For each , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in - seq analyses ( n = 3 ) . Enrichment of genes ( TSS ) closest to each canonical HIF - α / β binding site ( i . e . identified in both HIF - 1α ( or HIF - 2α ) both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment ( ES = enrichment score , NES = normalized enrichment score )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)


Immunoblots show progressive induction of HIF - 1α HIF - 2α with increasing severity of

Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxia


HIF - 1α ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for canonical HIF - binding sites that bound at one oxygen or other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen no new sites were

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


( C ) HIF - 2α ( D ) HIF - 1β ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for canonical HIF - binding sites that bound at one oxygen or other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen no new sites were

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


Frequency distribution of ChIP - seq signals at 0 . 5 % oxygen compared to 3 % oxygen . of total HIF - α signal was unimodally distributed ( purple ) . Upper tertile HIF - α sites ( solid red ) had a significantly ( p = 10 - 7 , Wilcox rank sum ) higher of HIF - 1β signal ( dotted red ) vice versa ( blue lines )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)


of HIF - 1α to HIF - 2α ChIP - seq signal for canonical HIF - binding sites at 0 . 5 % oxygen was plotted against that at 3 % oxygen . Strong correlation in HIF - 1α to HIF - 2α was observed between two oxygen

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations


Immunoblots show induction of HIF - 1α HIF - 2α level , which is highest at 6 hours falls by 48

O O O O O O O O O O O O O O O O O O O O O

Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours


HIF - 1α , ( C ) HIF - 2α ( D ) HIF - 1β ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 16 hours is plotted against that at 6 hours for canonical HIF - binding sites that bound at any of three timepoints . ChIP - seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia , but correlated well between two timepoints no novel sites were observed at either timepoint . ( E - F ) same plots comparing ( E ) HIF - 1α , ( F ) HIF - 2α ( G ) HIF - 1β ChIP - seq signals at 48 hours 6 hours hypoxia . ChIP - seq signal at 48 hours correlated well with that at 6 hours again no novel sites were observed at either

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepoint


Frequency distribution of ChIP - seq signals at 48 hours compared to 6 hours hypoxia . of total HIF - α signal had a unimodal distribution ( purple ) . However , upper tertile HIF - α sites ( solid red ) had a comparable of HIF - 1β signal ( dotted red ) to of HIF - 1β signal in lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Frequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological difference


of HIF - 1α to HIF - 2α ChIP - seq signal for canonical HIF - binding sites at ( I ) 16 hours hypoxia at ( J ) 48 hours hypoxia was plotted against that at 6 hours hypoxia . Strong correlation in HIF - 1α to HIF - 2α was observed between various

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints


were incubated in 0 . 5 % ambient oxygen for 16 hours . Immunoblots show HIF - 1α , HIF - 2α HIF - 1β in wild type single clones of CRISPR - Cas9 engineered

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Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines


HIF - 1α ChIP - seq signal in HIF - 2α KO was plotted against that in wild - type for canonical HIF - 1 binding sites in either

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell type


HIF - 2α ChIP - seq signal in HIF - 1α KO was plotted against that in wild - type for canonical HIF - 2 binding sites in either type . No significant increase ( Wilcoxon rank sum ) in binding of either isoform was observed following deletion of

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the other


Box - - whisker plots showing : ( D ) effect of HIF - 2α inactivation on HIF - 1α ChIP - seq signal at sites that specifically bound HIF - 2α , but not HIF - 1α in wild - type showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank ) decrease rather than increase ( compare red boxes ) consistent with reduction in HIF - 1α observed in panel

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Box-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel A


effect of HIF - 1α inactivation on HIF - 2α ChIP - seq signal ( compare blue boxes ) at sites that specifically bound HIF - 1α , but not HIF - 2α in wild - type showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test)


effect , at sites that bind both isoforms in wild - type , of ( F ) HIF - 2α inactivation on HIF - 1α binding showing a similar small but significant reduction rather than increase ( p = 2 x 10 - 16 , Wilcoxon Signed rank ) as

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as abov


effect , at sites that bind both isoforms in wild - type , o HIF - 1α inactivation on HIF - 2α binding showing no significant effect ( p = 0 . 4 , Wilcoxon signed rank )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The effect, at sites that bind both isoforms in wild-type cells, o HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test)


Canonical HIF - 1 HIF - 2 binding sites were determined by overlap of HIF - α HIF - 1β binding sites identified by MACS peak caller as for Figure 4 . from each binding site to nearest annotated gene promoter was determined plotted as a frequency distribution for ( A ) HIF - 1 ( B ) HIF - 2 sites in HKC - 8 , ( C ) HIF - 1 ( D ) HIF - 2 sites in RCC4 , ( E ) HIF - 1 ( F ) HIF - 2 sites in HepG2 . distribution of HIF - 1 binding sites was consistently more promoter proximal than HIF - 2 sites despite sites themselves differing between

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types


distribution of accessible HRE motifs was also plotted in HepG2 differed significantly from that of both HIF - 1 ( p ~ 10 - 50 ) HIF - 2 ( p ~ 10 - 80 ) binding sites ( Chi - squared )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)


Immunoblots showing HIF - 1α , HIF - 2α HIF - 1β in HKC - 8 , HepG2 , RCC4 MCF - 7 lines in normoxia following 16 hours incubation at 0 . 5 %

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen


Sites that bound ( B ) canonical HIF - 1 ( i . e . identified in both HIF - 1α both HIF - 1β ChIP - seq experiments ) or ( C ) canonical HIF - 2 ( i . e . identified in both HIF - 2α both HIF - 1β ChIP - seq experiments ) were identified from ChIP - seq datasets in HepG2 HKC - 8 following 16 hours incubation in 0 . 5 % oxygen from normoxic RCC4 compared with previously published data from MCF - 7 . ( D ) proportion of total HIF - 1 or HIF - 2 sites that were shared between 2 , 3 or 4 lines is plotted . For sites that are shared between ( E ) HepG2 HKC - 8 , ( F ) HKC - 8 RCC4 , or ( G ) RCC4 HepG2 , of HIF - 1α / HIF - 2α signal for one was plotted against other . of HIF - 1α / HIF - 2α in one correlates well with that in other . ChIP - seq signal is averaged across two independent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments


Heatmaps showing average ( n = 2 , independent ChIP - seq experiments ) ChIP - seq signal ( read per million mapped reads , CPM ; expressed as colour ) at canonical HIF - 1 HIF - 2 binding sites across flanking ± 5kb regions ( x - axis ) in HepG2 incubated in 0 . 5 % oxygen for 16 hours . Sites are ordered on y - axis according to HIF signal derived from HepG2 incubated in hypoxia . ChIP - seq signal for histone modifications was obtained from ENCODE was derived from HepG2 incubated in

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Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia


Bar chart showing enrichment of transcription factor binding sites ( TFBS ) at canonical HIF - 1 HIF - 2 sites in HepG2 ( identified as in Figure 4 ) . TFBSs for 61 additional - binding were determined from ENCODE ChIP - seq data in normoxic HepG2 . significance of overlap between each set of binding sites canonical HIF - 1 HIF - 2 sites , was determined using a hypergeometric . Accessible sites determined from ENCODE DNAse - seq data in HepG2 was used as a negative

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control


Flow cytometric evaluation of CXCR4 surface expression using an anti - CXCR4 - PE antibody . Left : positive ; right : negative ; representative data are shown . gating strategy is depicted in Supplementary Fig S1 .

Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.


Representative histograms revealing magnitude of CXCR4 expression in myeloma as compared to indicated bone marrow subtype .

Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.


Median of surface CXCR4 expression relative to isotype control ( n = 7 - 10 ) . Horizontal bars indicate mean of values ; asterisks indicate statistical significance ( plasma vs B : P = 0 . 0082 ; plasma vs T : P = 0 . 0091 ; plasma vs CD34 + : P = 0 . 011 ; plasma vs monocytes : P = 0 . 0423 ; Student ' s t - to compare mean plasmacell relative expression with mean relative expression of each further indicated subtype ) . MM judged positive for CXCR4 expression were selected for this .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7-10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.


Representative MM bone marrow of a positive for MM CXCR4 expression : hematoxylin eosin ( H & amp ; amp ; E ) . Immunohistochemistry for CD138 , CXCR4 , chain kappa , chain lambda .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&amp;E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.


Real - time PCR of cxcr4 transcript expression in HeLa ( negative control ) in MM lines MM . 1S OPM - 2 . Shown is mean relative expression ± ( n = 3 independent experiments ) . Values are normalized to expression of ubiquitin ( Ub ) . asterisk indicates statistically significant differences ( HeLa vs MM . 1S : P < 0 . 001 ; HeLa vs OPM - 2 : P < 0 . 0001 ; Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).


CXCR4 expression assessed by immunoblotting ( one representative blot out of 3 is shown ) .

CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).


Binding of [ 68Ga ] Pentixafor to MM . 1S OPM - 2 after indicated incubation periods ( n = 3 per time point ) . Shown is mean ± . difference between OPM - 2 groups is statistically significant ; * P < 0 . 0017 ( one - way ANOVA ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).


Mean - to - background ( TBR ) for [ 18F ] FDG ( left ) for [ 68Ga ] Pentixafor ( right ) in MM . 1S OPM - 2 xenograft - bearing NOD SCID mice . Shown is mean ± , n = 8 tumors ( 4 mice ) ; * P = 0 . 0111 for [ 18F ] FDG * P = 0 . 0113 for [ 68Ga ] Pentixafor ( Student ' s t - ) . One - way ANOVA revealed significant differences between groups ; P < 0 . 0001 ( not graphically shown ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).


Representative [ 68Ga ] Pentixafor PET of three mice bearing MM . 1S ( right shoulder ) OPM - 2 ( left shoulder ) tumors .

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Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.


Flow cytometric of CXCR4 surface expression on resected MM . 1S OPM - 2 tumors . Data are mean ± , n = 4 . * P = 0 . 0286 ; Mann - Whitney U - .

Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test.


Correlation of [ 68Ga ] Pentixafor PET mean TBR CXCR4 surface expression assessed by flow cytometry . n = 8 tumors were analyzed .

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Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.


Mice ( n = 4 ) bearing OPM - 2 MM . 1S xenografts were coinjected with AMD3100 ( right image , one representative mouse ) or not pretreated ( left image ) before undergoing [ 68Ga ] PentixaforPET . arrows point to bladder . is shown in Supplementary Fig S3A . Source data are available online for this figure .

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

Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.


[ 68Ga ] PentixaforPET / CT [ 18F ] FDGPET / CTA - D Maximum projections ( MIP ) of [ 68Ga ] Pentixafor ( A ) [ 18F ] FDGPET / CT ( B ) of a 68 - year - old with histologically proven multiple myeloma indicating better lesion - to - background contrast for [ 68Ga ] Pentixafor in corresponding myeloma manifestations . Trans - axial views of upper thorax ( C ) pelvis ( D ) underline higher uptake values of bone manifestations ( yellow arrows ) of [ 68Ga ] Pentixafor compared to [ 18F ] FDG .

O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O

[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.


Visual comparison of [ 18F ] FDG - [ 68Ga ] Pentixafor PET scansNumber of with visual positivity for indicated PET tracer ( total : n = 14 ) . Number of ( total n = 14 ) for whom imaging with [ 18F ] FDG PET ( FDG , n = 2 ) or [ 68Ga ] Pentixafor PET ( Pentixafor , n = 7 ) was superior , with comparable positivity ( comparable , n = 3 ) , with dual imaging providing complementary visual information ( complementary , n = 2 ) .

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Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).


[ 68Ga ] PentixaforPET / MRimagesA - C Coronal views of [ 68Ga ] Pentixafor ( A ) , T2 STIR weighted MRI ( B ) CTbone window ( C ) of a maleuptake with histologically proven multiple myeloma . increased [ 68Ga ] Pentixaforbone correlates with hyperintense T2 STIR signal ; however , myeloma manifestations are underestimated in corresponding bone window CT .

O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.


( A ′ ) HeLa transfected with pEGFP - RAB11 were fixed in basal conditions stained for mATG9 . Pearson ' s coefficient : 0 . 452 ± 0 . 034 ( ) . ( A ″ ) HeLa were transfected with ATG9L1 - pEGFP , fixed in basal condition , labeled for RAB11 . Pearson ' s coefficient : 0 . 413 ± 0 . 020 ( ) .

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(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson's coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson's coefficient: 0.413 ± 0.020 (SEM).


( B ) HeLa were incubated for 1 hr at 37 ° C or 18 ° C to reach correct , loaded with transferrin Alexa - 555 for 1 hr , fixed , labeled with anti - ATG9 anti - TGN46 ( marker for Trans Golgi Network ) . Histogram shows Pearson ' s coefficient between ATG9 transferrin ( Tf ) or TGN46 . Error bar , . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification .

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(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson's coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.


( C ) HeLa were incubated in serum - free medium containing Dynasore 100 μM or DMSO for 2 hr loaded for 30 min with transferrin Alexa - 555 . were then fixed labeled for mATG9 TGN46 . Pearson ' s coefficient between mATG9 transferrin or TGN46 was quantified . Error bar , . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification .

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(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.


( D ) treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed then analyzed by TIRF microscopy . Pictures are at same magnification brightness . transfected with Dynamin mutant were identified by abnormal transferrin accumulation at plasma .

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(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.


( E ) HeLa were incubated for 1 hr at 4 ° C with ice - cold NHS - LC - Biotin ( Pierce ) solution , rinsed , lysed in RIPA buffer . Biotinylated were precipitated using streptavidin - agarose beads ( Pierce ) blotted for mATG9 to assess localization at plasma .

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(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.


