d . Nonhuman primate model development study . The y - axis shows urinary oxalate normalized to creatine . X - axis shows baseline urinary oxalate levels after overnight fast , and urinary oxalate measured 6 hours post administration of either a spinach preparation ( triangle , pink ) or water ( circle , blue ) ( n = 6 for each group ) . Individual dots represent individual animals . Error bars are calculated as SEM . ( Two - way ANOVA followed by Sidak ' s multiple comparison analysis , * * * * p < 0 . 0001 ) .

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d. Nonhuman primate model development study. The y-axis shows urinary oxalate normalized to creatine. X-axis shows baseline urinary oxalate levels after overnight fast, and urinary oxalate measured 6 hours post administration of either a spinach preparation (triangle, pink) or water (circle, blue) (n=6 for each group). Individual dots represent individual animals. Error bars are calculated as SEM. (Two-way ANOVA followed by Sidak's multiple comparison analysis, **** p < 0.0001).


f . Urinary recovery of oxalate in nonhuman primates . The y - axis shows change in urinary oxalate from vehicle control . The x - axis shows vehicle ( control , grey ) and increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatment groups ) . Individual dots represent normalized urinary oxalate for each individual animal . Error bars are calculated as SEM . ( One - way ANOVA followed by Tukey ' s multiple comparison analysis , * p < 0 . 05 )

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f. Urinary recovery of oxalate in nonhuman primates. The y-axis shows change in urinary oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatment groups). Individual dots represent normalized urinary oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, * p < 0.05)


g . Urinary recovery of 13C2 oxalate in nonhuman primates . The y - axis shows change in urinary 13C2 - oxalate from vehicle control . The x - axis shows vehicle ( control , grey ) and increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatmentgroups ) . Individual dots represent normalized urinary 13C2 - oxalate for each individual animal . Error bars are calculated as SEM . ( One - way ANOVA followed by Tukey ' s multiple comparison analysis , * * p < 0 . 01 )

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g. Urinary recovery of 13C2 oxalate in nonhuman primates. The y-axis shows change in urinary 13C2-oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatmentgroups). Individual dots represent normalized urinary 13C2-oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, ** p < 0.01)


G ) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates ( n = 3 ) . Β - actin serves as an internal loading control .

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G) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates (n=3). Β-actin serves as an internal loading control.


B ) Images represent localization of KTR for ERK , JNK , or p38 under basal growth conditions . Scales bars = 20µm .

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B) Images represent localization of KTR for ERK, JNK, or p38 under basal growth conditions. Scales bars=20µm.


C - E ) Kinase activity for ERK ( C ) , JNK ( D ) , or p38 ( E ) in three representative single cells ( upper panels ) in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) as measured by C / N ratio . Lower panel shows mean C / N ratio in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) . Thick line represents mean with shaded area = SD . N for each condition and kinase is shown in population average plots .

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C-E) Kinase activity for ERK (C), JNK (D), or p38 (E) in three representative single cells (upper panels) in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red) as measured by C/N ratio. Lower panel shows mean C/N ratio in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red). Thick line represents mean with shaded area=SD. N for each condition and kinase is shown in population average plots.


C - E ) Mean kinase activation for ERK € , JNK ( D ) , or p38 ( E ) in both live ( grey ) and dead ( red ) cells measured over 24 - hours in response to H2O2 . Shaded areas = SD . N for each condition is shown .

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C-E) Mean kinase activation for ERK €, JNK (D), or p38 (E) in both live (grey) and dead (red) cells measured over 24-hours in response to H2O2. Shaded areas=SD. N for each condition is shown.


D ) Mean JNK activity as measured by C / N ratio ( bold line ) ± SD ( shaded area ) in response to 300 µM H2O2 with or without p38 inhibition ( grey ) . Bar graph shows integrated JNK activity ( C / N ratio ) over 24 - hours . Data represents mean ± SD . The number of analyzed cells , n , is shown for each condition . Statistics performed using 2 - tailed t - test . Data information : * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant .

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D) Mean JNK activity as measured by C/N ratio (bold line)±SD (shaded area) in response to 300 µM H2O2 with or without p38 inhibition (grey). Bar graph shows integrated JNK activity (C/N ratio) over 24-hours. Data represents mean±SD. The number of analyzed cells, n, is shown for each condition. Statistics performed using 2-tailed t-test. Data information: ** p<0.01, *** p<0.001, ns=not significant.


( B ) Representative images of immunostaining demonstrate that FKRP - iPSCs express specific pluripotency - associated markers , including NANOG , OCT4 , Tra - 1 - 60 and SSEA4 . Data information : Scale bars , 50 μm .

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(B) Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60 and SSEA4. Data information: Scale bars, 50 μm.


( C ) FKRP - iPSCs have a normal karyotype . Data information : Scale bars , 50 μm .

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(C) FKRP-iPSCs have a normal karyotype. Data information: Scale bars, 50 μm.


