A Double immunofluorescence for GFP ( green ) thymidine analog BrdU ( magenta , combined with DAPI ( ) , of 57 days - old human ( top panels ) chimpanzee ( bottom panels ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( upper panels ) or ARHGAP11A220 expression plasmid ( lower panels ) . Tick marks indicate borders of VZ SVZ / NL ; arrowheads indicate examples of GFP + BrdU + double - positive . Scale bar , 50 µm . B of proportion of GFP + in SVZ / NL that are BrdU + in 57 days - old human ( blue ) chimpanzee ( red ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( blue red ) or ARHGAP11A220 expression plasmid ( blue red ) . Data are mean of 12 human control , 12 human ARHGAP11A220 - transfected , 9 chimpanzee control 8 chimpanzee ARHGAP11A220 - transfected cerebral organoids ( grown from one human one chimpanzee iPSC , respectively ) of six ( human ) four ( chimpanzee ) independent batches each ; error bars indicate SD ; n . s . , not significant ; * * , P < 0 . 01 ( Kruskal Wallis ) .

O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O B-TISSUE O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O B-ORGANISM O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM B-GENEPROD O O O O B-ORGANISM O O B-ORGANISM B-GENEPROD O O B-TISSUE O O O O O B-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O 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 Double immunofluorescence for GFP (green) and the thymidine analog BrdU (magenta, combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and BrdU+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL that are BrdU+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 12 human control, 12 human ARHGAP11A220-transfected, 9 chimpanzee control and 8 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of six (human) and four (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; **, P <0.01 (Kruskal Wallis test).


C Double immunofluorescence for GFP ( green ) TBR2 ( yellow ) , combined with DAPI ( ) , of 57 days - old human ( top panels ) chimpanzee ( bottom panels ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( upper panels ) or ARHGAP11A220 expression plasmid ( lower panels ) . Tick marks indicate borders of VZ SVZ / NL ; arrowheads indicate examples of GFP + TBR2 + double - positive . Scale bar , 50 µm . D of proportion of GFP + in SVZ / NL that are TBR2 + in 57 days - old human ( blue ) chimpanzee ( red ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( blue red ) or ARHGAP11A220 expression plasmid ( blue red ) . Data are mean of 9 human control , 9 human ARHGAP11A220 - transfected , 10 chimpanzee control 10 chimpanzee ARHGAP11A220 - transfected cerebral organoids ( grown from one human one chimpanzee iPSC , respectively ) of four ( human ) three ( chimpanzee ) independent batches each ; error bars indicate SD ; n . s . , not significant ; * , P < 0 . 05 ( Kruskal Wallis ) .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O B-ORGANISM O O O B-ORGANISM O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM B-GENEPROD O O O O B-ORGANISM O O B-ORGANISM B-GENEPROD O O B-TISSUE O O O O O B-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O 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 Double immunofluorescence for GFP (green) and TBR2 (yellow), combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are TBR2+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 9 human control, 9 human ARHGAP11A220-transfected, 10 chimpanzee control and 10 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of four (human) and three (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05 (Kruskal Wallis test).


A Double immunofluorescence for GFP ( green ) bRG marker PTPRZ1 ( magenta ) , combined with DAPI ( ) , of 60 days - old human forebrain organoids , generated from ARHGAP11A plus ARHGAP11B double - knockout clone 16 . were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids ( ARHGAP11A + B - rescue , referred to as Control ) , ARHGAP11B expression plasmid only ( ARHGAP11B - rescue , referred to as ARHGAP11A - KO ) , or ARHGAP11A expression plasmid only ( ARHGAP11A - rescue , referred to as ARHGAP11B - KO ) . Tick marks indicate borders of VZ SVZ / NL ; arrowheads indicate examples of GFP + PTPRZ1 + double - positive . Scale bar , 50 µm B of proportion of GFP + in SVZ / NL that are PTPRZ1 + in 60 days - old ARHGAP11A plus ARHGAP11B double - knockout ( 11A + 11B KO ) human forebrain organoids . Organoids were generated from clone 16 ( left ) clone j ( right ) , data were obtained 10 days after indicated electroporation with GFP expression plasmid plus either ARHGAP11A ( 11A ) plus ARHGAP11B ( 11B ) expression plasmids ( referred to as Control , as this constitutes an ARHGAP11A + B double - rescue in 11A + 11B KO organoids ; blue ) , ARHGAP11B ( 11B ) expression plasmid only ( referred to as 11A - KO , as this constitutes a selective ARHGAP11B rescue in 11A + 11B KO organoids ; blue ) , or ARHGAP11A ( 11A ) expression plasmid only ( referred to as 11B - KO , as this constitues a selective ARHGAP11A rescue in 11A + 11B KO organoids ; red ) . For clone 16 , data are mean of 8 control , 8 11A - KO 8 11B - KO human forebrain organoids of two independent batches each ; for clone j , data are mean of 9 control , 10 11A - KO 8 11B - KO human forebrain organoids of three independent batches each ; error bars indicate SD ; n . s . , not significant ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ( Kruskal Wallis )

