Figure 5 ( B ) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT . Values are normalized to protein expression . ( C ) Western blot on cytoplasmic , nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT . Both panels represent the same blot . The blot was cut in two parts and antibodies against HSP90 and Lamin A / C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins . For AR visualization ( upper panel ) , longer exposure time was used . # = residual AR expression ( lower panel ) .

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Figure 5(B) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT. Values are normalized to protein expression.(C) Western blot on cytoplasmic, nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT. Both panels represent the same blot. The blot was cut in two parts and antibodies against HSP90 and Lamin A/C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins. For AR visualization (upper panel), longer exposure time was used. # = residual AR expression (lower panel).


Figure 6 ( B ) AR occupancy ( blue ) and levels of the histone marks H3K27ac ( green ) and H3K27me3 ( red ) at ARBS within the regulatory regions of Fkbp5 , Kap ( both androgen - regulated in kidney ) and Tox3 ( androgen - regulated in prostate but not in kidney and hence used as a negative control ) were assessed by ChIP - qPCR in kidneys from 13 - week - old ARLmon / Y mice and WT males . ( C ) Representative pictures of immunofluorescence AR staining ( green ) in kidneys of castrated WT males and ARLmon / Y mice supplemented with vehicle or T . Nuclei are shown in blue .

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Figure 6(B) AR occupancy (blue) and levels of the histone marks H3K27ac (green) and H3K27me3 (red) at ARBS within the regulatory regions of Fkbp5, Kap (both androgen-regulated in kidney) and Tox3 (androgen-regulated in prostate but not in kidney and hence used as a negative control) were assessed by ChIP-qPCR in kidneys from 13-week-old ARLmon/Y mice and WT males.(C) Representative pictures of immunofluorescence AR staining (green) in kidneys of castrated WT males and ARLmon/Y mice supplemented with vehicle or T. Nuclei are shown in blue.


Figure 7 ( A ) Double hybrid assay in COS cells transfected with WT LBD , W752R LBD or empty vector ( negative control ) in the presence of the LxxLL fragment of SRC1 . ( B ) Confocal fluorescence microscopy images of AR - WT - BirA * and AR W752R - BirA * expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle ( ethanol ) and with or without 0 . 03 µg / ml TET as indicated . AR - BirA * s were detected with anti - AR ( red ) and biotinylated proteins with fluorescently labeled streptavidin ( green ) . Nuclei were visualized using DAPI .

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Figure 7(A) Double hybrid assay in COS cells transfected with WT LBD, W752R LBD or empty vector (negative control) in the presence of the LxxLL fragment of SRC1.(B) Confocal fluorescence microscopy images of AR-WT-BirA* and AR W752R-BirA*expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle (ethanol) and with or without 0.03 µg/ml TET as indicated. AR-BirA*s were detected with anti-AR (red) and biotinylated proteins with fluorescently labeled streptavidin (green). Nuclei were visualized using DAPI.


Figure 3A . STAT3 immunohistochemistry - staining of low STAT3 and high STAT3 PCa samples . Red arrows indicate transformed PCa glands , black arrows indicate pre - transformed normal prostate glands . Scale bar = 100µm . # = sample - IDs .

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Figure 3A. STAT3 immunohistochemistry- staining of low STAT3 and high STAT3 PCa samples. Red arrows indicate transformed PCa glands, black arrows indicate pre-transformed normal prostate glands. Scale bar = 100µm. # = sample-IDs.


Figure 4A . Differentially expressed proteins in PtenStat3pc - / - vs . Ptenpc - / - proteomic samples . Colors indicate adj . p - value and log2 - FC . ( Black = Log2 - FC ≤ 1 & adj . p - value ≥ 0 . 05 , orange = Log2 - FC > 1 & adj . p - value ≥ 0 . 05 , red = Log2 - FC > 1 & adj . p - value < 0 . 05 ) . FC = fold change . B . STAT3 immunohistochemistry - staining of wild type , Ptenpc - / - , PtenStat3pc - / - mouse prostates . Scale bar = 100µm . E . Metabolite concentrations in nmol / µg of 5 metabolites in WT , Ptenpc - / - and PtenStat3pc - / - prostates . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents biological replicates . ANOVA tests and Tukey multiple comparisons were applied . Red = PtenStat3pc - / - ( n = 3 ) , blue = Ptenpc - / - ( n = 5 ) , grey = wild type ( WT , n = 5 ) , q = adj . p . - value , n . s . = not significant .

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Figure 4A. Differentially expressed proteins in PtenStat3pc-/- vs. Ptenpc-/- proteomic samples. Colors indicate adj.p-value and log2-FC. (Black = Log2-FC ≤ 1 & adj. p-value ≥ 0.05, orange = Log2-FC > 1 & adj. p-value ≥ 0.05, red = Log2-FC > 1 & adj. p-value < 0.05). FC = fold change.B. STAT3 immunohistochemistry- staining of wild type, Ptenpc-/-, PtenStat3pc-/- mouse prostates. Scale bar = 100µm.E. Metabolite concentrations in nmol/µg of 5 metabolites in WT, Ptenpc-/- and PtenStat3pc-/- prostates. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents biological replicates. ANOVA tests and Tukey multiple comparisons were applied. Red = PtenStat3pc-/- (n = 3), blue = Ptenpc-/- (n = 5), grey = wild type (WT, n=5), q = adj. p.-value, n.s. = not significant.


Figure 5A . SDHB protein expression levels in a TMA ( n = 83 ) detected by immunohistochemistry ( IHC ) . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant . TMA = Tissue Micro Array . B . Representative IHC - staining of SDHB in normal prostate glands and Gleason grade ( GL ) 3 - 5 PCa glands . Scale bar = 100µm . C . IDH2 protein expression levels in a TMA ( n = 83 ) detected by IHC . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant . D . Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands . Scale bar = 100µm . E . Nuclear ( N ) and cytoplasmatic ( C ) STAT3 protein expression levels in a TMA ( n = 83 ) detected by IHC . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant .

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Figure 5A. SDHB protein expression levels in a TMA (n = 83) detected by immunohistochemistry (IHC). Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant. TMA = Tissue Micro Array.B. Representative IHC- staining of SDHB in normal prostate glands and Gleason grade (GL) 3 - 5 PCa glands. Scale bar = 100µm.C. IDH2 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.D. Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands. Scale bar = 100µm.E. Nuclear (N) and cytoplasmatic (C) STAT3 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.


Figure 6B . Genes of the Hallmark gene set " Oxidative phosphorylation " that are differentially expressed in low STAT3 vs . high STAT3 tumors . Dotted lines show Log2 - FC = ± 1 ( x - axis ) and adj . p - value = - log10 ( 0 . 05 ) ( y - axis ) . Orange = up - regulated genes , green = down - regulated genes . DE = differentially expressed . C . - D . Kaplan - Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors ( C ) and in primary and metastatic tumors combined ( D ) in the MSKCC PCa GSE21032 data set . Groups were generated by a median split . P - values were estimated by Log - rank test ( C - D ) and adjusted with Benjamini - Hochberg method ( D ) . + = censored .

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Figure 6B. Genes of the Hallmark gene set "Oxidative phosphorylation" that are differentially expressed in low STAT3 vs. high STAT3 tumors. Dotted lines show Log2-FC = ±1 (x-axis) and adj. p-value = -log10(0.05) (y-axis). Orange = up-regulated genes, green = down-regulated genes. DE = differentially expressed.C.- D. Kaplan-Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors (C) and in primary and metastatic tumors combined (D) in the MSKCC PCa GSE21032 data set. Groups were generated by a median split. P-values were estimated by Log-rank test (C-D) and adjusted with Benjamini-Hochberg method (D). + = censored.