( F ) HeLa treated as in ( C ) were processed for immunogold labeling on cryosections stained with anti - mATG9 antibody . Arrows show clathrin - coated structures . Scale bar in a c , 150 nm ; scale bar in b , 100 nm .

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(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.


( G ) HeLa transfected with GFP - ATG16L1 were fixed , labeled for mATG9 , then analyzed by TIRF microscopy . Control were cotransfected with GFP - ATG16L1 Straw - ATG16L1 . Pictures are at same magnification brightness . See Figure S1J for .

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(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.


( H ) HeLa treated with Dynasore were processed for immunogold labeling on cryosections double labeled with ATG16L1 ( 10 nm ) mATG9 ( 15 nm ) . Scale bar , 100 nm .

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(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.


( I ) HeLa treated as in ( H ) were processed for triple labeling on cryosections ( clathrin , 5 nm ; ATG16L1 , 10 nm ; mATG9 , 15 nm ) . Scale bar , 150 nm . ( J ) Table shows of numbers of clathrin - coated structures carrying mATG9 ATG16L1 alone or together or neither . A minimum of 50 clathrin - coated structures were examined from three different experiments . We constructed a 2 × 2 table with 0 or 1 - 3 gold particles for mATG9 or ATG16L1 for a X2 to avoid statistical with 0 . X2 = 10 . 4 , df = 1 , p = 0 . 001 .

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(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1-3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.


( A ) HeLa transfected with GFP - ATG16L1 for 24 hr were incubated for 15 min with 2 . 5 μg / ml with HRP - cholera toxin subunit B at 4 ° C then for 10 min at 37 ° C . were then fixed treated for immunogold labeling on cryosections . ATG16L1 was detected with anti - ATG16L1 antibody ( 15 nm ) , cholera toxin was detected using anti - HRP antibody . Scale bar , 150 nm .

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(A) HeLa cells transfected with GFP-ATG16L1 for 24 hr were incubated for 15 min with 2.5 μg/ml with HRP-cholera toxin subunit B at 4°C and then for 10 min at 37°C. Cells were then fixed and treated for immunogold labeling on cryosections. ATG16L1 was detected with anti-ATG16L1 antibody (15 nm), and cholera toxin was detected using anti-HRP antibody. Scale bar, 150 nm.


( B ) HeLa were transiently transfected with GFP - ATG16L1 for 20 hr . in basal conditions were fixed labeled with anti - RAB11 antibody .

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(B) HeLa cells were transiently transfected with GFP-ATG16L1 for 20 hr. Cells in basal conditions were fixed and labeled with anti-RAB11 antibody.


( C ) HeLa transfected with mStrawberry - ATG16L1 ( 20 hr ) starved ( 4 hr ) were processed for immunogold labeling on cryosections . Double labeling was performed with rabbit anti - ATG16L1 ( 10 nm gold ) rabbit anti - RAB11 ( 15 nm gold ) . Scale bar , 150 nm . Schematic diagram shows outlines from EM . Green lines indicate structures positive for both ATG16L1 RAB11 , whereas red lines are only positive for RAB11 . Histogram shows by EM of ATG16L1 tubulovesicular structures that carry RAB11 . 281 tubulovesiclular structures from different experiments were analyzed . Error bars , SD .

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(C) HeLa cells transfected with mStrawberry-ATG16L1 (20 hr) and starved (4 hr) were processed for immunogold labeling on cryosections. Double labeling was performed with rabbit anti-ATG16L1 (10 nm gold) and rabbit anti-RAB11 (15 nm gold). Scale bar, 150 nm. Schematic diagram shows membrane outlines from EM. Green lines indicate structures positive for both ATG16L1 and RAB11, whereas red lines are only positive for RAB11. Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11. 281 tubulovesiclular structures from different experiments were analyzed. Error bars, SD.


( D ) HeLa were transfected with EGFP - ATG16L1 mCherry - RAB11 , 5 min movies were recorded . Representative from movies are shown .

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(D) HeLa cells were transfected with EGFP-ATG16L1 and mCherry-RAB11, and 5 min movies were recorded. Representative images from movies are shown.


( E ) HeLa were loaded with mouse anti - transferrin receptor antibody ( 5E9C11 ) for 30 min fixed for immunogold labeling for cryosections . sample was labeled with rabbit anti - mouse antibody to recognize transferrin receptor ( 5 nm ) , anti - ATG16L1 ( 10 nm ) , anti - mATG9 ( 15 nm ) . Scale bar , 150 nm .

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(E) HeLa cells were loaded with mouse anti-transferrin receptor antibody (5E9C11) for 30 min and fixed for immunogold labeling for cryosections. The sample was labeled with rabbit anti-mouse antibody to recognize transferrin receptor (5 nm), anti-ATG16L1 (10 nm), and anti-mATG9 (15 nm). Scale bar, 150 nm.


( F ) HeLa were transfected with pmStrawberry - ATG16L1 ATG9L1 - pEGFP , 5 min movies were recorded . Representative from movies are shown .

(F) HeLa cells were transfected with pmStrawberry-ATG16L1 and ATG9L1-pEGFP, and 5 min movies were recorded. Representative images from the movies are shown.


( G ) HeLa were transfected with pmStrawberry - ATG16L1 pEGFP - LC3 , starved for 2 hr , fixed , labeled for mATG9 .

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(G) HeLa cells were transfected with pmStrawberry-ATG16L1 and pEGFP-LC3, starved for 2 hr, fixed, and labeled for mATG9.


( A B ) ( A ) HeLa were incubated for 4 hr at 37 ° C or 18 ° C processed for LC3II western blot . Some were incubated overnight with 20 mM NH4Cl in full medium ( to block lysosomal degradation ) at 37 ° C or 18 ° C ( B ) .

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(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).


( C ) HeLa were transfected with pEGFP - LC3 for 20 hr incubated for 4 hr at 37 ° C or 18 ° C .

(C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C.


( D E ) ( D ) HeLa were transfected with pEGFP - ATG16L1 for 20 hr incubated for 4 hr at 37 ° C or 18 ° C . number ( E ) of vesicles were scored ( a minimum of 20 were examined for each condition ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 ∗ ∗ p < 0 . 01 .

(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.


( F G ) ( F ) HeLa were transfected with pEGFP - ATG16L1 for 20 hr , incubated for 4 hr at 37 ° C or 18 ° C , labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes . Histogram in ( G ) shows amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment ( Manders ' coefficient ) . Error bar , . NS , not significant ; ∗ ∗ p < 0 . 01 .

(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders' coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01.


( H ) HeLa were transfected with pmStrawberry - ATG16L1 for 20 hr , fixed , labeled for mATG9 . histograms show of colocalization of vesicles labeled with ATG16L1 mATG9 at 37 ° C or 18 ° C . Error bar , . ∗ ∗ ∗ p < 0 . 001 ∗ ∗ p < 0 . 01 .

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(H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.


