( C ) Gene expression of indicated stress - induced genes in YSC5106 wild - type , nat4 ∆ H4S1D strains . Expression were normalized to RPP0 whose expression remains unchanged . Error bars , of 3 independent experiments . p - values were obtained by comparing nat4 ∆ H4S1D to wild - type values calculated by unpaired two - Student ' s t : * 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 of indicated stress - induced genes in BY4742 wild - type , nat4 ∆ nat4 ∆ [ hNAA40 ] strains . Expression 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 for YSC5106 wild - type , nat4 ∆ , H4R3K double mutant strains . Values in parenthesis indicate mean lifespan . Statistical significance was determined by one - way ANOVA : * * * * p ≤ 0 . 0001 .

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(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 of indicated stress - induced genes in YSC5106 WT , nat4 ∆ , H4R3K double mutant strains . Expression were normalized to RPP0 , whose expression remains unchanged .

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

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(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 for WT , nat4 ∆ , pnc1 ∆ double mutant strains in BY4741 genetic background . Values in parenthesis ( here hereafter ) indicate mean lifespan . Statistical significance ( here hereafter ) was determined by one - way ANOVA : * 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 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 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 performed in BY4741 wild - type , nat4Δ Pste5 - NAT4 strains grown under NCR CR conditions . was immunoprecipitated using antibodies against N - acH4 Histone H4 , analyzed by qRT - PCR with indicated primers . PNC1 - P ( Promoter ) , PNC1 - S ( Transcriptional Start ) , PNC1 - E ( End of gene ) . enrichment from 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 / 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 supernatant at days 0 , 4 7 was detected by ELISA . Bottom , MDM were harvested stained for HIV p17 . Histograms are shown for a representative donor .

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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 , were lysed after 3 h exposure to infectious agents or at completion of infection phase . Intracellular mycobacteria were harvested assayed for mycobacterial growth at day 0 day 7 . Bottom , MDM were harvested 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 ) lysates from panel B were diluted run in a MGIT 960 . Bar 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 / or M . tuberculosis ( TB ) infected MDM were treated for 7 days with 100 pmol / L 1 , 25D3 . ( A ) were lysed 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 ) 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 of saponin - resistant LC3B - II in macrophages after 1 , 25D3 treatment for 7 d . Representative histograms of displaying saponin - resistant LC3B - II from three donors are shown .

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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 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 displaying saponin - resistant LC3B - II from three donors are shown .

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(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 addition of WST - 1 were tested for lactate dehydrogenase ( LDH ) spectrophotometrically using LDHPLUS . For last hour were incubated with WST - 1 , reduction of 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 M . tuberculosis . HIV ( A ) , M . tuberculosis ( TB ) ( B ) , HIV / TB dual infected MDM ( C ) were treated for 7 days with 100 pmol / L 1 , 25D3 , harvested stained for HIVp17 , mycobacteria , 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 stained for HIVp17 , mycobacteria , 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 ) selected using puromycin resistance . Five days later , were incubated with 100 pmol / L 1 , 25D3 or vehicle control for 4 h before infection with HIV / or M . tuberculosis ( TB ) for 3 h . were then washed incubated with 100 pmol / L 1 , 25D3 or vehicle control for 7 days . ( A ) Immunoblot performed using antibodies raised to Beclin - 1 or β - actin after initial pathogen exposure ( Day 0 ) or after 7 days .

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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 assayed for mycobacterial growth by cfu enumeration at day 0 day 7 .

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


D ) Intracellular mycobacteria harvested assayed for viability on time to positivity ( 75 growth units ) at day 0 day 7 using MGIT 960 . bar 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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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 ) selected using puromycin resistance . Five days later , were incubated with 100 pmol / L 1 , 25D3 or vehicle control for 4 h before infection with HIV / or M . tuberculosis ( TB ) for 3 h . were then washed incubated with 100 pmol / L 1 , 25D3 or vehicle control for 7 days . ( A ) Immunoblot performed using antibodies raised to ATG5 or β - actin after initial pathogen exposure ( Day 0 ) or after 7

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O 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 assayed for mycobacterial growth by cfu enumeration at day 0 day 7 . (

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


( D ) Intracellular mycobacteria harvested assayed for viability on time to positivity ( 75 growth units ) at day 0 day 7 using MGIT 960 . bar 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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(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 subsequent infection with HIV / or M . tuberculosis ( TB ) . ( A ) ELISA performed for HIV p24 antigen release at days 0 , 4 7 .

