c nude mice bearing G1202R Ba/F3 cells xenograft tumors were administrated with vehicle or XMU-MP-5 by tail vein injection twice daily. Lorlatinib was used as a positive control by oral administration. Tumor volume (E) and tumor weight (F) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (E), Vehicle vs XMU-MP-5 30 mg/kg, *P = 0.0433; Vehicle vs XMU-MP-5 30 mg/kg BID, **P = 0.0093; Vehicle vs Lorlatinib, **P = 0.0029. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (F). Vehicle vs XMU-MP-5 30 mg/kg, ***P = 0.0008; Vehicle vs XMU-MP-5 30 mg/kg BID, ***P = 0.0003; Vehicle vs Lorlatinib, ***P = 0.0002.


A Representative CT images ( upper panel ) and H & E staining ( lower panel ) of lungs from EML4 - ALK tumor - bearing mice are shown . The complete set of CT images refers to Appendix Figure S10A . Mice were treated with 30 mg / kg XMU - MP - 5 twice daily for 3 weeks . H : heart ; arrow indicated tumor . Scale bars , 200 μm . B IHC analyses of lungs from EML4 - ALK transgenic mice . pALK and Cl - caspase 3 were probed . Scale bars , 100 μm .

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

A Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10A. Mice were treated with 30 mg/kg XMU-MP-5 twice daily for 3 weeks. H: heart; arrow indicated tumor. Scale bars, 200 μm. B IHC analyses of lungs from EML4-ALK transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.


C Representative CT images ( upper panel ) and H & E staining ( lower panel ) of lungs from EML4 - ALK ( L1196M ) tumor - bearing mice are shown . The complete set of CT images refers to Appendix Figure S10B . Mice were treated with 40 mg / kg XMU - MP - 5 twice daily for 1 week . H : heart ; arrow indicated tumor . Scale bars , 200 μm . D IHC analyses of lungs from EML4 - ALK ( L1196M ) transgenic mice . pALK and Cl - caspase 3 were probed . Scale bars , 100 μm .

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

C Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK(L1196M) tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10B. Mice were treated with 40 mg/kg XMU-MP-5 twice daily for 1 week. H: heart; arrow indicated tumor. Scale bars, 200 μm. D IHC analyses of lungs from EML4-ALK(L1196M) transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.


B Inhibition zone - based assay for synergy between AMSIN and HNP1 & 2 or lysozyme C ( abbreviated as hLZc ) to the two oral Streptococcus ( SSAN and SSAL ) . Inset ( upper panel , boxed ) , a representative example of the inhibition - zone test showing the synergy . Peptide doses used here were 0 . 05 nmol / well for HNP1 & 2 and hLZc at which no visible inhibition zones were observed for both of them . At the same dose , AMSIN produced a small inhibition zone . For synergetic observation , two peptides were applied to the same well according to the doses indicated . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test ( * * * P < 0 . 001 Note : the nanomolar concentration of HNP1 & 2 was calculated from the average molecular weight of HNP1 and HNP2 .

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B Inhibition zone-based assay for synergy between AMSIN and HNP1&2 or lysozyme C (abbreviated as hLZc) to the two oral Streptococcus (SSAN and SSAL). Inset (upper panel, boxed), a representative example of the inhibition-zone test showing the synergy. Peptide doses used here were 0.05 nmol/well for HNP1&2 and hLZc at which no visible inhibition zones were observed for both of them. At the same dose, AMSIN produced a small inhibition zone. For synergetic observation, two peptides were applied to the same well according to the doses indicated. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test (***P<0.001 Note: the nanomolar concentration of HNP1&2 was calculated from the average molecular weight of HNP1 and HNP2.


C The FIC index assay for synergy between AMSIN and HNP1 & 2 or hLZc to SSAL and SSAN . The dashed line represents the cutoff of the FIC index ( 0 . 5 ) for synergy .

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C The FIC index assay for synergy between AMSIN and HNP1&2 or hLZc to SSAL and SSAN. The dashed line represents the cutoff of the FIC index (0.5) for synergy.


A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets . The peptide or the compounds used were 4 × CL . VAN , vancomycin ; RIF , rifampicin ; KAN , kanamycin . OD600 were calculated as measured values subtracting the background value from broth medium ( OD600 = 0 . 356 ) .

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A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets. The peptide or the compounds used were 4×CL. VAN, vancomycin; RIF, rifampicin; KAN, kanamycin. OD600 were calculated as measured values subtracting the background value from broth medium (OD600 = 0.356).


D Scanning electron microscopic observation of AMSIN - induced BM deformation . The doses used were 10 × CL for AMSIN and vancomycin .

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D Scanning electron microscopic observation of AMSIN-induced BM deformation. The doses used were 10×CL for AMSIN and vancomycin.


E Cytoplasmic accumulation of the soluble cell - wall precursor UMP in vancomycin - treated ( blue line ) , AMSIN - treated ( red dashed line ) or not treated ( green line ) culture of MRSA P1374 . Inset : UMP identified by UPLC - HRMS with singlely and doubly protonated m / z observed .

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E Cytoplasmic accumulation of the soluble cell-wall precursor UMP in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP identified by UPLC-HRMS with singlely and doubly protonated m/z observed.


H ESI - MS detecting the non - covalent complex between AMSIN and UMP . The quadruply protonated complex ( m / z 1304 . 8 ) is observed . Inset : comparison of intensities of the complex between two ratios of UMP : AMSIN Here , AMSIN is abbreviated as AMS due to space limitation .

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H ESI-MS detecting the non-covalent complex between AMSIN and UMP. The quadruply protonated complex (m/z 1304.8) is observed. Inset: comparison of intensities of the complex between two ratios of UMP:AMSIN Here, AMSIN is abbreviated as AMS due to space limitation.


J The dose - response unit curve of UMP binding to AMSIN , which is fitted by the BIAevaluation v2 . 0 . 1 . software using 1 : 1 Langmuir binding model .

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J The dose-response unit curve of UMP binding to AMSIN, which is fitted by the BIAevaluation v2.0.1. software using 1:1 Langmuir binding model.


B Hemolysis by AMSIN . Meucin - 18 was used as a positive control . Mean ± SD of three technical replicates is displayed .

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B Hemolysis by AMSIN. Meucin-18 was used as a positive control. Mean ± SD of three technical replicates is displayed.