Figure 7A . STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set . Pearson correlation between STAT3 and PDK4 is shown . Kaplan - Meier plot shows stratified subgroups . P - values were estimated by Log - rank test and adjusted with Benjamini - Hochberg method . Hi = high , lo = low . B . Western blot of STAT3 , PDK4 and β - TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3 . Ctrl = scrambled control , shSTAT3 = short hairpin knockdown of STAT3 . C . ChIP assay from IL - 6 stimulated or non - stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody ( blue shades ) and IgG antibody as negative control ( orange shades ) followed by qPCR with a promoter - specific primer pair for the PDK4 gene . Bars represent mean ± SD from 2 technical replicates . Precipitated DNA is presented as % of input . One representative experiment is shown . Result of qPCR using primer pair 2 is shown . Ctrl = scrambled control , shSTAT3 = short hairpin knockdown of STAT3 , + IL - 6 = IL - 6 stimulated . D . Correlation of STAT3 , c - MYC and HIF - 1α with PDK1 - 4 , PDC - genes ( PDHA1 , PDHB , PDHX , DLAT , DLD ) and TCA / OXPHOS genes ( CS , IDH2 , IDH3A , SDHB , SDHC , ATP5A1 , NDUFS1 ) in MSKCC PCa ( GSE21032 ) . Dot colors represent Pearson correlation ( 1 = red , - 1 = blue ) , dot sizes represent adj . p - values ≤ 0 . 05 . Only significant correlations are shown . P - values were adjusted with Benjamini - Hochberg method .

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Figure 7A. STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set. Pearson correlation between STAT3 and PDK4 is shown. Kaplan-Meier plot shows stratified subgroups. P-values were estimated by Log-rank test and adjusted with Benjamini-Hochberg method. Hi = high, lo = low.B. Western blot of STAT3, PDK4 and β-TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3.C. ChIP assay from IL-6 stimulated or non-stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody (blue shades) and IgG antibody as negative control (orange shades) followed by qPCR with a promoter- specific primer pair for the PDK4 gene. Bars represent mean ± SD from 2 technical replicates. Precipitated DNA is presented as % of input. One representative experiment is shown. Result of qPCR using primer pair 2 is shown. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3, +IL-6 = IL-6 stimulated.D. Correlation of STAT3, c-MYC and HIF-1α with PDK1-4, PDC- genes (PDHA1, PDHB, PDHX, DLAT, DLD) and TCA/OXPHOS genes (CS, IDH2, IDH3A, SDHB, SDHC, ATP5A1, NDUFS1) in MSKCC PCa (GSE21032). Dot colors represent Pearson correlation (1 = red, -1 = blue), dot sizes represent adj. p-values ≤ 0.05. Only significant correlations are shown. P-values were adjusted with Benjamini-Hochberg method.


figf1 ( a ) Fluorescence microscopy of GFP - LC3 - transduced HeLa cells left unstimulated ( Unstim ) or stimulated for 4 h with C12 - iEDAP ( 2 . 5 μg / ml ) or for 2 h with rapamycin ( Rapa ; 50 μg / ml ) . Green , GFP - LC3 ; blue , DAPI ( DNA - intercalating dye ) . Scale bar , 5 μm . ( b ) Quantification of dot - or ring - shaped GFP - LC3 signals ( representing autophagosomes ) in HeLa cells stimulated as described in a . ( c ) Confocal microscopy of wild - type and Nod2 - deficient BMDMs transduced with GFP - LC3 ( green ) and left unstimulated or stimulated for 4 h with LPS ( 1 μg / ml ) or MDP ( 100 μg / ml ) or for 2 h with rapamycin ( 50 μg / ml ) . Scale bar , 15 μm . ( d ) Quantification of dot - or ring - shaped GFP - LC3 ( green ) in BMDMs stimulated as described in c . ( e ) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate - injected wild - type , Nod1 - and Nod2 - deficient mice 4 h after intraperitoneal injection of MDP ( 300 μg / ml ) , FK565 ( 300 μg / ml ) or rapamycin ( 300 μg / ml ) ; after centrifugation by cytospin , cells were stained for endogenous LC3 ( red ) and with DAPI ( blue ) . Scale bar , 10 μm . ( f ) Quantification of dot - or ring - shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c . Data are representative of four ( a - d ) or two ( e , f ) independent experiments with at least 100 cells per condition ( error bars ( b , d , f ) , s . d . ) .

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figf1(a) Fluorescence microscopy of GFP-LC3-transduced HeLa cells left unstimulated (Unstim) or stimulated for 4 h with C12-iEDAP (2.5 μg/ml) or for 2 h with rapamycin (Rapa; 50 μg/ml). Green, GFP-LC3; blue, DAPI (DNA-intercalating dye). Scale bar, 5 μm. (b) Quantification of dot- or ring-shaped GFP-LC3 signals (representing autophagosomes) in HeLa cells stimulated as described in a.(c) Confocal microscopy of wild-type and Nod2-deficient BMDMs transduced with GFP-LC3 (green) and left unstimulated or stimulated for 4 h with LPS (1 μg/ml) or MDP (100 μg/ml) or for 2 h with rapamycin (50 μg/ml). Scale bar, 15 μm. (d) Quantification of dot- or ring-shaped GFP-LC3 (green) in BMDMs stimulated as described in c.(e) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate-injected wild-type, Nod1- and Nod2-deficient mice 4 h after intraperitoneal injection of MDP (300 μg/ml), FK565 (300 μg/ml) or rapamycin (300 μg/ml); after centrifugation by cytospin, cells were stained for endogenous LC3 (red) and with DAPI (blue). Scale bar, 10 μm. (f) Quantification of dot- or ring-shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c. Data are representative of four (a-d) or two (e,f) independent experiments with at least 100 cells per condition (error bars (b,d,f), s.d.).


figf2 ( a ) Confocal microscopy of wild - type and Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri M90T - RFP . ( b ) Quantification of the colocalization of GFP - LC3 signals with bacteria in a . ( c ) Confocal microscopy of wild - type and Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with S . flexneri ΔIcsB - RFP . ( d ) Quantification of the colocalization of GFP - LC3 signals with bacteria in c . ( e , f ) Plate assay of bacteria in wild - type or Nod1 - deficient MEFs infected for 0 . 5 h with S . flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 ( LY ) or protease inhibitors ( PI ) , followed by 5 . 5 h of incubation in the presence of gentamicin . ( g - j ) Confocal microscopy ( g , i ) and quantification of the colocalization of GFP - LC3 signals with M90T and ΔIcsB ( h , j ) in wild - type and RIP2 - deficient ( Ripk2 − / − ) GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri ( M90T ; g , h ) or ΔIcsB S . flexneri ( i , j ) . ( k ) Enzyme - linked immunosorbent assay of KC secretion by wild - type and RIP2 - deficient MEFs infected for 0 . 5 h with M90T and incubated for 5 . 5 h in the presence of gentamicin . ( l , m ) Confocal microscopy ( l ) and quantification of the colocalization of GFP - LC3 signals ( green ) with M90T and ΔIcsB ( m ) of wild - type and NEMO - deficient ( Ikbkg − / − ) GFP - LC3 - expressing MEFs infected for 0 . 5 h with wild - type S . flexneri ( M90T ) or S . flexneri ΔIcsB and incubated for 1 . 5 h in the presence of gentamicin . Red , RFP . Arrows indicate colocalization of GFP - LC3 signals with S . flexneri . Scale bars , 10 μm . * P 0 . 05 , and * * P 0 . 01 ( t - test ) . Data represent three independent experiments with at least 100 cells each ( error bars , s . e . m . ) .