( D ) In vitro differentiation of FKRP - iPSCs to cells representing ectoderm ( β - III Tubulin , Tuj1 ) , mesoderm ( SMA , smooth muscle actin ) and endoderm ( AFP , α - fetoprotein ) . Data information : Scale bars , 50 μm .

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(D) In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (β-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin) and endoderm (AFP, α-fetoprotein). Data information: Scale bars, 50 μm.


( C ) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones ( 5D17 , 5D23 and 3B17 ) , compared with their parental FKRPA455D - iPSCs . Selection cassette excision sites are identified in the corrected - iPSC lines .

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(C) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones (5D17, 5D23 and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines.


( D ) CRISPR / Cas9 corrected - iPSC lines show a normal karyotype .

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(D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.


( A and B ) Representative images of NSCs derived from FKRP - and corrected - iPSC lines expressing SOX1 , SOX2 and nestin . ( C and D ) Quantification of percentage of SOX1 + ( C ) and SOX2 + ( D ) cells in culture . The efficiency of neural induction is more than 99 % in FKRP - and corrected - iPSC ( 5D17 , 5D23 and 3B17 ) lines . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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(A and B) Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2 and nestin. (C and D) Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23 and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.


( E and F ) FKRP - and corrected NSC lines can be further differentiated to cortical neural progenitor cells , expressing PAX6 , OTX2 and vimentin . ( G - I ) Quantification of percentage of PAX6 + ( G ) and OTX2 + ( H ) cells in culture . About 91 - 98 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express PAX6 ( G ) . About 93 - 96 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express OTX2 ( H ) . Of the OTX2 + population , about 60 - 67 % cells are also Ki67 + cycling progenitors ( I ) . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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(E and F) FKRP- and corrected NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2 and vimentin. (G-I) Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.


( J and K ) Glutamatergic projection neurons derived from FKRP and corrected ( 5D17 , 5D23 and 3B17 ) progenitor cells . The vast majority of neurons contain vGlut1 + punctae in their neurites ( labelled by Tuj1 ) . Right panels are enlarged images from the insets of left panels . Data information : Scale bars , 50 μm .

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(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.


( A ) Molecular weight of glycosylated α - dystroglycan ( IIH6 epitope ) in WT - iPSC derived cortical neurons is similar to that in the mouse brain ( ~ 120 kDa ) and lower than that in the mouse muscle ( ~ 156kDa ) . IIH6 reactivity was almost not detected in cortical neurons derived from FKRP - iPSCs . Note that the β - Actin antibody does not cross - react with the muscle sample .

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(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.


( B ) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC - derived cortical neurons . Intensities of IIH6 reactivity are normalized to β - Actin expression , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * * p < 0 . 01 )

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(B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)


( C ) Representative laminin overlays show that targeted gene correction restores laminin - binding activity in iPSC - derived cortical neurons . Intensities of laminin - binding activity are normalized to β - dystroglycan , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * p < 0 . 05 ) .

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(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).


( C ) Dose response curve . H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed , stained and imaged as described above . The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non - linear regression curve was fitted for the 3 compounds showing the highest increase in α - dystroglycan glycosylation ( IIH6 immunostaining ) . Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown . HPFPTzone : ( 5Z ) - 5 - [ ( 3 - ethoxy - 4 - hydroxy - phenyl ) methylene ] - 3 - ( 4 - fluorophenyl ) - 2 - thioxo - thiazolidin - 4 - one . 4BPPNit : 4 - ( 4 - bromophenyl ) - 6 - ethylsulfanyl - 2 - oxo - 3 , 4 - dihydro - 1H - pyridine - 5 - carbonitrile . DPPTzone : ( 5E ) - 5 - [ ( 2 , 5 - dimethyl - 1 - phenyl - pyrrol - 3 - yl ) methylene ] - 3 - phenyl - 2 - thioxo - thiazolidin - 4 - one . Regression curve and EC50 values were calculated using GraphPad Prism 7 software . In images shown , green represents glycosylated α - dystroglycan and blue Hoechst staining . Scale bar , 50 µm .

(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.


( E ) Flow cytometry analysis confirmed that 4BPPNit treatment ( 20 µM ) significantly increased IIH6 - reactive glycan in mouse H2K 2B4 cells , compared with untreated cells . There was no significant difference in the percentage of IIH6 - positive cells . Values indicate mean ± s . e . m . ( n = 6 , triplicates of 2 independent experiments ; t - test ; NS , not significant ; * * p < 0 . 01 ) .

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(E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).


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

(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 .

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

(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 ) .

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

(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 .

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

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


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

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


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

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


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

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

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 .

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

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


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

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

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


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

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

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


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

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

(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 .

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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

(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 .

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

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


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

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

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


( 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 ) .

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

(B) Representative 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 .

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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 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-ORGANISM O O B-ORGANISM B-CELL_TYPE O O O O O O 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) 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 .

O O O B-EXP_ASSAY O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O 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) 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 B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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.