C Comparison of cytotoxic effects of AMSIN and LL - 37 on HL - 60 cells . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk ; * P < 0 . 05 , * * * P < 0 . 001 , ns : no

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C Comparison of cytotoxic effects of AMSIN and LL-37 on HL-60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk; *P<0.05, ***P<0.001, ns: no significance


D An inhibition - zone assay showing the inability of AMSIN to bind DNA . The bacterium used was MRSA P1374 and the peptide dose was 1 . 0 nmol / well . Mean ± SD of three biological replicates is displayed . P values were obtained by Mann - Whitney U ( ns : no

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D An inhibition-zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significance


E The effect of AMSIN on human ion channels expressed in the CNS and heart . The asterisks mark steady state current traces after administering 100 μM AMSIN .

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E The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady state current traces after administering 100 μM AMSIN.


F Peptide - induced IL - 8 release . A549 cells were treated with AMSIN or LL - 37 for 24 h . IL - 8 levels in culture supernatants from the FBS - containing medium ( left ) or the serum - free medium ( right ) were measured with an ELISA , as described in Materials and Methods . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significance G Peptide - induced MCP - 1 release . HL - 60 cells were treated with AMSIN or LL - 37 for 24 h . MCP - 1 levels in culture supernatants from the FBS - containing medium ( left ) or the serum - free medium ( right ) were measured with an ELISA . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , ns : no

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

F Peptide-induced IL-8 release. A549 cells were treated with AMSIN or LL-37 for 24 h. IL-8 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ***P<0.001, ns: no significance G Peptide-induced MCP-1 release. HL-60 cells were treated with AMSIN or LL-37 for 24 h. MCP-1 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ns: no significance


J Survival after peritoneal infection with MRSA P1374 . The survival fraction of the vehicle - treated control group was zero out of 11 mice and the treatment group contained six mice and all survived .

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J Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle-treated control group was zero out of 11 mice and the treatment group contained six mice and all survived.


A , B Functional freeMitos association with PMN . Overlay dot plot population , representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN ( anti - CD11b - PE ) and freeMitos . Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed . CD11b - labeled PMN were incubated in absence ( A ) or presence ( B ) of freeMitos at a 1 : 5 ratio ( PMN : mitochondria ) .

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A, B Functional freeMitos association with PMN. Overlay dot plot population, representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN (anti-CD11b-PE) and freeMitos. Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed. CD11b-labeled PMN were incubated in absence (A) or presence (B) of freeMitos at a 1:5 ratio (PMN:mitochondria).


C Confocal microscopy imaging of PMN cell membrane ( red ) labeled with CellMask Orange in absence ( upper panel ) or presence ( lower panel ) of MitoTrackerTM Deep Red - labeled freeMitos ( magenta ) . PMN nuclei were stained with DAPI ( cyan ) . The white scale bars shown in the lower right of the images represent 10 μm in size .

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C Confocal microscopy imaging of PMN cell membrane (red) labeled with CellMask Orange in absence (upper panel) or presence (lower panel) of MitoTrackerTM Deep Red-labeled freeMitos (magenta). PMN nuclei were stained with DAPI (cyan). The white scale bars shown in the lower right of the images represent 10 μm in size.


D , E FreeMitos associate with PMN and modulate gene expression . Volcano plot , of 4 biological replicates , representing the gene expression modulation induced by mitochondrial DAMPs preparation ( D ) and freshly isolated functional freeMitos ( E ) in PMN .

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D, E FreeMitos associate with PMN and modulate gene expression. Volcano plot, of 4 biological replicates, representing the gene expression modulation induced by mitochondrial DAMPs preparation (D) and freshly isolated functional freeMitos (E) in PMN.


FreeMitos effect on the respiratory state of PMN . O2 consumption rates of resting or activated ( incubated with TNF - a and GM - CSF ) PMN were measured using high - resolution O2 respirometry ( Oroboros Oxygraph ) and revealed no significant differences between the basal state ( left panel ) , leak state ( middle panel ) and the maximum electron transport system capacity ( ETS , right panel ) of cellular respiration ( F ) . Data information : For panels F data are shown as means ± SEM , of 3 biological replicates . Two - way ANOVA tests followed by Tukey ' s multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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FreeMitos effect on the respiratory state of PMN. O2 consumption rates of resting or activated (incubated with TNF-a and GM-CSF) PMN were measured using high-resolution O2 respirometry (Oroboros Oxygraph) and revealed no significant differences between the basal state (left panel), leak state (middle panel) and the maximum electron transport system capacity (ETS, right panel) of cellular respiration (F). Data information: For panels F data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different (P < 0.05).


Immunoblot of mitochondrial cytochrome c oxidase ( COX IV ) and histone H3 as a loading control of PMN after co - incubations with freeMitos ( G ) with subsequent densitometry analysis ( H ) . Data information : For panels H , data are shown as means ± SEM , of 3 biological replicates . Two - way ANOVA tests followed by Tukey ' s multiple comparisons tests for each sample was performed for panels H . Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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Immunoblot of mitochondrial cytochrome c oxidase (COX IV) and histone H3 as a loading control of PMN after co-incubations with freeMitos (G) with subsequent densitometry analysis (H). Data information: For panels H, data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels H. Values without at least one common superscript letter are different (P < 0.05).


A , B FreeMitos induce PMN intracellular calcium release in PMN . Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co - incubated with 5 x 106 mitochondria ( A ) . Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo - 3 AM . The gray line represents PMN with the vehicle ( HBSS ) while the dark line represents PMN incubated with mitochondria . PMN intracellular calcium mobilization induced by either the vehicle ( HBSS ) , freeMitos or mitochondrial DAMPs and quantified by flow cytometry ( B ) . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel B , One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels B , Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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A, B FreeMitos induce PMN intracellular calcium release in PMN. Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co-incubated with 5 x 106 mitochondria (A). Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo-3 AM. The gray line represents PMN with the vehicle (HBSS) while the dark line represents PMN incubated with mitochondria. PMN intracellular calcium mobilization induced by either the vehicle (HBSS), freeMitos or mitochondrial DAMPs and quantified by flow cytometry (B). Data information: Data are shown as means ± SEM of 3 biological replicates (panel B, One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels B, Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