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figf2(a) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri M90T-RFP. (b) Quantification of the colocalization of GFP-LC3 signals with bacteria in a.(c) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with S. flexneri ΔIcsB-RFP. (d) Quantification of the colocalization of GFP-LC3 signals with bacteria in c.(e,f) Plate assay of bacteria in wild-type or Nod1-deficient MEFs infected for 0.5 h with S. flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 (LY) or protease inhibitors (PI), followed by 5.5 h of incubation in the presence of gentamicin.(g-j) Confocal microscopy (g,i) and quantification of the colocalization of GFP-LC3 signals with M90T and ΔIcsB (h,j) in wild-type and RIP2-deficient (Ripk2−/−) GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri (M90T; g,h) or ΔIcsB S. flexneri (i,j).(k) Enzyme-linked immunosorbent assay of KC secretion by wild-type and RIP2-deficient MEFs infected for 0.5 h with M90T and incubated for 5.5 h in the presence of gentamicin.(l,m) Confocal microscopy (l) and quantification of the colocalization of GFP-LC3 signals (green) with M90T and ΔIcsB (m) of wild-type and NEMO-deficient (Ikbkg−/−) GFP-LC3-expressing MEFs infected for 0.5 h with wild-type S. flexneri (M90T) or S. flexneri ΔIcsB and incubated for 1.5 h in the presence of gentamicin. Red, RFP. Arrows indicate colocalization of GFP-LC3 signals with S. flexneri. Scale bars, 10 μm. *P 0.05, and **P 0.01 (t-test). Data represent three independent experiments with at least 100 cells each (error bars, s.e.m.).


figf3 ( a ) Immunofluorescence of HeLa cells expressing hemagglutinin - tagged Nod1 ( HA - Nod1 ( red ) ; top ) or HA - Nod2 ( red ; bottom ) . Blue , DAPI . ( b ) Immunofluorescence of HeLa cells expressing Myc - tagged FL - ATG16L1 ( Myc - FL - ATG16L1 ) . ( c , d ) Immunofluorescence of HeLa cells coexpressing HA - Nod1 ( red ; c ) or HA - Nod2 ( red ; d ) and Myc - FL - ATG16L1 ( green ) . Blue , DAPI . ( e ) Immunofluorescence of HeLa cells expressing a Flag - tagged truncated form of ATG16L1 ( Flag - ΔN85 - ATG16L1 ; green ) . Blue , DAPI . ( f ) Immunofluorescence of HeLa cells coexpressing HA - Nod2 ( red ) and Flag - ΔN85 - ATG16L1 ( green ) . Blue , DAPI . ( g ) Immunofluorescence microscopy of Mode - K cells stained for endogenous Nod2 ( green ) and ATG16L1 ( red ) and with DAPI ( blue ) . For a - g , arrows indicate membrane colocalization of ATG16 and Nod2 ; area outlined at left is enlarged at right ( a , b , e ) or in other images ( c , d , f , ) ; scale bars , 8 μm . ( h , i ) Immunoprecipitation ( IP ) and immunoblot ( IB ) analysis of HEK293 cells transfected with Flag - ΔN85 - ATG16L1 ( h ) or Myc - FL - ATG16L1 ( i ) , together with HA - Nod1 , HA - Nod2 or HA - tagged enhanced GFP ( EGFP - HA ) . Ig , immunoglobulin ( loading control ) . Results are representative of one of at least three independent experiments ( a - g ) or one of three independent experiments ( h , i ) .

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figf3(a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom). Blue, DAPI.(b) Immunofluorescence of HeLa cells expressing Myc-tagged FL-ATG16L1 (Myc-FL-ATG16L1).(c,d) Immunofluorescence of HeLa cells coexpressing HA-Nod1 (red; c) or HA-Nod2 (red; d) and Myc-FL-ATG16L1 (green). Blue, DAPI.(e) Immunofluorescence of HeLa cells expressing a Flag-tagged truncated form of ATG16L1 (Flag-ΔN85-ATG16L1; green). Blue, DAPI.(f) Immunofluorescence of HeLa cells coexpressing HA-Nod2 (red) and Flag-ΔN85-ATG16L1 (green). Blue, DAPI.(g) Immunofluorescence microscopy of Mode-K cells stained for endogenous Nod2 (green) and ATG16L1 (red) and with DAPI (blue). For a-g, arrows indicate membrane colocalization of ATG16 and Nod2; area outlined at left is enlarged at right (a,b,e) or in other images (c,d,f,); scale bars, 8 μm.(h,i) Immunoprecipitation (IP) and immunoblot (IB) analysis of HEK293 cells transfected with Flag-ΔN85-ATG16L1 (h) or Myc-FL-ATG16L1 (i), together with HA-Nod1, HA-Nod2 or HA-tagged enhanced GFP (EGFP-HA). Ig, immunoglobulin (loading control). Results are representative of one of at least three independent experiments (a-g) or one of three independent experiments (h,i).


figf4Immunofluorescence microscopy of wild - type and RIP2 - deficient MEFs expressing Flag - tagged ΔN85 - ATG16L1 and HA - Nod1 ( a ) or HA - Nod2 ( b ) . Scale bars , 10 μm . Area outlined at far left is enlarged at right . Data are representative one of two independent experiments .

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figf4Immunofluorescence microscopy of wild-type and RIP2-deficient MEFs expressing Flag-tagged ΔN85-ATG16L1 and HA-Nod1 (a) or HA-Nod2 (b). Scale bars, 10 μm. Area outlined at far left is enlarged at right. Data are representative one of two independent experiments.


figf5 ( a ) Fluorescence microscopy of immortalized GFP - LC3 - transduced lymphoblasts from donors homozygous for the normal ATG16L1 * 300T allele ( 300T ) or risk allele ATG16L1 * 300A ( 300A ) , left unstimulated or treated for 2 h with rapamycin ( 50 μg / ml ) or for 4 h with MDP ( 20 μg / ml ) . Green , GFP - LC3 . Scale bar , 5 μm . ( b ) Induction of GFP - LC3 signals by rapamycin , MDP or Gram - positive peptidoglycan ( PG + ; 20 μg / ml ) in cells treated as described in a , presented relative to GFP - LC3 signals in unstimulated control cells . * P 0 . 05 ( t - test ) . Data represent three independent experiments ( error bars ( b ) , s . d . ) .

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figf5(a) Fluorescence microscopy of immortalized GFP-LC3-transduced lymphoblasts from donors homozygous for the normal ATG16L1*300T allele (300T) or risk allele ATG16L1*300A (300A), left unstimulated or treated for 2 h with rapamycin (50 μg/ml) or for 4 h with MDP (20 μg/ml). Green, GFP-LC3. Scale bar, 5 μm. (b) Induction of GFP-LC3 signals by rapamycin, MDP or Gram-positive peptidoglycan (PG+; 20 μg/ml) in cells treated as described in a, presented relative to GFP-LC3 signals in unstimulated control cells. *P 0.05 (t-test). Data represent three independent experiments (error bars (b), s.d.).


figf6 ( a ) Immunofluorescence microscopy of HeLa cells transfected with HA - Nod2 ( red ) and Flag - ΔN85 - ATG16L1 ( green ) and infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria . Scale bars , 5 μm . Data are representative of one of three independent experiments . ( b ) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode - K cells and infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria . Scale bars , 5 μm . Data are representative of one of three independent experiments .