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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 assayed for mycobacterial growth by cfu enumeration at day 0 7 .

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


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

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

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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 assayed for mycobacterial growth by cfu enumeration at day 0 7 .

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


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

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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 TB co - infected were harvested 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 .

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


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

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

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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.


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

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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.


of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker ß3 - tubulin as morphological marker . Total neurite ( 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.


of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker ß3 - tubulin as morphological marker . ChgB 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.


of striatal neurons ( DIV14 ) immunostained for ChgB as DCV marker ß3 - tubulin as morphological marker . DCV pool measured as number of ChgB 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.


of striatal neurons ( DIV14 ) immunostained for ChgB as DCV marker ß3 - tubulin as morphological marker . ChgB 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 per neuron total neurite for hippocampal ( green ) striatal ( red ) neurons .

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


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

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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. 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.


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

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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. 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


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

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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. 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


of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in thalamus post - hoc immunostained for ChgA as DCV marker . ChgA per μm axon from mouse tissue , compared to distribution of ChgB 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 dSTORM strategy to increase resolution . Electron micrograph of axon ( green ) dendrite ( red ) containing DCVs , defined on . In conventional immunofluorescence confocal microscopy , DCVs cannot be separated ( right zoom , green signal representing signal using confocal ) . Super - resolution dSTORM imaging resolves point - spread function ( PSF ) resulting in single DCV localizations . Scale bars : 100 nm .

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

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

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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.


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

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

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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.


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

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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.


of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in thalamus post - hoc immunostained for ChgA as DCV marker . ( H ) Representative confocal ( 100x magnification ) image of mCherry labeled axon ( blue ) with ChgA confocal signal ( red ) 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 ) of single ChgA within mCherry labeled axons with corresponding number of overlaying STED . Data shows average ± . N = 2 mice ( 7 STED , 70 single observations ) .

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

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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 between ChgB , ß3 - tubulin MAP2 .

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


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

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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 per neuron in dendrites , axons total pool ( dendrites + axons ) . Wilcoxon matched - pairs signed rank : 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 axonal ( mm ) per neuron . Wilcoxon matched - pairs signed rank : * * * * 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 per μm dendrite axon per neuron . Lines next to data points show mean ± . Wilcoxon matched - pairs signed rank : * * * * 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 per in total neuron ( dendrites + axons ) in dendrites only . Wilcoxon matched - pairs signed rank : * * * * 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 of ChgB in total neuron ( dendrites + axons ) 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 per μm dendrite axon per neuron . Lines next to data points show mean ± . Wilcoxon matched - pairs signed rank : * * * * p = < 0 . 0001 . Mean ChgB per in dendrites axons . Wilcoxon matched - pairs signed rank : * * * * 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 axons of sparse - labeled network cultures . ( P ) ChgB per μm dendrite or axon . Lines next to data points show mean ± . Mann - Whitney U : * * * * p = < 0 . 0001 . ( Q ) Mean ChgB per in dendrites axons . Unpaired t - : * * * 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.


of DCVs in axons dendrites of mCherry labeled mouse Dentate Gyrus ( DG ) granule tissue after stereotaxic viral injections post - hoc immunostained for ChgA as DCV marker . ( R ) Representative mouse brain slice with labeled DG granule ( mCherry - filler , red ) immunostained for ChgA ( green ) . ChgA were measured in dendrites axons at indicated areas . Scale bar : 250 µm . ( S ) ChgA per μm dendrite or axon in DG granule . 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 zoom of a single isolated hippocampal neuron ( DIV14 ) immunostained for endogenous DCV marker ChgB ( green ) , synaptic VGLUT1 ( red ) dendritic marker MAP2 ( blue ) . of zoom ( B ) is indicated in ( A ) . Scale bars : 20 µm ( A ) , 2 µm ( B ) . Reconstructed ChgB localizations of dSTORM imaging of zoomed in ( B ) ( top ) . Middle panel shows overlay between dSTORM ChgB localizations 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 of synaptic sections containing DCVs . AZ indicates 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 of synapses . Inset : number of DCVs per synaptic section .