Functional freeMitos induce the release of PMN - derived microvesicles . A dose - response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles . PMN microvesicles labeling was performed with either anti - CD66b - FITC labelling ( C , left panel ) or anti - CD11b - PE ( C , right panel ) . Data information : Data are shown as means ± SEM of 4 biological replicates ( panel C two - way ( panel C ) ANOVA tests , followed by Dunnett ' s multiple comparisons test were performed and compared to the vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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Functional freeMitos induce the release of PMN-derived microvesicles. A dose-response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles. PMN microvesicles labeling was performed with either anti-CD66b-FITC labelling (C, left panel) or anti-CD11b-PE (C, right panel). Data information: Data are shown as means ± SEM of 4 biological replicates (panel C two-way (panel C) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


PMN - derived microparticle gating strategy is shown in panel D . Flow cytometry panel established to distinguish the PMNL - MVs from the background using the leucocyte specific integrin alpha - M beta - 2 ( CD11b ) , the granulocyte specific carcinoembryonic antigen - related cell adhesion molecule 8 ( CD66b ) , and size reference beads . PMN were either incubated with the vehicle ( Tyrode ' s Buffer , upper panels ) or freeMitos ( lower panels ) . Following co - incubation , PMN were washed twice and resuspended in HBSS before processing on flow cytometer . Only single events , identified by the singlets gate ( left upper and lower panels ) using side scatter area ( SSC - A ) vs side scatter heights ( SSC - H ) were analyzed . Light scatter ( side scatter ( SSC ) and forward scatter ( FSC ) ) approach for determining the background noise in HBSS . Histogram displaying the fluorescence intensity of samples labelled with either CD11b - FITC or CD66b - PE . Overlay dot plot of PMNL - MVs population and size reference beads , representative of 4 biological replicates , each performed in 2 technical replicates .

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PMN-derived microparticle gating strategy is shown in panel D. Flow cytometry panel established to distinguish the PMNL-MVs from the background using the leucocyte specific integrin alpha-M beta-2 (CD11b), the granulocyte specific carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b), and size reference beads. PMN were either incubated with the vehicle (Tyrode's Buffer, upper panels) or freeMitos (lower panels). Following co-incubation, PMN were washed twice and resuspended in HBSS before processing on flow cytometer. Only single events, identified by the singlets gate (left upper and lower panels) using side scatter area (SSC-A) vs side scatter heights (SSC-H) were analyzed. Light scatter (side scatter (SSC) and forward scatter (FSC)) approach for determining the background noise in HBSS. Histogram displaying the fluorescence intensity of samples labelled with either CD11b-FITC or CD66b-PE. Overlay dot plot of PMNL-MVs population and size reference beads, representative of 4 biological replicates, each performed in 2 technical replicates.


E Visual confirmation of PMN and PMN - derived microvesicles . Transmission electron microscopy ( TEM ) imaging of PMN - derived microvesicles ( black arrowhead ) , representative of 3 biological replicates , from resting or activated PMN ( 106 cells ) incubated with either mitochondria , mitochondrial DAMPs or fMLP as positive control . The black scale bars are shown in the lower right of the images for the vehicles ( 400 nm ) , mitochondria ( 600 nm ) , DAMPs ( 600 nm ) and fMLP ( 500 nm ) conditions .

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E Visual confirmation of PMN and PMN-derived microvesicles. Transmission electron microscopy (TEM) imaging of PMN-derived microvesicles (black arrowhead), representative of 3 biological replicates, from resting or activated PMN (106 cells) incubated with either mitochondria, mitochondrial DAMPs or fMLP as positive control. The black scale bars are shown in the lower right of the images for the vehicles (400 nm), mitochondria (600 nm), DAMPs (600 nm) and fMLP (500 nm) conditions.


F Quantification of resting PMN microvesicles produced by PMN ( 106 cells ) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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F Quantification of resting PMN microvesicles produced by PMN (106 cells) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


G Quantification of calpain activity , by fluorescence assay , in cell lysate of either resting ( left panel ) or activated ( right panel ) PMN ( 2 x 105 cells ) incubated in presence of freeMitos for 2 hr . Data information : Data are shown as means ± SEM of 4 biological replicates ( panel G ) . One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels G . Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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G Quantification of calpain activity, by fluorescence assay, in cell lysate of either resting (left panel) or activated (right panel) PMN (2 x 105 cells) incubated in presence of freeMitos for 2 hr. Data information: Data are shown as means ± SEM of 4 biological replicates (panel G). One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels G. Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


H ELISA quantification of IL - 8 concentrations present in the supernatant following the incubation of PMN ( 106 cells ) with freeMitos ( 107 ) , DAMPs ( 25 µg ) or fMLP ( 1 µM ) for 2 hr . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel H ) One - way ( panel H ) ANOVA tests , followed by Dunnett ' s multiple comparisons test were performed and compared to the vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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H ELISA quantification of IL-8 concentrations present in the supernatant following the incubation of PMN (106 cells) with freeMitos (107), DAMPs (25 µg) or fMLP (1 µM) for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel H) One-way (panel H) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


A Structure of autoinhibited ScV1 . The catalytic A ( pink ) and non catalytic B ( green ) subunits are arranged as a heterohexamer around the central rotor subunits D ( red ) and F ( cyan ) . The three peripheral stalks ( EG heterodimers 1 - 3 , blue and orange , respectively ) are bound at the periphery of the B subunits . The inhibitory H subunit ( yellow ) is unique to the eukaryotic V - ATPase . Unless otherwise stated , the coloring scheme used here for the V1 subunits will be used from here on .

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A Structure of autoinhibited ScV1. The catalytic A (pink) and non catalytic B (green) subunits are arranged as a heterohexamer around the central rotor subunits D (red) and F (cyan). The three peripheral stalks (EG heterodimers 1-3, blue and orange, respectively) are bound at the periphery of the B subunits. The inhibitory H subunit (yellow) is unique to the eukaryotic V-ATPase. Unless otherwise stated, the coloring scheme used here for the V1 subunits will be used from here on.


B Interaction of the peripheral stalks with the catalytic hexamer . Each peripheral stalk crosses a non - catalytic A / B interface on its way from the top of the V1 to the base of the catalytic core . Representative view of the interaction is shown for EG3 .

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B Interaction of the peripheral stalks with the catalytic hexamer. Each peripheral stalk crosses a non-catalytic A/B interface on its way from the top of the V1 to the base of the catalytic core. Representative view of the interaction is shown for EG3.


