( A ) FKRP - NSCs treated with 4BPPNit showed increased IIH6 reactivity , compared with untreated control cells . Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR - corrected NSCs . Under the same exposure time , the signal intensity of IIH6 reactivity is saturated in corrected - NSCs . Data information : Intensities of glycosylation are normalized to GAPDH protein expression .

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(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression.


( B ) Representative immunoblots with the IIH6 antibody using FKRP - and corrected - NSCs treated with 4BPPNit for 24 , 48 and 72 h , and DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time .

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(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time.


( C ) Quantification of α - dystroglycan glycosylation ( IIH6 ) , compared with DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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(C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( D ) Representative immunoblots of laminin - binding analysis using FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( E ) Quantification of laminin - binding activities , compared with DMSO only controls . Data information : Intensities of laminin - binding activity are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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(D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( F ) Representative immunoblots with the AF6868 antibody detecting both α - dystroglycan core protein and β - dystroglycan in FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( G ) Quantification of α - dystroglycan core protein and β - dystroglycan expression , compared with DMSO only controls . Data information : Intensities of protein expression are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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

(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( A ) DAG1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 .

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(A) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01


( B ) FKRP gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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(B) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( C ) LARGE1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

(C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


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 .

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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 , 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 .

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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 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 .

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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 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 .

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 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 O O O O O O O O O O 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 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 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

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 .

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

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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 ) 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

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

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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 and subsequent infection with HIV and / or M . tuberculosis ( TB ) . ( A ) ELISA performed for HIV p24 antigen release at days 0 , 4 and 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 and assayed for mycobacterial growth by cfu enumeration at day 0 and 7 .

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(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 .

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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 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 .

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

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 .

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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 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 .

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 .

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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.


( F ) Quantification of the highlighted genes enriched in either the somata or processes at the transcript or protein level . ( Upper row : Scatterplots of transcript level ; n = 2 ; crossbar represents mean . Lower row : Boxplot of protein levels ; n = 4 ) .

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(F) Quantification of the highlighted genes enriched in either the somata or processes at the transcript or protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4).


( A ) Volcano plots representing transcript down - ( red ) or up - regulation ( yellow ) after 48 hours PTX in the somatic and process compartment . Representative transcripts are highlighted and labelled . Linear model was used to fit transcriptome data and subsequently contrasted using likelihood ratio testing for PTX - treatment effects across compartments .

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(A) Volcano plots representing transcript down- (red) or up-regulation (yellow) after 48 hours PTX in the somatic and process compartment. Representative transcripts are highlighted and labelled. Linear model was used to fit transcriptome data and subsequently contrasted using likelihood ratio testing for PTX-treatment effects across compartments.


( A ) Real - time quantitative PCR ( RT - qPCR ) of transcripts changing differentially between compartments using the compartmentalized cultures after 48 hours PTX - treatment . n = 8 independent biological replicates ( except for Plk2 ( n = 7 ) , where one measurement couldn ' t be performed due to insufficient cDNA ) ; PTX - effect was assessed by three - way ANOVA followed by Tukey ' s post - hoc multiple comparison test ; * p < 0 . 05 ; * * p < 0 . 01 .

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(A) Real-time quantitative PCR (RT-qPCR) of transcripts changing differentially between compartments using the compartmentalized cultures after 48 hours PTX-treatment. n=8 independent biological replicates (except for Plk2 (n=7), where one measurement couldn't be performed due to insufficient cDNA); PTX-effect was assessed by three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01.


( B ) Representative images of single - molecule FISH ( smFISH ) in either control or 48 h PTX - treated rat hippocampal neurons ( DIV20 ) using probes specific for Add2 , Dnajc6 and Sort1 ( green ) . MAP2 immunostaining ( red ) was used to visualize neuronal somata and dendrites . Inserts at higher magnification illustrate PTX - dependent changes in dendritic RNA puncta . Scale bar = 10 μm . ( C ) Quantification of B . ( n = 3 - 4 independet biological replicates with 8 - 10 cells averaged per condition and replicate ; two - sample Student ' s t - test ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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(B) Representative images of single-molecule FISH (smFISH) in either control or 48 h PTX-treated rat hippocampal neurons (DIV20) using probes specific for Add2, Dnajc6 and Sort1 (green). MAP2 immunostaining (red) was used to visualize neuronal somata and dendrites. Inserts at higher magnification illustrate PTX-dependent changes in dendritic RNA puncta. Scale bar = 10 μm. (C) Quantification of B. (n=3-4 independet biological replicates with 8-10 cells averaged per condition and replicate; two-sample Student's t-test; *p < 0.05; **p < 0.01). B


( D ) Representative plots of genes changing after PTX at the transcript and protein level . ( Upper row : Scatterplots of transcript level ; n = 2 ; crossbar represents mean . Lower row : Boxplot of protein levels ; n = 4 ) . Boxplots : central line : median ; box : 25th to 75th percentile ; whiskers : until last data point within 1 . 5x interquartile range ( IQR ) .

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(D) Representative plots of genes changing after PTX at the transcript and protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4). Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).