( I ) HeLa were transfected with pEGFP - RAB11 or pEGFP - Myosin Vb tail ( MVb ) incubated during last 16 hr with Bafilomycin A1 ( Baf ) or DMSO processed for LC3 - II western blot . Control were transfected with pEGFP empty vector .

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(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.


( J ) HeLa were transfected with ATG16L1 - mStrawberry RAB11 - EGFP or MVb tail - EGFP . of ATG16L1 vesicles was scored in RAB11 MVb tail overexpression conditions . inserts show just ATG16L1 vesicles as quantified in histogram . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

(J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ∗∗∗p<0.001.


( K ) HeLa were transfected as in ( C ) loaded with transferrin Alexa - 647 for 30 min . Histogram inserts show correlation between ATG16L1 loaded transferrin ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( L ) HeLa were transfected as in ( C ) labeled for mATG9 . Histogram inserts show correlation between ATG16L1 mATG9 in RAB11 MVb tail overexpression conditions ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

(L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( M ) HeLa were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium , loaded with transferrin Alexa - 488 ( Tf ) . Pictures histogram show correlation between ATG16L1 transferrin in basal starving condition ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( N ) HeLa were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium then fixed labeled with anti - mATG9 antibody . Pictures histogram show correlation between ATG16L1 mATG9 in basal starved ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( A ) HeLa were transfected with mStrawberry - ATG16 for 20 hr treated for 10 min with N - ethylmaleimide ( NEM ; 100 μM ) in full medium , fixed , stained for mATG9 . Histogram shows correlation between mATG9 ATG16L1 ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( C D ) ( C ) Representative fields of in vitro fusion of ATG16L1 mATG9 vesicles . in ( D ) was performed on ten fields ( similar to that shown in figure ) per experiment . is expressed as Pearson ' s coefficient . Error bar , . ∗ ∗ p < 0 . 001 .

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(C and D) (C) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles. The quantification in (D) was performed on ten fields (similar to that shown in the figure) per experiment. The quantification is expressed as Pearson's coefficient. Error bar, SEM. ∗∗p < 0.001.


( A ) HeLa were transfected with siRNAs for different R - SNAREs , we assessed of autophagy substrate p62 versus actin .

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(A) HeLa cells were transfected with siRNAs for different R-SNAREs, and we assessed the levels of the autophagy substrate p62 versus actin.


( B ) HeLa transfected with control or VAMP3 siRNA ( oligo A ) for 4 days were treated during last 16 hr with Bafilomycin A1 ( Baf A1 ) or DMSO ( basal ) processed for LC3 - II western blots .

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(B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots.


( C ) Endogenous LC3 immunocytochemistry in HeLa transfected with control or VAMP3 siRNA for 4 days . histogram shows number of vesicles ( LC3 positive ) in a minimum of 30 . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(C) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days. The histogram shows the number of vesicles (LC3 positive) in a minimum of 30 cells. Error bar, SEM. ∗∗∗p < 0.001.


( D ) HeLa were transfected with VAMP3 - HA for 20 hr stained for EEA1 or RAB11 . Histogram shows colocalization between VAMP3 - HA EEA1 or VAMP3 - HA RAB11 ( Manders ' coefficient ) . Error bar , .

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(D) HeLa cells were transfected with VAMP3-HA for 20 hr and stained for EEA1 or RAB11. Histogram shows colocalization between VAMP3-HA and EEA1 or VAMP3-HA and RAB11 (Manders' coefficient). Error bar, SEM.


( E ) HeLa were transfected for 20 hr with VAMP3 - HA , incubated for 1 hr at 37 ° C or 18 ° C , fixed , labeled for HA mATG9 . Histogram shows of colocalization between VAMP3 with mATG9 ( Manders ' coefficient ) . Error bar , . NS , not significant .

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(E) HeLa cells were transfected for 20 hr with VAMP3-HA, incubated for 1 hr at 37°C or 18°C, fixed, and labeled for HA and mATG9. Histogram shows the percentage of colocalization between VAMP3 with mATG9 (Manders' coefficient). Error bar, SEM. NS, not significant.


( A - C ) HeLa transfected with control , VAMP3 siRNA for 4 days were transfected during last 20 hr with ATG9L1 - pEGFP labeled with anti - EEA1 ( for early endosomes ) or anti - RAB11 ( for recycling endosomes ) . histogram in ( C ) shows of colocalization of mATG9 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , . ∗ ∗ ∗ p < 0 . 001 .

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(A-C) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1-pEGFP and labeled with anti-EEA1 (for early endosomes) or anti-RAB11 (for recycling endosomes). The histogram in (C) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001.


( D - F ) HeLa were treated as in ( A ) - ( C ) transfected during last 20 hr with pEGFP - ATG16L1 labeled with anti - EEA1 ( early endosomes ) or anti - RAB11 ( recycling endosomes ) . Histogram in ( D ) shows of colocalization of ATG16L1 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) HeLa cells were treated as in (A)-(C) and transfected during the last 20 hr with pEGFP-ATG16L1 and labeled with anti-EEA1 (early endosomes) or anti-RAB11 (recycling endosomes). Histogram in (D) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001; NS, not significant.


( A ) HeLa transfected with control , VAMP3 siRNA for 4 days were transfected during last 20 hr with mStrawberry - ATG16L1 labeled for mATG9 . correlation between mATG9 ATG16L1 was quantified ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry-ATG16L1 and labeled for mATG9. The correlation between mATG9 and ATG16L1 was quantified (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( B ) HeLa were transfected with control or VAMP3 siRNA then with mStrawberry - ATG16 , 5 min movies were recorded . Homotypic fusion events between mStrawberry - ATG16 vesicles were assessed in control VAMP3 knockdown . Error bar , . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry-ATG16, and 5 min movies were recorded. Homotypic fusion events between mStrawberry-ATG16 vesicles were assessed in control and VAMP3 knockdown cells. Error bar, SEM. NS, not significant.


( C ) In vitro fusion of ATG16L1 mATG9 vesicles . HeLa were transfected with control or VAMP3 siRNA for 4 days separately , on fourth day , either transfected with mStrawberry - ATG16L1 or ATG9L1 - pEGFP . PNS of expressing two constructs were mixed for 1 hr at 37C ° observed by confocal microscopy . Histogram shows correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 . total number of structures per field ( ATG16L1 ATG9L1 ) is not significantly different in control KD samples ( control , 37 ± 3 ; VAMP3 KD , 45 ± 3 ; ± ; p = 0 . 106 ) .

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(C) In vitro fusion assay of ATG16L1 and mATG9 vesicles. HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and, on the fourth day, either transfected with mStrawberry-ATG16L1 or ATG9L1-pEGFP. PNS of cells expressing the two constructs were mixed for 1 hr at 37C° and observed by confocal microscopy. Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. The total number of structures per field (ATG16L1 and ATG9L1) is not significantly different in the control and KD samples (control, 37 ± 3; VAMP3 KD, 45 ± 3; ± SEM; p = 0.106).