C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer . The peripheral stalks are bound to the top of the B subunits via their C - termini ( boxed ) . The electron density ( contoured at 1 . 2 σ ) for the C - terminal domains of EG1 and EG2 is shown in the panels to the left and right . D The structure of autoinhibited ScV1rotated 90 ° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3 . Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the " bulge " in subunit G ( indicated with an arrow ) . The box shows electron density ( contoured at 1 . 2 σ ) for the EG3 N - termini contacting the C - terminal domain of the A subunit of a closed catalytic site ( see asterisk ) . The horizontal lines on the structure indicate the sections shown in panels E ( upper two lines ) and F ( lower two lines ) .

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

C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer. The peripheral stalks are bound to the top of the B subunits via their C-termini (boxed). The electron density (contoured at 1.2 σ) for the C-terminal domains of EG1 and EG2 is shown in the panels to the left and right.D The structure of autoinhibited ScV1rotated 90° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3. Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the "bulge" in subunit G (indicated with an arrow). The box shows electron density (contoured at 1.2 σ) for the EG3 N-termini contacting the C-terminal domain of the A subunit of a closed catalytic site (see asterisk). The horizontal lines on the structure indicate the sections shown in panels E (upper two lines) and F (lower two lines).


E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks ( EG1 - 3 ) . View is rotated 90 ° from the view in A , looking up the central rotor . The A / B pairs forming the three catalytic sites are designated ( AB ) 1 - 3 according to which of the three peripheral stalks is bound to each B subunit . For example , ( AB ) 1 has peripheral stalk EG1 bound to its B subunit . The black arrowheads indicate the non - catalytic A / B interfaces .

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E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks (EG1-3). View is rotated 90° from the view in A, looking up the central rotor. The A/B pairs forming the three catalytic sites are designated (AB)1-3 according to which of the three peripheral stalks is bound to each B subunit. For example, (AB)1 has peripheral stalk EG1 bound to its B subunit. The black arrowheads indicate the non-catalytic A/B interfaces.


F Section through the ScV1 electron density map at the level of the N - termini of the peripheral stalks . Note the change in position of the N - termini of the peripheral stalks , which have each crossed a non - catalytic interface ( black arrowheads in E ) and are now in proximity to the C - termini of the catalytic A subunits ( ACT ) of the adjacent A / B pairs . While EG1 and EG2 are now near ACT from ( AB ) 3 and ( AB ) 1 , respectively ( see double headed arrows ) , only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from ( AB ) 2 ( see asterisk ) .

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F Section through the ScV1 electron density map at the level of the N-termini of the peripheral stalks. Note the change in position of the N-termini of the peripheral stalks, which have each crossed a non-catalytic interface (black arrowheads in E) and are now in proximity to the C-termini of the catalytic A subunits (ACT) of the adjacent A/B pairs. While EG1 and EG2 are now near ACT from (AB)3 and (AB)1, respectively (see double headed arrows), only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from (AB)2 (see asterisk).


A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed , highlighting the core conserved catalytic complex labeled as " Catalytic core " . The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1 , whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex . Notably , the prokaryotic V1 is an active ATP hydrolase ( Arai et al , 2009 ) . The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left .

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

A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as "Catalytic core". The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left.


B Conformational differences in the catalytic A subunits . The catalytic A subunits are shown aligned by their N - terminal β barrels , highlighting the change in conformation of C - terminal α helical bundles associated with the closed ( ( AB ) 2 and ( AB ) 3 ; dark and light gray , respectively ) and open ( ( AB ) 1 ; magenta ) catalytic sites . Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis .

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B Conformational differences in the catalytic A subunits. The catalytic A subunits are shown aligned by their N-terminal β barrels, highlighting the change in conformation of C-terminal α helical bundles associated with the closed ((AB)2 and (AB)3; dark and light gray, respectively) and open ((AB)1; magenta) catalytic sites. Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis.


C ScV1 ' s catalytic hexamer contains one open and two closed catalytic sites . Side view of the three catalytic hexamer A / B pairs ( shown in surface representation ) . Note that the two closed pairs ( AB ) 2 and 3 are non - equivalently closed .

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C ScV1's catalytic hexamer contains one open and two closed catalytic sites. Side view of the three catalytic hexamer A/B pairs (shown in surface representation). Note that the two closed pairs (AB)2 and 3 are non-equivalently closed.


D Alignment of the A3B3DF from ScV1 and nucleotide - free EhV1 by the catalytic hexamers . The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2 , EhV1 is in blue . The alignment shows the difference in position of the central rotor subunit , D . Note that the C - terminus of subunit D ( DCT ) is longer in eukaryotic V - ATPase ( see electron density contoured at 1 . 2 σ in the box to the right ) . E ScV1 and EhV1are halted in different rotational positions . Alignment of the A3B3DF of ScV1 and nucleotide - free EhV1 ( blue , with dashed lines drawn through the A subunits ) by the central stalk subunit D , reveals that the two catalytic cores are halted in different rotational positions . The autoinhibited ScV1 requires 40 ° rotation to overlay its catalytic hexamer with that of the EhV1 .

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

D Alignment of the A3B3DF from ScV1 and nucleotide-free EhV1 by the catalytic hexamers. The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2, EhV1 is in blue. The alignment shows the difference in position of the central rotor subunit, D. Note that the C-terminus of subunit D (DCT) is longer in eukaryotic V-ATPase (see electron density contoured at 1.2 σ in the box to the right).E ScV1 and EhV1are halted in different rotational positions. Alignment of the A3B3DF of ScV1 and nucleotide-free EhV1 (blue, with dashed lines drawn through the A subunits) by the central stalk subunit D, reveals that the two catalytic cores are halted in different rotational positions. The autoinhibited ScV1 requires 40° rotation to overlay its catalytic hexamer with that of the EhV1.


F Comparison of the D subunits of ScV1 ( red ) and nucleotide free EhV1 ( blue ) . HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue .

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F Comparison of the D subunits of ScV1 (red) and nucleotide free EhV1 (blue). HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue.


A ScV1 - H subunit interactions . Region of electron density ( contoured at 1 . 2 σ ) outlined by the box in the side view of ScV1 shown to the right . The H subunit N - terminal domain ( HNT ) is bound to the N - terminal α helices of peripheral stalk EG1 while its globular C - terminal domain ( HCT ) is bound at the base of the catalytic hexamer .