( A ) Representative pictures of nascent Camk2a - peptides in mock - and PTX - treated neurons . Upper Panels : Representative overview images with merged channels ( grey : MAP2 - staining , red : Duolink - PLA signal , blue : Hoechst nuclear staining ) . Middle Panels : Magnifications of boxed areas marked in ( A ) . MAP2 signal was used to outline somata and dendritic processes ( white lines ) . Lower Panels : Negative control of puromycin - untreated cells with representative Duolink - signal in dendrite and soma , respectively . ( Scale bars : in overview images 20 μm , in inserts of higher magnification 5 μm ) . ( B ) Same as A with nascent Synapsin1 - peptides . (

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(A) Representative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink-signal in dendrite and soma, respectively. (Scale bars: in overview images 20 μm, in inserts of higher magnification 5 μm). (B) Same as A with nascent Synapsin1-peptides. (


( C ) Enrichment analysis for RBP motifs in down - and up - regulated genes in either compartment . Fisher ' s exact test was used to perform enrichment analysis ; FDR - corrected .

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(C) Enrichment analysis for RBP motifs in down- and up-regulated genes in either compartment. Fisher's exact test was used to perform enrichment analysis; FDR-corrected.


a . b . c . d . Chest computed tomographic scans of Patient 1 were obtained on the day of admission ( day 6 after the onset of illness ) . Bilateral focal consolidation , lobar consolidation , and patchy consolidation were clearly observed , especially in the lower lung .

a. b. c. d. Chest computed tomographic scans of Patient 1 were obtained on the day of admission (day 6 after the onset of illness). Bilateral focal consolidation, lobar consolidation, and patchy consolidation were clearly observed, especially in the lower lung.


e . Chest radiograph of patient 1 was obtained on day 5 after admission ( day 11 after the onset of illness ) . Bilateral diffuse patchy and fuzzy shadow were observed .

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e. Chest radiograph of patient 1 was obtained on day 5 after admission (day 11 after the onset of illness). Bilateral diffuse patchy and fuzzy shadow were observed.


( A ) miRNA abundance in RNA - sequencing samples from murine , porcine , and bovine oocytes Each graph shows on the y - axis reads per million of 19 - 32 nt reads for miRNAs and on the x - axis miRNAs ordered by name .

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(A) miRNA abundance in RNA-sequencing samples from murine, porcine, and bovine oocytes Each graph shows on the y-axis reads per million of 19-32 nt reads for miRNAs and on the x-axis miRNAs ordered by name.


( C ) qPCR quantification of ssc - miR - 205 and bta - miR - 10b miRNAs in murine ( M . m . ) , porcine ( S . s . ) , and bovine ( B . t . ) oocytes , respectively . Shown are a mean values from three independent experiments . Error bars = SD . Detailed information concerning miRNA quantification is provided in the Methods section .

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(C) qPCR quantification of ssc-miR-205 and bta-miR-10b miRNAs in murine (M.m.), porcine (S.s.), and bovine (B.t.) oocytes, respectively. Shown are a mean values from three independent experiments. Error bars = SD. Detailed information concerning miRNA quantification is provided in the Methods section.


( A ) bta - miR - 10b and ssc - miR - 205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary ( 1x perfect ) miRNA binding site or four partially complementary ( bulged ) miRNA binding sites . Error bars = SD . Each data point represents a value obtained from five microinjected oocytes . Data were collected in two microinjection sessions , each microinjection session used a new batch of in vitro transcribed reporter mRNAs . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) bta-miR-10b and ssc-miR-205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary (1x perfect) miRNA binding site or four partially complementary (bulged) miRNA binding sites. Error bars = SD. Each data point represents a value obtained from five microinjected oocytes. Data were collected in two microinjection sessions, each microinjection session used a new batch of in vitro transcribed reporter mRNAs. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


( B ) De - repression of predicted ssc - miR - 205 targets . In three independent microinjection experiments , ssc - miR - 205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT - qPCR . Error bars = SD . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(B) De-repression of predicted ssc-miR-205 targets. In three independent microinjection experiments, ssc-miR-205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT-qPCR. Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


( C ) Inhibition of ssc - miR - 205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage . Shown is development of microinjected oocytes to the blastocyst stage relative to water - injected oocytes . Five independent experiments ( represented by individual data points ) were performed , ~ 50 porcine oocytes were microinjected in each group in each experiment . Difference between let - 7a and miR - 205 inhibitors effects is statistically significant ( two - tailed paired t - test p - value = 0 . 016 ) . Error bars = SD . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(C) Inhibition of ssc-miR-205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage. Shown is development of microinjected oocytes to the blastocyst stage relative to water-injected oocytes. Five independent experiments (represented by individual data points) were performed, ~50 porcine oocytes were microinjected in each group in each experiment. Difference between let-7a and miR-205 inhibitors effects is statistically significant (two-tailed paired t-test p-value = 0.016). Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


( A ) ssc - miR - 205 turnover . Fully - grown transcriptionally quiescent oocytes were cultured for indicated periods of time and selected miRNAs were quantified by RT - qPCR in four ( ssc - let - 7a ) , five ( ssc - miR - 22 ) or six ( ssc - mir10b and ssc - miR - 205 ) independent experiments . Error bars = SD .

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(A) ssc-miR-205 turnover. Fully-grown transcriptionally quiescent oocytes were cultured for indicated periods of time and selected miRNAs were quantified by RT-qPCR in four (ssc-let-7a), five (ssc-miR-22) or six (ssc-mir10b and ssc-miR-205) independent experiments. Error bars = SD.


( B ) Comparison of mean relative changes of miRNA level at 20 h of culture ssc - miR - 205 in oocytes is less than ssc - miR - 10b , ssc - miR - 22 , or ssc - let - 7a . Error bars = SD . Asterisks indicate statistical significance ( p - value ) of one - tailed paired t - test , ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(B) Comparison of mean relative changes of miRNA level at 20 h of culture ssc-miR-205 in oocytes is less than ssc-miR-10b, ssc-miR-22, or ssc-let-7a. Error bars = SD. Asterisks indicate statistical significance (p-value) of one-tailed paired t-test, (*p < 0.05, **p < 0.01).