O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM B-TISSUE O O 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-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O 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-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the bRG marker PTPRZ1 (magenta), combined with DAPI staining (white), of 60 days-old human forebrain organoids, generated from ARHGAP11A plus ARHGAP11B double-knockout clone 16. Images were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids (ARHGAP11A+B-rescue, referred to as Control), ARHGAP11B expression plasmid only (ARHGAP11B-rescue, referred to as ARHGAP11A-KO), or ARHGAP11A expression plasmid only (ARHGAP11A-rescue, referred to as ARHGAP11B-KO). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and PTPRZ1+ double-positive cells. Scale bar, 50 µm B Quantification of the proportion of GFP+ cells in the SVZ/NL that are PTPRZ1+ in 60 days-old ARHGAP11A plus ARHGAP11B double-knockout (11A+11B KO) human forebrain organoids. Organoids were generated from clone 16 (left) and clone j (right), and data were obtained 10 days after the indicated electroporation with GFP expression plasmid plus either ARHGAP11A (11A) plus ARHGAP11B (11B) expression plasmids (referred to as Control, as this constitutes an ARHGAP11A+B double-rescue in the 11A+11B KO organoids; dark blue), ARHGAP11B (11B) expression plasmid only (referred to as 11A-KO, as this constitutes a selective ARHGAP11B rescue in the 11A+11B KO organoids; light blue), or ARHGAP11A (11A) expression plasmid only (referred to as 11B-KO, as this constitues a selective ARHGAP11A rescue in the 11A+11B KO organoids; dark red). For clone 16, data are the mean of 8 control, 8 11A-KO and 8 11B-KO human forebrain organoids of two independent batches each; for clone j, data are the mean of 9 control, 10 11A-KO and 8 11B-KO human forebrain organoids of three independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05; **, P <0.01; ***, P <0.001 (Kruskal Wallis test)


( A - F ) Atg1 ‐ induced myosin II activation depends on kinase activity of Atg1 . Third ‐ instar wing imaginal discs from ptc ‐ GAL4 UAS ‐ GFP controls or flies expressing indicated transgenes were stained with phospho ‐ MRLC ( blue ) TRITC ‐ labelled phalloidin ( red ) . Low level of phospho ‐ MRLC was observed in controls ( A ) overexpressing kinase ‐ deficient Atg1 ‐ KR ( C ) , whereas a robust increase in phospho ‐ MRLC was found in overexpressing Atg1 ( B ) . Co ‐ expression of Atg1 spaghetti ‐ squash ( A20A21 ) exhibited low level of phospho ‐ MRLC ( D ) . Atg1 ‐ induced high level of phospho ‐ MRLC was not suppressed by expression of caspase inhibitor p35 ( E ) or by depletion of Atg12 ( Atg12RNAi ) ( F ) . Bar , 20 μm .

O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(A-F) Atg1‐induced myosin II activation depends on the kinase activity of Atg1. Third‐instar wing imaginal discs from ptc‐GAL4 UAS‐GFP controls or flies expressing indicated transgenes were stained with phospho‐MRLC (blue) and TRITC‐labelled phalloidin (red). Low level of phospho‐MRLC staining was observed in controls (A) and cells overexpressing kinase‐deficient Atg1‐KR (C), whereas a robust increase in phospho‐MRLC was found in cells overexpressing Atg1 (B). Co‐expression of Atg1 and spaghetti‐squash (A20A21) exhibited low level of phospho‐MRLC staining (D). Atg1‐induced high level of phospho‐MRLC staining was not suppressed by expression of caspase inhibitor p35 (E) or by depletion of Atg12 (Atg12RNAi) (F). Bar, 20 μm.