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A ScV1-H subunit interactions. Region of electron density (contoured at 1.2 σ) outlined by the box in the side view of ScV1 shown to the right. The H subunit N-terminal domain (HNT) is bound to the N-terminal α helices of peripheral stalk EG1 while its globular C-terminal domain (HCT) is bound at the base of the catalytic hexamer.


B Interaction between HNT and peripheral stalk EG1 . View of HNT as in panel A rotated 110 ° along the horizontal , showing the α helical HEAT repeats of HNT forming a pocket for binding the N - termini of peripheral stalk EG1 . HNT ' s α helices forming the pocket are labeled as in ( Sagermann et al , 2001 ) . Pro197 on HNT ( red sphere ) has been previously shown to cross - link subunit E ( Jefferies Forgac , 2008 ) . C Interaction between HCT and the base of the catalytic hexamer . The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT ( highlighted in magenta ) that is seen to contact two short segments of the C - terminal domain of the subunit G from the open catalytic site ( AB ) 1 ( highlighted in purple ) and two turns of the N - terminal a helix of the central rotor subunit D ( highlighted in blue ) .

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B Interaction between HNT and peripheral stalk EG1. View of HNT as in panel A rotated 110° along the horizontal, showing the α helical HEAT repeats of HNT forming a pocket for binding the N-termini of peripheral stalk EG1. HNT's α helices forming the pocket are labeled as in (Sagermann et al, 2001). Pro197 on HNT (red sphere) has been previously shown to cross-link subunit E (Jefferies Forgac, 2008).C Interaction between HCT and the base of the catalytic hexamer. The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT (highlighted in magenta) that is seen to contact two short segments of the C-terminal domain of the subunit G from the open catalytic site (AB)1 (highlighted in purple) and two turns of the N-terminal a helix of the central rotor subunit D (highlighted in blue).


D HCT binds to the open ( AB ) 1 pair . View of ScV1 looking along the central rotor subunit D towards the cytoplasm , showing the position of the H subunit binding at the interface of the open A / B pair ( AB ) 1 . The catalytic hexamer is shown in surface representation and the central rotor , peripheral stalks and subunit H are shown as ribbons .

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D HCT binds to the open (AB)1 pair. View of ScV1 looking along the central rotor subunit D towards the cytoplasm, showing the position of the H subunit binding at the interface of the open A/B pair (AB)1. The catalytic hexamer is shown in surface representation and the central rotor, peripheral stalks and subunit H are shown as ribbons.


C Growth phenotype of H subunit mutants . Deletion of V - ATPase subunits from yeast results in a conditional lethal phenotype ( Vma - ) characterized by an inability to grow at pH 7 . 5 in the presence of 60 mM CaCl2 ( upper panel ) , but growth is observed at pH 5 ( lower panel ) . Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed . Both humanH subunit isoforms ( HsH1 , HsH2 ) , a chimeric H subunit ( HChim ) containing ScHNT and HsHCT , mutations in yeast intended to mimic the length of the human H loop sequence and accessibility ( Hloop ) , and a point mutation of the conserved aspartic acid residue in the loop sequence ( HD410A ) are shown compared to the H subunit deletion ( ΔH ) and wild type H on a plasmid ( H WTpl ) . All grow on pH 5 medium , but the human isoforms failed to complement the deletion phenotype .

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

C Growth phenotype of H subunit mutants. Deletion of V-ATPase subunits from yeast results in a conditional lethal phenotype (Vma-) characterized by an inability to grow at pH 7.5 in the presence of 60 mM CaCl2 (upper panel), but growth is observed at pH 5 (lower panel). Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed. Both humanH subunit isoforms (HsH1, HsH2), a chimeric H subunit (HChim) containing ScHNT and HsHCT, mutations in yeast intended to mimic the length of the human H loop sequence and accessibility (Hloop), and a point mutation of the conserved aspartic acid residue in the loop sequence (HD410A) are shown compared to the H subunit deletion (ΔH) and wild type H on a plasmid (H WTpl). All grow on pH 5 medium, but the human isoforms failed to complement the deletion phenotype.


D ATPase activity of H subunit mutants . The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH , for comparison of MgATPase activity . Average specific activities are shown plotted + / - s . e . m from two independent purifications . The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity ( Parra et al , 2000 ; Zhang et al , 2003 ) , whereas ScV1containing plasmid - borne wild type H ( H WTpl ) exhibits low levels of activity likely due to reduced expression from the low copy plasmid . The HChim and HLoop mutants lead to a loss of activity silencing , displaying nearly the same activity as ScV1ΔH .

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D ATPase activity of H subunit mutants. The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH, for comparison of MgATPase activity. Average specific activities are shown plotted +/- s.e.m from two independent purifications. The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity (Parra et al, 2000; Zhang et al, 2003), whereas ScV1containing plasmid-borne wild type H (H WTpl) exhibits low levels of activity likely due to reduced expression from the low copy plasmid. The HChim and HLoop mutants lead to a loss of activity silencing, displaying nearly the same activity as ScV1ΔH.


E SDS - PAGE and Western Blot analysis of ScV1 preparations . Left panel , 15 % SDS PAGE of ScV1 purified from the subunit H mutant and WT strains . Note that H WTch is purified from a yeast strain deleted for the C subunit ( used for crystallization ) . The remaining strains contain subunit C , which is co - purified with ScV1 in varying levels . Note that while H WTch ScV1 , which is expressed from a strain deleted for subunit C , displays no detectable MgATPase activity , the H subunit mutants co - purified with the most C subunit display the least activity , suggesting that absence or presence of subunit C has no effect on MgATPase activity . Right panel , immunoblot probed with an antibody directed against the N - terminal Myc tagged mutant H subunits expressed from a plasmid . Note that the levels of mutant H expression does not appear to vary from strain to strain .

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

E SDS-PAGE and Western Blot analysis of ScV1 preparations. Left panel, 15% SDS PAGE of ScV1 purified from the subunit H mutant and WT strains. Note that H WTch is purified from a yeast strain deleted for the C subunit (used for crystallization). The remaining strains contain subunit C, which is co-purified with ScV1 in varying levels. Note that while H WTch ScV1, which is expressed from a strain deleted for subunit C, displays no detectable MgATPase activity, the H subunit mutants co-purified with the most C subunit display the least activity, suggesting that absence or presence of subunit C has no effect on MgATPase activity. Right panel, immunoblot probed with an antibody directed against the N-terminal Myc tagged mutant H subunits expressed from a plasmid. Note that the levels of mutant H expression does not appear to vary from strain to strain.