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


of DCVs to active zone ( nm ) , measured from center of DCV to middle of active zone . Histogram of of DCVs to middle of active zone ( nm ) . Inset : shortest of DCVs to 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 of of synaptic vesicles ( grey ) to active zone corresponding of DCVs ( black ) to 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 ) synaptic vesicle cluster . Scale bars : 100 nm . of DCVs outside or inside synaptic vesicle cluster . Histogram of ( nm ) of DCVs to border of 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 ) corresponding plot showing overlap between NPY - pHluorin 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 ChgB per neuron .

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


Total ChgB per neuron in uninfected ( control ) NPY - pHluorin infected neurons ( NPY - pH ) . Student ' s t - : n . s . = not significant , p = 0 . 90 . Mean ChgB per per neuron in uninfected ( control ) NPY - pHluorin infected neurons ( NPY - pH ) . Student ' s t - : 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 per neuron total neurite ( mm ) for uninfected control ( grey ) NPY - pHluorin infected ( green ) neurons . Linear regression , inset shows goodness of fit ( r2 ) 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 ( - green ) , superfusion with 60 mM KCl Tyrode solution for 30 s . ( - green ) 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 ) 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 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 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 negative hippocampus neurons ( DIV14 ) upon repetitive electrical stimulation ( 16x50AP at 50 Hz ) . Mann - Whitney U : 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 dendritic areas of ( B ) , with location of fusion events ( middle ) . Average decrease upon NPY - mCherry labeled DCV fusion in axons 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 - targeted EGFP ( mEGFP ) ( black ) 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 ) 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 dendritic fusion events per neuron . of axonal fusion events per .

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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 ΔnlpI were heated at a range of temperatures soluble components were labelled by TMT , combined quantified by LC - MS , using published 2D - TPP protocol ( Mateus et al . , 2018 ) . Shown are volcano plots of two replicates depicting changes in : abundance ( A ) thermostability ( B ) . A local FDR ( false discovery rate ) < 0 . 01 was set as a threshold for significance . Highlighted : outer ( OMPs , green ) , β - barrel assembly machinery ( BAMs , red ) , PG synthases / regulators ( green ) , PG hydrolases regulators ( blue ) Tol - Pal ( violet ) . other were colored grey not labelled to increase 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 . extracts from E . coli were incubated in low high salt binding conditions ( 50 400 mM NaCl , respectively ) , then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label - free LC - MS . plot shows log2 fold enriched when compared to those eluted from a parallel empty column control , versus log10 p - value , in low ( 4 replicates ) ( C ) high ( 2 replicates ) ( D ) salt . Highlighted points are interactions with PG enzymes their regulators , as well as members of divisome . other were colored grey not labelled to increase plot clarity ; many were non - physiological interactions with abundant .

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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 ) . 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 mean data + / - SD of three independent experiments . Fl , fluorescently labelled ; FNorm , normalized .

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

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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 PBP7 as shown by ability of labelled MepS to bind preformed NlpI - PBP7 ( D ) complexes by a fixed MST . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , data were fit using a linear model . Calculated means were compared using Tukey ' s HSD , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in 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)


A . HPLC - PG digestion representing EPase activity . graph shows relative of muropeptide TetraTetra present at end of incubation period for each as described in materials methods . MepM PBP4 were incubated with sacculi , whilst MepS PBP7 were incubated with soluble muropeptides , both from E . coli MC1061 , respectively . Values are mean ± standard deviation of three independent experiments . To calculate significance , data were fit using a linear model . Calculated means were compared using Tukey ' s HSD , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

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A. HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± standard deviation of three 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 . Genetic interactions of nlpI with EPase genes . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from wildtype , single mutants double mutants . An example of a 384 plate is shown . Double mutants were made twice , swapping resistance markers to two single mutants . Colony integral opacity was quantified as a fitness readout , using image software Iris ( Kritikos et al . , 2017 ) . Bar plots shows averaged values of 2 biological experiments , each having 96 technical replicates ( i = 2 , n = 192 ) . error bars represent 95 % confidence interval .

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B. Genetic interactions of nlpI with EPase genes. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants. An example of a 384 plate is shown. Double mutants were made twice, swapping the resistance markers to the two single mutants. Colony integral opacity was quantified as a fitness readout, using the image analysis software Iris (Kritikos et al., 2017). Bar plots shows the averaged values of 2 biological experiments, each having 96 technical replicates (i=2, n=192). The error bars represent the 95% confidence interval.


