figf6 ( a ) Immunoblot for the indicated proteins in homogenates ( Hom ) and purified PM from fed ( F ) and 24 h starved mice ( Stv ) . Starvation - induced changes in the PM content relative to fed levels were as follows : Vps15 ( 1 . 2 ± 0 . 2 ) , Beclin - 1 ( 0 . 7 ± 0 . 5 ) , Vps34 ( 0 . 9 ± 0 . 1 ) , Atg16 ( 1 . 1 ± 0 . 3 ) , Atg14 ( 2 . 5 ± 0 . 2 ) and only Atg14 were significant for P 0 . 01 . ( b ) Immunoblot of Cx43immunoprecipitates ( IP ) in MEFs maintained in the presence or absence of serum . I , input ; FT , flow through . ( c ) Immunoblot of Cx43 IP in MEFs from WT or Atg5 − / − mice grown in serum - supplemented media . ( d ) Immunoblot of Cx43 IP in WT or Atg16 knockdown ( KD ) MEFs maintained in the presence / absence of serum for 4 h . ( e ) Co - staining for Atg14 and Cx43 in NRK cells maintained in the presence / absence of serum . Merged channels are shown and single channels and broad - field images are shown in Supplementary Fig . 5d . Insets : higher magnification . ( f ) Maximum - intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and EGFP - ATG14 followed by a single - plane time - lapse acquisition of the outlined area ; cells were incubated in serum - deprived medium for 1 h before imaging . ( g ) Immunoblot of Atg14 IP in WT or Atg5 − / − MEFs maintained in the presence of serum . ( h ) Immunoblot in total homogenates and PM purified from WT or Cx43 − / − MOBs . ( i ) Immunofluorescence for e - cadherin and Vps34 in NRK cells WT or knocked down ( KD ) for Cx43 . Single channel and merge ( left ) and z - stack surface reconstruction and higher magnification of the outlined region ( right ) . ( j ) Immunostaining for Cx43 and e - cadherin in NRK cells expressing a tandem FYVE domain ( 2xFYVE ) tagged to GFP and maintained in serum - free media for 4 h . Single channels ( inverted black and white ) and the merged image of the outlined area ( colour ) . Inset : higher magnification image . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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

figf6(a) Immunoblot for the indicated proteins in homogenates (Hom) and purified PM from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2 ± 0.2), Beclin-1 (0.7 ± 0.5), Vps34 (0.9 ± 0.1), Atg16 (1.1 ± 0.3), Atg14 (2.5 ± 0.2) and only Atg14 were significant for P 0.01.(b) Immunoblot of Cx43immunoprecipitates (IP) in MEFs maintained in the presence or absence of serum. I, input; FT, flow through.(c) Immunoblot of Cx43 IP in MEFs from WT or Atg5−/− mice grown in serum-supplemented media.(d) Immunoblot of Cx43 IP in WT or Atg16 knockdown (KD) MEFs maintained in the presence/absence of serum for 4 h.(e) Co-staining for Atg14 and Cx43 in NRK cells maintained in the presence/absence of serum. Merged channels are shown and single channels and broad-field images are shown in Supplementary Fig. 5d. Insets: higher magnification.(f) Maximum-intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and EGFP-ATG14 followed by a single-plane time-lapse acquisition of the outlined area; cells were incubated in serum-deprived medium for 1 h before imaging.(g) Immunoblot of Atg14 IP in WT or Atg5−/− MEFs maintained in the presence of serum.(h) Immunoblot in total homogenates and PM purified from WT or Cx43−/− MOBs.(i) Immunofluorescence for e-cadherin and Vps34 in NRK cells WT or knocked down (KD) for Cx43. Single channel and merge (left) and z-stack surface reconstruction and higher magnification of the outlined region (right).(j) Immunostaining for Cx43 and e-cadherin in NRK cells expressing a tandem FYVE domain (2xFYVE) tagged to GFP and maintained in serum-free media for 4 h. Single channels (inverted black and white) and the merged image of the outlined area (colour). Inset: higher magnification image. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


figf7 ( a ) Co - immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum ( top ) . Single and merged channels of the 3D reconstruction of the outlined regions are shown ( bottom ) . Nuclei are highlighted with DAPI . ( b , c ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of Cx43 in NRK cells maintained in the presence or absence of serum ( b ) or treated or not with 3 - methyladenine ( c ) . Atg5 is shown as the negative control for immunoprecipitation . ( d ) Co - staining for Atg9 and e - cadherin in control NRK cells and those knocked down for Cx43 . Single and merged channels at higher magnification areas are shown . ( e ) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15 . ( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg14 ( f ) maintained in the presence or absence of serum . ( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg9 ( g ) maintained in the presence or absence of serum . E - cadherin staining ( red ) is shown to highlight PM in g and h . ( h ) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14 , treated ( or not ) with tamoxifen or lindane ( left ) . E - cadherin staining ( red ) is shown to highlight PM in g and h . Full fields are shown in Supplementary Fig . 6h . Quantification of intracytoplasmic Cx43 - positives vesicles ( right ; n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . Values are mean + s . e . m and significant for * P 0 . 01 and * * P 0 . 001 using analysis of variance + Bonferroni test . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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

figf7(a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the outlined regions are shown (bottom). Nuclei are highlighted with DAPI.(b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation.(d) Co-staining for Atg9 and e-cadherin in control NRK cells and those knocked down for Cx43. Single and merged channels at higher magnification areas are shown.(e) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15.(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg14 (f) maintained in the presence or absence of serum.(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg9 (g) maintained in the presence or absence of serum. E-cadherin staining (red) is shown to highlight PM in g and h.(h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


figf8 ( a ) Immunoblot for the indicated proteins in control MEFs or those knocked down ( KD ) for the three different Cx . ( b ) Immunofluorescence of LC3 in control MEFs ( Ctr ) or those knocked down ( KD ) for three different Cx maintained in the presence or absence of serum . Left : representative fields and higher magnification insets . Nuclei are highlighted with DAPI . Right : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . ( c ) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated ( − ) or treated with inhibitors of lysosomal proteases ( PI ) for the indicated times . GAPDH is shown as the loading control . ( d ) Representative images of control MEFs or those knocked down ( KD ) for the indicated proteins transfected with the mCherry - GFP - LC3 tandem reporter . Right : quantification of the average number per cell of puncta positive for GFP and mCherry ( autophagosomes ; APG ) and those positive only for mCherry ( autophagolysosomes ; APL ; n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . ( e ) Electron micrographs of control MEFs or those knocked down for the indicated Cx . Insets show autophagic vesicles . Bottom : Quantification of the average number of AV ( left ) and percentage of cytosolic area ( right ) occupied by AVs ( n = 4 wells , 4 independent experiments , > 10 micrographs per well ) . ( f ) Immunoblot for indicated proteins in immunoprecipitates ( IP ) for Cx26 or Cx32 ( left ) or Atg16 ( right ) from liver homogenates . Inp : input , FT : flow through . BIP is shown as the negative control for immunoprecipitation . All values are mean + s . e . m and significant for * P 0 . 01 ( analysis of variance + Bonferroni test was used in b ) . Scale bars : 5 μm in fluorescence images ; 1 μm and 0 . 1 μm in top panels and insets of e . Uncropped images of blots are shown in Supplementary Fig . 9 .