A Overlay of ScV1 and the cryo - EM structure of the V1Vo holoenzyme in " state 2 " ( Zhao et al , 2015 ) . The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers , thus highlighting structural differences in the central rotor and peripheral stator subunits . Coloring of ScV1Vo ( shown as semi - transparent ribbons ) is as used throughout for ScV1 for all but the A3B3F ( tan ) , subunit H ( dark green ) , subunit C and Vo domain ( gray ) . The EG1 - HNT interaction is boxed . Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo .

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

A Overlay of ScV1 and the cryo-EM structure of the V1Vo holoenzyme in "state 2" (Zhao et al, 2015). The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers, thus highlighting structural differences in the central rotor and peripheral stator subunits. Coloring of ScV1Vo (shown as semi-transparent ribbons) is as used throughout for ScV1 for all but the A3B3F (tan), subunit H (dark green), subunit C and Vo domain (gray). The EG1-HNT interaction is boxed. Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo.


B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo . The boxed view in A is shown enlarged . The N - terminal α helices of peripheral stalk EG1 and HNT change in conformation , likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation .

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B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo. The boxed view in A is shown enlarged. The N-terminal α helices of peripheral stalk EG1 and HNT change in conformation, likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation.


C View as in A rotated 150º around the vertical . Comparisons of peripheral stalk EG3 and HCT are boxed . D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo . In ScV1Vo , EG3 binds to the C subunit head domain ( Chead ; gray ) whereas in ScV1 , EG3 is bent towards the C - terminal domain of a catalytic A subunit ( enlarged view of box labeled EG3 in panel C ) . E Comparison of HCT ' s conformation in ScV1 versus ScV1Vo . Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT ; for clarity only aNT , HCT and the central rotor ( DF ) are shown . In ScV1Vo , the most C - terminal α helix ( dark green ) of HCT ( green ) binds to aNT ( light gray ) , with the inhibitory loop ( green spheres ) rotated away and inaccessible for binding the B subunit and the central stalk ( transparent red ) . In ScV1 , the HCT ( yellow ) inhibitory loop ( yellow spheres ) binds to the central stalk subunit D ( red ) and the B subunit of the open ( AB ) 1 catalytic site . The difference in conformation between ScV1Vo and ScV1 involves a ~ 150 ° rotation of HCT . Note that the HCT loop ( spheres ) is rotated away from the central stalk in ScV1Vo whereas in ScV1 , the C - terminal α helix ( orange ) involved in binding aNT is rotated away and unavailable for rebinding to the membrane .

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C View as in A rotated 150º around the vertical. Comparisons of peripheral stalk EG3 and HCT are boxed.D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo. In ScV1Vo, EG3 binds to the C subunit head domain (Chead; gray) whereas in ScV1, EG3 is bent towards the C-terminal domain of a catalytic A subunit (enlarged view of box labeled EG3 in panel C).E Comparison of HCT's conformation in ScV1 versus ScV1Vo. Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT; for clarity only aNT, HCT and the central rotor (DF) are shown. In ScV1Vo, the most C-terminal α helix (dark green) of HCT (green) binds to aNT (light gray), with the inhibitory loop (green spheres) rotated away and inaccessible for binding the B subunit and the central stalk (transparent red). In ScV1, the HCT (yellow) inhibitory loop (yellow spheres) binds to the central stalk subunit D (red) and the B subunit of the open (AB)1 catalytic site. The difference in conformation between ScV1Vo and ScV1 involves a ~150° rotation of HCT. Note that the HCT loop (spheres) is rotated away from the central stalk in ScV1Vo whereas in ScV1, the C-terminal α helix (orange) involved in binding aNT is rotated away and unavailable for rebinding to the membrane.


F HCT conformational changes . Available subunit H structures as obtained for isolated H ( Sagermann et al , 2001 ) ( purple ) , in ScV1Vo ( Zhao et al , 2015 ) ( green ) and in ScV1 ( yellow ) are shown aligned by their N - terminal domains ( HNT ) illustrating the conformational flexibility of HCT . The loop involved in inhibition of ScV1 is shown as red spheres , highlighting the 180º rotation of HCT when going from the conformation of isolated H ( HCT 1ho8 ) to the inhibitory conformation in ScV1 ( HCT ScV1 ) .

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F HCT conformational changes. Available subunit H structures as obtained for isolated H (Sagermann et al, 2001) (purple), in ScV1Vo (Zhao et al, 2015) (green) and in ScV1 (yellow) are shown aligned by their N-terminal domains (HNT) illustrating the conformational flexibility of HCT. The loop involved in inhibition of ScV1 is shown as red spheres, highlighting the 180º rotation of HCT when going from the conformation of isolated H (HCT 1ho8) to the inhibitory conformation in ScV1(HCT ScV1).


The rapZ mutants were expressed from plasmids under control of the PBAD promoter ( induction with arabinose , repression by glucose ) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in ( b ) Northern blotting experiment detecting GlmZ ( top panel ) and 5S rRNA ( bottom panel ; loading control ) to assess the degree of GlmZ cleavage triggered by the RapZ variants . Processed GlmZ is indicated with an asterisk . Strains Z8 ( wild - type , lanes 1 , 2 ) and Z28 ( ∆ rapZ , all other lanes were used . Strain Z28 carried plasmid pBAD33 ( lanes 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of the indicated RapZ

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The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (b) Northern blotting experiment detecting GlmZ (top panel) and 5S rRNA (bottom panel; loading control) to assess the degree of GlmZ cleavage triggered by the RapZ variants. Processed GlmZ is indicated with an asterisk. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


The rapZ mutants were expressed from plasmids under control of the PBAD promoter ( induction with arabinose , repression by glucose ) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in ( c ) GlmS protein ( MW = 66 . 9 kDa ) levels in the same cultures as revealed by SDS - PAGE of total protein extracts followed by Coomassie blue staining . Strains Z8 ( wild - type , lanes 1 , 2 ) and Z28 ( ∆ rapZ , all other lanes were used . Strain Z28 carried plasmid pBAD33 ( lanes 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of the indicated RapZ