( H ) Sqa , but not Atg1 , directly phosphorylated Sqh in vitro . Flag - Atg1 , Flag - Atg1 ‐ KR , HA - Sqa HA - Sqa ‐ KA were immunoprecipated from lysate of transfected incubated in an in vitro kinase reaction mixture containing [ γ ‐ 32P ] ATP bacterially expressed recombinant wild ‐ type Sqh or SqhA20A21 . As shown on autoradiogram ( top panel ) , wild ‐ type but not kinase ‐ deficient Atg1 ( Atg1 ‐ KR ) Sqa ( Sqa ‐ KA ) was autophosphorylated . No phosphorylation was seen with SqhA20A21 . equal input of His ‐ fusion is shown on Coomassie . Anti ‐ Flag anti ‐ HA immunoblottings ( IBs ) were used as controls to quantify amount of precipitated .

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

(H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag-Atg1, Flag-Atg1‐KR, HA-Sqa and HA-Sqa‐KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ‐32P]ATP and bacterially expressed recombinant wild‐type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild‐type but not the kinase‐deficient Atg1 (Atg1‐KR) and Sqa (Sqa‐KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His‐fusion proteins is shown on the Coomassie staining. Anti‐Flag and anti‐HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated.


( A - K ) Genetic interactions between Atg1 , Sqa , spaghetti ‐ squash ( Sqh ) . Compared with ptc ‐ GAL4 controls ( A ) , expression of Atg1 or Sqa by ptc ‐ GAL4 resulted in missing anterior cross ‐ vein ( B , C ) . However , RNAi ‐ mediated downregulation of Atg1 ( Atg1RNAi ) or Sqa ( SqaRNAi ) did not cause wing vein defects ( D , E ) . Depletion of Atg1 Sqa suppressed Atg1 Sqa ‐ induced wing vein defects , respectively ( F , I ) . Atg1 ‐ induced wing defects were modulated by depletion of Sqa ( G ) or by co ‐ expression of SqhA20A21 ( H ) . Nevertheless , Sqa ‐ induced wing vein defects were suppressed by co ‐ expression of SqhA20A21 ( J ) but not Atg1RNAi ( K ) .

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(A-K) Genetic interactions between Atg1, Sqa, and spaghetti‐squash (Sqh). Compared with the ptc‐GAL4 controls (A), expression of Atg1 or Sqa by ptc‐GAL4 resulted in missing anterior cross‐vein phenotypes (B, C). However, RNAi‐mediated downregulation of Atg1 (Atg1RNAi) or Sqa (SqaRNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa‐induced wing vein defects, respectively (F, I). Atg1‐induced wing defects were modulated by depletion of Sqa (G) or by co‐expression of SqhA20A21 (H). Nevertheless, Sqa‐induced wing vein defects were suppressed by co‐expression of SqhA20A21 (J) but not Atg1RNAi (K).


( M ) 293T were transfected with HA ‐ tagged Sqa WT ( wild ‐ type ) or KA , together with Flag ‐ tagged Atg1 WT or KR . were lysed 48 h after transfection immunoprecipitated ( IP ) with anti ‐ Flag antibodies . immunoprecipitated total lysates ( TCL ) were analysed by immunoblotting ( IB ) with antibodies as indicated .

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(M) 293T cells were transfected with HA‐tagged Sqa WT (wild‐type) or KA, together with Flag‐tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti‐Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated.


( N ) 293T transfected with Flag - Atg1 ‐ KR various HA ‐ tagged Sqa contracts were subjected to immunoprecipitations with anti ‐ HA antibody . immunoprecipitated total lysates were analysed by immunoblotting with antibodies as indicated .

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(N) 293T cells transfected with Flag-Atg1‐KR and various HA‐tagged Sqa contracts were subjected to immunoprecipitations with anti‐HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated.


( O ) Atg1 directly phosphorylated Sqa in vitro . Flag ‐ tagged Atg1 WT or KR immunoprecipated from lysate of transfected was used to phosphorylate bacterially expressed recombinant Sqa ‐ K1 , Sqa ‐ K2 , Sqa ‐ C in an in vitro kinase . lower panels represent equal input of His ‐ fusion Atg1 immunoprecipitates .

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(O) Atg1 directly phosphorylated Sqa in vitro. Flag‐tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa‐K1, Sqa‐K2, and Sqa‐C in an in vitro kinase assay. The lower panels represent equal input of His‐fusion proteins and Atg1 immunoprecipitates.


( A ) Characterization of Atg1 ‐ dependent phosphorylation sites on Sqa . 293T transfected with Flag ‐ tagged Atg1 or Atg1 ‐ KR were subjected to immunoprecipitation with anti ‐ Flag antibodies , followed by in vitro kinase assays with bacterially expressed Sqa ‐ K2 , Sqa ‐ K2 ‐ T194A , Sqa ‐ K2 ‐ T239A , Sqa ‐ K2 ‐ T279A as substrates . Atg1 but not Atg1 ‐ KR was autophosphorylated ( top panel ) . Relative phosphorylation of substrates were quantified . Data are represented as mean ± s . e . of triplicates . ( B - D ) Phosphorylation of Sqa at Thr ‐ 279 is critical for kinase activity of Sqa .