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figf6(a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag-ΔN85-ATG16L1(green) and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.(b) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode-K cells and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


figf7 ( a , b ) Immunofluorescence microscopy of HeLa cells expressing Flag - ΔN85 - ATG16L1 ( green ; a ) or Myc - FL - ATG16L1 ( green ; b ) and HA - tagged Nod2 L1007fsinsC ( HA - Nod2 , 1007fs ) . Area outlined at left is enlarged in other images . ( c , d ) Immunofluorescence microscopy of HeLa cells expressing Flag - ΔN85 - ATG16L1 ( c ) or Myc - FL - ATG16L1 ( d ) together with HA - tagged Nod2fs ( red ) . Blue , DAPI . Scale bar , 8 μm . ( c , d ) Immunofluorescence microscopy of HeLa cells expressing Flag - ΔN85 - ATG16L1 ( c ) or Myc - FL - ATG16L1 ( d ) together with HA - tagged Nod2fs ( red ) . Blue , DAPI . Scale bar , 8 μm . ( e ) Immunofluorescence microscopy of wild - type and Nod2fs - KI BMDMs transduced with GFP - LC3 ( green ) and infected for 0 . 5 h with S . flexneri M90T - RFP ( red ) , followed by incubation for 1 h with gentamicin . Arrows indicate no colocalization of Nod2 with ΔN85 - ATG16L1 or FL - ATG16L1 . Scale bar , 15 μm . ( f ) Quantification of the colocalization of GFP - LC3 signals with S . flexneri M90T - RFP in experiments as described in e . Data are representative of one of three independent experiments ( error bars ( f ) , s . d . ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a,b) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (green; a) or Myc-FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA-Nod2, 1007fs). Area outlined at left is enlarged in other images.(c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm.(c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm.(e) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).


Figure 1 ( A ) Assessment of vacuole ( arrows ) formation in neutrophils after stimulation with PMA ( II ) and rapamycin ( III ) , compared with control cells ( I : treated with PBS and serum ) ( magnification 1000 × ) . ( B ) ( I ) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin ( RAP ) compared with control cells ( PBS ) and cells treated with PMA and 3 ‐ MA ( magnification 400 and 1000 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . Data are representative of six independent experiments and are presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 : PMA ( bar 2 : 86 . 16 ± 6 . 49 % ) , rapamycin ( bar 3 : 65 . 83 ± 8 . 47 % ) treated cells , cells treated with PMA and 3 ‐ MA ( bar 4 : 9 . 17 ± 0 . 75 % ) and cells treated with rapamycin and 3 ‐ MA ( bar 5 : 8 . 5 ± 1 . 87 % ) compared with control conditions ( bar 1 : 15 . 67 ± 3 . 50 % ) . The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3 ‐ MA ( bar 4 ) or rapamycin and 3 ‐ MA ( bar 5 ) was significantly lower ( p < 0 . 05 ) compared with cells treated with PMA ( bar 2 ) and rapamycin ( bar 3 ) , respectively . ( C ) Analysis of LC3B ‐ I to LC3B ‐ II conversion in cells treated with PMA ( lane IV ) and rapamycin ( lane II ) , as observed by LC3B immunoblotting . Effect of 3 ‐ MA in both PMA ( lane V ) and rapamycin ( lane I ) ‐ treated cells . Lane III : control cells , and lane VI : cells treated only with 3 ‐ MA . One representative out of six independent experiments is shown . ( D ) Impact of PI ‐ 3K inhibition using LY294002 on LC3B conversion after PMA challenge ( lane III ) , ( lane I : control cells , lane II : PMA , lane IV : LY294002 only ) . One representative out of four independent experiments is shown . ( E ) Immunofluorescence confocal microscopy staining of LC3B . ( I ) Control cells treated with PBS and serum , ( II ) PMA ‐ treated cells . 1 . DAPI : blue , 2 . LC3B : red and 3 . merge . ( III ) Percentage of PMN with LC3B aggregates . Control conditions ( bar 1 : 17 . 7 ± 2 . 4 % ) , 3 ‐ MA ( bar 2 : 3 ± 1 . 01 % ) , PMA ( bar 3 : 69 ± 2 . 1 % ) , PMA and 3 ‐ MA ( bar 4 : 4 ± 1 . 9 % ) , rapamycin ( bar 5 : 56 ± 1 . 6 % ) . Data are representative of five independent experiments and presented as mean ± SD ; Student ' s t ‐ test , * p < 0 . 01 compared to the control ( bar 1 ) or the indicated comparision ( bar 3 vs 4 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×).(B) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively.(C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown.(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.(E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).


Figure 2ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin . ATG3 mRNA expression analysis ( fold ) after PMA ( 2 . 44 ± 0 . 78 ) and rapamycin ( RAP : 1 . 81 ± 0 . 41 ) stimulation . LC3B gene alterations ( PMA : 4 . 98 ± 0 . 59 ) and rapamycin ( RAP : 1 . 7 ± 0 . 55 ) . Effect of 3 ‐ MA pre ‐ treatment , for both stimuli , was also assessed ( PMA + 3 ‐ MA : ATG3 : 1 . 36 ± 0 . 64 , LC3B : 1 . 5 ± 0 . 49 and rapamycin + 3 ‐ MA : ATG3 : 1 . 28 ± 0 . 46 , LC3B : 1 . 24 ± 0 . 57 ) . No other significant alteration in ATG genes mRNA levels was detected . Representative data of ATG7 mRNA expression analysis after treatment with PMA ( 1 . 21 ± 0 . 42 ) and rapamycin ( 0 . 94 ± 0 . 64 ) . Data are representative of six independent experiments and presented as mean relative expression ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 and n . s . = not significant compared with control conditions ( dot line ) as indicated in the figure . Relative expression ( folds ) of each gene is derived from substitution of DCT values ( normalized by GAPDH DCT value ) in 2DDCT equation 36 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.


Figure 3 ( A ) ( I ) MDC staining in neutrophils treated with LPS , LTA , peptidoglycan ( PGN ) , loxorubin ( LOX ) and R848 ( 400 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . LPS ( 50 ng / mL ) ( bar 2 : 69 . 67 ± 8 . 21 % ) , LTA ( bar 3 : 75 . 17 ± 9 . 80 % ) , peptidoglycan ( bar 4 : 77 ± 6 . 96 % ) , loxorubin ( bar 5 : 66 . 67 ± 5 . 61 % ) , R848 ( bar 6 : 69 . 5 ± 7 . 45 % ) , poly I : C ( bar 7 : 16 . 33 ± 3 . 14 % ) and flagellin ( bar 8 : 19 . 83 ± 5 . 16 % ) − treated cells compared with control conditions ( bar 1 : 14 . 5 ± 4 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched − pairs test , * p < 0 . 05 , n . s . = not significant . ( B ) LC3B lipidation status of cells treated with LPS ( lane I ) , LTA ( lane II ) , peptidoglycan ( lane III ) , loxoribin ( lane IV ) and R848 ( lane V ) , detected by immunoblotting . One representative out of six independent experiments is shown . ( C ) LC3B − II / LC3B - I ratio as obtained from integrated optical density measurements . LTA ( bar 2 : 1 . 75 ± 0 . 84 ) , peptidoglycan ( bar 3 : 1 . 82 ± 1 . 17 ) , LPS ( bar 4 : 1 . 9 ± 1 . 7 ) , loxorubin ( bar 5 : 1 . 32 ± 0 . 49 ) and R848 ( bar 6 : 1 . 4 ± 0 . 77 ) compared with control neutrophils ( bar 1 : 0 . 76 ± 0 . 30 ) . Data are representative of six independent experiments and are presented as mean ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 . ( D ) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists . LPS ( bar 1 : 2 . 27 ± 0 . 43 ) , peptidoglycan ( bar 2 : 2 . 07 ± 0 . 42 ) , LTA ( bar 3 : 2 . 1 ± 0 . 49 ) and R848 ( bar 4 : 1 . 89 ± 0 . 35 ) compared with medium − treated control cells . Dotted line represents LC3B mRNA levels of medium - treated control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 . ( E ) Assessment of ATG gene expression levels in PMN after LPS stimulation ( 50 ng / mL ) . ATG3 ( bar 1 : 2 . 94 ± 0 . 52 ) , ATG4 ( bar 2 : 1 . 72 ± 0 . 42 ) , ATG5 ( bar 3 : 1 . 84 ± 0 . 34 ) , ATG6 ( bar 4 : 1 . 8 ± 0 . 3 ) , ATG7 ( bar 5 : 3 . 13 ± 0 . 64 ) and LC3B ( bar 6 : 2 . 27 ± 0 . 43 ) compared with control cells . Dotted line represents mRNA levels of medium - treated cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant.(B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown.(C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05.(D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.(E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