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The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (c) GlmS protein (MW = 66.9 kDa) levels in the same cultures as revealed by SDS-PAGE of total protein extracts followed by Coomassie blue staining. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


The rapZ mutants were expressed from plasmids under control of the PBAD promoter ( induction with arabinose , repression by glucose ) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in ( d ) β - Galactosidase activities produced by the same cultures and replicates ( mean values and standard deviations are shown ) . Cells carry a glmS ' - lacZ fusion on the chromosome , which requires base - pairing with sRNA GlmZ for high expression . Strains Z8 ( wild - type columns 1 , 2 ) and Z28 all other columns ) were used . Strain Z28 carried plasmid pBAD33 columns 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of the indicated RapZ

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The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (d) β-Galactosidase activities produced by the same cultures and replicates (mean values and standard deviations are shown). Cells carry a glmS'-lacZ fusion on the chromosome, which requires base-pairing with sRNA GlmZ for high expression. Strains Z8 (wild-type columns 1, 2) and Z28 all other columns) were used. Strain Z28 carried plasmid pBAD33 columns 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


C ) MXE saturation analysis . Whereas increasing amounts of RNA - seq reads should lead to the confirmation of further MXE candidates , more RNA - seq reads might also result in the rejection of previously validated MXEs . The green curves show the number of validated MXEs in relation to the percentage of total RNA - seq reads used for validation . The orange curves indicate the number of initially ' validated MXEs ' that were rejected with increasing amounts of reads . Grey dashed lines indicate the point of saturation , which is defined as the point where a two - fold increase in reads leads to rejection of less than 1 % of the validated MXEs . Of note , whereas the rejection of validated MXEs saturates with 20 % of the data the amount of novel MXE validations is still rapidly increasing .

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C) MXE saturation analysis. Whereas increasing amounts of RNA-seq reads should lead to the confirmation of further MXE candidates, more RNA-seq reads might also result in the rejection of previously validated MXEs. The green curves show the number of validated MXEs in relation to the percentage of total RNA-seq reads used for validation. The orange curves indicate the number of initially 'validated MXEs' that were rejected with increasing amounts of reads. Grey dashed lines indicate the point of saturation, which is defined as the point where a two-fold increase in reads leads to rejection of less than 1% of the validated MXEs. Of note, whereas the rejection of validated MXEs saturates with 20% of the data the amount of novel MXE validations is still rapidly increasing.


E ) Size and distribution of multi - cluster MXEs .

E) Size and distribution of multi-cluster MXEs.


A ) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM . Here , we used the Gini coefficient , which is a measure of the inequality among values of a frequency distribution ( Ceriani & Verme , 2012 ) and has successfully been used to determine tissue - enriched gene sets ( Zhang et al , 2017 ) , to determine highly tissue - specific MXEs ( maximum normalized Gini index of cluster ) and MXEs with a broad tissue expression distribution ( minimum Gini index ) . For each MXE cluster the percent - spliced - in ( PSI ) value of the ubiquitous MXE ( minimum Gini index ) is subtracted from the PSI value of the specific MXE ( maximum Gini index of cluster ) ( delta PSI value ) and scaled between - 1 ( broad tissue distribution ) and 1 ( highly tissue specific ) . Each column represents an MXE pair and each row represents MXE expression in a tissue , cell - type , or at a developmental time point . The bar graph summarizes counts where the specific MXE is 1 . 5 fold more spliced in than the ubiquitous MXE .

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A) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM. Here, we used the Gini coefficient, which is a measure of the inequality among values of a frequency distribution (Ceriani & Verme, 2012) and has successfully been used to determine tissue-enriched gene sets (Zhang et al, 2017), to determine highly tissue-specific MXEs (maximum normalized Gini index of cluster) and MXEs with a broad tissue expression distribution (minimum Gini index). For each MXE cluster the percent-spliced-in (PSI) value of the ubiquitous MXE (minimum Gini index) is subtracted from the PSI value of the specific MXE (maximum Gini index of cluster) (delta PSI value) and scaled between -1 (broad tissue distribution) and 1 (highly tissue specific). Each column represents an MXE pair and each row represents MXE expression in a tissue, cell-type, or at a developmental time point. The bar graph summarizes counts where the specific MXE is 1.5 fold more spliced in than the ubiquitous MXE.


C ) Heatmap showing the the delta PSI values ( PSI value of the non - SNP - containing MXE subtracted from the PSI value of the SNP - containing MXE ) of MXE clusters containing pathogenic SNPs scaled between - 1 and 1 ( blue = high expression non - SNP - containing MXE , red = high expression SNP - containing MXE ) . Columns represent MXE clusters and rows tissues , cell types , and developmental stages . The column bar graph summarizes counts where the SNP - containing MXE is 1 . 5 fold more expressed than the non - SNP - containing MXE , whereas the row bar graph shows this for each tissue , cell type , and developmental stage .

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C) Heatmap showing the the delta PSI values (PSI value of the non-SNP-containing MXE subtracted from the PSI value of the SNP-containing MXE) of MXE clusters containing pathogenic SNPs scaled between -1 and 1 (blue = high expression non-SNP-containing MXE, red = high expression SNP-containing MXE). Columns represent MXE clusters and rows tissues, cell types, and developmental stages. The column bar graph summarizes counts where the SNP-containing MXE is 1.5 fold more expressed than the non-SNP-containing MXE, whereas the row bar graph shows this for each tissue, cell type, and developmental stage.


B atg19Δ cells containing HTB ‐ Atg19 were grown to mid ‐ log phase . Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping . Phosphorylation sites : enlarged ; lysine substitutions : gray ; Atg11 binding region : green .

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B atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green.


C atg19Δ cells containing GFP ‐ Atg19 wild ‐ type or mutants as indicated were grown to mid ‐ log phase . Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild ‐ type . Atg19 expression was assessed by anti ‐ GFP Western blotting .

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C atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild‐type. Atg19 expression was assessed by anti‐GFP Western blotting.


D atg19Δ GFP ‐ Atg11 Atg1 ‐ TAP cells containing myc ‐ Atg19 as indicated were grown to log phase . Atg11 was immunoprecipitated , and bound Atg19 was analyzed by Western blotting .

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D atg19Δ GFP‐Atg11 Atg1‐TAP cells containing myc‐Atg19 as indicated were grown to log phase. Atg11 was immunoprecipitated, and bound Atg19 was analyzed by Western blotting.