( C ) Treatment by sodium periodate followed by qPCR does not support 2 ' - OH modification of either ssc - miR - 205 or bta - miR - 10b miRNA . Shown are relative levels of the sodium periodate - treated samples to non - treated samples , which were set to one . The experiment was performed three times . Error bars = SD . Efficiency of periodate treatment was confirmed using non - methylated and methylated miR - 221 RNA

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(C) Treatment by sodium periodate followed by qPCR does not support 2'-OH modification of either ssc-miR-205 or bta-miR-10b miRNA. Shown are relative levels of the sodium periodate-treated samples to non-treated samples, which were set to one. The experiment was performed three times. Error bars = SD. Efficiency of periodate treatment was confirmed using non-methylated and methylated miR-221 RNA oligonucleotides


( D ) 3 ′ tailing of small RNAs in murine , bovine , and porcine oocytes Shown are percentages of miRNAs tailed at 3 ′ end with mono - and oligonucleotides .

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(D) 3′ tailing of small RNAs in murine, bovine, and porcine oocytes Shown are percentages of miRNAs tailed at 3′ end with mono- and oligonucleotides.


B . Jac1488 was imported into purified mitochondria for indicated times and treated with Proteinase K ( PK ) or left untreated as indicated . For comparison , 47 pmol of precursor were loaded ( lane 1 ) . Prec . , purified precursor protein ; p , precursor ; m , mature protein ; ∆ ψ , membrane potential .

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B. Jac1488 was imported into purified mitochondria for indicated times and treated with Proteinase K (PK) or left untreated as indicated. For comparison, 47 pmol of precursor were loaded (lane 1). Prec., purified precursor protein; p, precursor; m, mature protein; ∆ψ, membrane potential.


C . Jac1488 import into mitochondria as in B , right side ; Jac1488 protein dilution series , left side .

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C. Jac1488 import into mitochondria as in B, right side; Jac1488 protein dilution series, left side.


D . Quantification of import of Jac1488 ( maximal signal at 15 min import time , 100 % ) ; error bars indicate ( SEM ) ( n = 3 ) . E . Quantification of absolute imported amounts in picomoles Jac1488 per µg of mitochondria ; error bars indicate SEM ( n = 3 ) .

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D. Quantification of import of Jac1488 (maximal signal at 15 min import time, 100%); error bars indicate (SEM) (n=3). E. Quantification of absolute imported amounts in picomoles Jac1488 per µg of mitochondria; error bars indicate SEM (n=3).


A . Steady state protein levels in wild - type ( WT ) and Tim50 - depleted mitochondria visualized by Western blotting and immunodetection with indicated antisera .

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A. Steady state protein levels in wild-type (WT) and Tim50-depleted mitochondria visualized by Western blotting and immunodetection with indicated antisera.


B . Jac1488 was imported into wild - type ( WT ) and Tim50 - depleted mitochondria for indicated times and samples were treated with Proteinase K . p ,

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B. Jac1488 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples were treated with Proteinase K. p, precursor


C . Quantification of Jac1488 import into wild - type ( WT ) and Tim50 - depleted mitochondria . The amount of imported protease - protected protein in WT mitochondria at 15 min was set to 100 % ; error bars indicate SEM ( n = 3 ) .

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C. Quantification of Jac1488 import into wild-type (WT) and Tim50-depleted mitochondria. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=3).


D . [ 35S ] Jac1 was imported into wild - type ( WT ) and Tim50 - depleted mitochondria for indicated times and samples were treated with Proteinase K . p , precursor ; m , mature .

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D. [35S]Jac1 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples were treated with Proteinase K. p, precursor; m, mature.


E . Quantification of [ 35S ] Jac1 import into wild - type ( WT ) and Tim50 - depleted mitochondria . The amount of imported protease - protected protein in WT mitochondria at 15 min was set to 100 % ; error bars indicate SEM ( n = 3 ) .

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E. Quantification of [35S]Jac1 import into wild-type (WT) and Tim50-depleted mitochondria. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=3).


F . Steady state protein levels in wild - type ( WT ) and tim44 - 804 mitochondria visualized by Western blotting and immunodetection with indicated antisera .

F. Steady state protein levels in wild-type (WT) and tim44-804 mitochondria visualized by Western blotting and immunodetection with indicated antisera.


B . Purified pAmJac1 was conjugated with different fluorophores as indicated . Detected without bleed through under fluorescence imaging conditions .

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B. Purified pAmJac1 was conjugated with different fluorophores as indicated. Detected without bleed through under fluorescence imaging conditions.


C . Jac1488 and pAmJac1 conjugated to DyLight 488 , 680 , and 800 were imported into wild - type mitochondria for indicated times and samples were treated with Proteinase K . Prec . , purified precursor protein ; p , precursor ; m , mature .

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C. Jac1488 and pAmJac1 conjugated to DyLight 488, 680, and 800 were imported into wild-type mitochondria for indicated times and samples were treated with Proteinase K. Prec., purified precursor protein; p, precursor; m, mature.


E . Jac1488 and pImJac1 conjugated to DyLight 488 , 680 and 800 were imported into wild - type mitochondria for indicated times and samples treated with Proteinase K . Prec . , purified precursor protein ; p , precursor ; m , mature .

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E. Jac1488 and pImJac1 conjugated to DyLight 488, 680 and 800 were imported into wild-type mitochondria for indicated times and samples treated with Proteinase K. Prec., purified precursor protein; p, precursor; m, mature.