( A ) THP - 1 were subjected to knockdowns as indicated treated with 100 ng / mL LPS overnight , followed by 0 . 25 mM LLOMe for 3 h , of IL - 1β were determined in supernatants .

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(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants.


( B ) THP - 1 were subjected to GABARAP knockdown processed as in A .

(B) THP-1 cells were subjected to GABARAP knockdown and processed as in A.


( C ) IL - 1β in supernatants of THP - 1 subjected to TRIM knockdowns treated with LPS then with LLOMe .

(C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.


( D ) Confocal microscopy of THP - 1 treated sequentially with LPS LLOMe , stained for LC3B TRIM16 . tracings correspond to arrows . Arrowheads indicate colocalization . Scale bars , 5 μm .

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(D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm.


( E ) IL - 1β in supernatants of THP - 1 subjected to TRIM knockdowns treated with LPS then with LLOMe .

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

(E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.


( F ) of IL - 1β were determine in supernatants from THP - 1 subjected to knockdown as indicated , treated with 100 ng / mL LPS for overnight then with 0 . 25mM of Silica , Alum , or monosodium urate ( MSU ) .

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

(F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU).


( G ) IL - 1β in supernatants of human macrophages ( MDM ) subjected to knockdowns sequentially treated with LPS LLOMe . Data , means ± ; n ≥ 5 ( except for B where a representative data set from 3 repeats ) . * P < 0 . 05 ( t or ANOVA ) .

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

(G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).


( A B ) of IL - 1β in supernatants from THP - 1 that were subjected to knockdown as indicated were treated with LPS , then ( A ) treated with LLOMe or ( B ) starved in EBSS .

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

(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS.


( C ) IL - 1β in supernatants of human macrophages ( MDM ) subjected to knockdowns sequentially treated with LPS LLOMe .

(C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.


( D ) Co - immunoprecipitation ( co - IP ) of flag - galectin - 8 ( Gal - 8 ) GFP - TRIM16 ( T16 ) in HEK293T .

O O O O O O O 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) Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells.


( E ) Co - IP of endogenous galectin - 8 TRIM16 in complexes from LLOMe - treated THP - 1 .

O O O O O O O O O O O O O O O O O O O O O O

(E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells.


( F ) Confocal microscopy of THP - 1 treated with LPS LLOMe , stained for TRIM16 galectin - 8 . tracings correspond to arrowed dashed lines . Pearson correlation coefficient was calculated using SlideBook as described previously . Scale bars , 5 μm .

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

(F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm.


( G ) In vitro translated radiolabeled [ 35S ] myc - HA - TRIM16 was incubated with GST - galectin - 8 in presence ( + ) or absence ( - ) of flag - ULK1 cold ATP , GST pulldowns were performed , [ 35S ] radiolabeled Myc - HA - TRIM16 in pulled - down material detected by PAGE autoradiography . Amounts of GST fusion are shown in coommassie brilliant blue ( CBB ) - stained gels . Data , means  ; n ≥ 5 , except for immunoblot quantifications where n ≥ 3 . * P < 0 . 05 , † P ≥ 0 . 05 ( t or ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).


( A B ) Immunoblot analyses of indicated fractions prepared by differential centrifugation . HeLa reconstituted with flag - pro - IL - 1β myc - pro - Caspase - 1 were treated with 1mM LLOMe for 1h , lysates were subjected to differential centrifugations at 3 , 000xg ( 3k ) , 25 , 000xg ( 25k ) 100 , 000xg ( 100k ) as depicted in cartoon . Arrows , two ways membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B - D , or by further sucrose OptiPrep density separation of 25k pellets in panels F G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation. HeLa cells reconstituted with flag-pro-IL-1β and myc-pro-Caspase-1 were treated with 1mM LLOMe for 1h, and lysates were subjected to differential centrifugations at 3,000xg (3k), 25,000xg (25k) and 100,000xg (100k) as depicted in the cartoon. Arrows, two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B-D, or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G.


( C ) IL - 1β in 25k fractions ( pellets ) from HeLa their CRISPR knockout derivatives ( TRIM16KO ) that were reconstituted for IL - 1β secretion treated with 1mM LLOMe for 1 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IL-1β levels in 25k membrane fractions (pellets) from HeLa cells and their CRISPR knockout derivatives (TRIM16KO) that were reconstituted for IL-1β secretion and treated with 1mM LLOMe for 1 h.


( D ) of analyses in ( B ) . RI , relative .

(D) Quantification of analyses in (B). RI, relative intensity.


( E ) Whole lysates ( immunoblots ) analyzed in F G .

(E) Whole cell lysates (immunoblots) analyzed in F and G.


( F , G ) Sucrose density gradient of 25k membranes . Note that mature IL - 1β ( mIL - 1β ) is recruited to co - fractionates with TRIM16 + , Sec22b + , LC3 - II + membranes in TRIM16wt HeLa reconstituted with pro - IL - 1β pro - caspase 1 , but not in TRIM16KO mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,G) Sucrose density gradient analysis of 25k membranes. Note that mature IL-1β (mIL-1β) is recruited to and co-fractionates with TRIM16+, Sec22b+, and LC3-II+ membranes in TRIM16wt HeLa cells reconstituted with pro-IL-1β and pro-caspase 1, but not in TRIM16KO mutant cells.


( H ) Confocal of LC3 in HeLa expressing GFP - Sec22b treated with LLOMe or untreated ( control ) . Arrowheads , colocalization . Scale bars , 5 μm . ( I ) GFP - Sec22b LC3 overlapping obtained by high content microscopy .

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

(H) Confocal images of LC3 in HeLa cells expressing GFP-Sec22b treated with LLOMe or untreated (control). Arrowheads, colocalization. Scale bars, 5 μm. (I) GFP-Sec22b and LC3 overlapping area obtained by high content microscopy.


( J ) Co - IP between flag - TRIM16 with GFP - Sec22b or GFP - syntaxin - 17 co - expressed in HEK293T .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Co-IP analysis between flag-TRIM16 with GFP-Sec22b or GFP-syntaxin-17 co-expressed in HEK293T cells.


( K ) Co - IP of endogenous , TRIM16 Sec22b , in lysates from THP - 1 treated with LLOMe .

(K) Co-IP analysis of endogenous proteins, TRIM16 and Sec22b, in lysates from THP-1 cells treated with LLOMe.


( L ) Co - IP of interactions between deletion variants of flag - TRIM16 with GFP - Sec22b in HEK293T .

(L) Co-IP analysis of interactions between deletion variants of flag-TRIM16 with GFP-Sec22b in HEK293T cells.


( M N ) IL - 1β secretion complementation experiments of TRIM16KO with TRIM16 Sec22b - interacting its Sec22b - nonbinding mutant shown .

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(M and N) IL-1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b-interacting and its Sec22b-nonbinding mutant shown.


