( A ) Flag ‐ tagged DAPK ( 100 ng ) was incubated with GST ( 900 ng ) or GST - beclin ‐ 1 ( 750 ng ) in the presence of Ca2 + , calmodulin and [ γ ‐ 33P ] ATP for 30 min or 60 min . Phosphorylated proteins were visualized by X ‐ ray film exposure , and GST / GST - beclin ‐ 1 levels were visualized by Ponceau S staining . The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples .

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

(A) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST-beclin‐1 (750 ng) in the presence of Ca2+, calmodulin and [γ‐33P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST-beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples.


( B ) Flag ‐ tagged DAPK ( 60 ng ) was incubated with Flag ‐ tagged beclin 1 ( 250 ng ) , which was purified from HEK293T cells , and a kinase assay was performed for 60 min . Where indicated ( + LiCl ) , beclin ‐ 1 ‐ bound beads were first washed stringently in 0 . 5 M LiCl and 0 . 5 M KCl . Phosphorylated proteins were visualized by X ‐ ray film exposure , and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies . DAPK , death ‐ associated protein kinase ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney .

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

(B) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney.


( Aa ) Protein extracts from COS7 cells , HEK293T cells , or no extracts were added to bacterially produced GST or GST - beclin ‐ 1 . The pulled ‐ down proteins , as well as the total cell extracts , were blotted with the indicated antibodies . ( Ab ) Ponceau S staining of GST and GST - beclin ‐ 1 to which the extracts were added .

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

(Aa) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST-beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (Ab) Ponceau S staining of GST and GST-beclin‐1 to which the extracts were added.


( B ) HEK293 cells were co ‐ transfected with Flag ‐ tagged beclin 1 or with Flag ‐ tagged beclin 1 lacking the Bcl ‐ 2 ‐ binding domain ( ΔBD ) together with Bcl ‐ XL and HA ‐ tagged DAPK . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted with DAPK , Bcl ‐ XL and beclin 1 antibodies .

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

(B) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐XL and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐XL and beclin 1 antibodies.


( C ) HEK293 cells were transfected with Flag - beclin ‐ 1 or Flag - GFP , and the Flag ‐ tagged proteins were immunoprecipitated using Flag antibodies , and eluted with an excess of Flag peptides . The blot was reacted with DAPK antibodies or with Flag antibodies at different time

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

(C) HEK293 cells were transfected with Flag-beclin‐1 or Flag-GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures


( A ) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30 ° C with increasing concentrations ( 5 - 10 nmol ) of a peptide corresponding to the BH3 domain of beclin 1 ( aa 108 - 127 ) and with the same peptide in which Thr 119 was substituted by alanine . An in vitro kinase assay was performed , and the reactions were applied to Whatman filters . Total levels of TCA ‐ insoluble counts were measured and plotted against substrate concentration . Data are the mean ± s . d . of three experiments .

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

(A) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5-10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108-127) and with the same peptide in which Thr 119 was substituted by alanine. An in vitro kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments.


( C ) Flag ‐ tagged DAPK ( 60 ng ) was incubated with GST - WT beclin 1 or with GST - T119A beclin 1 ( 1000 ng ) in the presence of Ca2 + , calmodulin and ATP for 30 min . GST ‐ beclin 1 levels were visualized by Ponceau S staining , and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody ( Western blot ) .

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

(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).


( D ) HEK293 cells were co ‐ transfected with Flag ‐ tagged beclin 1 with or without ΔCaM DAPK . After 24 h , beclin 1 was immunoprecipitated from cells , using Flag antibodies , and the immunoprecipitates were reacted with phosphoThr 119 antibodies . The Ponceau staining shows equal amounts of immunoprecipitated beclin 1 . The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney ; TCA , trichloro ‐ acetic acid .

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

(D) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.


( A ) HEK293 cells were transfected with Flag ‐ tagged beclin 1 and Bcl ‐ XL with or without haemagglutinin ‐ tagged DAPK . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies .

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

(A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.


( B ) HEK293 cells were co ‐ transfected with Flag ‐ tagged T119A or T119E beclin 1 mutants and Bcl ‐ XL . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies .

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

(B) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐XL. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.


( C ) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP - LC3 plasmid . After 24 h , cells were counted and lysates were prepared . ( a ) Representative GFP - LC3 staining . ( b ) The percentage of cells with punctate GFP - LC3 fluorescence per total GFP - LC3 ‐ positive cells was quantified . Data presented are the mean ± s . d . from a triplicate experiment with 100 transfected cells . The asterisks denote significance level : * P = 0 . 01 ; * * P = 0 . 005 . ( c ) Western blot analysis was performed using the indicated antibodies .

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

(C) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP-LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (a) Representative GFP-LC3 staining. (b) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: *P=0.01; **P=0.005. (c) Western blot analysis was performed using the indicated antibodies.


( D ) HEK293 cells were transfected with Bcl ‐ XL , Flag ‐ tagged beclin 1 ( WT ) or Flag ‐ tagged T119A beclin 1 mutant with or without haemagglutinin ‐ tagged activated DAPK ( ΔCaM ) . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies . Levels of Bcl ‐ XL were quantified using NIH image software , and the ratio between immunoprecipitated and expressed Bcl ‐ XL was calculated .

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

(D) HEK293 cells were transfected with Bcl‐XL, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐XL were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐XL was calculated.


( E ) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl ‐ XL , Flag ‐ tagged beclin 1 and ΔCaM or pcDNA3 ‐ luciferase as a control . The images in ( c ) and ( d ) were taken at higher magnifications of the ΔCaM treatment ( see the scale bars ) . ' AV ' indicates autophagic vacuoles . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GFP , green fluorescent protein ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney ; NIH , National Institutes of Health .

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

(E) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐XL, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (c) and (d) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). 'AV' indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.


( A ) Expression levels of NAT4 analyzed by qRT - PCR using total RNA extracted from a wild - type ( BY4741 ) and Pste5 - NAT4 strain grown in 2 % ( NCR ) , or 0 . 1 % glucose ( CR ) . NAT4 levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM ( Standard Error of the Mean ) of 3 independent experiments . * * p ≤ 0 . 01 ; calculated by unpaired two - tailed Student ' s t - test .

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

(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student's t-test.