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

figf8(a) Immunoblot for the indicated proteins in control MEFs or those knocked down (KD) for the three different Cx.(b) Immunofluorescence of LC3 in control MEFs (Ctr) or those knocked down (KD) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).(c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (−) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as the loading control.(d) Representative images of control MEFs or those knocked down (KD) for the indicated proteins transfected with the mCherry-GFP-LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL; n = 3 wells, 3 independent experiments, >50 cells per experiment).(e) Electron micrographs of control MEFs or those knocked down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n = 4 wells, 4 independent experiments, >10 micrographs per well).(f) Immunoblot for indicated proteins in immunoprecipitates (IP) for Cx26 or Cx32 (left) or Atg16 (right) from liver homogenates. Inp: input, FT: flow through. BIP is shown as the negative control for immunoprecipitation. All values are mean + s.e.m and significant for *P 0.01 (analysis of variance + Bonferroni test was used in b). Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of e. Uncropped images of blots are shown in Supplementary Fig. 9.


Figure 1A . Relative abundance of miR species in human naive CD4 T cells . Cell purity was > 97 % . B , C . Average miR expression levels in unstimulated naive CD4 T cells non - infected vs HIV - 1NL4 - 3 ( B ) or HIV - 2ROD ( C ) infected after 24h ( n = 2 , three pooled samples per library ) . Mean normalized read counts across libraries are shown . Library normalization was performed using the mean normalization method available in the Deseq package of Bioconductor ( Anders Huber , 2010 ) . Lines indicate changes of + / - 1 log2 fold between samples .

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

Figure 1A. Relative abundance of miR species in human naive CD4 T cells. Cell purity was >97%.B, C. Average miR expression levels in unstimulated naive CD4 T cells non-infected vs HIV-1NL4-3 (B) or HIV-2ROD (C) infected after 24h (n=2, three pooled samples per library). Mean normalized read counts across libraries are shown. Library normalization was performed using the mean normalization method available in the Deseq package of Bioconductor (Anders Huber, 2010). Lines indicate changes of +/- 1 log2 fold between samples.


Figure 2A . Average miR expression levels in naive CD4 T cells ( purity > 97 % ) before and after 72h TCR - stimulation . Lines indicate changes of + / - 1 log2 fold between samples ( n = 2 , paired pools of three and nine individual samples per library ) . Library normalization performed using the mean normalization method available in Deseq package of Bioconductor ( Anders Huber , 2010 ) . miRs with significant changes are highlighted ( black ) and with insets presenting mean read counts in unstimulated samples ( mean ) , log2 fold change values in stimulated samples ( log FC ) and corresponding adjusted p value ( p ; Fisher test implemented in Deseq ) . The two most highly expressed miRs ( mir - 21 - 5p and miR - 146b - 5p ) are also indicated . B . Relative miR - 34c - 5p and miR - 155 - 5p expression levels ( 2deltaCt normalized to RNU48 ) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries . mean ± SEM and p - value for differences in miR - 155 - 5p expression ( paired two tailed t - test ) are shown . Comparison could not be performed for miR - 34c - 5p as it is undetermined in unstimulated samples . C . Time course quantification of miR - 34c - 5p expression ( top panel , 2deltaCt values ) in three different donors . C . Time course quantification of cell proliferation ( middle panel ) and cell activation ( bottom panel ) markers by flow cytometry , in three different donors .

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

Figure 2A. Average miR expression levels in naive CD4 T cells (purity >97%) before and after 72h TCR-stimulation. Lines indicate changes of +/- 1 log2 fold between samples (n=2, paired pools of three and nine individual samples per library). Library normalization performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). miRs with significant changes are highlighted (black) and with insets presenting mean read counts in unstimulated samples (mean), log2 fold change values in stimulated samples (log FC) and corresponding adjusted p value (p; Fisher test implemented in Deseq). The two most highly expressed miRs (mir-21-5p and miR-146b-5p) are also indicated.B. Relative miR-34c-5p and miR-155-5p expression levels (2deltaCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. mean ± SEM and p-value for differences in miR-155-5p expression (paired two tailed t-test) are shown. Comparison could not be performed for miR-34c-5p as it is undetermined in unstimulated samples.C. Time course quantification of miR-34c-5p expression (top panel, 2deltaCt values) in three different donors.C. Time course quantification of cell proliferation (middle panel) and cell activation (bottom panel) markers by flow cytometry, in three different donors.


Figure 3A , B . Comparison of the mean miR expression level by in TCR - stimulated naive CD4 T cell small RNA - seq libraries from uninfected and HIV - 1NL4 - 3 ( A ) or HIV - 2ROD ( B ) infected samples ( 24h ) . Only miRNAs with a minimum of 10 normalized read counts . Library normalization was performed using the mean normalization method available in Deseq package of Bioconductor ( Anders Huber , 2010 ) . Lines indicate changes of + / - 1 log2 fold between samples ( n = 3 , paired pools of three individual samples per library ) . miR - 34c - 5p is highlighted in black . C . Quantification of miR - 34c - 5p expression levels of the individual samples pooled to generate the sequencing libraries by qPCR ( mean ± SEM 2deltaCt values and p value from a paired two tailed t test ; n = 5 ) .

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

Figure 3A, B. Comparison of the mean miR expression level by in TCR-stimulated naive CD4 T cell small RNA-seq libraries from uninfected and HIV-1NL4-3 (A) or HIV-2ROD (B) infected samples (24h). Only miRNAs with a minimum of 10 normalized read counts. Library normalization was performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). Lines indicate changes of +/- 1 log2 fold between samples (n=3, paired pools of three individual samples per library). miR-34c-5p is highlighted in black.C. Quantification of miR-34c-5p expression levels of the individual samples pooled to generate the sequencing libraries by qPCR (mean±SEM 2deltaCt values and p value from a paired two tailed t test; n=5).


Figure 4A . Cell - associated viral DNA levels ( left ) , Tat mRNA levels ( center ) , and RT activity levels ( right ) in cell culture supernatants in infected J34c cells vs . infected JØ cells at 24 and 48 hours post - infection . * p0 . 05 ( paired two tailed t test ) . B . Representative flow cytometry plot of intracellular HIV - 1 Gag protein staining ( KC57 ) versus side scatter ( SSC ) in JØ and J34c cells , mock infected or infected with HIV - 1NL4 - 3 , after 48 hours of culture and corresponding fold change ( J34c / JØ ) plot in four biological replicate assays . Differences to control were found to be significant with a paired two tailed t test ( p0 . 05 ) . C . Fold change levels for miR - 34c - 5p , cell - associated viral DNA level , Tat mRNA , and RT activity levels in J34c cells treated with miR - 34c - 5p antagomiR versus control antagomiR , 48 hours post - infection . Differences to control were found to be significant with a paired two tailed t test ( p0 . 05 ) .

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

Figure 4A. Cell-associated viral DNA levels (left), Tat mRNA levels (center), and RT activity levels (right) in cell culture supernatants in infected J34c cells vs. infected JØ cells at 24 and 48 hours post-infection. * p0.05 (paired two tailed t test).B. Representative flow cytometry plot of intracellular HIV-1 Gag protein staining (KC57) versus side scatter (SSC) in JØ and J34c cells, mock infected or infected with HIV-1NL4-3, after 48 hours of culture and corresponding fold change (J34c/JØ) plot in four biological replicate assays. Differences to control were found to be significant with a paired two tailed t test (p0.05).C. Fold change levels for miR-34c-5p, cell-associated viral DNA level, Tat mRNA, and RT activity levels in J34c cells treated with miR-34c-5p antagomiR versus control antagomiR, 48 hours post-infection. Differences to control were found to be significant with a paired two tailed t test (p0.05).