C . nlpI deletion changes of EPase - mutant strains . graph shows width of single double deletions strains ( 800 < n < 2000 ) . box has a medium between 25 75 % . whiskers with upper lower vertical indicating 95 5 % . dots are points outside 5 95 % range . Above graph are representative of lacking MepS or PBP4 in combination with a deletion of NlpI . scale bar equals 2 µm . Gene encoding legend : nlpI encodes NlpI , dacB encodes PBP4 , pbpG encode PBP7 , mepM encodes MepM , meps encodes MepS .

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C. nlpI deletion changes the morphology of EPase-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, dacB encodes PBP4, pbpG encode PBP7, mepM encodes MepM, meps encodes MepS.


D . Inducible mepS expression system ( pBAD30 ) strongly overproduces MepS . Strains were grown in LB at 30 ° C were collected at OD600 ≈ 0 . 4 . level of MepS contained in fraction was detected using purified anti - MepS antibody .

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D. Inducible mepS expression system (pBAD30) strongly overproduces MepS. Strains were grown in LB at 30°C and cells were collected at OD600≈0.4. The level of MepS contained in the membrane fraction was detected using purified anti-MepS antibody.


E . Visualization of effect of MepS absence or its overexpression on width by phase contrast microscopy . Cultures were grown in LB at 30 ° C aliquots of culture were taken at OD600 ≈ 0 . 1 . scale bar equals 5 μm .

E. Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy. Cultures were grown in LB at 30°C and aliquots of culture were taken at OD600≈0.1. The scale bar equals 5 μm.


F . MepS level modulates width . graph shows distribution of mean width for each , with n corresponding to number of measured for each median width for population being indicated by a dotted referred to as w .

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F. MepS level modulates cell width. The graph shows the distribution of mean cell width for each cell, with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w.


G . Relative fitness of ΔnlpI , ΔmepS ΔnlpIΔmepS mutants . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10 % sucrose , or LB agar plates containing 0 mM or 500 mM NaCl . Plates were incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from wildtype , single mutants double mutants .

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G. Relative fitness of ΔnlpI, ΔmepS and ΔnlpIΔmepS mutants. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10% sucrose, or LB agar plates containing 0 mM or 500 mM NaCl. Plates were incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants.


H . of wildtype ( WT ) , ΔnlpI , ΔmepS ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to envelope integrity . CPRG ( yellow ) cannot penetrate intact Gram - negative envelopes . Its conversion by intracellular β - galactosidase to CPR ( red ) indicates loss of envelope integrity .

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H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.


A . Phase contrast image corresponding SIM image of BW25113 that have been grown in LB at 37 ° C immunolabelled with specific antibodies against NlpI . Scale bar equals 2 µm .

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A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm.


B . Genetic interactions of NlpI with PG machineries . An example of a 384 plate is shown . Bar plots shows averaged values of 2 experiments ( i = 2 , n = 192 ) . Error bars denote 95 % confidence interval of mean .

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B. Genetic interactions of NlpI with PG machineries. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean.


C . NlpI deletion exacerbates morphological defects of PBP1B / LpoB - mutant strains . graph shows width of single double deletions strains ( 800 < n < 2000 ) . box has a medium between 25 75 % . whiskers with upper lower vertical indicating 95 5 % . dots are points outside 5 95 % range . Representative of strains are shown above graph . scale bar equals 2 µm . Gene encoding legend : nlpI encodes NlpI , mrcA encodes PBP1A , lpoA encodes LpoA , mrcB encodes PBP1B , lpoB encodes LpoB .

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C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB.


D . Dissociation constants for interactions between PBP1A LpoA with NlpI , MepM , MepS , PBP4 PBP7 as determined by MST . values are mean ± standard deviation of three independent experiments . 1PBP1A was used as fluorescently labelled in assays . 2LpoA was used as unlabelled ligand in combinations , except with MepM PBP4 .

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D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4.


A - B . PBP4 has different interaction sites for PBP1A / LpoA NlpI as shown by a single MST . Plots show FNorm or values of fluorescently labelled PBP1A or LpoA with or without PBP4 , NlpI or PBP4 - NlpI . Values are mean ± standard deviation of three independent experiments .