Figure 4 ( A ) ( I ) Formation of acidified autophagosomes after stimulation with 10 ng / mL of IL ‐ 1β , compared with medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . IL ‐ 1β ‐ treated cells ( bar 2 : 65 . 17 ± 10 . 23 % ) compared with control conditions ( bar 1 : 15 . 83 ± 6 . 08 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 . ( B ) LC3B conversion in neutrophils treated with 10 ng / mL ( lane II ) and 100 ng / mL ( lane III ) of IL ‐ 1β . ( lane I : medium ‐ treated control cells ) . One representative out of five independent experiments is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) (I) Formation of acidified autophagosomes after stimulation with 10 ng/mL of IL‐1β, compared with medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. IL‐1β‐treated cells (bar 2: 65.17±10.23%) compared with control conditions (bar 1: 15.83±6.08%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.(B) LC3B conversion in neutrophils treated with 10 ng/mL (lane II) and 100 ng/mL (lane III) of IL‐1β. (lane I: medium‐treated control cells). One representative out of five independent experiments is shown.


Figure 5 ( A ) Effect of 3 ‐ MA on neutrophil phagocytic capacity of opsonized E . coli as observed by flow cytometry . Percentage of phagocytosing neutrophils after 3 ‐ MA pretreatment ( III ) compared with cells incubated with opsonized bacteria ( II ) . Data are representative of six independent experiments and are presented as mean ± SD , ( I : control unstimulated group ) , ( IV ) MFI representation ( bar 1 : control : 1 ± 1 . 9 , bar 2 : E . coli : 387 . 7 ± 33 . 3 , bar 3 : E . coli + 3 ‐ MA : 25 . 13 ± 18 , Wilcoxon matched ‐ pairs test , * p < 0 . 05 ; bar 2 compared with bar 1 and bar 3 compared to bar 2 ) . ( B ) ( I ) Presence of acidified structures in E . coli ‐ phagocytosing neutrophils and medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli ‐ phagocytosing cells ( bar 2 : 79 . 33 ± 7 . 15 % ) compared with control conditions ( bar 1 : 14 . 17 ± 4 . 79 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 . ( ( C ) LC3B conversion analysis in E . coli ‐ phagocytosing neutrophils ( lane II ) , control neutrophils ( lane I ) and E . coli ‐ phagocytosing neutrophils after 3 ‐ MA pretreatment ( lane III ) . One representative out of six independent experiments is shown . ( D ) Effect of 3 ‐ MA administration to E . coli ‐ phagocytosing neutrophils 15 min after the initiation of phagocytosis ( lane III ) as detected by LC3B conversion analysis . Lane II represents E . coli ‐ phagocytosing neutrophils and lane I control neutrophils . One representative out of four independent experiments is shown . ( E ) ( I ) Percentage of neutrophils with LC3B punctuated structures . E . coli ( bar 1 : 84 ± 2 . 2 % ) compared with E . coli + 3 ‐ MA ( bar 2 : 4 ± 1 . 95 % ) . Data are representative of six independent experiments and presented as mean ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 . ( II ) Formation of LC3B puncta in phagocytosing neutrophils and ( III ) in cells treated with 3 ‐ MA before incubation with opsonized E . coli , as observed by immunofluorescence confocal microscopy ( 1 . LC3B : red ; 2 . FITC conjugated E . coli : green ; 3 . DAPI : blue ; and 4 . merge ) . Solid line arrows point the colocalization of LC3B puncta with E . coli bacteria . Dashed line arrows point LC3B aggregates that are not colocalized with E . coli .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Effect of 3‐MA on neutrophil phagocytic capacity of opsonized E. coli as observed by flow cytometry. Percentage of phagocytosing neutrophils after 3‐MA pretreatment (III) compared with cells incubated with opsonized bacteria (II). Data are representative of six independent experiments and are presented as mean±SD, (I: control unstimulated group), (IV) MFI representation (bar 1: control: 1±1.9, bar 2: E. coli: 387.7±33.3, bar 3: E. coli+3‐MA: 25.13±18, Wilcoxon matched‐pairs test, *p<0.05; bar 2 compared with bar 1 and bar 3 compared to bar 2).(B) (I) Presence of acidified structures in E. coli‐phagocytosing neutrophils and medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli‐phagocytosing cells (bar 2: 79.33±7.15%) compared with control conditions (bar 1: 14.17±4.79%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05. ((C) LC3B conversion analysis in E. coli‐phagocytosing neutrophils (lane II), control neutrophils (lane I) and E. coli‐phagocytosing neutrophils after 3‐MA pretreatment (lane III). One representative out of six independent experiments is shown.(D) Effect of 3‐MA administration to E. coli‐phagocytosing neutrophils 15 min after the initiation of phagocytosis (lane III) as detected by LC3B conversion analysis. Lane II represents E. coli‐phagocytosing neutrophils and lane I control neutrophils. One representative out of four independent experiments is shown.(E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.


Figure 6Alterations in ATG gene expression after phagocytosis of opsonized E . coli . ATG3 ( 2 . 05 ± 0 . 27 ) , ATG4 ( 2 . 20 ± 0 . 27 ) , ATG5 ( 2 . 89 ± 0 . 42 ) , ATG6 ( 2 . 04 ± 0 . 40 ) , ATG7 ( 1 . 98 ± 0 . 30 ) and LC3B ( 2 . 46 ± 0 . 39 ) gene expression ( fold ) in E . coli ‐ phagocytosing neutrophils compared with control cells . Dotted line represents mRNA expression levels of control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Alterations in ATG gene expression after phagocytosis of opsonized E. coli. ATG3 (2.05±0.27), ATG4 (2.20±0.27), ATG5 (2.89±0.42), ATG6 (2.04±0.40), ATG7 (1.98±0.30) and LC3B (2.46±0.39) gene expression (fold) in E. coli‐phagocytosing neutrophils compared with control cells. Dotted line represents mRNA expression levels of control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD Wilcoxon matched‐pairs test, *p<0.05.