D . Quantification of picomoles of pAmJac1680 imported per µg mitochondria as assessed in 96 - well format ; error bars indicate SEM ( n = 8 ) .

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D. Quantification of picomoles of pAmJac1680 imported per µg mitochondria as assessed in 96-well format; error bars indicate SEM (n=8).


E . pAmJac1680 was imported into purified mitochondria and samples analyzed by SDS - PAGE or in 96 - well format . The amount of imported , protease - protected samples at 15 min was set to 100 % in each case ; error bars indicate SEM ( n = 8 for 96 - well plate analysis , n = 3 for SDS - PAGE ) .

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E. pAmJac1680 was imported into purified mitochondria and samples analyzed by SDS-PAGE or in 96-well format. The amount of imported, protease-protected samples at 15 min was set to 100% in each case; error bars indicate SEM (n=8 for 96-well plate analysis, n=3 for SDS-PAGE).


G . pAmJac1680 was imported into wild - type ( WT ) and Tim50 - depleted mitochondria for indicated times and samples treated with proteinase K . Samples were analyzed in 96 - well format and by SDS - PAGE . The amount of imported protease - protected protein in WT mitochondria at 15 min was set to 100 % ; error bars indicate SEM ( n = 5 for 96 - well plate analysis , n = 4 for SDS - PAGE ) .

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G. pAmJac1680 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples treated with proteinase K. Samples were analyzed in 96-well format and by SDS-PAGE. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=5 for 96-well plate analysis, n=4 for SDS-PAGE).


A . Atp5488 ( left panel ) or [ 35S ] Atp5 ( right panel ) were imported into mitochondria for indicated times and in the presence or absence of a membrane potential ( ∆ ψ ) . After proteinase K treatment , samples were separated by SDS - Page and analyzed by fluorescence scanning or digital autoradiography . Prec . , purified precursor protein ; p , precursor ; m , mature protein .

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A. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were separated by SDS-Page and analyzed by fluorescence scanning or digital autoradiography. Prec., purified precursor protein; p, precursor; m, mature protein.


B . Import of Atp5488 with increasing concentrations of CCCP to study dependency of import on membrane potential ( ∆ ψ ) . P , precursor ; m , mature protein .

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B. Import of Atp5488 with increasing concentrations of CCCP to study dependency of import on membrane potential (∆ψ). P, precursor; m, mature protein.


D . [ 35S ] Atp5 was imported with increasing CCCP concentrations as described in B . Samples were separated by SDS - PAGE and analyzed by digital autoradiography . E . Quantification of import of [ 35S ] Atp5 with increasing CCCP concentrations . The amount of imported protease - protected protein in the absence of CCCP was set to 100 % ; error bars indicate SEM ( n = 3 ) .

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D. [35S]Atp5 was imported with increasing CCCP concentrations as described in B. Samples were separated by SDS-PAGE and analyzed by digital autoradiography. E. Quantification of import of [35S]Atp5 with increasing CCCP concentrations. The amount of imported protease-protected protein in the absence of CCCP was set to 100%; error bars indicate SEM (n=3).


G . Atp5488 ( left panel ) or [ 35S ] Atp5 ( right panel ) were imported into wild - type and atp21 ∆ mitochondria for indicated times and in the presence or absence of a membrane potential ( ∆ ψ ) . After proteinase K treatment , samples were solubilized in digitonin buffer and separated by BN - PAGE . Aliquots of the samples was analyzed by SDS - PAGE ( lower panels ) . Subsequently proteins were visualized by fluorescence scanning or digital autoradiography . Complex V dimer , ( CVD ) and monomer ( CVM ) . Prec . , purified precursor ; p , precursor ; m , mature protein .

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G. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into wild-type and atp21∆ mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were solubilized in digitonin buffer and separated by BN-PAGE. Aliquots of the samples was analyzed by SDS-PAGE (lower panels). Subsequently proteins were visualized by fluorescence scanning or digital autoradiography. Complex V dimer, (CVD) and monomer (CVM). Prec., purified precursor ; p, precursor; m, mature protein.


A Co ‐ localization of endogenous TBC1D5 and VPS35 with mCherry ‐ ATG9A , in steady state and autophagy induced by mTOR inhibitor KU0063794 ( 6 h ) .

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A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).


B , C Quantification of co ‐ localization experiments presented in ( A ) .

B, C Quantification of co‐localization experiments presented in (A).


D Co ‐ immunoprecipitation of HA ‐ Flag ‐ TBC1D5 with myc ‐ VPS29 , transiently overexpressed in 293T cells , DMSO or KU0063794 ( 6 h ) treated .

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D Co‐immunoprecipitation of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated.


E , F Co ‐ immunoprecipitation of endogenous TBC1D5 and ATG9A with myc ‐ VPS29 ( E ) transiently overexpressed in 293T cells or with HA ‐ Flag ‐ VPS35 ( F ) transiently overexpressed in 293T cells , treated with DMSO or KU0063794 ( 6 h ) .

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E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h).


G Co ‐ immunoprecipitation of endogenous VPS29 and ATG9A with HA ‐ Flag ‐ TBC1D5stably expressed in 293T cells , treated with DMSO or KU0063794 ( 6 h ) .

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G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).


H Co ‐ immunoprecipitation of endogenous ULK1 and pATG13 ( Ser318 ) with HA ‐ Flag ‐ TBC1D5 , stably expressed in T ‐ REx 293T cells , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

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H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


I Magnified still images extracted from Supplementary Movie S2 . mCherry ‐ TBC1D5 co ‐ localizes with GFP ‐ ATG9 upon autophagy induction by mTOR inhibitor , KU0063794 .