( B ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown in the same conditions as in ( A ) . Chromatin was immunoprecipitated using an antibody against N - acH4 and analyzed by qRT - PCR using primers for the different rDNA regions . Enrichment was normalized to Histone H4 levels . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 compared values to wild - type NCR conditions ; arrowhead p ≤ 0 . 05 compared values to wild - type CR

O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown in the same conditions as in (A). Chromatin was immunoprecipitated using an antibody against N-acH4 and analyzed by qRT-PCR using primers for the different rDNA regions. Enrichment was normalized to Histone H4 levels. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01 compared values to wild-type NCR conditions; arrowhead p ≤ 0.05 compared values to wild-type CR conditions


( C ) Replicative lifespan ( RLS ) for BY4741 wild - type and nat4Δ strains . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

(C) Replicative lifespan (RLS) for BY4741 wild-type and nat4Δ strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( D ) Mean RLS for wild - type and nat4 ∆ strains using different genetic backgrounds . Values outside of parenthesis indicate mean lifespan , while values in parenthesis indicate the number of cells examined .

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

(D) Mean RLS for wild-type and nat4∆ strains using different genetic backgrounds. Values outside of parenthesis indicate mean lifespan, while values in parenthesis indicate the number of cells examined.


( E ) Mean RLS for BY4741 wild - type and nat4Δ strains under NCR and CR conditions .

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

(E) Mean RLS for BY4741 wild-type and nat4Δ strains under NCR and CR conditions.


( F ) Mean RLS for BY4741 wild - type and Pste5 - NAT4 strains under NCR and CR conditions .

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

(F) Mean RLS for BY4741 wild-type and Pste5-NAT4 strains under NCR and CR conditions.


( B ) ChIP experiments were performed in SF10 wild - type , nat4 ∆ , NET1 - TAP and NET1 - TAP nat4 ∆ strains using an anti - IgG antibody . The enrichment from the antibody was normalized to input . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * * p ≤ 0 . 01 compared nat4 ∆ to WT values .

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

(B) ChIP experiments were performed in SF10 wild-type, nat4∆, NET1-TAP and NET1-TAP nat4∆ strains using an anti-IgG antibody. The enrichment from the antibody was normalized to input. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared nat4∆ to WT values.


( C ) Quantitative real - time PCR analysis of rDNA copy number for young and old cells in BY4741 wild - type , nat4Δ and fob1Δ strains . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * * p ≤ 0 . 01 compared to corresponding WT values .

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

(C) Quantitative real-time PCR analysis of rDNA copy number for young and old cells in BY4741 wild-type, nat4Δ and fob1Δ strains. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared to corresponding WT values.


( D ) Polysome profiling analysis performed in BY4741 wild - type and nat4Δ strains . The plots are representative of two independent experiments .

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

(D) Polysome profiling analysis performed in BY4741 wild-type and nat4Δ strains. The plots are representative of two independent experiments.


( C ) Gene expression analysis of selected upregulated stress - response genes ( according to results in 3A ) in BY4741 wild - type and nat4 ∆ strains grown under NCR and CR conditions . Expression levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM of 3 independent experiments . p - values were obtained comparing results to wild - type under NCR conditions , and calculated by unpaired two - tailed Student ' s t - test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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 B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene expression analysis of selected upregulated stress-response genes (according to results in 3A) in BY4741 wild-type and nat4∆ strains grown under NCR and CR conditions. Expression levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained comparing results to wild-type under NCR conditions, and calculated by unpaired two-tailed Student's t-test: * p ≤ 0.05; ** p ≤ 0.01.


( D ) Gene expression analysis showing fold induction levels ( Log2 ) of selected genes comparing BY4741 wild - type and STE5p - NAT4 strains under NCR ( 2 % glucose ) and CR ( 0 . 1 % glucose ) conditions . Expression levels were normalized to TAF10 , whose expression remains unchanged .

O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O

(D) Gene expression analysis showing fold induction levels (Log2) of selected genes comparing BY4741 wild-type and STE5p-NAT4 strains under NCR (2% glucose) and CR (0.1% glucose) conditions. Expression levels were normalized to TAF10, whose expression remains unchanged.


( A ) RLS analysis for BY4741 wild - type and nat4 - E186A strains . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

(A) RLS analysis for BY4741 wild-type and nat4-E186A strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: *** p ≤ 0.001; **** p ≤ 0.0001.


( B ) Mean RLS for YSC5106 wild - type , nat4 ∆ and H4S1D strains .

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

(B) Mean RLS for YSC5106 wild-type, nat4∆ and H4S1D strains.


( C ) Gene expression analysis of the indicated stress - induced genes in YSC5106 wild - type , nat4 ∆ and H4S1D strains . Expression levels were normalized to RPP0 whose expression remains unchanged . Error bars , SEM of 3 independent experiments . p - values were obtained by comparing nat4 ∆ and H4S1D to wild - type values and calculated by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(C) Gene expression analysis of the indicated stress-induced genes in YSC5106 wild-type, nat4∆ and H4S1D strains. Expression levels were normalized to RPP0 whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained by comparing nat4∆ and H4S1D to wild-type values and calculated by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01.


( D ) Gene expression analysis of the indicated stress - induced genes in BY4742 wild - type , nat4 ∆ and nat4 ∆ [ hNAA40 ] strains . Expression levels were normalized to ACT1 , whose expression remains unchanged .

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(D) Gene expression analysis of the indicated stress-induced genes in BY4742 wild-type, nat4∆ and nat4∆[hNAA40] strains. Expression levels were normalized to ACT1, whose expression remains unchanged.


( A ) RLS analysis for YSC5106 wild - type , nat4 ∆ , H4R3K and double mutant strains . Values in parenthesis indicate mean lifespan . Statistical significance was determined by one - way ANOVA test : * * * * p ≤ 0 . 0001 .

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

(A) RLS analysis for YSC5106 wild-type, nat4∆, H4R3K and double mutant strains. Values in parenthesis indicate mean lifespan. Statistical significance was determined by one-way ANOVA test: **** p ≤ 0.0001.


( B ) Gene expression analysis of the indicated stress - induced genes in YSC5106 WT , nat4 ∆ , H4R3K and double mutant strains . Expression levels were normalized to RPP0 , whose expression remains unchanged .

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

(B) Gene expression analysis of the indicated stress-induced genes in YSC5106 WT, nat4∆, H4R3K and double mutant strains. Expression levels were normalized to RPP0, whose expression remains unchanged.