Figure 7 ( A ) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E . coli stimulation ( lane I : PMA , lane II : PMA + BHA , lane III : E . coli , lane IV : E . coli + BHA , lane V : control cells ) . One representative out of four independent experiments is shown . ( B ) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E . coli − phagocytosing neutrophils ( lane I : PBS , lane II : E . coli , lane III : E . coli + DPI ) . One representative out of four independent experiments is shown . ( C ) ( I ) Effect of NADPH ‐ oxidase inhibition with DPI in the formation of acidified autophagosomes in E . coli ‐ phagocytosing neutrophils . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli − phagocytosing PMN ( 77 . 17 ± 8 . 33 % ) and PMN treated with DPI before incubation with opsonized E . coli ( 21 . 67 ± 4 . 37 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 ( D ) ( I ) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan ( PGN ) , compared with cells treated with PGN . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . PGN ‐ treated neutrophils ( 74 . 83 ± 9 . 09 % ) and neutrophils pretreated with BNP before the addition of PGN ( 17 . 33 ± 2 . 07 % ) compared with control conditions ( 14 . 67 ± 3 . 20 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 , n . s . = not significant : ( E ) Alterations in LC3B conversion in BNP pretreated , peptidoglycan ‐ stimulated neutrophils ( lane III ) , ( lane I : medium ‐ treated cells , lane II : peptidoglycan ‐ treated cells ) . One representative out of four independent experiments is shown . ( F ) Effect of PI3K inhibition with 3 ‐ MA in the percentage of PMA ‐ induced oxidizing cells . PMA ( 99 . 41 ± 0 . 33 % ) and PMA + 3 ‐ MA ( 15 . 82 ± 3 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E. coli stimulation (lane I: PMA, lane II: PMA+BHA, lane III: E. coli, lane IV: E. coli+BHA, lane V: control cells). One representative out of four independent experiments is shown.(B) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E. coli−phagocytosing neutrophils (lane I: PBS, lane II: E. coli, lane III: E. coli+DPI). One representative out of four independent experiments is shown.(C) (I) Effect of NADPH‐oxidase inhibition with DPI in the formation of acidified autophagosomes in E. coli‐phagocytosing neutrophils. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli−phagocytosing PMN (77.17±8.33%) and PMN treated with DPI before incubation with opsonized E. coli (21.67±4.37%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05(D) (I) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan (PGN), compared with cells treated with PGN. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. PGN‐treated neutrophils (74.83±9.09%) and neutrophils pretreated with BNP before the addition of PGN (17.33±2.07%) compared with control conditions (14.67±3.20%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05, n.s.=not significant:(E) Alterations in LC3B conversion in BNP pretreated, peptidoglycan‐stimulated neutrophils (lane III), (lane I: medium‐treated cells, lane II: peptidoglycan‐treated cells). One representative out of four independent experiments is shown.(F) Effect of PI3K inhibition with 3‐MA in the percentage of PMA‐induced oxidizing cells. PMA (99.41±0.33%) and PMA+3‐MA (15.82±3.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


Figure 2 ( A ) Recombinant proteins were mixed , as indicated , in the presence ( lane 5 ) or absence ( lane 4 ) of ATP and incubated for 1 h at 30 ° C before being separated by SDS - PAGE and analyzed by western blotting using the α - His antibody . Components present ( + ) or absent ( − ) from the assay are indicated . ( B ) Recombinant proteins were mixed , as indicated , and incubated as above . The reactions were stopped , subjected to SDS - PAGE and stained with Coomassie Blue . ( C ) Recombinant proteins were mixed , as indicated , incubated as above and analyzed with α - His antibody .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Recombinant proteins were mixed, as indicated, in the presence (lane 5) or absence (lane 4) of ATP and incubated for 1 h at 30°C before being separated by SDS-PAGE and analyzed by western blotting using the α-His antibody. Components present (+) or absent (−) from the assay are indicated.(B) Recombinant proteins were mixed, as indicated, and incubated as above. The reactions were stopped, subjected to SDS-PAGE and stained with Coomassie Blue.(C) Recombinant proteins were mixed, as indicated, incubated as above and analyzed with α-His antibody.


Figure 3 ( A ) The occurrence of mC - ATG5 puncta in WT L . major promastigotes expressing mC - ATG5 incubated in nutrient - rich medium at log phase at 26 ° C . ( B ) The multiplicity of puncta in promastigotes expressing mC - ATG5 after starvation for up to 1 h in PBS at 26 ° C . The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken . ( C ) Co - labelling of puncta with mC - ATG5 and GFP - ATG12 co - expressed in promastigotes incubated in PBS for 2 h at 26 ° C . ( D ) In nutrient - rich medium , GFP - ATG8 punctum without mC - ATG5 staining is arrowed . ( E ) Incubated in PBS for 30 min at 26 ° C . ( F ) In nutrient - rich medium at 26 ° C . ( G ) Incubated in PBS for 30 min at 26 ° C , the small panels are enlargements of the merged panels . ( H - I ) Promastigotes in nutrient - rich medium . ( J - K ) Promastigotes at late log phase in nutrient - rich conditions . ( L ) Promastigotes at stationary phase in nutrient - rich conditions . Scale bar throughout , 10 µm .

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Figure 3(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C.(B) The multiplicity of puncta in promastigotes expressing mC-ATG5 after starvation for up to 1 h in PBS at 26°C. The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken.(C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H-I) Promastigotes in nutrient-rich medium. (J-K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.


Figure 4 ( A ) Growth curve of Leishmania promastigotes in HOMEM medium at 26 ° C . * , Δatg5 differed significantly from WT ( p < 0 . 05 ) . ( B ) The occurrence of GFP - ATG8 puncta in promastigotes after incubation in nutrient - deprived ( PBS , ND ) and nutrient - rich ( HOMEM medium , NR ) conditions for 2 h at 26 ° C . Scale bar , 10 µm ( C ) Occurrence of GFP - ATG8 puncta in promastigotes when incubated in the conditions detailed in ( B ) . Means ± SD from four independent experiments . * and * * , occurrence of GFP - ATG8 puncta in Δatg5 were significantly different from in WT in nutrient - deprived and nutrient - rich conditions ( p < 0 . 05 ) . ( D ) Western blot analysis of extracts of promastigotes expressing GFP - ATG8 at logarithmic growth under standard conditions and probed with α - GFP antibody . The faster migrating , lipidated band is labelled GFP - ATG8 - II while the un - lipidated band migrating more slowly is labelled GFP - ATG8 - I .

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Figure 4(A) Growth curve of Leishmania promastigotes in HOMEM medium at 26°C. *, Δatg5 differed significantly from WT (p<0.05).(B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm(C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05).(D) Western blot analysis of extracts of promastigotes expressing GFP-ATG8 at logarithmic growth under standard conditions and probed with α-GFP antibody. The faster migrating, lipidated band is labelled GFP-ATG8-II while the un-lipidated band migrating more slowly is labelled GFP-ATG8-I.


Figure 5 ( A ) Enlarged ( EM ) and swollen ( WM ) mitochondria seen by transmission electron microscopy ( TEM ) in Δatg5 promastigotes under standard growth conditions . WT is shown at bottom right . Scale bar , 500 nm . ( B ) Fluorescent intensity from MitoTracker Red ( MTR , 0 . 1 µM ) and MitoTracker Green ( MTG , 0 . 2 µM ) in 2 × 106promastigotes after 30 min incubation at 26 ° C . Values shown are the means ± SD from three independent experiments . * and * * , fluorescence was significantly different between WT and Δatg5 ( p < 0 . 05 ) . ( C ) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP - GFP . Scale bar , 10 µm . ( D ) Differential staining of promastigotes with both MTR ( 0 . 1 µM ) and MTG ( 0 . 2 µM ) . Scale bar , 10 µm . ( E ) Viability , as measured by Alamar Blue reduction , of promastigotes . All data are means ± SD from three independent experiments . * , Alamar blue reduction was significantly different ( p < 0 . 05 ) . ( F ) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2 × 106 / ml with H2DCFDA at 0 . 1 mM for 2 h at 26 ° C . Values shown are the means ± SD from three independent experiments . * , DCF fluorescence was significantly different ( p < 0 . 05 ) .