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I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794.


H Co - immunoprecipitation of endogenous ULK1 and pATG13 ( Ser318 ) with HA - Flag - TBC1D5 , stably expressed in T - REx 293T cells , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5 . Cells were treated with DMSO or KU0063794 ( 6 h ) , lysed in RIPA buffer ( 1 % SDS ) and lysates were subjected to SDS - PAGE .

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A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS-PAGE.


B U2OS cells stably expressing mCherry ‐ ATG9 were depleted of VPS29 and TBC1D5 using shRNAs , treated with DMSO or KU0063794 ( 6 h ) , fixed with 2 % PFA , and immunostained with anti ‐ LAMP1 antibody .

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B U2OS cells stably expressing mCherry‐ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti‐LAMP1 antibody.


C Quantification of ATG9 vesicular structures in cells from ( B ) under non ‐ stimulated conditions .

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C Quantification of ATG9 vesicular structures in cells from (B) under non‐stimulated conditions.


D Quantification of ATG9 and LAMP1 co ‐ localization in cells presented in ( B ) .

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D Quantification of ATG9 and LAMP1 co‐localization in cells presented in (B).


E Quantification of ATG9 and LAMP1 co − localization in cells stably depleted for TBC1D5 ( shRNA # 1 ) or VPS29 ( shRNA # 2 ) , transiently transfected with myc − TBC1D5 plasmid resistant to TBC1D5 shRNA # 1 . Cells were treated with KU0063794 ( 6 h ) , 20 h post − transfection , fixed , and immunostained with anti − LAMP1 and anti − myc antibodies .

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E Quantification of ATG9 and LAMP1 co−localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc−TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post−transfection, fixed, and immunostained with anti−LAMP1 and anti−myc antibodies.


F Control shRNA and U2OS cell lysates depleted for TBC1D5 ( shRNA # 1 ) , transfected with empty plasmid or with shRNA ‐ resistant myc ‐ TBC1D5 .

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F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA‐resistant myc‐TBC1D5.


G Immunofluorescence of TBC1D5 ( shRNA # 1 ) cells transiently transfected with shRNA ‐ resistant myc ‐ TBC1D5 , treated with KU0063794 ( 6 h ) . 20 h post ‐ transfection cells were treated , subsequently fixed , and immunostained with anti ‐ myc and anti ‐ LAMP1 antibodies .

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G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA‐resistant myc‐TBC1D5, treated with KU0063794 (6 h). 20 h post‐transfection cells were treated, subsequently fixed, and immunostained with anti‐myc and anti‐LAMP1 antibodies.


A HA ‐ Flag ‐ TBC1D5 expression was induced in T ‐ REx HeLa cells for 20 h , lysates were subjected to GSTpull ‐ down , − AP2M1 . Co ‐ precipitatedTBC1D5 was detected with anti − Flag antibody .

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A HA‐Flag‐TBC1D5 expression was induced in T‐REx HeLa cells for 20 h, lysates were subjected to GSTpull‐down, −AP2M1. Co‐precipitatedTBC1D5 was detected with anti−Flag antibody.


B Control cells and TBC1D5 ( shRNA # 1 ) U2OS cell lysates subjected to SDS - PAGE , subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1 .

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B Control cells and TBC1D5 (shRNA#1) U2OS cell lysates subjected to SDS-PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1.


C Immunofluorescent staining of AP2A1 in U2OS shRNA control , or shRNA # 1 TBC1D5 cells treated with KU0063974 or DMSO ( 6 h ) .

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C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).


D U2OS cells stably expressing HA ‐ Flag ‐ ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5 . Regions of co ‐ localization are indicated with arrows .

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D U2OS cells stably expressing HA‐Flag‐ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co‐localization are indicated with arrows.


E U2OS cells stably expressing HA ‐ Flag ‐ ATG9A were starved in EBSS ( 4 h ) or treated with KU0063794 ( 6 h ) , subsequently fixed and stained with anti ‐ AP2A1 antibody .

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E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody.


F U2OS cells stably expressing mCherry ‐ ATG9A were treated with KU0063794 ( 6 h ) , lysed in co ‐ immunoprecipitation buffer . Lysates from DMSO ‐ or KU0063794 ‐ treated cells were split and equal volumes were incubated with RFP ‐ Trap beads or GFP ‐ Trap beads , or agarose as a negative control , overnight at 4 ° C . Precipitated proteins were analyzed by SDS - PAGE .

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F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.


A U2OS cells stably expressing HA ‐ Flag ‐ ATG9A transfected with siRNA targeting AP2A1 ( 40 nM ) , or siRNA AllStar control ( 40 nM ) were treated with KU0063794 or DMSO ( 6 h ) 4 days post ‐ transfection , fixed , and stained with anti ‐ HA antibody .

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A U2OS cells stably expressing HA‐Flag‐ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post‐transfection, fixed, and stained with anti‐HA antibody.


B Co ‐ localization of HA ‐ Flag ‐ ATG9A with TGN46 .

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B Co‐localization of HA‐Flag‐ATG9A with TGN46.


C Lysates of U2OS cells transfected with siRNA targeting AP2A1 , or siRNA control AllStars analyzed by SDS - PAGE .

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C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS-PAGE.


D U2OS cells stably expressing mCherry − ATG9 were transiently transfected with GFP − Dynamin 2 or GFP − Dynamin 2 ( K44A ) , 20 h post − transfection cells were fixed and with anti − TBC1D5 antibody . ATG9A and Dynamin 2co − localization is indicated by arrows .