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

(A) Characterization of Atg1‐dependent phosphorylation sites on Sqa. 293T cells transfected with Flag‐tagged Atg1 or Atg1‐KR were subjected to immunoprecipitation with anti‐Flag antibodies, followed by in vitro kinase assays with bacterially expressed Sqa‐K2, Sqa‐K2‐T194A, Sqa‐K2‐T239A, and Sqa‐K2‐T279A as substrates. Atg1 but not Atg1‐KR was autophosphorylated (top panel). Relative phosphorylation levels of substrates were quantified. Data are represented as mean±s.e. of triplicates. (B-D) Phosphorylation of Sqa at Thr‐279 is critical for the kinase activity of Sqa.


( B ) HA ‐ tagged Sqa or Sqa ‐ T279A immunoprecipated from lysate of transfected was used to phosphorylate bacterially expressed recombinant spaghetti ‐ squash ( Sqh ) WT A20A21 , in an in vitro kinase .

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(B) HA‐tagged Sqa or Sqa‐T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti‐squash (Sqh) WT and A20A21, in an in vitro kinase assay.


( C ) Clonal expression of Sqa but not Sqa ‐ T279A ( GFP ‐ positive ) in larval wing imaginal discs resulted in a marked increase in phospho ‐ MRLC ( blue ) actin reorganization ( red ) . Bar , 20 μm .

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(C) Clonal expression of Sqa but not Sqa‐T279A (GFP‐positive cells) in the larval wing imaginal discs resulted in a marked increase in phospho‐MRLC staining (blue) and actin reorganization (red). Bar, 20 μm.


( D ) larval fat of denoted genotypes were dissected , lysed , subjected to western blot using anti ‐ phospho ‐ MRLC anti ‐ Sqh antibodies . For , of MRLC phosphorylation in each genotype were measured with ImageJ normalized to Sqh

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(D) The larval fat body of denoted genotypes were dissected, lysed, and subjected to western blot analysis using anti‐phospho‐MRLC and anti‐Sqh antibodies. For quantification, the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh levels


( A , B ) Activation of myosin II on nutrient deprivation . larval fat of denoted genotypes under fed or starved conditions were dissected , lysed , subjected to western blot using antibodies specific for phospho ‐ myosin regulatory chain ( MRLC ) total MRLC . Rheb , SqaRNAi , Sqa ‐ T279A , spaghetti ‐ squash ( SqhA20A21 ) , Atg7RNAi transgenes were expressed under control of hs - GAL4 driver ( B ) . For , relative phosphorylation of MRLC were quantified as in Figure 3D . Data are represented as mean ± s . e . of triplicates .

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(A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho‐myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa‐T279A, spaghetti‐squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs-GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates.


( C - J ) Starvation ‐ induced autophagosome formation was compromised by inhibition of myosin II activation . Compared with fed condition ( C ) , starvation induced a robust formation of GFP - Atg8a in larval fat ‐ ( D ) . Clonal expression of Atg12RNAi ( E ) , Sqa ‐ T279A ( F ) , SqaRNAi ( G ) , or SqhA20A21 ( H ) markedly suppressed GFP - Atg8a formation during starvation . autophagic defects caused by Sqa ‐ T279A SqaRNAi were rescued by co ‐ expression of constitutively active SqhE20E21 ( I ) SqhD20D21 ( J ) , respectively . Bar , 20 μm .

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(C-J) Starvation‐induced autophagosome formation was compromised by inhibition of myosin II activation. Compared with the fed condition (C), starvation induced a robust formation of GFP-Atg8a puncta in larval fat‐body cells (D). Clonal expression of Atg12RNAi (E), Sqa‐T279A (F), SqaRNAi (G), or SqhA20A21 (H) markedly suppressed GFP-Atg8a puncta formation during starvation. The autophagic defects caused by Sqa‐T279A and SqaRNAi were rescued by co‐expression of the constitutively active SqhE20E21 (I) and SqhD20D21 (J), respectively. Bar, 20 μm.


( K ) average number of GFP - Atg8a marked autophagosomes per is shown ( data are represented as mean ± s . e . of 20 fat ‐ samples imaged per genotype ; * * * P0 . 001 ) .