Figure 5A . Hierarchical clustering of the 82 differentially expressed ( DE ) genes identified in Jurkat cell lines overexpressing miR - 34c - 5p ( J34c - 2 and J34c - 5 ) vs control ( JØ - 1 , 2 and 3 clones ) . Color scale represents mean centered log2 fold expression level across samples . Pie charts represent the relative proportion of predicted miR - 34c - 5p binding sites by the Miranda or PITA algorithms ( or both ) in down - regulated ( top ) and up - regulated ( bottom ) gene sets . B . qRT - PCR validation of selected genes . Plot represents the average percentage of target gene mRNA ( normalized to GAPDH ) in J34c vs JØ cells ( all differences were found to be significant with a paired two tailed t test , P < 0 . 05 ; n = 5 ) .

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

Figure 5A. Hierarchical clustering of the 82 differentially expressed (DE) genes identified in Jurkat cell lines overexpressing miR-34c-5p (J34c-2 and J34c-5) vs control (JØ-1, 2 and 3 clones). Color scale represents mean centered log2 fold expression level across samples. Pie charts represent the relative proportion of predicted miR-34c-5p binding sites by the Miranda or PITA algorithms (or both) in down-regulated (top) and up-regulated (bottom) gene sets.B. qRT-PCR validation of selected genes. Plot represents the average percentage of target gene mRNA (normalized to GAPDH) in J34c vs JØ cells (all differences were found to be significant with a paired two tailed t test, P<0.05; n=5).


Figure 7B . Western - blot of PCAF / KAT2B and histone H3 in JØ or J34c ( top ) , confirming decrease in protein levels . B . Bottom : antagomiR to miR - 34c - 5p results in increased KAT2B mRNA levels compared to control - treated cells . Plot presents relative expression values for mRNA in 3 biological replicates ; bars represent mean and SEM . Differences were found to be significant with p < 0 . 05 ( paired two tailed t test ) . C . Predicted target sites for miR - 34c - 5p in the PCAF / KAT2B mRNA ( black bars ) and miR - target hybridization for the two strongest sites ( top ) ; Luciferase assay with the pEZX renilla / firefly - PCAF / KAT2B 3 ' UTR reporter in HEK293 cells co - transfected with either the miR - 34c - 5p overexpression vector or pcDNA3 empty control ( bottom ) . Differences were found to be significant with p < 0 . 05 ( paired two tailed t test , n = 5 ) .

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Figure 7B. Western-blot of PCAF/KAT2B and histone H3 in JØ or J34c (top), confirming decrease in protein levels.B. Bottom: antagomiR to miR-34c-5p results in increased KAT2B mRNA levels compared to control-treated cells. Plot presents relative expression values for mRNA in 3 biological replicates; bars represent mean and SEM. Differences were found to be significant with p<0.05 (paired two tailed t test).C. Predicted target sites for miR-34c-5p in the PCAF/KAT2B mRNA(black bars) and miR-target hybridization for the two strongest sites (top); Luciferase assay with the pEZX renilla/firefly-PCAF/KAT2B 3'UTR reporter in HEK293 cells co-transfected with either the miR-34c-5p overexpression vector or pcDNA3 empty control (bottom). Differences were found to be significant with p<0.05 (paired two tailed t test, n=5).


Figure 1B . RT - PCR analysis for forebrain ( FOXG1 ) , optic cup ( VSX2 ) , dorsal forebrain ( PAX6 and EMX2 ) and ventral forebrain ( NKX2 - 1 ) identity in organoids . All expression values ( 2 - ΔCt ) calculated relative to reference gene TBP . Analysis was performed on 5 - 6 organoids from multiple independent differentiations - control ( 7 ) , dorsal ( 7 ) , extended ventral ( 9 ) and short ventral ( 5 ) . Significance values , * = < 0 . 05 , * * = < 0 . 01 , * * * = < 0 . 001 . Statistical analysis was performed using one - way ANOVA and post - hoc Tukey ' s comparison of means . The central band depicts the median , the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values . F . Magnified ( 60x ) dorsal regions from organoids fusions at day 60 ; Co - immunostaining of GFP with markers of interneuronal ( GABA ) , GABAergic ( DLX2 ) and mature neuronal ( NeuN ) identity . Scale bars , 10μm . White arrows indicate GFP + cells with positive labeling . G . Quantification of co - expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60 . Quantification was performed on a total of 3 organoid fusions ( technical replicates ) from two independent differentiations each ( biological replicates ) , with 2 regions of interest ( ROI ; 10x field of view ) in the dorsal and ventral regions of each fusion analyzed . Each dot represents the quantification of co - expression for both ROI analyzed per organoid fusion . Significance values , * = < 0 . 05 , * * = < 0 . 01 , * * * = < 0 . 001 . Statistical analysis was performed using an unpaired two - tailed Student ' s t - test . The central band displays the mean and error bars depict the SD .

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Figure 1B. RT-PCR analysis for forebrain (FOXG1), optic cup (VSX2), dorsal forebrain (PAX6 and EMX2) and ventral forebrain (NKX2-1) identity in organoids. All expression values (2-ΔCt) calculated relative to reference gene TBP. Analysis was performed on 5-6 organoids from multiple independent differentiations - control (7), dorsal (7), extended ventral (9) and short ventral (5). Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's comparison of means. The central band depicts the median, the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values.F. Magnified (60x) dorsal regions from organoids fusions at day 60; Co-immunostaining of GFP with markers of interneuronal (GABA), GABAergic (DLX2) and mature neuronal (NeuN) identity. Scale bars, 10μm. White arrows indicate GFP+ cells with positive labeling. G. Quantification of co-expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60. Quantification was performed on a total of 3 organoid fusions (technical replicates) from two independent differentiations each (biological replicates), with 2 regions of interest (ROI; 10x field of view) in the dorsal and ventral regions of each fusion analyzed. Each dot represents the quantification of co-expression for both ROI analyzed per organoid fusion. Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using an unpaired two-tailed Student's t-test. The central band displays the mean and error bars depict the SD.


Figure 2C . UMAPs depicting the expression of GABAergic markers DLX2 and DLX5 , progenitor marker TOP2A , MGE marker SOX6 , CGE marker NR2F2 , and striatal marker ZFHX3 .

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Figure 2C. UMAPs depicting the expression of GABAergic markers DLX2 and DLX5, progenitor marker TOP2A, MGE marker SOX6, CGE marker NR2F2, and striatal marker ZFHX3.


Figure 3C . UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5 . Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis . D . UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis .

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Figure 3C. UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5. Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis. D. UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis.


Figure 4B . Illustration of saltatory tangential migration performed by an interneuron . Nucleus is marked with a red asterisk . All images taken on a spinning disc confocal microscope at 10x magnification .

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Figure 4B. Illustration of saltatory tangential migration performed by an interneuron. Nucleus is marked with a red asterisk. All images taken on a spinning disc confocal microscope at 10x magnification.


Figure 5C . Heatmap depicting the control - normalized values ( log2 - scaled ) for selected parameters ( from Figure S9A ) for the 8 receptors analyzed in 5B . Grey values indicated non - significant values . In total 3979 moving cell tracks with the following number of tracks per group : Control - 696 , Bicuculline - Methiodide - 1189 , Saclofen - 527 , TPMPA - 138 , AP - 5 - 429 , CNQX - 500 , Strychnine - 98 , Serotonin - 180 , and SR57227 - 222 , were analyzed .

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Figure 5C. Heatmap depicting the control-normalized values (log2-scaled) for selected parameters (from Figure S9A) for the 8 receptors analyzed in 5B. Grey values indicated non-significant values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed.


Figure 6C . Mean square displacement ( MSD ) in μm2 over the delay in minutes ( min ) for all cell tracks in a cluster . The curves are color - coded to denote the corresponding cluster . For visualization purposes , the MSD is shown for a time delay corresponding to 24 hours of imaging . F . Bar plot depicting the fold - change in composition of cluster groups - directed , exploratory or confined - of treatment groups relative to the control distribution . Bars are color - coded to denote the corresponding cluster group . The dotted line represents the control baseline for visualization of the fold changes between the groups .