( A ) Gene expression analysis of the indicated stress - induced genes in young and old cells isolated from YSC5106 WT , nat4 ∆ and H4R3K nat4 ∆ strains . Expression levels were normalized to ACT1 , whose expression remains unchanged . Error bars , SEM of 3 independent experiments . P - values were calculated by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 compared to young wild - type ; arrowhead p ≤ 0 . 05 compared to old wild - type .

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

(A) Gene expression analysis of the indicated stress-induced genes in young and old cells isolated from YSC5106 WT, nat4∆ and H4R3K nat4∆ strains. Expression levels were normalized to ACT1, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. P-values were calculated by unpaired two-tailed Student's t test: * p ≤ 0.05 compared to young wild-type; arrowhead p ≤ 0.05 compared to old wild-type.


( B ) RLS analysis for WT , nat4 ∆ , pnc1 ∆ and double mutant strains in BY4741 genetic background . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ns = non - significant .

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(B) RLS analysis for WT, nat4∆, pnc1∆ and double mutant strains in BY4741 genetic background. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. ns = non-significant.


( C ) Mean RLS in BY4741 wild - type , msn2 ∆ msn4 ∆ double and msn2 ∆ msn4 ∆ nat4 ∆ triple mutant strains .

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(C) Mean RLS in BY4741 wild-type, msn2∆ msn4∆ double and msn2∆ msn4∆ nat4∆ triple mutant strains.


( D ) Mean RLS in BY4741 wild - type , nat4 ∆ , fob1 ∆ sir2 ∆ double and fob1 ∆ sir2 ∆ nat4 ∆ triple mutant strains .

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(D) Mean RLS in BY4741 wild-type, nat4∆, fob1∆ sir2∆ double and fob1∆ sir2∆ nat4∆ triple mutant strains.


( E ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown under NCR and CR conditions . Chromatin was immunoprecipitated using antibodies against N - acH4 and Histone H4 , and analyzed by qRT - PCR with the indicated primers . PNC1 - P ( Promoter ) , PNC1 - S ( Transcriptional Start ) , PNC1 - E ( End of gene ) . The enrichment from the antibody was normalized to Histone H4 .

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(E) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown under NCR and CR conditions. Chromatin was immunoprecipitated using antibodies against N-acH4 and Histone H4, and analyzed by qRT-PCR with the indicated primers. PNC1-P (Promoter), PNC1-S (Transcriptional Start), PNC1-E (End of gene). The enrichment from the antibody was normalized to Histone H4.


MDM were incubated with 1 , 25D3 for 4 h before infection with HIV and / or M . tuberculosis ( TB ) for 3 h , washed then incubated with or without 1 , 25D3 for 7 days . ( A ) Top , extracellular release of HIV p24 antigen into the cell supernatant at days 0 , 4 and 7 was detected by ELISA . Bottom , MDM were harvested and stained for HIV p17 . Histograms are shown for a representative donor .

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

MDM were incubated with 1,25D3 for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h, washed then incubated with or without 1,25D3 for 7 days. (A) Top, extracellular release of HIV p24 antigen into the cell supernatant at days 0, 4 and 7 was detected by ELISA. Bottom, MDM were harvested and stained for HIV p17. Histograms are shown for a representative donor.


( B ) Top , cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase . Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7 . Bottom , MDM were harvested and stained for mycobacterium . Histograms are shown for a representative donor .

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(B) Top, cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase. Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7. Bottom, MDM were harvested and stained for mycobacterium. Histograms are shown for a representative donor.


( C ) Cell lysates from panel B were diluted and run in a MGIT 960 . Bar and line graphs are reported as mean ± s . e . m . of three independent experiments performed in triplicate . § P < 0 . 05 ; * P < 0 . 001 .

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(C) Cell lysates from panel B were diluted and run in a MGIT 960. Bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.


HIV and / or M . tuberculosis ( TB ) infected MDM were treated for 7 days with 100 pmol / L 1 , 25D3 . ( A ) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β - actin performed .

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HIV and/or M. tuberculosis (TB) infected MDM were treated for 7 days with 100 pmol/L 1,25D3. (A) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β-actin performed.


B ) Cells were incubated with 10 µg / mL pepstatin A for 4 h on day 7 prior to lysis . Immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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B) Cells were incubated with 10 µg/mL pepstatin A for 4 h on day 7 prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


C ) Flow cytometry analysis of saponin - resistant LC3B - II in macrophages after 1 , 25D3 treatment for 7 d . Representative histograms of cells displaying saponin - resistant LC3B - II from three donors are shown .

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

C) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.


( D ) Flow cytometry analysis of saponin - resistant LC3B - II in macrophages after 1 , 25D3 treatment for 7 d followed by 10 µg / mL pepstatin A for a further 4 h . Representative histograms of cells displaying saponin - resistant LC3B - II from three donors are shown .

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

(D) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d followed by 10 µg/mL pepstatin A for a further 4 h. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.


( E ) Immunoblots of p62 using antibody to p62 or β - actin 7 d after macrophages treated with 1 , 25D3 .

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(E) Immunoblots of p62 using antibody to p62 or β-actin 7 d after macrophages treated with 1,25D3.


( F ) At 7 d post - infection , aliquots of supernatant taken before the addition of WST - 1 were tested for lactate dehydrogenase ( LDH ) spectrophotometrically using the LDHPLUS assay . For the last hour cells were incubated with WST - 1 , and the reduction of the WST - 1 reagent to its formazan product was monitored spectrophotometrically .

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(F) At 7 d post-infection, aliquots of supernatant taken before the addition of WST-1 were tested for lactate dehydrogenase (LDH) spectrophotometrically using the LDHPLUS assay. For the last hour cells were incubated with WST-1, and the reduction of the WST-1 reagent to its formazan product was monitored spectrophotometrically.


1 , 25D3 induces autophagy in human macrophages co - infected with HIV and M . tuberculosis . HIV ( A ) , M . tuberculosis ( TB ) ( B ) , and HIV / TB dual infected MDM ( C ) were treated for 7 days with 100 pmol / L 1 , 25D3 , harvested and stained for HIVp17 , mycobacteria , and saponin - resistant LC3B - II . Representative density plots from three donors are shown . ( D ) HIV / M . tuberculosis infected MDM ( C ) were treated for 7 days with 100 pmol / L 1 , 25D3 , harvested and stained for HIVp17 , mycobacteria , and saponin - resistant LC3B - II . Left , representative density plots from three donors from HIV / M . tuberculosis infected MDM are shown for HIV / M . tuberculosis co - infection . Right , histograms of saponin - resistant LC3B - II in macrophages that were HIV + M . tuberculosis − or HIV + day 7 + at day 7 post - infection .