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D U2OS cells stably expressing mCherry−ATG9 were transiently transfected with GFP−Dynamin 2 or GFP−Dynamin 2 (K44A), 20 h post−transfection cells were fixed and with anti−TBC1D5 antibody. ATG9A and Dynamin 2co−localization is indicated by arrows.


A U2OS cells stably expressing HA − Flag − ATG9A treated with DMSO or Dynasore ( 100 μM ) 15 min , fixed and immunostained with anti − HA and anti − TBC1D5 antibodies .

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A U2OS cells stably expressing HA−Flag−ATG9A treated with DMSO or Dynasore (100 μM) 15 min, fixed and immunostained with anti−HA and anti−TBC1D5 antibodies.


B Quantification of ATG9Aand AP2A1co ‐ localization from experiment in ( A ) , pictures from 3 independent experiments were analyzed and co ‐ localization quantified , statistics is calculated as described in Supplementary Materials and Methods .

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B Quantification of ATG9Aand AP2A1co‐localization from experiment in (A), pictures from 3 independent experiments were analyzed and co‐localization quantified, statistics is calculated as described in Supplementary Materials and Methods.


C RGB ‐ line profile across the vesicle indicated with arrow in ( B ) ; red - AP2A1 ; green - ATG9A ; blue - TBC1D5 .

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C RGB‐line profile across the vesicle indicated with arrow in (B); red-AP2A1; green-ATG9A; blue-TBC1D5.


D Quantification of ATG9A and AP2 co ‐ localization . Cells were starved in EBSS media ( 45 min ) or pretreated with Dynasore ( 15 min ) and subsequently starved for 45 min in combination with Dynasore . Control cells were treated with DMSO .

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D Quantification of ATG9A and AP2 co‐localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO.


E Quantification of LC3 puncta in cells stably expressing HA ‐ Flag ‐ ATG9A , treated and presented as in ( D ) . 100 cells were quantified in 3 independent experiments .

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E Quantification of LC3 puncta in cells stably expressing HA‐Flag‐ATG9A, treated and presented as in (D). 100 cells were quantified in 3 independent experiments.


F T ‐ REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo # 1 targeting ( 40 nM ) . 72 h post − transfection , expression of HA ‐ Flag − TBC1D5 was induced with doxycycline ( 50 ng / ml ) , and 96 h post − transfection cells were treated with Dynasore for 15 min , or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min . Cells were lysed in co ‐ immunoprecipitation buffer , and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4 ° C . Beads were washed 3 times with incubation buffer and subjected to SDS ‐ PAGE .

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F T‐REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting (40 nM). 72 h post−transfection, expression of HA‐Flag−TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post−transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co‐immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS‐PAGE.


( A ) Clone P15 induces HA - LC3 lipidation . 293T cells were transfected with P15 plus plasmids expressing HA - LC3A and / or the apoptotic inhibitor p35 ( as shown ) , and lysed for western blotting against the indicated molecules ( anti ‐ HA for HA - LC3A ) . The figure shows that expression of P15 induces HA - LC3A conversion to a lower molecular weight form indicative of protein lipidation . This activity remains unchanged by p35 .

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(A) Clone P15 induces HA-LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA-LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti‐HA for HA-LC3A). The figure shows that expression of P15 induces HA-LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35.


( B ) P15 induces GFP - LC3 translocation to a vesiculated pattern . 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP - LC3A and p35 . The known autophagic inducer bNIP3L constituted a positive control . Representative confocal microscopy pictures are shown .

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(B) P15 induces GFP-LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP-LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal microscopy pictures are shown.


( C ) TMEM59 induces HA - LC3 lipidation in different cell lines lacking the SV40 large T − antigen plasmid amplification system . Cells were transfected with the shown plasmids , vectors expressing HA - LC3A ( left ) or HA - LC3B ( right ) and GST ( as transfection control ) , and lysed for western blotting against the indicated molecules .

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(C) TMEM59 induces HA-LC3 lipidation in different cell lines lacking the SV40 large T−antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA-LC3A (left) or HA-LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules.


( D ) TMEM59 induces GFP - LC3 activation . 293 cells were transfected with the indicated plasmids and GFP - LC3A ( top ) or GFP - LC3B ( bottom ) . Representative confocal microscopy pictures are shown .

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(D) TMEM59 induces GFP-LC3 activation. 293 cells were transfected with the indicated plasmids and GFP-LC3A (top) or GFP-LC3B (bottom). Representative confocal microscopy pictures are shown.


( E ) Quantification of the phenotype in D . The number of GFP - LC3 ‐ positive vesicles per transfected cell was scored for at least fifty cells . Data are expressed as means ± s . d . of one representative experiment of three repetitions . Source data for this figure is available on the online supplementary information page .

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(E) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.


( A ) TMEM59 ID is required for HA - LC3 lipidation . 293 cells were transfected with full ‐ length TMEM59 ( FL ) or a deleted version lacking the ID ( ΔID , Δ263 - 323 ) , HA - LC3A ( left ) or HA - LC3B ( right ) and GST . Cells were lysed for western blotting against the shown molecules .

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(A) TMEM59 ID is required for HA-LC3 lipidation. 293 cells were transfected with full‐length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263-323), HA-LC3A (left) or HA-LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules.


( B ) The ID is necessary for GFP - LC3 activation . 293 cells were transfected with the indicated TMEM59 constructs and GFP - LC3A or GFP - LC3B ( as indicated ) . Representative confocal pictures are shown .

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(B) The ID is necessary for GFP-LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP-LC3A or GFP-LC3B (as indicated). Representative confocal pictures are shown.