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Figure 6C. Mean square displacement (MSD) in μm2 over the delay in minutes (min) for all cell tracks in a cluster. The curves are color-coded to denote the corresponding cluster. For visualization purposes, the MSD is shown for a time delay corresponding to 24 hours of imaging.F. Bar plot depicting the fold-change in composition of cluster groups - directed, exploratory or confined - of treatment groups relative to the control distribution. Bars are color-coded to denote the corresponding cluster group. The dotted line represents the control baseline for visualization of the fold changes between the groups.


Figure 1A Fluorescence microscopy showing UBQLN2 ( Alexa Fluor 647 ) in solutions contained 50 µM FL UBQLN2 with increasing amounts of K48 - and K63 - linked chains of different lengths in 20 mM NaPhosphate and 200 mM NaCl ( pH 6 . 8 ) at 30 ºC . [ Ub ] : [ UBQLN2 ] reflects the ratio between Ub monomers and UBQLN2 molecules . The same image is used for the 0 : 1 condition . Scale bar , 5 µm . Yellow cartoons illustrate one major solution conformation of each chain . B Representative results from spectrophotometric turbidity assay comparing the effects of varying amounts of K48 - and K63 - linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2 .

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Figure 1A Fluorescence microscopy showing UBQLN2 (Alexa Fluor 647) in solutions contained 50 µM FL UBQLN2 with increasing amounts of K48- and K63-linked chains of different lengths in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. The same image is used for the 0:1 condition. Scale bar, 5 µm. Yellow cartoons illustrate one major solution conformation of each chain.B Representative results from spectrophotometric turbidity assay comparing the effects of varying amounts of K48- and K63-linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2.


Figure 2A CSPs for UBQLN2 450 - 624 residues at 1 : 1 ratio with Ub4 . The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS ( Dao et al , 2018 ) . B Amide peak intensities between the unbound and Ub4 - bound forms of UBQLN2 450 - 624 with the 555 - 570 region highlighted in gray . C CSPs for residues in UBQLN2 487 - 624 at 1 : 1 ratio with Ub4 . D CSPs for residues in UBQLN2 450 - 624 lacking residues 551 - 569 ( red / gray region ) at 1 : 1 ratio with Ub4 . E Normalized titration curves of 11 amide resonances for UBQLN2 450 - 624 . F Comparison of binding affinity ( Kd ) measurements obtained from NMR and fluorescence anisotropy . Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis . For visual comparison , gray dashed line is Kd of Ub binding to UBQLN2 450 - 624 as determined from NMR and FA experiments ( green bars ) . G Fluorescence anisotropy values for fluorescently - labeled UBQLN2 450 - 624 in the presence of increasing K48 - or K63 - Ub4 . Error bars represent the standard deviation over three biological trials . H Fluorescence anisotropy values for fluorescently - labeled K48 - or K63 - Ub4 in the presence of increasing UBQLN2 450 - 624 . Error bars represent the standard deviation over three biological trials .

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Figure 2A CSPs for UBQLN2 450-624 residues at 1:1 ratio with Ub4. The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS (Dao et al, 2018). B Amide peak intensities between the unbound and Ub4-bound forms of UBQLN2 450-624 with the 555-570 region highlighted in gray. C CSPs for residues in UBQLN2 487-624 at 1:1 ratio with Ub4. D CSPs for residues in UBQLN2 450-624 lacking residues 551-569 (red/gray region) at 1:1 ratio with Ub4. E Normalized titration curves of 11 amide resonances for UBQLN2 450-624.F Comparison of binding affinity (Kd) measurements obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing K48- or K63-Ub4. Error bars represent the standard deviation over three biological trials.H Fluorescence anisotropy values for fluorescently-labeled K48- or K63-Ub4 in the presence of increasing UBQLN2 450-624. Error bars represent the standard deviation over three biological trials.


Figure 3B Proximal Ub resonances in Ub4 were compared to monoUb , as noted in schematic . CSPs > 0 . 05 ppm ( orange dashed line ) are mapped onto the proximal Ub unit of Ub4 in panel A . C 15N - 1H SOFAST - HMQC NMR spectra of proximal Ub of K48 - Ub4 or K63 - Ub4 at 50 µM in the absence and presence of UBQLN2 450 - 624 at a 2 : 1 UBQLN2 : Ub4 ratio . Unbound monoUb spectrum ( black ) is overlaid for comparison . NMR spectra collected and processed under identical conditions . D CSPs of the same proximal Ub resonances in K63 - Ub4 vs K48 - Ub4 . Gray bars indicate K48 - Ub4 resonances that were too weak to be detected .

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Figure 3B Proximal Ub resonances in Ub4 were compared to monoUb, as noted in schematic. CSPs > 0.05 ppm (orange dashed line) are mapped onto the proximal Ub unit of Ub4 in panel A.C 15N-1H SOFAST-HMQC NMR spectra of proximal Ub of K48-Ub4 or K63-Ub4 at 50 µM in the absence and presence of UBQLN2 450-624 at a 2:1 UBQLN2:Ub4 ratio. Unbound monoUb spectrum (black) is overlaid for comparison. NMR spectra collected and processed under identical conditions.D CSPs of the same proximal Ub resonances in K63-Ub4 vs K48-Ub4. Gray bars indicate K48-Ub4 resonances that were too weak to be detected.


Figure 4B Normalized pair distance distribution function P ( r ) from SAXS data for four Ub4 chain types . C CSPs at 1 : 1 Ub4 : UBQLN2 for all chain types , but clustered into two categories for clarity . F Normalized titration curves of UBA resonances showing more pronounced sigmoidal behaviors upon titration of K48 - Ub4 into UBQLN2 450 - 624 than upon titrations of K11 - Ub4 , K63 - Ub4 , M1 - Ub4 and HOTag6 - Ub . G Fluorescence anisotropy values for fluorescently - labeled UBQLN2 450 - 624 in the presence of increasing amounts of different Ub4 chains . Error bars represent the standard deviation over three biological trials . H Comparison of Kd values obtained from NMR and fluorescence anisotropy . Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis . For visual comparison , gray dashed line is Kd of Ub binding to UBQLN2 450 - 624 as determined from NMR and FA experiments ( green bars ) .

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Figure 4B Normalized pair distance distribution function P(r) from SAXS data for four Ub4 chain types.C CSPs at 1:1 Ub4:UBQLN2 for all chain types, but clustered into two categories for clarity.F Normalized titration curves of UBA resonances showing more pronounced sigmoidal behaviors upon titration of K48-Ub4 into UBQLN2 450-624 than upon titrations of K11-Ub4, K63-Ub4, M1-Ub4 and HOTag6-Ub.G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing amounts of different Ub4 chains. Error bars represent the standard deviation over three biological trials.H Comparison of Kd values obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).


Figure 5A Fluorescence microscopy showing enhancement or inhibition of UBQLN2 droplet formation at increasing amounts of chains at 50 µM UBQLN2 in 20 mM NaPhosphate and 200 mM NaCl ( pH 6 . 8 ) at 30 ºC . [ Ub ] : [ UBQLN2 ] reflects the ratio between Ub monomers and UBQLN2 molecules . Solution is spiked with Alexa Fluor 647 - labeled UBQLN2 . The same image is used for the 0 : 1 condition . For visual comparison , images for K48 - Ub4 and K63 - Ub4 are identical to those shown in Figure 1A . Scale bar , 5 µm . B Representative results from spectrophotometric turbidity assay as a function of temperature at 30 μM UBQLN2 . E Fluorescence microscopy showing the distribution of Ub4 ( spiked with Dylight 488 - labeled chains ) inside and outside droplets containing 50 µM UBQLN2 ( spiked with Alexa Fluor 647 - labeled UBQLN2 ) . Scale bar , 5 µm .

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Figure 5A Fluorescence microscopy showing enhancement or inhibition of UBQLN2 droplet formation at increasing amounts of chains at 50 µM UBQLN2 in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. Solution is spiked with Alexa Fluor 647-labeled UBQLN2. The same image is used for the 0:1 condition. For visual comparison, images for K48-Ub4 and K63-Ub4 are identical to those shown in Figure 1A. Scale bar, 5 µm.B Representative results from spectrophotometric turbidity assay as a function of temperature at 30 μM UBQLN2.E Fluorescence microscopy showing the distribution of Ub4 (spiked with Dylight 488-labeled chains) inside and outside droplets containing 50 µM UBQLN2 (spiked with Alexa Fluor 647-labeled UBQLN2). Scale bar, 5 µm.


Figure 6A SDS - PAGE gels monitoring the formation of K63 - linked polyUb chains and possibly ubiquitinated GST - Ubc13 over time , as indicated by asterisks and curly brackets . K63 - Ub3 migrates on the gel similarly as His - Mms2 , right below 20 kDa . Experimental conditions : ± 50 µM Ub and UBQLN2 , 30 nM Dylight 650 - labeled UBQLN2 , 1 µM mE1 , 2 µM His - Mms2 , 4 µM GST - Ubc13 , 10 mM ATP and MgCl2 , 3 mM TCEP , 50 mM Tris pH8 , 37 ºC . The K63 - linked polyUb chain reactions were done in the absence ( top ) and presence ( bottom ) of UBQLN2 . Since UBQLN2 competes with E1 and E2 to bind to Ub , the presence of UBQLN2 slowed down chain formation ( indicated by comparing disappearance rates of Ub ) . The band intensity for GST - Ubc13 decreased with time . The time - dependent appearance of species between 50 - 85 kDa were obstructed in the presence of UBQLN2 . These species are most likely ubiquitinated GST - Ubc13 , and not free polyUb chains , since these bands were observed with BioRad Stain - free gels ( right ) that detect proteins with tryptophan residues unlike Ub or polyUb chains .

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Figure 6A SDS-PAGE gels monitoring the formation of K63-linked polyUb chains and possibly ubiquitinated GST-Ubc13 over time, as indicated by asterisks and curly brackets. K63-Ub3 migrates on the gel similarly as His-Mms2, right below 20 kDa. Experimental conditions: ± 50 µM Ub and UBQLN2, 30 nM Dylight 650-labeled UBQLN2, 1 µM mE1, 2 µM His-Mms2, 4 µM GST-Ubc13, 10 mM ATP and MgCl2, 3 mM TCEP, 50 mM Tris pH8, 37 ºC. The K63-linked polyUb chain reactions were done in the absence (top) and presence (bottom) of UBQLN2. Since UBQLN2 competes with E1 and E2 to bind to Ub, the presence of UBQLN2 slowed down chain formation (indicated by comparing disappearance rates of Ub). The band intensity for GST-Ubc13 decreased with time. The time-dependent appearance of species between 50-85 kDa were obstructed in the presence of UBQLN2. These species are most likely ubiquitinated GST-Ubc13, and not free polyUb chains, since these bands were observed with BioRad Stain-free gels (right) that detect proteins with tryptophan residues unlike Ub or polyUb chains.


figf1 ( a ) Chemical structure of PIK - III . ( b ) The indicated kinases were incubated with different doses of PIK - III and IC50 values were determined . ( c ) U2OS cells expressing GFP - FYVE were incubated with the indicated compounds for 2 h , fixed , stained with Hoechst 33342 and imaged by automated epifluorescence microscopy . GFP - positive puncta per cell were calculated and used to determine IC50 values for each compound . Data points represent values from three wells of one experiment . ( d ) U2OS cells expressing GFP - FOXO3A were incubated with the indicated compounds for 2 h , fixed , stained with Hoechst 33342 and imaged by automated epifluorescence microscopy . The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound . Data points represent values from three wells of one experiment .

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figf1(a) Chemical structure of PIK-III. (b) The indicated kinases were incubated with different doses of PIK-III and IC50 values were determined.(c) U2OS cells expressing GFP-FYVE were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. GFP-positive puncta per cell were calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.(d) U2OS cells expressing GFP-FOXO3A were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.


figf2 ( a ) General domain structure of human VPS34 kinase comprising the helical domain ( yellow ) and the kinase domain ( green ) . The ATP - binding pocket and active site lie between the amino - terminal lobe and the carboxy - terminal lobe of the kinase domain . ( b ) Close - up view of the VPS34 active site with PIK - III bound in the ATP - binding pocket . PIK - III forms two hydrogen - bond interactions between the compound ' s donor / acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685 . A prominent hydrophobic pocket , bounded in part by the side chains of Phe 612 , Pro 618 and Phe 684 accommodates the cyclopropyl functional group . An extended solvent - mediated hydrogen - bonding network bridges interactions between the PIK - III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644 . ( c ) The binding properties surface of the VPS34 active site is superimposed on the solvent - accessible surface . Green surfaces are regions with hydrophobic binding properties ; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits . Blue and red surfaces represent hydrogen bond donor and acceptor regions , respectively . PIK - III is shown bound in the active site . ( d ) Superposition of the PIK - III - bound VPS34 pocket surface with a mesh ( white ) mapping the human PI ( 3 ) Kα active site . The displacement of the PI ( 3 ) Kα hydrophobic pocket away from the hinge ( relative to that of VPS34 ) is readily apparent .

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figf2(a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green). The ATP-binding pocket and active site lie between the amino-terminal lobe and the carboxy-terminal lobe of the kinase domain. (b) Close-up view of the VPS34 active site with PIK-III bound in the ATP-binding pocket. PIK-III forms two hydrogen-bond interactions between the compound's donor/acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685. A prominent hydrophobic pocket, bounded in part by the side chains of Phe 612, Pro 618 and Phe 684 accommodates the cyclopropyl functional group. An extended solvent-mediated hydrogen-bonding network bridges interactions between the PIK-III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644. (c) The binding properties surface of the VPS34 active site is superimposed on the solvent-accessible surface. Green surfaces are regions with hydrophobic binding properties; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits. Blue and red surfaces represent hydrogen bond donor and acceptor regions, respectively. PIK-III is shown bound in the active site. (d) Superposition of the PIK-III-bound VPS34 pocket surface with a mesh (white) mapping the human PI(3)Kα active site. The displacement of the PI(3)Kα hydrophobic pocket away from the hinge (relative to that of VPS34) is readily apparent.


figf3 ( a ) H4 cells expressing mCherry - GFP - LC3 were treated overnight with the indicated compounds , fixed , stained with Hoechst 33342 and imaged by automated acquisition . Representative images of an overlay of GFP ( green ) , mCherry ( red ) and Hoechst ( blue ) are shown with a scale bar equivalent to 10 μm . Bars represent the mean GFP - and mCherry - positive LC3 puncta per cell and data points represent values from 18 imaged fields in two wells from one experiment . b ) HeLa cells expressing GFP - Parkin were treated with PIK - III for 12 h followed by the addition of CCCP for 12 h , fixed , stained for endogenous Tom20 and imaged . Representative images of an overlay of Tom20 ( red ) and Hoechst ( blue ) are shown with a scale bar equivalent to 50 μm . Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment . ( c ) H4 cells expressing GFP - p62 were treated overnight with the indicated compounds , fixed , stained with Hoechst 33342 and imaged . Representative images of an overlay of GFP ( green ) and Hoechst ( blue ) are shown with a scale bar equivalent to 10 μm . Bars represent the mean GFP - positive puncta area per cell and data points represent values from 18 imaged fields in 2 wells from one experiment . ( d ) Steady - state levels of autophagy markers visualized by western blotting in H4 GFP - p62 cells treated overnight with 0 . 5 μM AZD8055 or 5 μM PIK - III . ( e ) Steady - state levels of p62 , LC3 and GAPDH visualized by western blotting in PSN - 1 cells treated for 48 h with the indicated doses of PIK - III . ( f ) Steady - state levels of p62 , LC3 and GAPDH visualized by western blotting in Panc10 . 05 cells treated for 48 h with the indicated doses of PIK - III . ( g ) RKO cells expressing inducible HA - ATG4B - C74A were treated with doxycycline ( DOX induction ) for five days and then incubated in DOX - free medium for 24 h ( DOX washout ) . Bafilomycin A1 was added with or without PIK - III at the beginning of the washout period ( lanes 5 and 6 ) . Steady - state levels of ATG4B , LC3 and GAPDH were visualized by western blotting . Uncropped images of blots are shown in Supplementary Fig . 7 .

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

figf3(a) H4 cells expressing mCherry-GFP-LC3 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged by automated acquisition. Representative images of an overlay of GFP (green), mCherry (red) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP- and mCherry-positive LC3 puncta per cell and data points represent values from 18 imaged fields in two wells from one experiment.b) HeLa cells expressing GFP-Parkin were treated with PIK-III for 12 h followed by the addition of CCCP for 12 h, fixed, stained for endogenous Tom20 and imaged. Representative images of an overlay of Tom20 (red) and Hoechst (blue) are shown with a scale bar equivalent to 50 μm. Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment.(c) H4 cells expressing GFP-p62 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged. Representative images of an overlay of GFP (green) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP-positive puncta area per cell and data points represent values from 18 imaged fields in 2 wells from one experiment.(d) Steady-state levels of autophagy markers visualized by western blotting in H4 GFP-p62 cells treated overnight with 0.5 μM AZD8055 or 5 μM PIK-III.(e) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in PSN-1 cells treated for 48 h with the indicated doses of PIK-III. (f) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in Panc10.05 cells treated for 48 h with the indicated doses of PIK-III.(g) RKO cells expressing inducible HA-ATG4B-C74A were treated with doxycycline (DOX induction) for five days and then incubated in DOX-free medium for 24 h (DOX washout). Bafilomycin A1 was added with or without PIK-III at the beginning of the washout period (lanes 5 and 6). Steady-state levels of ATG4B, LC3 and GAPDH were visualized by western blotting. Uncropped images of blots are shown in Supplementary Fig. 7.


figf4 . ( b ) Proteins represented by unique ubiquitylation sites with > 1 . 5 log2 ( fold change ) in both replicates after diglycyl - peptide enrichment were searched for > 1 . 5 log2 ( fold change ) at the proteome level in both replicates . The resulting 15 proteins are listed to the right in descending order of the average protein fold change in the flow - through replicate analyses . Two independent biological replicates are shown in crossplots . ( c ) Steady - state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK - III for 20 h . ( d ) The levels of NCOA4 and p62 in ATG7 wild - type ( + / + ) , heterozygous ( + / − ) or homozygous knockout ( − / − ) DLD1 clones 1 and 2 were determinede ) DLD1 cells cultured in rich medium ( Fed ) were deprived of serum and amino acids ( Starve ) for the indicated times ( hours , h ) and levels of NCOA4 , p62 , LC3 and GAPDH were determined by western blotting . ( f ) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4 , LC3 , LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK - III . Regions outlined with white dashed lines are magnified to the right of each panel . Scale bars in full panels on left , and zoomed panels on right correspond to 10 and 2 μm respectively . Uncropped images of blots are shown in Supplementary Fig . 7 .

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

figf4. (b) Proteins represented by unique ubiquitylation sites with >1.5 log2 (fold change) in both replicates after diglycyl-peptide enrichment were searched for >1.5 log2 (fold change) at the proteome level in both replicates. The resulting 15 proteins are listed to the right in descending order of the average protein fold change in the flow-through replicate analyses. Two independent biological replicates are shown in crossplots.(c) Steady-state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK-III for 20 h.(d) The levels of NCOA4 and p62 in ATG7 wild-type (+/+), heterozygous (+/−) or homozygous knockout (−/−) DLD1 clones 1 and 2 were determinede) DLD1 cells cultured in rich medium (Fed) were deprived of serum and amino acids (Starve) for the indicated times (hours, h) and levels of NCOA4, p62, LC3 and GAPDH were determined by western blotting.(f) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4, LC3, LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK-III. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full panels on left, and zoomed panels on right correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.


figf5 ( a ) Endogenous immunoprecipitation of NCOA4 with ferritin heavy ( FTH1 ) and light ( FTL ) chains from DLD1 cell lysates . b ) Steady - state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated ( hours , h ) . ( c ) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight , fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3 , LAMP2 or EEA1 . Regions outlined with white dashed lines are magnified to the right of each panel . Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively . ( d ) DLD1 parental or NCOA4 − / − cells ( clones 1 and 2 ; KO - 1 , KO - 2 ) were starved for 6 h and the levels of NCOA4 , FTH1 , FTL and GAPDH determined . ( e ) DLD1 parental or NCOA4 − / − cells were cultured in the presence or absence of bafilomycin A1overnight , fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin ( FTH1 - FTL ) and LAMP2 . Regions outlined with white dashed lines are magnified to the right of each panel . Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively . Uncropped images of blots are shown in Supplementary Fig . 7 .

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

figf5(a) Endogenous immunoprecipitation of NCOA4 with ferritin heavy (FTH1) and light (FTL) chains from DLD1 cell lysates.b) Steady-state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated (hours, h).(c) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3, LAMP2 or EEA1. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively.(d) DLD1 parental or NCOA4−/− cells (clones 1 and 2; KO-1, KO-2) were starved for 6 h and the levels of NCOA4, FTH1, FTL and GAPDH determined.(e) DLD1 parental or NCOA4−/− cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin (FTH1-FTL) and LAMP2. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.


figf6 ( a ) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot . Proteins elute in high - to low - molecular - weight fractions from 18 to 33 , respectively . Dashed lines are for visual comparison only . ( b ) DLD1 parental or NCOA4 − / − cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4 , FTH1 and FTL by western blot . ( c ) NCOA4 − / − cells were transfected with control or SBP - NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors , lysed and the occurrence of the faster - migrating FTH1 band was determined . ( d ) Endogenous NCOA4 immunoprecipitations from DLD1 parental , FTH1 − / − or FTL − / − ( clones 1 and 2 for each genotype ) protein extracts . The presence of FTH1 and FTL was detected by western blot . Uncropped images of blots are shown in Supplementary Fig . 7 .

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

figf6(a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot. Proteins elute in high- to low-molecular-weight fractions from 18 to 33, respectively. Dashed lines are for visual comparison only.(b) DLD1 parental or NCOA4−/− cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4, FTH1 and FTL by western blot.(c) NCOA4−/− cells were transfected with control or SBP-NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors, lysed and the occurrence of the faster-migrating FTH1 band was determined.(d) Endogenous NCOA4 immunoprecipitations from DLD1 parental, FTH1−/− or FTL−/− (clones 1 and 2 for each genotype) protein extracts. The presence of FTH1 and FTL was detected by western blot. Uncropped images of blots are shown in Supplementary Fig. 7.


figf7 ( a ) DLD1 cells were incubated in the presence or absence of DFX ( 30 μM ) and PIK - III ( 5 μM ) and the levels of FTH1 and FTL were analysed using western blotting . . ( b ) DLD parental or NCOA4 − / − cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined . ( c ) Ncoa4 − / − mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting . Note the complete absence of the lower Fth1 band consistent with earlier findings . ( d ) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4 − / − mice is shown and brown pigmented regions apparent in Ncoa4 − / − sections are indicated ( white arrows ) . ( e ) The presence of iron in spleens from control or Ncoa4 − / − mice was determined using Perls ' Prussian blue staining . Representative micrographs ( left panels ) or magnifications ( right panels ) are shown . Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively . ( f ) Quantitative image analysis of the data shown in e was performed as described in the Methods . Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group ( n = 6 mice per group ; ∗ P 0 . 0006 for unpaired t - test ) . Uncropped images of blots are shown in Supplementary Fig . 7 .

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

figf7(a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting.. (b) DLD parental or NCOA4−/− cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined.(c) Ncoa4−/− mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting. Note the complete absence of the lower Fth1 band consistent with earlier findings.(d) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4−/− mice is shown and brown pigmented regions apparent in Ncoa4−/− sections are indicated (white arrows).(e) The presence of iron in spleens from control or Ncoa4−/− mice was determined using Perls' Prussian blue staining. Representative micrographs (left panels) or magnifications (right panels) are shown. Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively. (f) Quantitative image analysis of the data shown in e was performed as described in the Methods. Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group (n = 6 mice per group; ∗P 0.0006 for unpaired t-test). Uncropped images of blots are shown in Supplementary Fig. 7.


Figure 1A Concentrations of amylase , lipase , CRP , and ANGPTL4 in the sera of patients ( n = 90 ) diagnosed with pancreatitis . The box plots show a typical display consisting of a median value depicted by the line in the center of the box ; an interquartile range ( IQR ; 25th to the 75th percentile ) depicted by the box ; and the maximum ( Q3 + 1 . 5 * IQR ) and minimum ( Q1 - 1 . 5 * IQR ) values depicted by the whisker . Statistical significance of Mann - Whitney U - tests is indicated ( * * P < 0 . 01 and * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA . The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient ( R ) . Each dot represents the levels of ANGPTL4 and CRP from an individual patient ( n = 90 ) . B Pancreatitis and adjacent normal regions ( n = 6 ) were stained by immunofluorescence for amylase and ANGPTL4 . Scale bar represents 30 µmC H & E staining , ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild ( AP ) and severe acute pancreatitis ( SAP ) for 2 h ( n = 15 , each group ) . Scale bar represents 50 µm . D Amylase , lipase , and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas ( n = 15 , each group ) .

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

Figure 1A Concentrations of amylase, lipase, CRP, and ANGPTL4 in the sera of patients (n = 90) diagnosed with pancreatitis. The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (** P < 0.01 and *** P < 0.001). Exact P values are shown in Appendix Table S2. ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA. The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient (R). Each dot represents the levels of ANGPTL4 and CRP from an individual patient (n = 90).B Pancreatitis and adjacent normal regions (n = 6) were stained by immunofluorescence for amylase and ANGPTL4. Scale bar represents 30 µmC H&E staining, ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild (AP) and severe acute pancreatitis (SAP) for 2 h (n = 15, each group). Scale bar represents 50 µm.D Amylase, lipase, and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas (n = 15, each group).


Figure 2A AP was induced by 5 injections ( IP ) of cerulein at a dosage of 50 μg / kg . SAP was induced by AP and one injection of lipopolysaccharide ( LPS , IP ) at a dosage of 20 mg / kg . AP + ANGPTL4 ( 2 mg / kg ) was induced by AP and one injection of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . ANGPTL4 ( 4 mg / kg ) was induced by two injections of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . Mice were sacrificed 6 h after pancreatitis induction . ( H & E and immunofluorescent staining for IL - 1β ( red ) and amylase ( green ) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues ( n = 10 , each group ) . Scale bar represents 50 µmB Concentrations of the inflammatory cytokines TGF - β , IL - 1β , and IFN - γ as well as ANGPTL4 in the sera ( n = 10 , each group ) . C TUNEL staining for apoptosis and vacuoles formation ( TEM ) . Red arrow , apoptotic cells ; white dotted line , vacuoles region . D T . E . M . ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP , SAP and ANGPTL4 - treated models ( n = 5 , each group ) .

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Figure 2A AP was induced by 5 injections (IP) of cerulein at a dosage of 50 μg/kg. SAP was induced by AP and one injection of lipopolysaccharide (LPS, IP) at a dosage of 20 mg/kg. AP+ANGPTL4 (2 mg/kg) was induced by AP and one injection of ANGPTL4 (IP) at a dosage of 2 mg/kg. ANGPTL4 (4 mg/kg) was induced by two injections of ANGPTL4 (IP) at a dosage of 2 mg/kg. Mice were sacrificed 6 h after pancreatitis induction. (H&E and immunofluorescent staining for IL-1β (red) and amylase (green) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues (n = 10, each group). Scale bar represents 50 µmB Concentrations of the inflammatory cytokines TGF-β, IL-1β, and IFN-γ as well as ANGPTL4 in the sera (n = 10, each group).C TUNEL staining for apoptosis and vacuoles formation (TEM). Red arrow, apoptotic cells; white dotted line, vacuoles region.D T.E.M. ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP, SAP and ANGPTL4-treated models (n = 5, each group).


Figure 3A AP and SAP were induced in wild type ( WT ) and ANGPTL4 - / - mice ( n = 15 , each group ) , as described in the material and methods . H & E staining , TUNEL staining for apoptosis , and immunofluorescence staining for ANGPTL4 ( red ) and amylase ( green ) were performed in the pancreatic tissues . ANGPTL4 and IL - 6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and - / - mice by Western blotting . Scale bar represents 100 µm . B Levels of ANGPTL4 , amylase , lipase , and MPO in serum ( n = 15 , each group ) . C Immunofluorescence staining of TNF - α and IL - 6 , and the levels of the inflammatory cytokines TGF - β , IFN - γ , and IL - 1β in the sera from ANGPTL4 WT and - / - mice ( n = 10 , each group ) . Scale bar represents 150

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Figure 3A AP and SAP were induced in wild type (WT) and ANGPTL4 -/- mice (n = 15, each group), as described in the material and methods. H&E staining, TUNEL staining for apoptosis, and immunofluorescence staining for ANGPTL4 (red) and amylase (green) were performed in the pancreatic tissues. ANGPTL4 and IL-6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and -/- mice by Western blotting. Scale bar represents 100 µm.B Levels of ANGPTL4, amylase, lipase, and MPO in serum (n = 15, each group).C Immunofluorescence staining of TNF-α and IL-6, and the levels of the inflammatory cytokines TGF-β, IFN-γ, and IL-1β in the sera from ANGPTL4 WT and -/- mice (n = 10, each group). Scale bar represents 150 µm


Figure 4A For macrophage depletion models , mice ( six weeks ) were injected with GdCl3 ( 15 mg / kg ) . After the induction of macrophage depletion in the mice , pancreatitis was induced by cerulein , LPS , and ANGPTL4 for 24 h ( n = 10 , each group ) . H & E staining of pancreatic tissue , and levels of amylase and lipase in the serum . Scale bar represents 100 µmB Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues . Levels of ANGPTL4 in the serum were measured in the AP , SAP , and ANGPTL4 ( 4 mg / kg ) groups of macrophage WT or depletion mice h ( n = 10 , each group ) . Scale bar represents 100 µmC Immunofluorescent staining of the F4 / 80 macrophage marker and quantification of macrophage - positive cells in the AP and SAP groups from the ANGPTL4 - / - and WT mice ( n = 10 ) . Scale bar represents 150

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Figure 4A For macrophage depletion models, mice (six weeks) were injected with GdCl3 (15 mg/kg). After the induction of macrophage depletion in the mice, pancreatitis was induced by cerulein, LPS, and ANGPTL4 for 24 h (n = 10, each group). H&E staining of pancreatic tissue, and levels of amylase and lipase in the serum. Scale bar represents 100 µmB Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues. Levels of ANGPTL4 in the serum were measured in the AP, SAP, and ANGPTL4 (4 mg/kg) groups of macrophage WT or depletion mice h (n = 10, each group). Scale bar represents 100 µmC Immunofluorescent staining of the F4/80 macrophage marker and quantification of macrophage-positive cells in the AP and SAP groups from the ANGPTL4 -/-and WT mice (n = 10). Scale bar represents 150 µm


Figure 5A Cell morphology and the mRNA levels of inflammatory cytokines , including and TNF - α , IL - 6 , and IL - 1β in mouse primary macrophages treated with LPS and ANGPTL4 ( 100 ng / ml ) using qPCR and RT - PCR , and levels of TGF - β and NO in the LPS - and ANGPTL4 - treated primary macrophages ( n = 5 ) . Scale bar represents 100 µmB Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2 - well silicone culture inserts that were placed in a cell culture μ - Dish and transwell at 24 h ( n = 5 ) . Scale bar represents 50 µmC Mouse primary acinar cells and macrophages were labelled with fluorescence dyes ( macrophages , red and acinar cells , green ) . Co - cultured cells treated with 100 ng / ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h . Scale bars represent 80 µm ( red ) and 40 µm ( white ) .

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Figure 5A Cell morphology and the mRNA levels of inflammatory cytokines, including and TNF-α, IL-6, and IL-1β in mouse primary macrophages treated with LPS and ANGPTL4 (100 ng/ml) using qPCR and RT-PCR, and levels of TGF-β and NO in the LPS- and ANGPTL4-treated primary macrophages (n = 5). Scale bar represents 100 µmB Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2-well silicone culture inserts that were placed in a cell culture μ-Dish and transwell at 24 h (n = 5). Scale bar represents 50 µmC Mouse primary acinar cells and macrophages were labelled with fluorescence dyes (macrophages, red and acinar cells, green). Co-cultured cells treated with 100 ng/ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h. Scale bars represent 80 µm (red) and 40 µm (white).


Figure 6A Macrophage cells were treated with ANGPTL4 ( 100 ng / ml ) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay ( n = 3 ) . Mean pixel density was quantified using ImageJ software analysis . B ANGPTL4 and C5a expression levels were measured in bone marrow - derived macrophages from pancreatitis - induced ANGPTL4 - / - and WT mice . C Phospho - kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1 - derived macrophage cells ( n = 3 ) . Cells were treated with ANGPTL4 ( 100 ng / ml ) for 6 h . After phospho - kinase array analysis , the expression of AMPKα , JNK , AKT , P70S6K , and EGFR was confirmed by western blotting . D To explore whether ANGPTL4 affects JNK , AMPK , and PI3K / AKT signaling , each inhibitor ( SP600125 , compound C , and HS - 173 , 1 µM ) was treated to in THP1 - derived macrophages for 2 h after ANGPTL4 treatment ( 100 ng / ml ) for 6 h . E C5a , C5aR , TNF - α , IL - 6 , and IL - 1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS ( 100 ng / ml ) . In the transwell experiment , mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 ( 100 ng / ml ) . Expression of ANGPTL4 , C5aR , and C5a was determined in siANGPTL4 - treated THP1 - derived macrophage after LPS activation . F In the transwell experiment , TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist ( W - 54011 , 10 nM ) pretreatment for 12 h prior to treatment with ANGPTL4 , LPS , and C5a ( 100 ng / ml ) for 24 h to macrophage in transwell . Scale bar represents 20

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Figure 6A Macrophage cells were treated with ANGPTL4 (100 ng/ml) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay (n=3). Mean pixel density was quantified using ImageJ software analysis.B ANGPTL4 and C5a expression levels were measured in bone marrow-derived macrophages from pancreatitis-induced ANGPTL4-/- and WT mice.C Phospho-kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1-derived macrophage cells (n=3). Cells were treated with ANGPTL4 (100 ng/ml) for 6 h. After phospho-kinase array analysis, the expression of AMPKα, JNK, AKT, P70S6K, and EGFR was confirmed by western blotting.D To explore whether ANGPTL4 affects JNK, AMPK, and PI3K/AKT signaling, each inhibitor (SP600125, compound C, and HS-173, 1 µM) was treated to in THP1-derived macrophages for 2 h after ANGPTL4 treatment (100 ng/ml) for 6 h.E C5a, C5aR, TNF-α, IL-6, and IL-1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS (100 ng/ml). In the transwell experiment, mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 (100 ng/ml). Expression of ANGPTL4, C5aR, and C5a was determined in siANGPTL4-treated THP1-derived macrophage after LPS activation.F In the transwell experiment, TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist (W-54011, 10 nM) pretreatment for 12 h prior to treatment with ANGPTL4, LPS, and C5a (100 ng/ml) for 24 h to macrophage in transwell. Scale bar represents 20 µm


Figure 7A Concentrations of C5a in the sera from pancreatitis patients , and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients ( n = 90 ) . The box plots show a typical display consisting of a median value depicted by the line in the center of the box ; an interquartile range ( IQR ; 25th to the 75th percentile ) depicted by the box ; and the maximum ( Q3 + 1 . 5 * IQR ) and minimum ( Q1 - 1 . 5 * IQR ) values depicted by the whisker . Statistical significance of Mann - Whitney U - tests is indicated ( * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient ( R ) . Each dot represents the levels of C5a and ANGPTL4 from an individual patient ( n = 90 ) . The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues . Scale bar represents 50 µmB Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 - / - and WT animals and macrophage depletion / WT models with pancreatitis . Scale bar represents 150 µm . Levels of C5a were measured in serum ( n = 7 ) . C After macrophages were activated with LPS ( 100 ng / ml ) , the cells were treated with either neutralizing ANGPTL4 or C5a antibody ( 100 ng / ml ) or an isotype control ( IgG ) . ANGPTL4 , C5a , TNF - α , and IL - 6 levels were measured in culture media ( n = 5 ) . D The neutralizing ANGPTL4 antibody ( 10 mg / kg ) was injected twice ( IP ) into the AP and SAP models . H & E staining of pancreatic tissues and the concentrations of ANGPTL4 , C5a , TNF - α , and amylase in the serum ( n = 10 , each group ) . Scale bar represents 50

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Figure 7A Concentrations of C5a in the sera from pancreatitis patients, and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients (n = 90). The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (*** P < 0.001). Exact P values are shown in Appendix Table S2. The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient (R). Each dot represents the levels of C5a and ANGPTL4 from an individual patient (n = 90). The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues. Scale bar represents 50 µmB Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 -/- and WT animals and macrophage depletion/WT models with pancreatitis. Scale bar represents 150 µm. Levels of C5a were measured in serum (n = 7).C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5).D The neutralizing ANGPTL4 antibody (10 mg/kg) was injected twice (IP) into the AP and SAP models. H&E staining of pancreatic tissues and the concentrations of ANGPTL4, C5a, TNF-α, and amylase in the serum (n = 10, each group). Scale bar represents 50 µm