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1,25D3 induces autophagy in human macrophages co-infected with HIV and M. tuberculosis.HIV (A), M. tuberculosis (TB) (B), and HIV/TB dual infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Representative density plots from three donors are shown. (D) HIV/M. tuberculosis infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Left, representative density plots from three donors from HIV/M. tuberculosis infected MDM are shown for HIV/M. tuberculosis co-infection. Right, histograms of saponin-resistant LC3B-II in macrophages that were HIV+M. tuberculosis− or HIV+day 7+ at day 7 post-infection.


MDM were transduced with non - specific scrambled shRNA ( shNS ) or Beclin - 1 shRNA ( shBCLN1 ) and selected using puromycin resistance . Five days later , cells were incubated with 100 pmol / L 1 , 25D3 or vehicle control for 4 h before infection with HIV and / or M . tuberculosis ( TB ) for 3 h . Cells were then washed and incubated with 100 pmol / L 1 , 25D3 or vehicle control for 7 days . ( A ) Immunoblot analysis performed using antibodies raised to Beclin - 1 or β - actin after initial pathogen exposure ( Day 0 ) or after 7 days .

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

MDM were transduced with non-specific scrambled shRNA (shNS) or Beclin-1 shRNA (shBCLN1) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to Beclin-1 or β-actin after initial pathogen exposure (Day 0) or after 7 days.


( B ) ELISA performed for HIV p24 antigen release over 7 d .

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(B) ELISA performed for HIV p24 antigen release over 7 d.


( C ) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7 .

O O O O B-ORGANISM O O O O O B-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O

(C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7.


D ) Intracellular mycobacteria harvested and assayed for viability based on time to positivity ( 75 growth units ) at day 0 and day 7 using the MGIT 960 . All bar and line graphs are reported as mean ± s . e . m . of three independent experiments performed in triplicate . § P < 0 . 05 ; * P < 0 . 001 .

O O O B-ORGANISM O O O O B-EXP_ASSAY 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

D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.


MDM were transduced with non - specific scrambled shRNA ( shNS ) or ATG5 shRNA ( shATG5 ) and selected using puromycin resistance . Five days later , cells were incubated with 100 pmol / L 1 , 25D3 or vehicle control for 4 h before infection with HIV and / or M . tuberculosis ( TB ) for 3 h . Cells were then washed and incubated with 100 pmol / L 1 , 25D3 or vehicle control for 7 days . ( A ) Immunoblot analysis performed using antibodies raised to ATG5 or β - actin after initial pathogen exposure ( Day 0 ) or after 7

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

MDM were transduced with non-specific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to ATG5 or β-actin after initial pathogen exposure (Day 0) or after 7 days


( C ) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7 . (

O O O O B-ORGANISM O O O O O B-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O

(C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. (


( D ) Intracellular mycobacteria harvested and assayed for viability based on time to positivity ( 75 growth units ) at day 0 and day 7 using the MGIT 960 . All bar and line graphs are reported as mean ± s . e . m . of three independent experiments performed in triplicate . § P < 0 . 05 ; * P < 0 . 001 .

O O O O B-ORGANISM O O O O B-EXP_ASSAY 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

(D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.


MDM were pretreated with bafilomycin A1 ( Baf A1 ) before treatment with 1 , 25D3 and subsequent infection with HIV and / or M . tuberculosis ( TB ) . ( A ) ELISA performed for HIV p24 antigen release at days 0 , 4 and 7 .

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

MDM were pretreated with bafilomycin A1 (Baf A1) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7.


( B ) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7 .

O O O O B-ORGANISM O O O O O B-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

(B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7.


( C ) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7 . All bar and line graphs are reported as mean ± s . e . m . of three independent experiments performed in triplicate . § P < 0 . 05 ; * P < 0 . 01 .

O O O O B-ORGANISM 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

(C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.


SID 26681509 inhibits the 1 , 25D3 mediated inhibition of HIV and M . tuberculosis replication . MDM were pretreated with SID 26681509 ( SID ) before treatment with 1 , 25D3 and subsequent infection with HIV and / or M . tuberculosis ( TB ) . ( A ) ELISA performed for HIV p24 antigen release at days 0 , 4 and 7 .

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

SID 26681509 inhibits the 1,25D3 mediated inhibition of HIV and M. tuberculosis replication.MDM were pretreated with SID 26681509 (SID) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7.


B ) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7 .

O O O B-ORGANISM O O O O O B-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7.


Inhibition of HIV and M . tuberculosis by 1 , 25D3 is CAMP and autophagy dependent . MDM were transduced with non - specific scrambled shRNA ( shNS ) or CAMP shRNA ( shCAMP ) and selected using puromycin resistance . Five days later , cells were incubated with 100 pmol / L 1 , 25D3 or vehicle control for 4 h before infection with HIV and / or M . tuberculosis ( TB ) for 3 h . Cells were then washed and incubated with 100 pmol / L 1 , 25D3 or vehicle control for 7 days . ( A ) Immunoblot analysis performed using antibodies raised to CAMP or β - actin after initial pathogen exposure ( Day 0 ) or after 7 days .

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

Inhibition of HIV and M. tuberculosis by 1,25D3 is CAMP and autophagy dependent.MDM were transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to CAMP or β-actin after initial pathogen exposure (Day 0) or after 7 days.


( B ) Day 7 HIV - 1Ba - L and TB co - infected cells were harvested and stained for saponin - resistant LC3B - II at day 7 . A representative histogram from three donors are shown .

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(B) Day 7 HIV-1Ba-L and TB co-infected cells were harvested and stained for saponin-resistant LC3B-II at day 7. A representative histogram from three donors are shown.


C ) ELISA performed for HIV p24 antigen release over 7 d .

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C) ELISA performed for HIV p24 antigen release over 7 d.


( D ) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7 . All bar and line graphs are reported as mean ± s . e . m . of three independent experiments performed in triplicate . * P < 0 . 001 .

O O O O B-ORGANISM O O O O O B-ORGANISM B-EXP_ASSAY 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

(D) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. * P<0.001.