( O ) Two color super - resolution image showing co - clustering of flag - TRIM16 ( red ) GFP - Sec22b ( green ) ; Scale bar , 200 nm . ( P ) Cross - correlation of flag - TRIM16 GFP - Sec22b super - resolution data showing a characteristic separation of ~ 70 nm .

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(O) Two color super-resolution image showing co-clustering of flag-TRIM16 (red) and GFP-Sec22b (green); Scale bar, 200 nm. (P) Cross-correlation analysis of flag-TRIM16 and GFP-Sec22b super-resolution data showing a characteristic separation of ~ 70 nm.


( A B ) IL - 1β in supernatants of THP - 1 subjected to knockdowns , treated with LPS , then ( A ) with LLOMe or ( B ) starved in EBSS .

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

(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS.


( C ) IL - 1β in supernatants of human macrophages ( MDM ) subjected to knockdowns sequentially treated with LPS LLOMe .

(C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.


( D ) Immunoblot analyses of p62 in THP - 1 subjected to knockdowns as indicated starved for 3 h .

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(D) Immunoblot analyses of p62 levels in THP-1 cells subjected to knockdowns as indicated and starved for 3 h.


( E ) mRFP - GFP overlapping areas of tandem HeLa reconstituted with flag - pro - IL - 1β myc - Caspase 1 starved for 3 h . Mask overlay , iDev software - defined objects ( objects , outlines ; internal secondary objects , GFP ( top ) , mRFP ( middle ) , or their overlapping ( bottom ) ) . Bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) mRFP-GFP overlapping areas of tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 and starved for 3 h. Mask overlay, iDev software-defined objects (primary objects, cell outlines; internal secondary objects, GFP (top), mRFP (middle), or their overlapping area (bottom)). Bars, 10 μm.


( F ) IL - 1β in supernatants of starved tandem HeLa reconstituted with flag - pro - IL - 1β myc - Caspase 1 as in E .

O O O O O 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) IL-1β levels in supernatants of starved tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 as in E.


( A - C ) IL - 1β in supernatants of THP - 1 subjected to knockdowns , treated with LPS , then with LLOMe ( A C ) or starved in EBSS ( B ) .

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(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B).


( A - C ) IL - 1β in supernatants of THP - 1 subjected to knockdowns , treated with LPS , then with LLOMe ( A C ) .

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

(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C).


( C ) Ferritin in supernatants of THP - 1 subjected to knockdowns as indicated sequentially treated with LPS LLOMe .

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(C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe.


( D ) Ferritin in supernatants of THP - 1 subjected to knockdowns as indicated infected with M . tuberculosis as indicated .

O O O O O O O O O O O O O O O O O O O O O O O

(D) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and infected with M. tuberculosis as indicated.


( E ) of ferritin were determine in supernatants from THP - 1 that were subjected to knockdown as indicated were sequentially treated with LPS LLOMe .

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

(E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe.


( F ) Confocal microscopy of HeLa expressing GFP - Sec22b were treated with LLOMe , stained for FTH1 . tracings correspond to arrows . Scale bars , 5 μm ;

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(F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;


( F ) Malate dehydrogenase ( MDH ; 0 . 2 µM ) was incubated at 40 ° C in absence ( black ) or presence of 0 . 5 µM WT HSP27 ( red ) or 0 . 5 µM P182L HSP27 ( blue ) . ( G ) Insulin ( 40 µM ) was incubated at 40 ° C in absence ( black ) or presence of 20 µM WT HSP27 ( red ) or 20 µM P182L HSP27 ( blue ) . y - axes depict absorbance at 340 nm due to formation of large . solid lines represent average of three replicates with filled reflecting ± one standard deviation .

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(F) Malate dehydrogenase (MDH; 0.2 µM) was incubated at 40 °C in the absence (black) or presence of 0.5 µM WT HSP27 (red) or 0.5 µM P182L HSP27 (blue). (G) Insulin (40 µM) was incubated at 40 °C in the absence (black) or presence of 20 µM WT HSP27 (red) or 20 µM P182L HSP27 (blue). The y-axes depict the absorbance at 340 nm due to the formation of large aggregates. The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation.


( A ) SEC - MALS data from WT HSP27 P182L variant , with cHSP27 included for comparison . samples were injected at 40 µM ( monomer ) . respective average molecular masses are 14 MDa , 470 kDa , 18 kDa for P182L , WT HSP27 , cHSP27 . normalized UV signals are plotted on left y - axis as solid lines molecular masses are plotted on right y - axis as circles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC-MALS data from WT HSP27 and the P182L variant, with cHSP27 included for comparison. All samples were injected at 40 µM concentration (monomer). The respective average molecular masses are 14 MDa, 470 kDa, and 18 kDa for P182L, WT HSP27, and cHSP27. The normalized UV signals are plotted on the left y-axis as solid lines and the molecular masses are plotted on the right y-axis as circles.


( B ) Fractionation of HeLa overexpressing either HSP27 or P182L mutant by a 10 % to 80 % sucrose gradient . Western blot against anti - V5 is shown .

O O O O O O O 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) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown.


( C ) Expression of V5 - epitope - tagged HSP27 ( WT or P182L mutant ) in HSP27 knock - out HeLa immunostaining of HSP27 . scale bars correspond to non - expanded dimensions , derived by dividing by sample expansion factor of 4 . 3 . ( D ) P182L mutation decreases density of HSP27 spots upon detection of local maxima of number of detected spots in expanded samples ( n = 30 ) . error bar is standard deviation for different regions . ( E ) Distribution of of spots ( n = 3342 ) after Gaussian fitting of in neighborhood of detected spots .

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(C) Expression of V5-epitope-tagged HSP27 (WT or P182L mutant) in HSP27 knock-out HeLa cells and immunostaining of HSP27. The scale bars correspond to the non-expanded dimensions, derived by dividing by the sample expansion factor of 4.3. (D) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples (n = 30). The error bar is the standard deviation for the different cytoplasmic regions. (E) Distribution of the intensity of individual spots (n = 3342) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots.


( F ) Immunostaining of HSP27 ( ) in motor neurons differentiated from CMT - derived iPSCs that harbor heterozygous P182L mutation reveals large , ( center right panels ) . control motor neurons that contain WT HSP27 do not show evidence of any . nucleus is stained with Hoechst ( blue ) . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining of HSP27 (white) in motor neurons differentiated from CMT patient-derived iPSCs that harbor the heterozygous P182L mutation reveals large, cytoplasmic aggregates (center and right panels). The control motor neurons that contain WT HSP27 do not show evidence of any aggregates. The nucleus is stained with Hoechst (blue). Scale bar, 10 µm.