E GST ‐ fused propeptide of Ape1 , Atg19 as indicated , and a His ‐ tagged fragment of Atg11 ( amino acids 685 - 1178 ) were purified from Escherichia coli , mixed , and propeptide ‐ bound proteins were isolated and analyzed by Coomassie staining . Quantification of three individual experiments is shown . Error bars represent standard deviation .

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E GST‐fused propeptide of Ape1, Atg19 as indicated, and a His‐tagged fragment of Atg11 (amino acids 685-1178) were purified from Escherichia coli, mixed, and propeptide‐bound proteins were isolated and analyzed by Coomassie staining. Quantification of three individual experiments is shown. Error bars represent standard deviation.


A GFP ‐ Atg11 Ape1 ‐ mRuby atg19Δ cells containing myc ‐ Atg19 as indicated and Cup1 ‐ Ape1 were grown to log phase . Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h , and autophagy was induced by treating cells for 1 h with rapamycin . Scale bar , 5 μm .

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A GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm.


B Ape1 ‐ mRuby atg19Δ cells containing GFP ‐ Atg19 wild ‐ type or GFP ‐ Atg19 ‐ 3A were analyzed in log phase . Scale bar , 5 μm .

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B Ape1‐mRuby atg19Δ cells containing GFP‐Atg19 wild‐type or GFP‐Atg19‐3A were analyzed in log phase. Scale bar, 5 μm.


C GFP ‐ Atg8 Ape1 ‐ mRuby atg19Δ cells containing myc ‐ Atg19 as indicated and Cup1 ‐ Ape1 were analyzed as in ( A ) . Scale bar , 5 μm .

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C GFP‐Atg8 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were analyzed as in (A). Scale bar, 5 μm.


B atg34Δ yeast cells containing HTB ‐ Atg34 were grown to mid ‐ log phase and treated with rapamycin . Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping . Phosphorylation sites : enlarged ; Atg11 binding region : green ; lysine substitution : gray .

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B atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray.


C Ams1 ‐ GFP atg19Δatg34Δ cells containing protein A ( Stag ) ‐ tagged Atg34 as indicated were starved for 4 h . Processing of endogenous Ams1 ‐ GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1 ‐ GFP normalized to the wild ‐ type . Expression of Atg34 proteins was assessed by anti ‐ protein A Western blotting .

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C Ams1‐GFP atg19Δatg34Δ cells containing protein A (Stag)‐tagged Atg34 as indicated were starved for 4 h. Processing of endogenous Ams1‐GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1‐GFP normalized to the wild‐type. Expression of Atg34 proteins was assessed by anti‐protein A Western blotting.


E GST , GST ‐ Atg19 , and GST ‐ Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1 ‐ TAP bound to IgG magnetic beads .

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E GST, GST‐Atg19, and GST‐Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1‐TAP bound to IgG magnetic beads.


F In vitro phosphorylation of recombinant Atg19 , Atg19 ‐ 3A , and Atg19 ‐ 3D using wild ‐ type and kinase ‐ dead Atg1 ‐ D211A as described in ( E ) .

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F In vitro phosphorylation of recombinant Atg19, Atg19‐3A, and Atg19‐3D using wild‐type and kinase‐dead Atg1‐D211A as described in (E).


A Wild ‐ type , hrr25 ‐ ts , and hrr25 ‐ ts cells containing HRR25 on a centromeric plasmid were grown to mid ‐ log phase at 18 ° C followed by 2 and 4 h of log ‐ phase growth at 37 ° C . Ape1 processing was analyzed by Western blotting .

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A Wild‐type, hrr25‐ts, and hrr25‐ts cells containing HRR25 on a centromeric plasmid were grown to mid‐log phase at 18°C followed by 2 and 4 h of log‐phase growth at 37°C. Ape1 processing was analyzed by Western blotting.


B atg19Δ and atg19Δ hrr25 ‐ ts cells containing GFP ‐ Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37 ° C .

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B atg19Δ and atg19Δ hrr25‐ts cells containing GFP‐Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37°C.


C GFP ‐ Atg11 , GFP ‐ Atg11 hrr25 ‐ ts , and GFP ‐ Atg11 atg1 ‐ D211A cells were analyzed for GFP ‐ Atg11 dot formation . Quantification of at least 70 cells in three individual experiments is shown . Error bars represent standard deviation . Scale bar , 3 μm .

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C GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg1‐D211A cells were analyzed for GFP‐Atg11 dot formation. Quantification of at least 70 cells in three individual experiments is shown. Error bars represent standard deviation. Scale bar, 3 μm.


D GFP ‐ Atg11 , GFP ‐ Atg11 hrr25 ‐ ts , and GFP ‐ Atg11 atg19Δ cells containing BFP ‐ Ape1 and Cup1 ‐ Ape1 were grown to log phase . Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h . coloc . : Cells with GFP ‐ Atg11 / BFP ‐ Ape1 co ‐ localization . n = 50 ( wt ) , 84 ( hrr25 ‐ ts ) , 127 ( atg19Δ ) . Scale bar , 3 μm .

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D GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg19Δ cells containing BFP‐Ape1 and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h. coloc.: Cells with GFP‐Atg11/BFP‐Ape1 co‐localization. n = 50 (wt), 84 (hrr25‐ts), 127 (atg19Δ). Scale bar, 3 μm.


E Hrr25 was immunoprecipitated from Hrr25 ‐ TAP cells and as a control from a non ‐ tagged wild ‐ type strain . In vitro phosphorylation of recombinant Atg19 , Atg19 ‐ 3A , Atg34 , and GST using the kinase ‐ bound beads was performed as described in Fig E . Both Atg19 panels are from the same gel with the same exposure .

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E Hrr25 was immunoprecipitated from Hrr25‐TAP cells and as a control from a non‐tagged wild‐type strain. In vitro phosphorylation of recombinant Atg19, Atg19‐3A, Atg34, and GST using the kinase‐bound beads was performed as described in Fig E. Both Atg19 panels are from the same gel with the same exposure.


( B ) Immunoblot analysis of the immunoprecipitated HA - SOX9 or FLAG - FBW7α and their pull - down products . The corresponding Western blotting from the whole cell extract ( WCE ) of the transfected HEK293 showed the levels of exogenous HA - SOX9 or FLAG - FBW7α proteins in the total input . Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation . Blots are representative from 3 independent experiments .

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(B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments.