( A ) Representative images of neurons after 4 days of culture in vitro . GFP expressing plasmid was electroporated into the E13 . 5 cerebral cortices of WT and KO mice to label neural progenitor cells . After 24 h , the GFP + cells were isolated and cultured in differentiation medium for 4 days . Scale bar represents 15 μm . ( B ) Graph shows that the total dendritic length of neurons is decreased when mH2A1 . 2 is deleted . n = 25 cells from four samples . ( C ) Statistics show that the number of branch points is reduced upon mH2A1 . 2 deletion . n = 25 cells from four samples . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) .

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

(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).


( F ) The brain sections were immunostained with CUX1 and CTIP2 ( marker of layer I ; marker of layer V ) at P0 . Scale bar represents 20 μm . n = 4 mice , independent replicates . ( G ) Statistics of CUX1 + cells in the upper layer . CUX1 + cells were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + cells in the deep layer . CTIP2 + cells were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 (

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(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(


( A ) Representative tracing path of WT mice and KO mice in the open field test . ( B ) The total traveling distance was not different between WT and KO mice . ( C ) KO mice spent less time in the center than WT mice . Date information : All mice were male , 8 - 12 weeks old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . Analysis was performed by researcher blinded to the experimental conditions .

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(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.


( E ) Graph shows the percentage of GFP + cells in the VZ / SVZ , IZ and CP . The number of GFP + cells in the VZ / SVZ region increased significantly , and the number of GFP + in the CP layer decreased significantly . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


( I ) Representative images of E16 . 5 cortices that had been electroporated with GFP or GFP + NKX2 . 2 into WT and KO brains at E13 . 5 . Scale bar represents 50 μm . ( J ) Graphs of the percentage of GFP + cells in the VZ / SVZ , IZ and CP . Overexpression of NKX2 - 2 restored the distribution of GFP + cells to the normal state . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm. (J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state. n=5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


A ) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , and hippocampus . The heatmap indicates the associated T - statistic for each voxel , with light blue representing the highest value .

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A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value.


Sites that bound either HIF - 1α ( A & B ) or HIF - 2α ( C & D ) in both replicates were identified by the MACS peak caller and ordered on the y - axis according to HIF - 1β signal intensity . Heatmaps show HIF ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity , averaged across two independent ChIP - seq experiments ) at HIF binding sites and across the flanking ± 5kb regions ( x - axis ) . ( A ) HIF - 1β signal intensity and ( B ) HIF - 1α signal intensity at HIF - 1α binding sites and ( C ) HIF - 1β signal intensity and ( D ) HIF - 2α signal intensity at HIF - 2α binding sites . HIF - 1β signal intensity above local background levels is observed at all HIF - 1α and HIF - 2α binding sites . ( E & F ) Line plots showing average ( n = 2 , independent ChIP - seq experiments ) HIF - 1β signal intensity ( solid blue lines ) within the MACS defined ( E ) HIF - 1α and ( F ) HIF - 2α binding sites compared to the average background HIF - 1β binding signal ( dashed black line ) at non - HIF - α - binding accessible sites ( defined by FAIRE - seq ) . Sites are ranked on the x - axis according to HIF - 1β signal . HIF - 1β signal intensity at both HIF - 1α and HIF - 2α binding sites was consistently above genome - wide background levels . ( G ) HIF - 1β signal intensity was plotted against total HIF - α ( HIF - 1α + HIF - 2α ) signal intensity for all sites that bound one or more HIF subunits . Strong correlation was observed between HIF - α and HIF - 1β signal intensities . ( H ) The ratio of total HIF - α signal to HIF - 1β signal ( x - axis ) was determined for each site and plotted as a frequency distribution ( y - axis ) showing a tight unimodal distribution . ( I ) The non - reproducibility of the total HIF - α / HIF - 1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate

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Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2


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 )

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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 and HIF - 2α protein levels with increasing severity of

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


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

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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 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

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(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 . 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 )

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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)


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

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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 and HIF - 2α protein level , which is highest at 6 hours and falls by 48

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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 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

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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 . 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

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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


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

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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


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

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

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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 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

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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 - 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

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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


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 )

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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)


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

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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


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 )

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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 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

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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


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 )

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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 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 %

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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 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

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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 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

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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 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

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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 patient ; right : negative patient ; representative data are shown . The gating strategy is depicted in Supplementary Fig S1 .

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

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


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 .

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

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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 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 ) .

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

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


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 ) .

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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 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 ) .

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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 images of three mice bearing MM . 1S ( right shoulder ) and 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 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 .

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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 and CXCR4 cell 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 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 .

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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 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 .

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[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 - 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 ) .

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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 ) 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 .

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[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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were transiently transfected with GFP - ATG16L1 for 20 hr . Cells in basal conditions were fixed and 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 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 .

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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 cells were transfected with EGFP - ATG16L1 and mCherry - RAB11 , and 5 min movies were recorded . Representative images 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 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 .

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

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

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

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


( 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 .

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(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 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 .

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(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 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 .

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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 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 .

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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 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 .

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(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 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 .

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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 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 .

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(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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were transfected with siRNAs for different R - SNAREs , and we assessed the levels of the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 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 .

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(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 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 .

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(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 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 ) .

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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 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 .

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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 cells were subjected to GABARAP knockdown and processed as in A .

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


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

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(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 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 .

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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 levels in supernatants of THP - 1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe .

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


( 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 ) .

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(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 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 ) .

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(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 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 .

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(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 primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe .

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(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 ) and GFP - TRIM16 ( T16 ) in HEK293T cells .

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


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

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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

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


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

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


( 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 .

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(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 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 .

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

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


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

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


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

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


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

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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 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 .

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

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(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 levels in THP - 1 cells subjected to knockdowns as indicated and 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 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 .

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

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(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 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 ) .

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

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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).


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

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


( 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 .

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(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 cells expressing GFP - Sec22b were treated with LLOMe , and stained for FTH1 . Line 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 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 .

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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 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 .

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(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 cells overexpressing either HSP27 or the 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 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

(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 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 .

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

(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.