( A ) sequences of WT peptide ( red ) P182L peptide ( blue ) . IxI / V motif is indicated with text . Below : regions of 2D 1H - 15N HSQC spectra of 15N - labeled cHSP27 ( black ) in presence of increasing amounts of WT ( red ) or P182L peptide ( blue ) . Resonances in β4 β8 strands broaden disappear upon peptide binding , indicative of intermediate exchange . ( B ) Zoomed - in regions of resonances during peptide titrations . color bars indicate amount of added WT or P182L peptide . ( C ) combined 1HN 15N chemical shift changes , denoted as chemical shift perturbation ( CSP ) , are plotted as a function of added peptide . ( D ) CSPs shown as a function of residue number for WT ( red ) P182L peptide ( blue ) . ( E ) X - ray structure of cHSP27 bound to WT peptide ( PDB : 4mjh ) , shown in sticks , with results from ( D ) plotted onto structure .

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(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.


( A , B ) i . 15N CPMG RD data for residues in β4 strand ( T113 ) β8 strand ( S154 ) in absence of added peptide . CPMG data are shown for same residues in presence of ii . ca . 2 % cHSP27 - WT peptide or iii . ca . 2 % of cHSP27 - P182L peptide . Uncertainties in R2 , eff values are derived from standard deviations of fitted peak intensities obtained from duplicated νCPMG values . ( C ) i . association ( kon ) dissociation ( koff ) rates for WT ( red ) P182L ( blue ) IxI / V peptide binding to ACD , calculated directly from CPMG RD data . Residues that are impacted by presence of a small amount of peptide are indicated by red ( ii . , WT ) blue ( iii . , P182L ) spheres . In both cases , residues in similar ACD regions show enhanced Rex , indicative of chemical exchange at similar sites .

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(A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites.


( B ) Volcano plot representing interactors that are enriched for P182L mutant versus GFP as a negative control , obtained with affinity - enrichment mass spectrometry . that co - immunoprecipitated significantly more ( p < 0 . 05 ) with P182L variant are shown in upper right quadrant . Interactors with one or more [ I / V ] - x - [ I / V ] motifs are displayed in red while other significantly enriched interactors are displayed in black .

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(B) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control, obtained with affinity-enrichment mass spectrometry. Proteins that co-immunoprecipitated significantly more (p<0.05) with the P182L variant are shown in the upper right quadrant. Interactors with one or more [I/V]-x-[I/V] motifs are displayed in red while other significantly enriched interactors are displayed in black.


( C - D ) Co - immunoprecipitation from HeLa stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) using anti - V5 beads . Co - immunoprecipitation was quantified presented as a relative to P182L variant ( n = 3 ) . mean ± one standard deviation ( n = 3 ) is shown . Right : location of Pro182 ( red ) in crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) in silico creation of P182L mutation ( blue ) . peptide is shown in sticks . P182L specifically weakens binding of HSP27 IxI / V motif .

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(C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif.


( E ) Co - immunoprecipitation from HeLa stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) transiently transfected with BAG3 - eGFP ( WT or IPV - mutant ) using anti - GFP beads . Western blots for anti - GFP ( BAG3 ) , V5 ( HSPB1 ) , α - tubulin . NS indicates a non - specific band . Note that detection of BAG3 ( anti - GFP ) HSP27 ( anti - V5 ) occurs with different antibodies . locations of IPV - to - GPG mutations in BAG3 are depicted below .

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(E) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) and transiently transfected with BAG3-eGFP (WT or IPV-mutant) using anti-GFP beads. Western blots for anti-GFP (BAG3), V5 (HSPB1), and α-tubulin. NS indicates a non-specific band. Note that detection of BAG3 (anti-GFP) and HSP27 (anti-V5) occurs with different antibodies. The locations of the IPV-to-GPG mutations in BAG3 are depicted below.


( F ) Co - immunoprecipitation from HeLa transiently transfected with V5 - epitope - tagged HSP27 ( P182L or S155Q / P182L mutant ) using anti - V5 beads ( n = 2 ) . Right : location of Ser155 ( red ) in crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) in silico creation of S155Q mutation ( purple ) showing a clash with bound peptide . peptide is shown in sticks . S155Q disrupts binding of IxI / V motifs .

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(F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.


B ) Western blot assays for expression level of TFAM in ten HCC lines , one normal liver ( LO2 ) two non - liver tissues . level was quantified as ratios between TFAM GAPDH ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

B) Western blot assays for the expression level of TFAM in ten HCC cell lines, one normal liver cell line (LO2) and two non-tumor liver tissues. The protein level was quantified as ratios between TFAM and GAPDH (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Western blot quantitative for expression of TFAM in HCC with TFAM stable knockdown control ( n = 3 independent experiments ) . shCtrl , control shRNA ; shTFAM , shRNA against TFAM ; EV , empty vector ; TFAM , expression vector encoding TFAM . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(C) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells (n = 3 independent experiments). shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( D , E ) Wound - healing migration ( D ) Transwell migration invasion assays ( E ) for HCC with treatment as indicated ( n = 3 independent experiments ) . Scale bars , 100μm . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(D, E) Wound-healing migration assay (D) and Transwell migration and invasion assays (E) for HCC cells with treatment as indicated (n = 3 independent experiments). Scale bars, 100μm. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( G ) Representative H & E of lung metastasis in mice used in ( F ) . Scale bars , 100μm . ( H , Number of metastatic nodules per lung ( H ) in mice used in ( F ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(G) Representative H&E images of lung metastasis in mice used in (F). Scale bars, 100μm. (H, Number of metastatic nodules per lung (H) in mice used in (F). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Immunofluorescence of TFAM Phalloidin in human HCC from paired clinical samples ( n = 100 ) . 3D reconstruction of z - stacks from region is showed in right side . Red : LifeAct , Blue : DAPI , Green : TFAM . Scale bars , 20μm for microscopy , 40μm for 3D reconstruction .

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(C) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.


( D ) Statistical for of F - actin in ( A ) , ( B ) ( C ) . are quantified from at least 30 for each group in ( A ) ( B ) . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 .

(D) Statistical analysis for fluorescent intensity of nuclear F-actin in (A), (B) and (C). Fluorescence intensity are quantified from at least 30 cells for each group in (A) and (B). One-way ANOVA. *P < 0.05; **P < 0.01.


( E ) Representative H & E of lung metastasis in mice used in ( D ) . Scale bars , 100μm . ( F G ) Number of metastatic nodules per lung in mice used in ( F ) incidence of lung metastasis ( G ) in mice used in ( D ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(E) Representative H&E images of lung metastasis in mice used in (D). Scale bars, 100μm. (F and G) Number of metastatic nodules per lung in mice used in (F) and the incidence of lung metastasis (G) in mice used in (D). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


F ) Western blot assays for expression of metastasis related gene in HCC ( n = 3 independent experiments ) tissues from mice ( n = 4 per group ) with treatments as indicated . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * P < 0 . 05 ; * * P < 0 . 01 .

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F) Western blot assays for protein expression levels of metastasis related gene in HCC cells (n = 3 independent experiments) and tumor tissues from mice (n =4 per group) with treatments as indicated. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( H Western blot assays of FN1 , ITGB3 in HCC with treatment as indicated ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * P < 0 . 05 ; * * P < 0 . 01 .