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A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.


A . Estimated number of molecules molarity of PBP1a / LpoA EPases . 1Numbers obtained by ribosomal profiling in rich growth medium ( Li et al . , 2014 ) . 2Concentration of monomer . 3Decreases in presence of NlpI ( Singh et al . , 2015 ) . 4 " no change detected " . periplasmic concentrations of were estimated for a with periplasmic volume of 3 . 33 × 10 - 16 L , where 1 molecule corresponds to 5 nM .

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A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.


( B ) Islet graft survival of mice receiving vehicle ( PBS ) or IL - 33 was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * * * P < 0 . 001 vs islet + vehicle . ( C ) Blood glucose level of mice treated with IL - 33 ( horizontal black indicates a BGL of 16 mmol / liter , threshold for rejection ) . Each represents one mouse .

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(B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle. (C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse.


( D ) Intraperitoneal glucose tolerance ( IPGTT ) was assessed in normal mice , islet transplant mice receiving vehicle ( on day when grafts were considered rejected ) , islet transplant mice treated with IL - 33 ( at day 30 day 80 post - islet transplantation ) . Data shown are mean ± ( n = 6 - 9 per group ) . ( E ) under curve ( AUC ) for IPGTT was assessed . Data shown are mean ± ( n = 6 - 9 per group ) a one - way ANOVA was performed , * * * P < 0 . 001 .

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(D) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group). (E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001.


( F ) Representative immunohistochemical for insulin in graft samples from mice receiving vehicle or IL - 33 . Scale bar = 100 µm .

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(F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.


C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( A , B ) Representative FACS showing proportion of Tregs ( CD4 + Foxp3 + ) in CD4 + T compartment from spleens ( A ) kidneys ( B ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) .

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C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).


C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( C , D ) Representative FACS showing proportion of ILC2s ( Lin - GATA - 3 + ) in CD45 + leukocyte compartment from spleens ( C ) kidneys ( D ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) .

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

C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).


( E , F ) proportion of Tregs ( E ) ILC2s ( F ) in spleen kidney in PBS - injected controls ( n = 4 ) at 1 - 12 after IL - 33 treatment ( n = 4 - 6 per IL - 33 - treated group ) . Data information : Data shown are mean ± ; Statistical was performed with an unpaired t - , * * P < 0 . 01 , * * * P < 0 . 001 .

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

(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.


( B - D ) Proportion or numbers of CD4 + Foxp3 + Tregs in spleens , kidneys islet grafts of normal , islet transplant mice receiving vehicle islet transplant mice with IL - 33 treatment ( at day 7 , 30 80 post - islet transplantation ) . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.


( E ) Representative confocal microscopy of immunostaining for CD4 , Foxp3 insulin in islet grafts . Scale bar = 50 µm .

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(E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm.


( F - H ) Proportion or numbers of ILC2s in spleens , kidneys islet graft of mice with or without IL - 33 treatment . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


( I ) Representative confocal microscopy of immunostaining for CD127 , ST2 , CD3 insulin in islet grafts . Scale bar = 50 µm .

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(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.


( J ) mRNA expression of IL - 25 , IL - 33 TSLP in islet grafts of mice with or without IL - 33 treatment was examined by qPCR , expressed relative to control of each experiment . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

(J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


Proportion of CD4 + Foxp3 + Tregs ( B ) from spleens kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are mean ± ( n = 4 - 5 per group ) a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 .

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

Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.


Proportion of Lin - GATA - 3 + ILC2s ( C ) from spleens kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are mean ± ( n = 4 - 5 per group ) a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 .

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

Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.


( D ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * 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 O O O O O O O O O O O O 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) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.


( E ) Data are shown as blood glucose measurement in islet transplant mice treated with IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 . horizontal black indicates a BGL of 16 mmol / liter , threshold for rejection . Each represents one mouse .

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(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.


( A , B ) ILC210 non - IL - 10 producing ILC2s were assessed in islet graft ( A ) kidney ( B ) by intracellular IL - 10 at day 5 post islet transplantation . Data shown are mean ± ( n = 5 per group ) an unpaired t - was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 .

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(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.


( C ) IL - 10 reporter C57BL / 6 mice were treated with PBS , IL - 33 alone or IL - 33 IL - 2C daily for 5 consecutive days . Proportion of lin - CD127 + ST2 + IL - 10 - GFP ILC210 from kidneys at day 3 after treatment in IL - 10 reporter C57BL / 6 mice . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 .