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Figure 5(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm.(B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05).(C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm.(D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm.(E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05).(F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).


Figure 6Phospholipid accumulation in Δatg5 promastigotes . Negative ion survey scans ( 650 - 900 m / z ) of WT ( A ) and Δatg5 ( B ) promastigotes extracted for lipids and analysed by ES - MS , as described in Materials and Methods . a = ( alkylacyl ) .

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Figure 6Phospholipid accumulation in Δatg5 promastigotes.Negative ion survey scans (650-900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).


Figure 7 ( A ) Proportion of metacyclic promastigotes in stationary phase cultures , assessed using the PNA assay . Values shown are the means ± SD from three independent experiments . * , differed significantly ( p < 0 . 05 ) . ( B ) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α - HASPB . α - Cysteine Synthase was used as a loading control [ 56 ] . ( C ) Infectivity and survival of promastigotes in peritoneal macrophages in vitro , infected at a ratio 5 ∶ 1 , with the infection rates being assessed after 1 and 5 days . * , differed significantly ( p < 0 . 01 ) . ( D ) Lesion progression in BALB / c mice inoculated with 5 × 105 stationary phase promastigotes . Values shown are the means ± SD from 5 mice . * , infection level of Δatg5 differed significantly from WT ( p < 0 . 01 ) and Δatg5 : : ATG5 ( p < 0 . 05 ) . ( E ) Morphologies of cells isolated from mouse lesions and analysed by SEM . Scale bar : 2 µm .

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Figure 7(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05).(B) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56].(C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01).(D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05).(E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.


Figure 8A ) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient - rich medium . Shown are ovoid and amastigote - like form ( left ) ; spindled - shaped form without an external flagellum ( centre left panel ) and with an external flagellum of varying lengths ( centre right and right panels ) . Scale bar , 10 µm . ( B ) Distribution of flagella lengths and body lengths of stationary phase promastigotes . Data represent measurements from ∼ 200 cells from each promastigote population .

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Figure 8A) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient-rich medium. Shown are ovoid and amastigote-like form (left); spindled-shaped form without an external flagellum (centre left panel) and with an external flagellum of varying lengths (centre right and right panels). Scale bar, 10 µm.(B) Distribution of flagella lengths and body lengths of stationary phase promastigotes. Data represent measurements from ∼200 cells from each promastigote population.


figf1 ( a ) Identification of biomarkers modulated upon VCP silencing . HeLa , HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h . Protein cell extracts were resolved by SDS - PAGE , and filters were probed with the indicated antibodies . The extent of biomarker modulation correlated with the efficiency of silencing ( oligo 4 versus pool ) . VCP silencing determined stabilization of cyclin E ; induction of the endoplasmic reticulum chaperone GRP78 , CHOP transcription factor and its downstream target GADD - 34 ; conversion of the autophagy regulator ATG8 / light chain 3 ( LC3B ) from the free to the lipidated form ; activation of the effector caspase - 3 ; and cleavage of the caspase target PARP - 1 . A graphical representation of the cell cycle distribution ( percentage of cells ) obtained from the cell cycle analysis ( Supplementary Fig . 3b ) is reported at the bottom of each sample . C , control ; NT , nontargeting oligo ; P , oligonucleotide pool . ( b ) Effect of VCP silencing on cell growth . HeLa , HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter ( data represent mean values of two experiments ) . ( c ) Combination of VCP siRNA with standard - of - care therapeutics . HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose - response analysis with a variety of chemotherapeutic agents .

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figf1(a) Identification of biomarkers modulated upon VCP silencing. HeLa, HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h. Protein cell extracts were resolved by SDS-PAGE, and filters were probed with the indicated antibodies. The extent of biomarker modulation correlated with the efficiency of silencing (oligo 4 versus pool). VCP silencing determined stabilization of cyclin E; induction of the endoplasmic reticulum chaperone GRP78, CHOP transcription factor and its downstream target GADD-34; conversion of the autophagy regulator ATG8/light chain 3 (LC3B) from the free to the lipidated form; activation of the effector caspase-3; and cleavage of the caspase target PARP-1. A graphical representation of the cell cycle distribution (percentage of cells) obtained from the cell cycle analysis (Supplementary Fig. 3b) is reported at the bottom of each sample. C, control; NT, nontargeting oligo; P, oligonucleotide pool.(b) Effect of VCP silencing on cell growth. HeLa, HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter (data represent mean values of two experiments).(c) Combination of VCP siRNA with standard-of-care therapeutics. HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose-response analysis with a variety of chemotherapeutic agents.


figf2 ( b ) LC / MS analysis of VCP after 1 h incubation at 20 ° C with 1 % DMSO ( top ) or 100 μM NMS - 859 ( bottom ) , showing a 313 - Da increase corresponding to the addition of NMS - 859 lacking the Cl atom .

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figf2(b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom.


figf4 ( a ) Activity of VCP inhibitors NMS - 249 and NMS - 873 on wild - type VCP and mutants . Data represent mean values of at least three independent experiments . ( b ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP in the presence of different allosteric inhibitors or ADP . ( c ) Binding of the EDA - ADP - ATTO 590 probe to wild - type ( WT ) VCP and the K615V and N616F mutants in the presence of NMS - 249 . Minor differences in the mP values are due to the use of different enzyme batches . ( d ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP and Walker A mutants in the presence of NMS - 249 . Data shown are representative results of one of three independent experiments : each determination was done in triplicate , data points denote the average value , and error bars represent s . d .

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figf4(a) Activity of VCP inhibitors NMS-249 and NMS-873 on wild-type VCP and mutants. Data represent mean values of at least three independent experiments.(b) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP in the presence of different allosteric inhibitors or ADP.(c) Binding of the EDA-ADP-ATTO 590 probe to wild-type (WT) VCP and the K615V and N616F mutants in the presence of NMS-249. Minor differences in the mP values are due to the use of different enzyme batches.(d) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP and Walker A mutants in the presence of NMS-249. Data shown are representative results of one of three independent experiments: each determination was done in triplicate, data points denote the average value, and error bars represent s.d.


figf5 ( a ) Western blot analysis of biomarker modulation in the HCT116 cell line . HCT116 cells were treated with the indicated doses of inhibitors for 8 h ( top ) or 24 h ( bottom ) , and protein cell extracts were resolved on SDS - PAGE gel . Filters were probed with the indicated antibodies . C , control ; B , bortezomib ( 0 . 01 μM ) ; T , thapsigargin ( 0 . 1 μM ) ; Mg , MG132 ( 0 . 1 μM ) . ( b ) NMS - 873 compound - mediated stabilization of Mcl - 1 in live cells . Top , confocal time - lapse images of live stable , inducible T - REx - 293 cells expressing a bicistronic construct of mCherry - BimS ( 2A ) ΔC ( red ) with eGFP - Mcl - 1 ( green ) after doxycycline treatment . Cells were treated with cycloheximide ( CHX , 50 μg ml − 1 ) ± 2 . 5 μM of NMS - 873 for 6 h . Bottom , total intensity of eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC was measured for time - lapse series shown at top . A decrease in eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control . WT , wild type . Scale bar , 10 μm . ( c ) High - content analysis of poly - Ub and CHOP induction upon treatment with VCP inhibitors . HeLa cells were treated with increasing doses of the indicated compounds . Cells were then fixed and immunostained with the indicated antibodies . Cell numbers and mean fluorescence intensity ( MFI ) were determined using the ArrayScan platform . Data were collected in triplicate ( n = 3 ) , and error bars represent s . d . AU , arbitrary units .