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(H Western blot assays levels of FN1, ITGB3 in HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( A ) Western blot analyses for level of key regulators of actin polymerization in cytoplasm nucleus of HCC with treatment as indicated . Tublin Lamin B1 were used as loading controls for cytoplasm nucleus , respectively ( n = 3 independent experiments ) .

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(A) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated. Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus, respectively (n = 3 independent experiments).


( B ) Western blot analyses for level in tissues of mouse liver induced by Akt / β - catenin ( n = 3 independent experiments ) .

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(B) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt/β-catenin (n = 3 independent experiments).


Representative of mDia2 translocation in human ( E ) HCC tissues . NT , non - liver tissues , n = 100 ; MT , metastatic tissues , n = 58 ; NMT , non - metastatic tissues n = 42 . Scale bars , 25μm . ( F ) Positive rates of mDia2 in nucleus cytoplasm was calculated on in ( E ) . Graphs show mean ± s . e . m . One - way ANOVA . * * P < 0 . 01 .

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Representative IHC images of mDia2 nuclear translocation in human (E) HCC tissues. NT, non-tumor liver tissues, n=100; MT, metastatic tumor tissues, n=58; NMT, non-metastatic tumor tissues n=42. Scale bars, 25μm. (F) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in (E). Graphs show mean ± s.e.m. One-way ANOVA. **P < 0.01.


( H I ) Spearman correlation between positive rates of mDia2 TFAM expression level ( or positive rates of F - actin ) on in ( E ) .

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(H and I) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level (or positive rates of nuclear F-actin) based on the IHC assay in (E).


( D ) Western blot of anti - mDia2 IPs whole - lysates ( WCL ) derived from HCC mouse tissues ( n = 3 independent experiments ) . NT , non - liver tissues ; MT , metastatic tissues ; NMT , non - metastatic tissues .

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(D) Western blot analysis of anti-mDia2 IPs and whole-cell lysates (WCL) derived from HCC cells and mouse tumor tissues (n = 3 independent experiments). NT, non-tumor liver tissues; MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues.


( E ) level of malonylated mDia2 . Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control was loaded in Western blot .

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(E) The level of malonylated mDia2. Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay.


( F ) Western blot for distribution of mDia2 in SNU - 368 incubated with malonyl - CoA ( 5 μM ) or ND - 630 ( 10 μM ) ( n = 3 independent experiments ) .

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(F) Western blot analysis for the distribution of mDia2 in SNU-368 cells incubated with malonyl-CoA (5 μM) or ND-630 (10 μM) (n = 3 independent experiments).


( H ) Representative H & E of lung metastasis in mice used in ( G ) . Scale bars , 100μm . ( I ) Number of metastatic nodules per lung in mice used in ( G ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - for , I ) . * P < 0 . 05 ; * * P < 0 . 01 .

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(H) Representative H&E images of lung metastasis in mice used in (G). Scale bars, 100μm. (I) Number of metastatic nodules per lung in mice used in (G). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for , I). *P < 0.05; **P < 0.01.


( A ) scores of TFAM in 429 paired tissues from HCC . relative expression of to non - was log2 - transformed . T , tissues ; NT , non - liver tissues .

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(A) IHC scores of TFAM in 429 paired tissues from HCC patients. The relative expression ratio of tumor to non-tumor was log2-transformed. T, tumor tissues; NT, non-tumor liver tissues.


( B ) mRNA level of TFAM in 50 HCC ' paired tissues from TCGA database . relative expression of to non - was log2 - transformed .

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(B) mRNA level of TFAM in 50 HCC patients' paired tissues from TCGA database. The relative expression ratio of tumor to non-tumor was log2-transformed.


( C ) Representative of of TFAM ( top panel ) scores ( bottom panel ) in HCC tissues . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 . MT , metastatic tissues ; NMT , non - metastatic tissues . Scale bars , 25μm . Boxes : first quartile to third quartile ; Whiskers : minimum value to maximum value ; Central band : median .

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(C) Representative images of IHC staining of TFAM (top panel) and scores (bottom panel) in HCC tumor tissues. One-way ANOVA. *P < 0.05; **P < 0.01. MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. Scale bars, 25μm. Boxes: first quartile to third quartile; Whiskers: minimum value to maximum value; Central band: median.


( D Kaplan - Meier curves for overall survival of HCC stratified by TFAM expression . ( F Kaplan - Meier curves for overall survival of HCC stratified by F - actin positive rates .

(D Kaplan-Meier curves for overall survival of HCC patients stratified by TFAM expression. (F Kaplan-Meier curves for overall survival of HCC patients stratified by nuclear F-actin positive rates.


E ) recurrence of HCC stratified by TFAM expression . G ) recurrence of HCC stratified by F - actin positive rates .

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E) the recurrence of HCC patients stratified by TFAM expression. G) the recurrence of HCC patients stratified by nuclear F-actin positive rates.


( H ) Representative of of TFAM , FN1 ITGB3 from two HCC . Scale bars , 25μm . ( I ) Spearman correlation between of TFAM FN1 , IL - 6 , TGFB1 or ITGB3 .

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(H) Representative images of IHC staining of TFAM, FN1 and ITGB3 from two HCC patients. Scale bars, 25μm. (I) Spearman correlation analysis between the levels of TFAM and FN1, IL-6, TGFB1 or ITGB3.


b , Representative confocal of NeuN ( green ) Npas4 ( red ) immunostaining performed 1h after last injection of cocaine or saline ( scale bar : 50 µm ) .

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b, Representative confocal images of NeuN (green) and Npas4 (red) immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).


c , Quantifications of Npas4 - positive ( fold change normalized to 30min saline control group ) in NAc dStr . Mean + ; n = 4 mice per group . 10 ( 5 per hemisphere ) per mouse for NAc 10 ( 5 per hemisphere ) for dStr . p * * < 0 . 01 , unpaired Student ' s t - .

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c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc and 10 images (5 per hemisphere) for the dStr. p** < 0.01, unpaired Student's t-test.


c , MSN cultures at DIV11 were treated for 2h with either 10 µM of D1 - agonist SKF - 38393 ( SKF ) alone or with SKF 10 µM of selective D1 - antagonist SCH - 23390 ( SCH ) . qPCR was performed to determine relative mRNA of immediate early genes cFos Arc . Mean + ; one - way ANOVA with Holm - Sidak post - hoc correction ; p * * < 0 . 01 , p * * * * < 0 . 0001 ; n = 3 independent cultures as biological replicates .

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c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.


d , MSNs were depolarized with potassium chloride ( HiK , 55 mM ) or treated with D1 - agonist SKF - 38393 ( 10 µM ) for 1h , 2h or 4h . Subsequently , qPCR was performed to assess immediate early gene mRNA . Mean + ; two - way ANOVA with Bonferroni post - hoc ; indicated p - values for induction over control for each treatment ; p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 ; n = 4 independent cultures as biological replicates .

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d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.