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(K) The average number of GFP-Atg8a marked autophagosomes per cell is shown (data are represented as mean±s.e. of 20 fat‐body samples imaged per genotype; ***P0.001).


( L ) Inhibition of myosin II activity increased sensitivity to starvation . Flies carrying transgenes under control of fat ‐ specific Cg - GAL4 driver were used for further . Histogram illustrating survival curve of adult flies of denoted genotypes when placed under starved conditions . Data are mean ± s . e . from triplicate experiments ( n = 100 flies / genotype / treatment ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . See Supplementary data for genotypes .

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(L) Inhibition of myosin II activity increased sensitivity to starvation. Flies carrying transgenes under the control of fat body‐specific Cg-GAL4 driver were used for further analysis. Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions. Data are mean±s.e. from triplicate experiments (n=100 flies/genotype/treatment). *P0.05, **P0.01, ***P0.001. See Supplementary data for genotypes.


( A ) Ulk1 interacted with ZIPK . 293T transfected with HA ‐ tagged ZIPK together with Flag ‐ tagged Ulk1 or Ulk1 ‐ KI were subjected to immunoprecipitations with anti ‐ Flag antibody . immunoprecipitated total lysates were analysed by immunoblotting with antibodies as indicated .

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(A) Ulk1 interacted with ZIPK. 293T cells transfected with HA‐tagged ZIPK together with Flag‐tagged Ulk1 or Ulk1‐KI were subjected to immunoprecipitations with anti‐Flag antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated.


( B ) HA ‐ tagged kinase ‐ inactive ZIPK ‐ KA ( K42A ) was expressed in 293T with Flag ‐ tagged Ulk1 or Ulk1 ‐ KI . lysates were incubated with or without alkaline phosphatase ( CIP ) analysed by immunoblotting with antibodies as indicated .

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(B) HA‐tagged kinase‐inactive ZIPK‐KA (K42A) was expressed in 293T cells with Flag‐tagged Ulk1 or Ulk1‐KI. Cell lysates were incubated with or without alkaline phosphatase (CIP) and analysed by immunoblotting with antibodies as indicated.


( C ) Myosin II was activated upon nutrient deprivation in MCF7 . MCF7 were cultured in serum containing DMEM medium ( nutrient ‐ rich condition ) or in Earle ' s balanced salt solution ( EBSS ; starved condition ) for 2 h . myosin II activity was significantly downregulated after EBSS ‐ starved were replenished with fresh DMEM medium containing 10 % FBS for 2 h . myosin II activity was quantified expressed as a fold changes compared with DMEM controls . Each value represents mean ± s . e . of triplicates .

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(C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates.


( D ) MCF7 stably infected with lentivirus expressing control ( shLuc ) , Ulk1 or ZIPK shRNA were cultured in nutrient ‐ rich DMEM medium ( F ) or EBSS ( S ) for 2 h . Effects of Ulk1 ZIPK knockdown on starvation ‐ induced myosin II activation were analysed by immunoblotting with antibodies as indicated . Data are mean ± s . e . of triplicates .

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(D) MCF7 cells stably infected with lentivirus expressing control (shLuc), Ulk1 or ZIPK shRNA were cultured in nutrient‐rich DMEM medium (F) or EBSS (S) for 2 h. Effects of Ulk1 and ZIPK knockdown on starvation‐induced myosin II activation were analysed by immunoblotting with antibodies as indicated. Data are mean±s.e. of triplicates.


( A ) MCF7 / GFP - LC3 stably infected with lentivirus expressing control ( shLuc ) , ZIPK , or non ‐ muscle myosin heavy chain ‐ IIA ( NMHC ‐ IIA ) shRNA were cultured in nutrient ‐ rich medium ( DMEM ) or starvation medium ( Earle ' s balanced salt solution ; EBSS ) in presence or absence of lysosomal inhibitor bafilomycin A1 ( BafA1 ) for 2 h . Depletion of ZIPK NMHC ‐ IIA markedly inhibited starvation ‐ induced GFP - LC3 formation . of number of GFP - LC3 dots per ( lower panel ) was shown ( data are represented as mean ± s . e . of 100 , * * * P0 . 001 ) .

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(A) MCF7/GFP-LC3 cells stably infected with lentivirus expressing control (shLuc), ZIPK, or non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) shRNA were cultured in nutrient‐rich medium (DMEM) or starvation medium (Earle's balanced salt solution; EBSS) in the presence or absence of lysosomal inhibitor bafilomycin A1 (BafA1) for 2 h. Depletion of ZIPK and NMHC‐IIA markedly inhibited starvation‐induced GFP-LC3 puncta formation. Quantification of the number of GFP-LC3 dots per cell (lower panel) was shown (data are represented as mean±s.e. of 100 cells, ***P0.001).