Representative composite confocal image of single cultured hippocampal mouse neurons , labeled with MAP2 ( green ) on islands of glial cells , labeled with GFAP ( red ) . Scale bar : 100 µm . Representative confocal image of single cultured hippocampal mouse neuron labeled with endogenous DCV cargo ChgB ( green ) and ß3 - tubulin ( red ) as neurite marker . Scale bar : 20 µm .

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

Representative composite confocal image of single cultured hippocampal mouse neurons, labeled with MAP2 (green) on islands of glial cells, labeled with GFAP (red). Scale bar: 100 µm. Representative confocal image of single cultured hippocampal mouse neuron labeled with endogenous DCV cargo ChgB (green) and ß3-tubulin (red) as neurite marker. Scale bar: 20 µm.


Analysis of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . DCV pool size measured as number of ChgB puncta per neuron .

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

Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.


Analysis of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . Total neurite length ( mm ) per neuron .

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Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. Total neurite length (mm) per neuron.


Analysis of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . ChgB puncta per μm neurite .

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Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. ChgB puncta per μm neurite.


Analysis of striatal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . DCV pool size measured as number of ChgB puncta per neuron .

Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.


Total neurite length ( mm ) per neuron .

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Total neurite length (mm) per neuron.


Analysis of striatal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . ChgB puncta per μm neurite .

Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. ChgB puncta per μm neurite.


Correlation between ChgB puncta per neuron and total neurite length for hippocampal ( green ) and striatal ( red ) neurons .

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Correlation between ChgB puncta per neuron and total neurite length for hippocampal (green) and striatal (red) neurons.


Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . Representative mouse brain slice with labeled axonal projections in cortical region ( mCherry - filler , red ) and immunostained for ChgA ( green ) . Scale bar : 1 mm . ( a - b ) Zoom of axonal projections in ( L ) , scale bars : 100 µm .

O O B-ORGANISM B-TISSUE O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O B-ORGANISM B-TISSUE O O O B-SUBCELLULAR O O B-TISSUE O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative mouse brain slice with labeled axonal projections in cortical region (mCherry-filler, red) and immunostained for ChgA (green). Scale bar: 1 mm. (a-b) Zoom of axonal projections in (L), scale bars: 100 µm.


Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . Representative confocal image and zoom of cortex tissue containing labeled axonal projections . Measurements of ChgA puncta ( black ) were performed within axonal projections ( mask , yellow ) . Zoomed areas are indicated in top , right image ( M ) . Scale bars : 10 µm ( M ) , 5 µm (

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

Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative confocal image and zoom of cortex tissue containing labeled axonal projections. Measurements of ChgA puncta (black) were performed within axonal projections (mask, yellow). Zoomed areas are indicated in top, right image (M). Scale bars: 10 µm (M), 5 µm (N


Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . Representative confocal image and zoom of cortex tissue containing labeled axonal projections . Measurements of ChgA puncta ( black ) were performed within axonal projections ( mask , yellow ) . Zoomed areas Arrows ( O ) indicate ChgA staining measured as individual ChgA puncta . Scale bars : , 5

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

Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative confocal image and zoom of cortex tissue containing labeled axonal projections. Measurements of ChgA puncta (black) were performed within axonal projections (mask, yellow). Zoomed areas Arrows (O) indicate ChgA staining measured as individual ChgA puncta. Scale bars: , 5 µm


Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . ChgA puncta per μm axon from mouse tissue , compared to the distribution of ChgB puncta in axons of cultured hippocampal

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Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. ChgA puncta per μm axon from mouse tissue, compared to the distribution of ChgB puncta in axons of cultured hippocampal neurons


Representation of resolution limit in confocal imaging and dSTORM strategy to increase resolution . Electron micrograph of axon ( green ) and dendrite ( red ) containing DCVs , defined based on morphology . In conventional immunofluorescence confocal microscopy , individual DCVs cannot be separated ( right zoom , green signal representing fluorescent signal using confocal ) . Super - resolution dSTORM imaging resolves the point - spread function ( PSF ) resulting in single DCV localizations . Scale bars : 100 nm .

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

Representation of resolution limit in confocal imaging and dSTORM strategy to increase resolution. Electron micrograph of axon (green) and dendrite (red) containing DCVs, defined based on morphology. In conventional immunofluorescence confocal microscopy, individual DCVs cannot be separated (right zoom, green signal representing fluorescent signal using confocal). Super-resolution dSTORM imaging resolves the point-spread function (PSF) resulting in single DCV localizations. Scale bars: 100 nm.


Representative confocal image and zoom of a single isolated hippocampal neuron ( DIV14 ) immunostained for the endogenous DCV marker ChgB ( green ) , axonal marker SMI312 ( red ) and dendritic marker MAP2 ( blue ) . Areas of zoom ( C ) is indicated in ( B ) . Scale bars : 20 µm ( B ) , 5 µm ( C ) .

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

Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), axonal marker SMI312 (red) and dendritic marker MAP2 (blue). Areas of zoom (C) is indicated in (B). Scale bars: 20 µm (B), 5 µm (C).


ChgB immunoreactivity of zoomed area indicated in ( C ) ( top panel ) . Reconstructed ChgB localizations of dSTORM imaging ( middle panel ) . Lower panel shows overlay of dSTORM ChgB localizations and ChgB immunoreactivity ( confocal , 40x ) . Calibration bar represents no ( 0 ) to high ( 255 ) clustering of dSTORM localizations . Scale bar : 2 µm .

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

ChgB immunoreactivity of zoomed area indicated in (C) (top panel). Reconstructed ChgB localizations of dSTORM imaging (middle panel). Lower panel shows overlay of dSTORM ChgB localizations and ChgB immunoreactivity (confocal, 40x). Calibration bar represents no (0) to high (255) clustering of dSTORM localizations. Scale bar: 2 µm.


Fluorescence intensity of ChgB immunoreactivity puncta ( confocal ) with corresponding number of dSTORM puncta for hippocampal ( green ) or striatal ( red ) cultured neurons . Squares show average ± SEM , dots represent individual observations . Hippocampus : N = 2 cells ( 89 puncta ) , striatum : N = 3 cells ( 37 puncta ) .

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

Fluorescence intensity of ChgB immunoreactivity puncta (confocal) with corresponding number of dSTORM puncta for hippocampal (green) or striatal (red) cultured neurons. Squares show average ± SEM, dots represent individual observations. Hippocampus: N = 2 cells (89 puncta), striatum: N = 3 cells (37 puncta).