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figf5(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM).(b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A)ΔC (red) with eGFP-Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP-Mcl-1 and mCherry-BimS(2A)ΔC was measured for time-lapse series shown at top. A decrease in eGFP-Mcl-1 and mCherry-BimS(2A)ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm.(c) High-content analysis of poly-Ub and CHOP induction upon treatment with VCP inhibitors. HeLa cells were treated with increasing doses of the indicated compounds. Cells were then fixed and immunostained with the indicated antibodies. Cell numbers and mean fluorescence intensity (MFI) were determined using the ArrayScan platform. Data were collected in triplicate (n = 3), and error bars represent s.d. AU, arbitrary units.


Figure 1B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in ( A ) . 1cs , 1 - cell stage ; 2cs , 2 - cell stage ; st6 , 32 - cell stage ; st8 . 5 , blastula stage . Blots are representative of 3 independent experiments . C Densitometry analysis of Hwa bands from the western blot in ( B ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 by unpaired t test . D Western blot analysis of exogenous Hwa in HEK293T cells . Treatment of cells with lysosome inhibitors bafilomycin A1 ( Baf - A1 ) and NH4Cl for 24 hours , DMSO was used as control . Dosage of Baf - A1 : 10 nM , 50 nM , 100 nM ; Dosage of NH4Cl : 2 . 5 mM , 5 mM , 10 mM . Hwa with modification is indicated by white arrow ; Degraded Hwa is indicated by black arrow . Blots are representative of 3 independent experiments . E Densitometry analysis of Hwa bands from the western blot in ( D ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , * * p < 0 . 01 by unpaired t test .

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Figure 1B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in (A). 1cs, 1-cell stage; 2cs, 2-cell stage; st6, 32-cell stage; st8.5, blastula stage. Blots are representative of 3 independent experiments. C Densitometry analysis of Hwa bands from the western blot in (B) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, *p < 0.05, **p < 0.01 by unpaired t test. D Western blot analysis of exogenous Hwa in HEK293T cells. Treatment of cells with lysosome inhibitors bafilomycin A1 (Baf-A1) and NH4Cl for 24 hours, DMSO was used as control. Dosage of Baf-A1: 10 nM, 50 nM, 100 nM; Dosage of NH4Cl: 2.5 mM, 5 mM, 10 mM. Hwa with modification is indicated by white arrow; Degraded Hwa is indicated by black arrow. Blots are representative of 3 independent experiments. E Densitometry analysis of Hwa bands from the western blot in (D) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, **p < 0.01 by unpaired t test.


Figure 2C Co - IP with HEK293T cells co - transfected with plasmids encoding Hwa - Flag and ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . Blots are representative of 3 independent experiments . D Co - IP with HEK293T cells co - transfected with plasmids encoding Hwa - Flag and ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - HA were immunoblotted with Flag and HA antibodies . Blots are representative of 3 independent experiments . E Colocalization of Hwa - Flag ( red ) and ZNRF3 - HA ( green ) or ZNRF3ΔRING - HA ( green ) in Hela cells is indicated by yellow dots . Baf - A1 , 50 nM , 12 hrs . Magnification : 100 × ; scale bar : 10 μm .

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Figure 2C Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments. D Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-HA were immunoblotted with Flag and HA antibodies. Blots are representative of 3 independent experiments. E Colocalization of Hwa-Flag (red) and ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm.


Figure 3B Whole mount in situ hybridization of Spemann organizer markers chrd , gsc , and xnr3 in gastrula embryos ( stage 10 . 5 ) . Oocytes were injected with AS or mRNAs as indicated , Dosage : znrf3 AS ( 3 ng AS4 + 3 ng AS9 ) , znrf3 mRNA 100 pg , znrf3ΔRING mRNA 50 pg . embryos were obtained through Host transfer technology . Scale bars : 1 mm . E Relative levels of β - catenin ( left ) and Axin ( right ) in ( D ) normalized to GAPDH . n = 3 independent experiments , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by unpaired t test . H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos ( stage 10 . 5 ) . Embryos were injected and obtained as descripted in ( G ) . Scale bars : 1 mm . I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl ( gfp mRNA ) , znrf3FL , and znrf3 truncations as indicated at 4 - cell stage . Duplicated axes ( red arrowhead ) . znrf3FL , full - length znrf3 ; znrf3ΔRING , znrf3 lacking the RING domain ; znrf31 - 320 , amino acids 1 - 320 of znrf3 ; znrf3320 - 606 , amino acids 320 - 606 of znrf3 ; znrf3606 - 854 , amino acids 606 - 854 of znrf3 . Scale bars : 1 mm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B Whole mount in situ hybridization of Spemann organizer markers chrd, gsc, and xnr3 in gastrula embryos (stage 10.5). Oocytes were injected with AS or mRNAs as indicated, Dosage: znrf3 AS (3 ng AS4 + 3 ng AS9), znrf3 mRNA 100 pg, znrf3ΔRING mRNA 50 pg. embryos were obtained through Host transfer technology. Scale bars: 1 mm.E Relative levels of β-catenin (left) and Axin (right) in (D) normalized to GAPDH. n = 3 independent experiments, mean ± SEM, *p < 0.05, ***p < 0.005, ****p < 0.0001 by unpaired t test.H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos (stage 10.5). Embryos were injected and obtained as descripted in (G). Scale bars: 1 mm.I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl (gfp mRNA), znrf3FL, and znrf3 truncations as indicated at 4-cell stage. Duplicated axes (red arrowhead). znrf3FL, full-length znrf3; znrf3ΔRING, znrf3 lacking the RING domain; znrf31-320, amino acids 1-320 of znrf3; znrf3320-606, amino acids 320-606 of znrf3; znrf3606-854, amino acids 606-854 of znrf3. Scale bars: 1 mm.


Figure 4B Hwa ubiquitination was analyzed under denaturing conditions . HEK293T cells co - transfected with wildtype His - HA - Ubiquitin ( His - HA - Ub WT ) or lysine - less mutant ( K0 ) , Hwa - Flag , and ZNRF3 - Myc . The lysates were immunoblotted with Myc , Flag , and α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . K0 , all lysine sites on ubiquitin were mutated . Blots are representative of 3 independent experiments . D Screen of ZNRF3 - mediated Hwa ubiquitination sites under denaturing conditions . HEK293T cells were co - transfected with His - HA - Ub , Hwa - Flag or Hwa10KR - Flag , and ZNRF3 or ZNRF3ΔRING . The lysate was immunoblotted with Myc , Flag , and α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . Hwa10KR - Flag mutant , all lysine sites in Hwa sequence were replaced with arginine . Denaturing conditions : before immunoprecipitation , lysates were denatured by adding SDS to a final concentration at 1 % and boiling at 95 ℃ for 15 min . Blots are representative of 3 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and ZNRF3-Myc. The lysates were immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. K0, all lysine sites on ubiquitin were mutated. Blots are representative of 3 independent experiments.D Screen of ZNRF3-mediated Hwa ubiquitination sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or Hwa10KR-Flag, and ZNRF3 or ZNRF3ΔRING. The lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa sequence were replaced with arginine. Denaturing conditions: before immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots are representative of 3 independent experiments.