( B ) as in ( A ) were cultured in nutrient ‐ rich medium ( F ) or EBSS ( S ) with or without BafA1 for 2 h . Effects of ZIPK NMHC ‐ IIA knockdown on starvation ‐ induced GFP - LC3 conversion were assessed by immunoblotting with anti ‐ LC3 , anti ‐ ZIPK , anti ‐ NMHC ‐ IIA , anti ‐ tubulin antibodies . relative of LC3II / LC3I is shown at right panel . Data are mean ± s . e . of triplicates .

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(B) Cells as in (A) were cultured in nutrient‐rich medium (F) or EBSS (S) with or without BafA1 for 2 h. Effects of ZIPK and NMHC‐IIA knockdown on starvation‐induced GFP-LC3 conversion were assessed by immunoblotting with anti‐LC3, anti‐ZIPK, anti‐NMHC‐IIA, and anti‐tubulin antibodies. The relative ratio of LC3II/LC3I is shown at the right panel. Data are mean±s.e. of triplicates.


( A ) fat of spaghetti ‐ squash ( sqhAX3 ) ; sqh - GFP larva under fed or starved conditions were dissected subjected to immunofluorescence . Sqh - GFP phospho ‐ myosin regulatory chain ( MRLC ) were enriched in - junction under nutrient ‐ rich ( fed ) condition . Under starvation conditions , Sqh - GFP phospho ‐ MRLC were localized to both - junction perinuclear region . Actin was stained with TRITC ‐ labelled phalloidin ( red ) nucleus was stained with DAPI ( blue ) . Bar , 20 μm .

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(A) The fat body of spaghetti‐squash (sqhAX3); sqh-GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh-GFP and phospho‐myosin regulatory light chain (MRLC) were enriched in cell-cell junction under nutrient‐rich (fed) condition. Under starvation conditions, Sqh-GFP and phospho‐MRLC were localized to both cell-cell junction and perinuclear region. Actin was stained with TRITC‐labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm.


( B ) Starvation induced redistribution of myosin II in MCF7 / GFP - LC3 . Both phospho ‐ MRLC MRLC were localized in peripheral region of in nutrient ‐ rich medium ( DMEM ) , whereas they redistributed to perinuclear region co ‐ localized with GFP - LC3 under starvation conditions ( Earle ' s balanced salt solution ; EBSS ) . Bar , 10 μm .

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(B) Starvation induced redistribution of myosin II in MCF7/GFP-LC3 cells. Both phospho‐MRLC and MRLC were localized in peripheral region of cells in nutrient‐rich medium (DMEM), whereas they redistributed to the perinuclear region and co‐localized with GFP-LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm.


( C ) MCF7 / GFP - LC3 cultured in nutrient ‐ rich ( DMEM ) or starved ( EBSS ) conditions were homogenized subjected to centrifugation , resulting post ‐ supernatant ( PNS ) was fractionated by high ‐ speed centrifugation into pellet cytosol . were resolved by SDS - PAGE immunoblotted with anti ‐ TGN46 antibody as a control for ‐ association , anti ‐ caspase ‐ 3 as a control for . of phospho ‐ MRLC MRLC in each fraction were quantified using ImageJ plotted relative to their amounts in PNS ( n = 3 ) . Each value represents mean ± s . e . of three experiments . * P0 . 05 , * * P0 . 01 .

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(C) MCF7/GFP-LC3 cells cultured in nutrient‐rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by high‐speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS-PAGE and immunoblotted with anti‐TGN46 antibody as a control for membrane‐association proteins, anti‐caspase‐3 as a control for cytosolic proteins. The levels of phospho‐MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. *P0.05, **P0.01.


( A ) Immunofluorescence of GFP - mAtg9 phospho ‐ myosin regulatory chain ( MRLC ) in MCF7 / GFP - mAtg9 . GFP - mAtg9 was enriched in trans ‐ Golgi network ( TGN ) labelled with anti ‐ TGN46 antibody phospho ‐ MRLC localized to peripheral in nutrient ‐ rich condition ( DMEM ) . GFP - mAtg9 redistributed co ‐ localized with phospho ‐ MRLC in starvation condition ( Earle ' s balanced salt solution ; EBSS ) .