( C ) Quantification of the phenotype in B . Scoring and data expression were as in Figure 1E .

(C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E.


( D ) TMEM59 ID ( amino acids 263 - 323 ) suffices for HA - LC3A lipidation . 293 cells were transfected with the indicated CD16 : 7 chimera ( Control : empty chimera ) and vectors encoding HA - LC3A and GST , subjected to aggregation with the shown amounts of anti ‐ CD16 antibody and lysed for western blotting . The right panel shows control WBs demonstrating equal loading ( ACTIN ) , transfection ( GST ) and chimera expression ( CD16 ) in unaggregated samples , as all experimental points per chimera derive from a single transfection .

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(D) TMEM59 ID (amino acids 263-323) suffices for HA-LC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HA-LC3A and GST, subjected to aggregation with the shown amounts of anti‐CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection.


( E ) Comparable surface levels of CD16 : 7 constructs . Transfected 293 cells were processed for anti ‐ CD16 flow cytometry . The graph displays percentages of positive cells ( left axis ) and means of fluorescence of positive cells ( MF , right axis ) obtained from triplicates . Data are expressed as means ± s . d . of the triplicates .

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(E) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti‐CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates.


( F ) The ID suffices for HA - LC3B lipidation .

(F) The ID suffices for HA-LC3B lipidation.


( G ) The ID suffices for GFP - LC3 activation . 293 cells were transfected with the indicated chimeras and GFP - LC3A or GFP - LC3B , aggregated and mounted . Representative confocal microscopy pictures are shown . Activated GFP - LC3 appears as a collapsed mass of indiscernible vacuoles .

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(G) The ID suffices for GFP-LC3 activation. 293 cells were transfected with the indicated chimeras and GFP-LC3A or GFP-LC3B, aggregated and mounted. Representative confocal microscopy pictures are shown. Activated GFP-LC3 appears as a collapsed mass of indiscernible vacuoles.


( H ) Quantification of the phenotype in G . As individual vesicles could not be counted , quantification was done as the percentage of transfected cells showing redistributed GFP - LC3 . At least 10 different fields were counted ( about 400 cells ) . The experiment was repeated three times . Data are expressed as means ± s . d . of the triplicates . Source data for this figure is available on the online supplementary information page .

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(H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFP-LC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page.


( A , B ) Ability of serial C ‐ terminal deletions of TMEM59 to activate LC3 . 293 cells were transfected with the indicated TMEM59 C ‐ terminal deletions , HA - LC3A ( A ) or HA - LC3B ( B ) and GST , and lysed for western blotting against the indicated molecules .

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(A, B) Ability of serial C‐terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C‐terminal deletions, HA-LC3A (A) or HA-LC3B (B) and GST, and lysed for western blotting against the indicated molecules.


( C ) Amino acids 263 - 281 retain the full potential of TMEM59 ID to promote HA - LC3 conversion . 293 cells were transfected with the shown CD16 : 7 chimeras , HA - LC3A or HA - LC3B ( as indicated ) and GST , and subjected to anti ‐ CD16 aggregation before lysing them for western blotting . The lower panels show control WBs ( unaggregated samples ) .

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(C) Amino acids 263-281 retain the full potential of TMEM59 ID to promote HA-LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA-LC3A or HA-LC3B (as indicated) and GST, and subjected to anti‐CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples).


D Amino acids 263 - 281 suffice for GFP - LC3 activation and retain the full potential of TMEM59 ID to promote GFP - LC3 activation . 293 cells were transfected with the indicated CD16 : 7 chimeras and GFP - LC3A or GFP - LC3B ( as indicated ) , and subjected to anti ‐ CD16 aggregation before fixing them for microscopy . The graph shows percentages of transfected cells exhibiting redistributed GFP - LC3 . Quantification and data expression were done as in Figure 2H .

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D Amino acids 263-281 suffice for GFP-LC3 activation and retain the full potential of TMEM59 ID to promote GFP-LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP-LC3A or GFP-LC3B (as indicated), and subjected to anti‐CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP-LC3. Quantification and data expression were done as in Figure 2H.


( E ) Amino acids 282 - 323 of TMEM59 lack LC3 activation potential . 293 cells were transfected with the shown chimeras , HA - LC3A or HA - LC3B and GST , and processed for western blotting against the indicated molecules . Control WBs of unaggregated samples are shown in the lower panels .

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(E) Amino acids 282-323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA-LC3A or HA-LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels.


( F ) Surface expression levels of CD16 : 7 chimeras . Procedures and data expression were as in Figure 2E . The figure shows that the functional differences observed between CD16 : 7 chimeras ( C - E ) are not caused by differential surface expression .

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(F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C-E) are not caused by differential surface expression.


( A ) Endocytic vesicles containing aggregated CD16 : 7 - 263 - 281 become labelled with GFP - LC3 . JAR cells were transfected with vectors expressing the indicated CD16 : 7 chimeras and GFP - LC3A , and subjected to anti ‐ CD16 aggregation for 8 h before staining for the endocytosed chimera ( red ) . Representative confocal pictures are shown . The inset highlights a vesicle where the peripheral GFP - LC3 staining is particularly distinguishable . The right panel displays control WBs showing that chimera aggregation for 8 h activates HA - LC3A conversion in JAR cells .

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(A) Endocytic vesicles containing aggregated CD16:7-263-281 become labelled with GFP-LC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFP-LC3A, and subjected to anti‐CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFP-LC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HA-LC3A conversion in JAR cells.