Average histogram of fluorescence intensity of ChgB puncta ( 40x confocal ) in neurons used for dSTORM imaging . Dotted lines indicate cut - off values of fluorescence intensity corresponding 1 , 2 , 3 or 4 dSTORM puncta . Data shows average ± SEM , Hippocampus : N = 2 cells , striatum : N = 3 cells .

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

Average histogram of fluorescence intensity of ChgB puncta (40x confocal) in neurons used for dSTORM imaging. Dotted lines indicate cut-off values of fluorescence intensity corresponding 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM, Hippocampus: N = 2 cells, striatum: N = 3 cells.


Percentage of ChgB puncta per neuron with corresponding fluorescence value of 1 , 2 , 3 or 4 dSTORM puncta . Data shows average ± SEM . Hippocampus : N = 2 cells , striatum : N = 3 cells .

O O B-GENEPROD B-SUBCELLULAR O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O

Percentage of ChgB puncta per neuron with corresponding fluorescence value of 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM. Hippocampus: N = 2 cells, striatum: N = 3 cells.


Analysis of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . ( H ) Representative confocal ( 100x magnification ) image of mCherry labeled axon ( blue ) with ChgA confocal signal ( red ) and corresponding super - resolution STED ChgA signal ( green ) . Zoomed areas are indicated by arrows . Scale bar merged image ( left ) : 1 µm , scale bars zooms : 100 nm . ( I ) Percentage of single ChgA puncta within mCherry labeled axons with corresponding number of overlaying STED puncta . Data shows average ± SEM . N = 2 mice ( 7 STED images , 70 single observations ) .

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

Analysis of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. (H) Representative confocal (100x magnification) image of mCherry labeled axon (blue) with ChgA confocal signal (red) and corresponding super-resolution STED ChgA signal (green). Zoomed areas are indicated by arrows. Scale bar merged image (left): 1 µm, scale bars zooms: 100 nm. (I) Percentage of single ChgA puncta within mCherry labeled axons with corresponding number of overlaying STED puncta. Data shows average ± SEM. N = 2 mice (7 STED images, 70 single observations).


Representative confocal images and zooms of a single isolated hippocampal neuron ( DIV14 ) immunostained for the endogenous DCV marker ChgB ( green ) , dendritic marker MAP2 ( red ) and ß3 - tubulin ( blue ) as neurite marker . Areas of zooms are indicated in ( A ) . Closed arrows : dendritic ChgB staining , open arrows : axonal ChgB staining . Scale bars : 20 µm ( A ) , 5 µm ( B , C ) . Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB . Closed arrows : synaptic ChgB staining , open arrows : extra - synaptic ChgB staining . Scale bar : 5 µm . Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I / II . Scale bar : 5 µm .

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

Representative confocal images and zooms of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), dendritic marker MAP2 (red) and ß3-tubulin (blue) as neurite marker. Areas of zooms are indicated in (A). Closed arrows: dendritic ChgB staining, open arrows: axonal ChgB staining. Scale bars: 20 µm (A), 5 µm (B, C). Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB. Closed arrows: synaptic ChgB staining, open arrows: extra-synaptic ChgB staining. Scale bar: 5 µm. Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I/II. Scale bar: 5 µm.


Manders ' coefficients of colocalization analysis between ChgB , ß3 - tubulin and MAP2 .

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Manders' coefficients of colocalization analysis between ChgB, ß3-tubulin and MAP2.


Manders ' coefficients of colocalization analysis between ChgB and VGLUT1 , with VGLUT1 - Synapsin I / II colocalization as positive control and VGLUT1 - Synapsin I / II rotated 90 ° as negative control .

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

Manders' coefficients of colocalization analysis between ChgB and VGLUT1, with VGLUT1-Synapsin I/II colocalization as positive control and VGLUT1-Synapsin I/II rotated 90° as negative control.


Number of ChgB puncta per neuron in dendrites , axons and the total pool ( dendrites + axons ) . Wilcoxon matched - pairs signed rank test : n . s . = not significant , p = 0 . 75 .

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Number of ChgB puncta per neuron in dendrites, axons and the total pool (dendrites + axons). Wilcoxon matched-pairs signed rank test: n.s. = not significant, p = 0.75.


Total dendritic and axonal length ( mm ) per neuron . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 .

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Total dendritic and axonal length (mm) per neuron. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.


ChgB puncta per μm dendrite and axon per neuron . Lines next to individual data points show mean ± SEM . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 .

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ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.


Mean ChgB fluorescence intensity per puncta in the total neuron ( dendrites + axons ) and in dendrites only . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 .

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Mean ChgB fluorescence intensity per puncta in the total neuron (dendrites + axons) and in dendrites only. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.


Histogram of fluorescence intensity of ChgB puncta in the total neuron ( dendrites + axons ) and in dendrites only .

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Histogram of fluorescence intensity of ChgB puncta in the total neuron (dendrites + axons) and in dendrites only.


ChgB puncta per μm dendrite and axon per neuron . Lines next to individual data points show mean ± SEM . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 . Mean ChgB fluorescence intensity per puncta in dendrites and axons . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 .

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ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Mean ChgB fluorescence intensity per puncta in dendrites and axons. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.


Distribution of DCVs in dendrites and axons of sparse - labeled network cultures . ( P ) ChgB puncta per μm dendrite or axon . Lines next to individual data points show mean ± SEM . Mann - Whitney U test : * * * * p = < 0 . 0001 . ( Q ) Mean ChgB fluorescence intensity per puncta in dendrites and axons . Unpaired t - test : * * * p = < 0 . 0001 .

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Distribution of DCVs in dendrites and axons of sparse-labeled network cultures. (P) ChgB puncta per μm dendrite or axon. Lines next to individual data points show mean ± SEM. Mann-Whitney U test: **** p = < 0.0001. (Q) Mean ChgB fluorescence intensity per puncta in dendrites and axons. Unpaired t-test: *** p = < 0.0001.


Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus ( DG ) granule cell tissue after stereotaxic viral injections and post - hoc immunostained for ChgA as DCV marker . ( R ) Representative mouse brain slice with labeled DG granule cells ( mCherry - filler , red ) and immunostained for ChgA ( green ) . ChgA puncta were measured in dendrites and axons at indicated areas . Scale bar : 250 µm . ( S ) ChgA puncta per μm dendrite or axon in DG granule cells . N = 1 mouse ( 6 dendrite regions , 7 axonal regions ) .