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

(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.


( D ) Kidney ILC2s isolated from normal IL - 10 reporter C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 IL - 2C for six days . Proportion of ILC210 was assessed by flow cytometry . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 .

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(D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.


( E ) secreted cytokine IL - 10 was analyzed via ELISA . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 .

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(E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.


( F ) Kidney ILC2s isolated from C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 IL - 2C for 30 minutes . expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry . p - STAT5 ( red lines ) isotype controls ( gray - filled areas ) are shown . Data represent mean ± of evaluations of MFI from each group ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 . MFI , mean .

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

(F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity.


( G ) kidney ILC2s were cultured with Medium , IL - 33 / IL - 2C , or IL - 33 / IL - 2C STAT5 inhibitor ( STAT5 - IN ) for three days . secreted cytokine IL - 10 was analyzed via ELISA . Data shown are mean ± ( n = 6 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 .

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

(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.


( A ) ILC210 non - ILC210 were isolated from ex vivo - expanded kidney ILC2 by flow sorting . ILC210 were transfected with control ( ILC210 - C ) or IL - 10 CRISPR - Cas9 ( ILC210 - IL - 10 ) . IL - 10 was measured in culture supernatant of ILC210 - C ILC210 - IL - 10 via ELISA . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 .

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

(A) ILC210 and non-ILC210 were isolated from ex vivo-expanded kidney ILC2 by flow sorting. ILC210 were transfected with control (ILC210-C) or IL-10 CRISPR-Cas9 (ILC210-IL-10). IL-10 was measured in culture supernatant of ILC210-C and ILC210-IL-10 via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.


( C ) Transfused ILC2s ( CFSE labeled , Green ) were observed in islet graft at day 5 post islet transplantation . Scale bar = 50 µm . numbers of CFSE - labeled ILC2s in islet graft were counted . Data shown are mean ± a one - way ANOVA was performed ( n = 4 per group ) . NS , not significant .

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(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.


( D ) mRNA expression of IL - 10 in islet grafts at day 5 post islet transplantation was examined by qPCR . Data shown are mean ± ( n = 4 per group ) a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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(D) The mRNA expression of IL-10 in islet grafts at day 5 post islet transplantation was examined by qPCR. Data shown are the mean ± SEM (n=4 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


( E ) Islet graft survival of four groups of mice was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.


( F ) CD4 + T isolated from C57BL / 6 splenocytes were cultured with irradiated splenocytes derived from BALB / c in presence of ILC210 at indicated ratios for 4 days . CD4 + T proliferation was assessed using [ 3H ] - thymidine incorporation assays . Data shown are mean ± ( n = 4 - 6 per group ) .

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(F) CD4+ T cells isolated from C57BL/6 splenocytes were cultured with irradiated splenocytes derived from BALB/c in the presence of ILC210 at the indicated ratios for 4 days. CD4+ T cells proliferation was assessed using [3H]-thymidine incorporation assays. Data shown are the mean ± SEM (n=4-6 per group).


( G ) Neutralizing anti - IL - 10 antibodies or genetic ablation of IL - 10 were used to block effect of ILC210 on CD4 + T proliferation . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed . * * * P < 0 . 001 .

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

(G) Neutralizing anti-IL-10 antibodies or genetic ablation of IL-10 were used to block the effect of ILC210 on CD4+ T cells proliferation. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed. ***P<0.001.


( B ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * * * P < 0 . 001 .

(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.


( C ) To track ILC210 in vivo , CD45 . 2 + ILC210 were co - transplanted with islets in diabetic CD45 . 1 + C57BL / 6 mice . Representative FACS showing proportion of locally transplanted ILC210 ( CD45 . 2 + ST2 + ) in total CD45 + compartment from islet graft at day 5 80 post - islet transplantation or at day when grafts were considered rejected .

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(C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected.


( D , E ) Proportion numbers of locally transplanted CD45 . 2 + ST2 + ILC210 in islet grafts of islet transplant mice over time . Data shown are mean ± ( n = 4 - 5 per group ) a one - way ANOVA was performed , * * P < 0 . 01 , * * * P < 0 . 001 .

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(D,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001.