( B ) Colocalization of endocytosed CD16 : 7 - 263 - 281 with EEA1 and CD63 . JAR cells were transfected with CD16 : 7 - 263 - 281 and subjected to aggregation before staining them for the endocytosed chimera ( red ) and EEA1 or CD63 ( green ; FITC ‐ coupled primary antibodies ) , as indicated . Representative confocal microscopy pictures are shown .

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(B) Colocalization of endocytosed CD16:7-263-281 with EEA1 and CD63. JAR cells were transfected with CD16:7-263-281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC‐coupled primary antibodies), as indicated. Representative confocal microscopy pictures are shown.


( C ) Immunoelectron microscopy assay showing that endocytosed CD16 : 7 - 263 - 281 localizes to LC3 − labelled , single − membrane vesicles . JAR cells were transfected with the CD16 : 7 - 263 - 281 chimera and a construct expressing human IgG1 fused to LC3A . Cells were subjected to anti − CD16 aggregation for 8 h and processed for immunoelectron microscopy . Thick gold signal ( 18 nm ) : aggregated , endocytosed chimera ; thin gold signal ( 12 nm ) : IgG1 - LC3 . Arrows indicate single membrane ( black ) or IgG1 - LC3A ( white ) . Scale bar : 400 nm .

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(C) Immunoelectron microscopy assay showing that endocytosed CD16:7-263-281 localizes to LC3−labelled, single−membrane vesicles. JAR cells were transfected with the CD16:7-263-281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti−CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1-LC3. Arrows indicate single membrane (black) or IgG1-LC3A (white). Scale bar: 400 nm.


( D ) Lysosomal inhibition increases HA - LC3II levels promoted by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimera and HA - LC3A , and subjected to anti − CD16 aggregation in the absence or presence of bafilomycin ( 200 nM , added 4 h post aggregation ) before lysing them for western blotting against the indicated molecules .

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(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.


( E ) Aggregation of CD16 : 7 - 263 - 281 promotes its own degradation . 293 cells were transfected with the indicated constructs , aggregated and lysed for western blotting . Shown are overexposed anti ‐ CD16 WBs .

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(E) Aggregation of CD16:7-263-281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti‐CD16 WBs.


( F ) Degradation of CD16 : 7 - 263 - 281 is inhibited by ATG5 depletion . 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16 : 7 constructs , aggregated and lysed for anti ‐ CD16 western blotting ( left panel ) . The right panel displays control WBs showing ATG5 depletion . Source data for this figure is available on the online supplementary information page .

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(F) Degradation of CD16:7-263-281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti‐CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page.


( A ) Identification of critical amino acids for HA - LC3 conversion . 293 cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and HA - LC3A , subjected to anti ‐ CD16 aggregation and lysed for western blotting against the indicated molecules . Asterisks mark mutations with reduced activity . Shown is one representative experiment of six repetitions .

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(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions.


( B ) Identification of critical amino acids for GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Preparations were scored by blindly counting the number of red vesicles ( chimera ) and green vesicles ( GFP - LC3A ) per cell , as well as the number of red vesicles colocalizing with green ones . The percentage of green vesicles colocalizing with red ones was close to 100 % for all mutants , that is , virtually no GFP - LC3A vesicles were unrelated to endocytosed chimera ( not shown ) . At least 50 cells were scored per experimental point . The experiment was repeated three times . The graph shows the number of GFP - LC3A vesicles per cell expressed as the percentage of the value obtained for the wild − type chimera ( left axis ) , and the percentage of chimera vesicles labelled with GFP - LC3A ( right axis ) . Data are expressed as means ± s . d . of the triplicates . Asterisks indicate significant differences with respect to wild − type values ( paired Student ' s t − test ; P < 0 . 01 ) .

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(B) Identification of critical amino acids for GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 mutants and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFP-LC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFP-LC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFP-LC3A vesicles per cell expressed as the percentage of the value obtained for the wild−type chimera (left axis), and the percentage of chimera vesicles labelled with GFP-LC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild−type values (paired Student's t−test; P<0.01).


( C ) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations . Procedures were as in B .

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(C) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations. Procedures were as in B.


( D ) Simultaneous mutation of the four essential amino acids to alanine blocks HA - LC3 conversion induced by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimeras ( 4M , quadruple mutant ) and HA - LC3A , aggregated and lysed for western blotting .

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(D) Simultaneous mutation of the four essential amino acids to alanine blocks HA-LC3 conversion induced by CD16:7-263-281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HA-LC3A, aggregated and lysed for western blotting.


( E ) Simultaneous mutation of the four essential amino acids blocks GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 constructs and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Representative confocal microscopy images are shown .

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(E) Simultaneous mutation of the four essential amino acids blocks GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 constructs and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal microscopy images are shown.


( F ) Quantification of the phenotype in E . Data gathering and expression were as in B . Source data for this figure is available on the online supplementary information page .

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(F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.


( A ) Apposition events between GFP - ATG16L1 and endocytosed CD16 : 7 - 263 - 281 . JAR cells were transfected with CD16 : 7 - 263 - 281 and GFP - ATG16L1 or GFP - BECLIN ( as indicated ) , aggregated for 4 h and stained for the endocytosed chimera ( red ) . Representative confocal microscopy pictures are shown . Two different examples are provided for GFP - ATG16L1 .

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(A) Apposition events between GFP-ATG16L1 and endocytosed CD16:7-263-281. JAR cells were transfected with CD16:7-263-281 and GFP-ATG16L1 or GFP-BECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal microscopy pictures are shown. Two different examples are provided for GFP-ATG16L1.