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(A) Immunofluorescence analysis of GFP-mAtg9 and phospho‐myosin regulatory light chain (MRLC) in MCF7/GFP-mAtg9 cells. GFP-mAtg9 was enriched in the trans‐Golgi network (TGN) labelled with anti‐TGN46 antibody and phospho‐MRLC localized to cell peripheral in nutrient‐rich condition (DMEM). GFP-mAtg9 redistributed and co‐localized with phospho‐MRLC in starvation condition (Earle's balanced salt solution; EBSS).


( B ) mAtg9 interacted with non ‐ muscle myosin heavy chain ‐ IIA ( NMHC ‐ IIA ) under starvation conditions . 293T ( right panel ) or 293T stably infected with lentivirus expressing control ( shLuc ) , UNC ‐ 51 ‐ like kinases ( Ulk1 ) or zipper ‐ interacting kinase ( ZIPK ) shRNA ( left panel ) were transfected with V5 ‐ tagged mAtg9 GFP - tagged NMHC ‐ IIA . At 48 h after transfection , were incubated in either serum ‐ containing medium ( DMEM ) or EBSS for 2 h with or without treatment of 50 μM ML ‐ 7 blebbistatin ( Blebb ) . lysates were immunoprecipitated with anti ‐ V5 antibody immunoblotted with indicated antibodies .

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(B) mAtg9 interacted with non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) under starvation conditions. 293T cells (right panel) or 293T cells stably infected with lentivirus expressing control (shLuc), UNC‐51‐like kinases (Ulk1) or zipper‐interacting protein kinase (ZIPK) shRNA (left panel) were transfected with V5‐tagged mAtg9 and GFP-tagged NMHC‐IIA. At 48 h after transfection, cells were incubated in either serum‐containing medium (DMEM) or EBSS for 2 h with or without the treatment of 50 μM ML‐7 and blebbistatin (Blebb). Cell lysates were immunoprecipitated with anti‐V5 antibody and immunoblotted with indicated antibodies.


( C ) MCF7 / GFP - mAtg9 were stably infected with lentivirus expressing control ( shLuc ) , ZIPK , or NMHC ‐ IIA shRNA . Under starved conditions , mAtg9 was redistributed from TGN to a dispersed peripheral pool in control ( shLuc ) . Starvation ‐ induced GFP - mAtg9 redistribution was blocked in ZIPK NMHC ‐ IIA knockdown . were analysed by quantifying mAtg9 localization in for either a TGN ‐ enriched or dispersed pattern . Data are represented as mean ± s . e . of 70 . * P0 . 05 , * * P0 . 01 . Bar , 10 μm .

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(C) MCF7/GFP-mAtg9 cells were stably infected with lentivirus expressing control (shLuc), ZIPK, or NMHC‐IIA shRNA. Under starved conditions, mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells (shLuc). Starvation‐induced GFP-mAtg9 redistribution was blocked in ZIPK and NMHC‐IIA knockdown cells. The images were analysed by quantifying mAtg9 localization in cells for either a TGN‐enriched or dispersed pattern. Data are represented as mean±s.e. of 70 cells. *P0.05, **P0.01. Bar, 10 μm.


( A ) Four fetuses ( named NW - 1 , NW - 2 , NW - 3 NW - 4 ) with different eye were retrieved at E44 by caesarean

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(A) Four fetuses (named NW-1, NW-2, NW-3 and NW-4) with different eye morphology were retrieved at E44 by caesarean section


( B ) DraI digestion of PCR products from multiple organs of four fetuses ( Upper ) . NC , negative control with no genomic loaded . Mut , MITFL247S / L247S genomic loaded . WT , LW genomic loaded . Multiple organs of four fetuses were examined for KIT expression by agarose gel electrophoresis ( Bottom ) . Mut , MITFL247S / L247S genomic loaded . WT , LW genomic

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(B) DraI digestion of PCR products from the multiple organs of the four fetuses (Upper). NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (Bottom). Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded


( A ) Representative microscopic appearances of retina RPE ( arrowhead ) of a chimeric , a WT a Mut at same gestational by H & E . WT , Bama WT . Mut , Bama MITFL247S / L247S . NW - 2 , chimeric . Scale bars , 100

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(A) Representative microscopic appearances of the retina and RPE cells (arrowhead) of a chimeric, a WT and a Mut fetus at the same gestational age by H&E staining. WT, Bama WT fetus. Mut, Bama MITFL247S/L247S fetus. NW-2, the chimeric fetus. Scale bars, 100 μm