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Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus (DG) granule cell tissue after stereotaxic viral injections and post-hoc immunostained for ChgA as DCV marker. (R) Representative mouse brain slice with labeled DG granule cells (mCherry-filler, red) and immunostained for ChgA (green). ChgA puncta were measured in dendrites and axons at indicated areas. Scale bar: 250 µm. (S) ChgA puncta per μm dendrite or axon in DG granule cells. N = 1 mouse (6 dendrite regions, 7 axonal regions).


Representative confocal image and zoom of a single isolated hippocampal neuron ( DIV14 ) immunostained for the endogenous DCV marker ChgB ( green ) , synaptic protein VGLUT1 ( red ) and dendritic marker MAP2 ( blue ) . Area of zoom ( B ) is indicated in ( A ) . Scale bars : 20 µm ( A ) , 2 µm ( B ) . Reconstructed ChgB localizations of dSTORM imaging of zoomed area in ( B ) ( top ) . Middle panel shows overlay between dSTORM ChgB localizations and VGLUT1 immunofluorescence ( confocal , 40x ) . Scale bar : 2 µm . Zooms of boxed areas containing synapses are visualized in lower panels ( a - c ) . Arrows indicate extra - synaptic DCVs . Scale bar ( a - c ) : 1 µm . Calibration bar represents n : o ( 0 ) to high ( 255 ) clustering of dSTORM localizations .

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Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), synaptic protein VGLUT1 (red) and dendritic marker MAP2 (blue). Area of zoom (B) is indicated in (A). Scale bars: 20 µm (A), 2 µm (B). Reconstructed ChgB localizations of dSTORM imaging of zoomed area in (B) (top). Middle panel shows overlay between dSTORM ChgB localizations and VGLUT1 immunofluorescence (confocal, 40x). Scale bar: 2 µm. Zooms of boxed areas containing synapses are visualized in lower panels (a-c). Arrows indicate extra-synaptic DCVs. Scale bar (a-c): 1 µm. Calibration bar represents n:o (0) to high (255) clustering of dSTORM localizations.


Representative electron micrographs of synaptic sections containing DCVs . AZ indicates the active zone , dashed lines represent DCV diameter measurements . Arrow indicates extra - synaptic DCV . Scale bars : 100 nm .

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Representative electron micrographs of synaptic sections containing DCVs. AZ indicates the active zone, dashed lines represent DCV diameter measurements. Arrow indicates extra-synaptic DCV. Scale bars: 100 nm.


Histogram of number of DCVs in percentage of synapses . Inset : number of DCVs per synaptic section .

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Histogram of number of DCVs in percentage of synapses. Inset: number of DCVs per synaptic section.


Distance of DCVs to the active zone ( nm ) , measured from center of DCV to the middle of the active zone . Histogram of distance of DCVs to the middle of the active zone ( nm ) . Inset : shortest distance of DCVs to the active zone ( nm ) .

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Distance of DCVs to the active zone (nm), measured from center of DCV to the middle of the active zone. Histogram of distance of DCVs to the middle of the active zone (nm). Inset: shortest distance of DCVs to the active zone (nm).


Histogram of normalized distance of percentage of synaptic vesicles ( grey ) to the active zone and corresponding distance of DCVs ( black ) to the active zone .

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Histogram of normalized distance of percentage of synaptic vesicles (grey) to the active zone and corresponding distance of DCVs (black) to the active zone.


Example electron micrograph zooms of synapses containing DCVs outside ( top ) or inside ( bottom ) the synaptic vesicle cluster . Scale bars : 100 nm . Percentage of DCVs outside or inside the synaptic vesicle cluster . Histogram of distance ( nm ) of DCVs to the border of the synaptic vesicle cluster , negative values are inside , positive values outside .

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Example electron micrograph zooms of synapses containing DCVs outside (top) or inside (bottom) the synaptic vesicle cluster. Scale bars: 100 nm. Percentage of DCVs outside or inside the synaptic vesicle cluster. Histogram of distance (nm) of DCVs to the border of the synaptic vesicle cluster, negative values are inside, positive values outside.


Schematic representation of NPY - pHluorin as optical reporter for DCV fusion . Inset below shows F / F0 of single DCV fusion event . Scale bar : 1 µm .

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Schematic representation of NPY-pHluorin as optical reporter for DCV fusion. Inset below shows F/F0 of single DCV fusion event. Scale bar: 1 µm.


Zoomed confocal image of NPY - pHluorin expressing ( green ) hippocampal neuron ( DIV14 ) immunostained for ChgB ( red ) and corresponding fluorescence intensity plot showing overlap between NPY - pHluorin and ChgB . Scale bar : 2 µm .

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Zoomed confocal image of NPY-pHluorin expressing (green) hippocampal neuron (DIV14) immunostained for ChgB (red) and corresponding fluorescence intensity plot showing overlap between NPY-pHluorin and ChgB. Scale bar: 2 µm.


Manders ' coefficients of colocalization between NPY - pHluorin and ChgB per neuron .

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Manders' coefficients of colocalization between NPY-pHluorin and ChgB per neuron.


Total ChgB puncta per neuron in uninfected ( control ) and NPY - pHluorin infected neurons ( NPY - pH ) . Student ' s t - test : n . s . = not significant , p = 0 . 90 . Mean ChgB fluorescence intensity per puncta per neuron in uninfected ( control ) and NPY - pHluorin infected neurons ( NPY - pH ) . Student ' s t - test : n . s . = not significant , p = 0 . 71 .

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Total ChgB puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.90. Mean ChgB fluorescence intensity per puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.71.


Correlation between ChgB puncta per neuron and total neurite length ( mm ) for uninfected control ( grey ) and NPY - pHluorin infected ( green ) neurons . Linear regression , inset shows goodness of fit ( r2 ) and significance ( p - value ) per group . Slopes are not significantly different , ANCOVA : p = 0 . 19 .

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Correlation between ChgB puncta per neuron and total neurite length (mm) for uninfected control (grey) and NPY-pHluorin infected (green) neurons. Linear regression, inset shows goodness of fit (r2) and significance (p-value) per group. Slopes are not significantly different, ANCOVA: p = 0.19.


Single isolated hippocampal neurons ( DIV14 ) were labeled with NPY - pHluorin . DCV fusion events were quantified per neuron upon different stimulation paradigms . Histogram of DCV fusion events per stimulation per time point . Stimulations include 1 action potential ( AP ) ( grey ) , 16 bursts of 50 AP at 50 Hz ( light - green ) , superfusion with 60 mM KCl Tyrode solution for 30 s . ( dark - green ) and superfusion with 5 μM Ionomycin for 30 s . ( blue ) . Inset : Typical ∆ F / F0 trace of rise in intracellular calcium ( Fluo5 - AM ) upon different stimulations . Traces are corrected for baseline ( first 10 frames ) and normalized .

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Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Histogram of DCV fusion events per stimulation per time point. Stimulations include 1 action potential (AP) (grey), 16 bursts of 50 AP at 50 Hz (light-green), superfusion with 60 mM KCl Tyrode solution for 30 s. (dark-green) and superfusion with 5 μM Ionomycin for 30 s. (blue). Inset: Typical ∆F/F0 trace of rise in intracellular calcium (Fluo5-AM) upon different stimulations. Traces are corrected for baseline (first 10 frames) and normalized.


Single isolated hippocampal neurons ( DIV14 ) were labeled with NPY - pHluorin . DCV fusion events were quantified per neuron upon different stimulation paradigms . Cumulative plot of fusion events per neuron for different stimulations .

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Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Cumulative plot of fusion events per neuron for different stimulations.


Single isolated hippocampal neurons ( DIV14 ) were labeled with NPY - pHluorin . DCV fusion events were quantified per neuron upon different stimulation paradigms . DCV fusion events per neuron per stimulation . Kruskal - Wallis with Dunn ' s correction : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . 1AP vs . 5 μM Ionomycin , 16x50 AP , 50Hz vs . 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non - significant , p > 0 . 05 . DCV release probability per neuron per stimulation . Kruskal - Wallis with Dunn ' s correction : * * p < 0 . 01 , * * * p < 0 . 001 . 1AP vs . 5 μM Ionomycin , 16x50 AP , 50Hz vs . 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non - significant , p > 0 . 05 .

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Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. DCV fusion events per neuron per stimulation. Kruskal-Wallis with Dunn's correction: *p < 0.05, **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p > 0.05. DCV release probability per neuron per stimulation. Kruskal-Wallis with Dunn's correction: **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p>0.05.


DCV release probability per neuron in CaMKII - positive and negative hippocampus neurons ( DIV14 ) upon repetitive electrical stimulation ( 16x50AP at 50 Hz ) . Mann - Whitney U test : n . s . = not significant , p = 0 . 12 .

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DCV release probability per neuron in CaMKII-positive and negative hippocampus neurons (DIV14) upon repetitive electrical stimulation (16x50AP at 50 Hz). Mann-Whitney U test: n.s. = not significant, p = 0.12.


Schematic representation of NPY - mCherry as optical reporter for DCV fusion with full cargo release ( top ) . Zoom of axonal and dendritic areas of ( B ) , with location of fusion events ( middle ) . Average fluorescence decrease upon NPY - mCherry labeled DCV fusion in axons and dendrites ( below ) .

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Schematic representation of NPY-mCherry as optical reporter for DCV fusion with full cargo release (top). Zoom of axonal and dendritic areas of (B), with location of fusion events (middle). Average fluorescence decrease upon NPY-mCherry labeled DCV fusion in axons and dendrites (below).


Representative neuron labeled with membrane - targeted EGFP ( mEGFP ) ( black ) and localization of axonal ( blue dots ) or dendritic ( purple dots ) events upon stimulation . Scale bar : 20 µm .

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Representative neuron labeled with membrane-targeted EGFP (mEGFP) (black) and localization of axonal (blue dots) or dendritic (purple dots) events upon stimulation. Scale bar: 20 µm.


Histogram of axonal ( blue ) and dendritic ( purple ) DCV fusion events upon repetitive electrical stimulation ( 16x50AP at 50 Hz ) .

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Histogram of axonal (blue) and dendritic (purple) DCV fusion events upon repetitive electrical stimulation (16x50AP at 50 Hz).


Cumulative plot of axonal or dendritic fusion events per neuron . Normalized cumulative plot of axonal and dendritic fusion events per neuron . Percentage of axonal fusion events per cell .

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Cumulative plot of axonal or dendritic fusion events per neuron. Normalized cumulative plot of axonal and dendritic fusion events per neuron. Percentage of axonal fusion events per cell.


A - B . Wild - type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT , combined and quantified by LC - MS , using the published 2D - TPP protocol ( Mateus et al . , 2018 ) . Shown are volcano plots of two replicates depicting changes in : protein abundance ( A ) and thermostability ( B ) . A local FDR ( false discovery rate ) < 0 . 01 was set as a threshold for significance . Highlighted proteins : outer membrane proteins ( OMPs , light green ) , β - barrel assembly machinery ( BAMs , red ) , PG synthases / regulators ( green ) , PG hydrolases and regulators ( blue ) and the Tol - Pal complex ( violet ) . All other proteins were colored grey and not labelled to increase the plot clarity .

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A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity.


C - D . Affinity chromatography with immobilised NlpI . Membrane extracts from E . coli were incubated in low and high salt binding conditions ( 50 and 400 mM NaCl , respectively ) , and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label - free LC - MS analysis . The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control , versus the log10 p - value , in low ( 4 replicates ) ( C ) and high ( 2 replicates ) ( D ) salt . Highlighted points are all interactions with PG enzymes and their regulators , as well as members of the divisome . All other proteins were colored grey and not labelled to increase the plot clarity ; many were non - physiological interactions with abundant cytoplasmic proteins .

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C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins.


A . Dissociation constants for interactions between NlpI with MepM , MepS , PBP4 , PBP7 as determined by Microscale thermophoresis ( MST ) . The values are mean ± standard deviation of three independent experiments . B . MepS - NlpI interaction by MST as an example plot for Fig 2A . MST curve plotted is the mean data + / - SD of three independent experiments . Fl , fluorescently labelled ; FNorm , normalized fluorescence .

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A. Dissociation constants for interactions between NlpI with MepM, MepS, PBP4, PBP7 as determined by Microscale thermophoresis (MST). The values are mean ± standard deviation of three independent experiments. B. MepS-NlpI interaction by MST as an example plot for Fig 2A. MST curve plotted is the mean data +/- SD of three independent experiments. Fl, fluorescently labelled; FNorm, normalized fluorescence.


NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI - PBP4 ( C ) complexes by a fixed concentration MST assay . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

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

NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI-PBP4 (C) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)


NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI - PBP7 ( D ) complexes by a fixed concentration MST assay . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI-PBP7 (D) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)