( B ) Representative immunofluorescence showed expression of multiple RPE markers Pax6 , MITF Bestrophin in chimeric fetuses . Blue , Hochst3342 . WT , Bama WT as positive control . NW - 2 , chimeric . MITFL247S / L247S as negative control . Scale bars , 50

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(B) Representative immunofluorescence images showed the expression of multiple RPE markers Pax6, MITF and Bestrophin in the chimeric fetuses. Blue, Hochst3342. WT, Bama WT fetus as positive control. NW-2, the chimeric fetus. MITFL247S/L247S fetus as negative control. Scale bars, 50 μm


( A ) Representative microscopic show that in vitro cultured RPE exhibited a hexagonal shape . D3 , D5 D11 , cultured for 3 days , 5 days 11 days , respectively . Scale bars , 100

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(A) Representative light microscopic images show that the in vitro cultured RPE cells exhibited a hexagonal shape. D3, D5 and D11, cultured for 3 days, 5 days and 11 days, respectively. Scale bars, 100 μm


Representative immunofluorescence showed expression of RPE - specific markers , including RPE65 ( B in cultured RPE . Scale bars , 50

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Representative immunofluorescence images showed the expression of RPE-specific markers, including RPE65 (B in the cultured RPE cells. Scale bars, 50 μm


Representative immunofluorescence showed expression of RPE - specific markers , includin ZO - 1 ( C ) Bestrophin ( D ) , respectively , in cultured RPE . Scale bars , 50

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Representative immunofluorescence images showed the expression of RPE-specific markers, includin ZO-1 (C) and Bestrophin (D), respectively, in the cultured RPE cells. Scale bars, 50 μm


( E ) Semi - quantitative RT - PCR of RPE functional markers in cultured porcine RPE . GAPDH was used to confirm cDNA quality of

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(E) Semi-quantitative RT-PCR analysis of RPE functional markers in cultured porcine RPE cells. GAPDH was used to confirm the cDNA quality of all the samples


( F ) Western blotting identified three RPE markers in porcine RPE . GAPDH served as a housekeeping gene

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(F) Western blotting identified three RPE markers in porcine RPE cells. GAPDH served as a housekeeping gene control


( G ) ELISA identified secreted VEGF PEDF in chimeric WT porcine RPE . Media were collected at 48 hours after were seeded onto 12 - well culture plates to be used for analyses . results are representative of three independent experiments were evaluated using a two - t - . No significant differences were identified . Data are presented as mean ± standard error mean ( ) . WT , RPE cultured from NW - 8 . CH , RPE cultured from chimeric NW - 7

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(G) ELISA identified secreted VEGF and PEDF in chimeric and WT porcine RPE cells. Media were collected at 48 hours after the cells were seeded onto 12-well cell culture plates to be used for analyses. The results are representative of three independent experiments and were evaluated using a two-tailed t-test. No significant differences were identified. Data are presented as the mean ± standard error mean (SEM). WT, RPE cells cultured from the NW-8 fetus. CH, RPE cells cultured from chimeric NW-7 fetus


( A ) full - term chimera ( NW - 16 ) piglet was born showed normal development , of

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(A) The full-term chimera (NW-16) piglet was born and showed normal development, size and morphology of the eyes


( B ) Digestion of MITF PCR products in multiple organs of full - term chimeric pig . NC , negative control with no genomic loaded . Mut , MITFL247S / L247S genomic loaded . WT , Bama GFP - labeled genomic loaded . In chimera piglet , GFP - specific primers were used to further confirm chimeric contribution to multiple organs in piglet . GAPDH was used to confirm quality of samples . NC , negative control with no genomic loaded . Mut , Bama MITFL247S / L247S genomic loaded . WT , Bama GFP - labeled genomic

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(B) Digestion of the MITF PCR products in multiple organs of the full-term chimeric pig. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded. In the chimera piglet, GFP-specific primers were used to further confirm the chimeric contribution to multiple organs in the piglet. GAPDH was used to confirm the DNA quality of all the samples. NC, negative control with no genomic DNA loaded. Mut, Bama MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded


( C ) Representative microscopic appearances of corneal epithelial of chimeric piglet by H & E . Scale bars , 100

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(C) Representative microscopic appearances of the corneal epithelial cells of the chimeric piglet staining by H&E. Scale bars, 100 μm


( D ) Representative microscopic appearances of retina of chimeric piglet by H & E . Scale bars , 100

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(D) Representative microscopic appearances of the retina cells of the chimeric piglet staining by H&E. Scale bars, 100 μm


( A ) Representative GFP - labeled blastomeres that contributed to chimeric porcine RPE are shown with immunofluorescence . Scale bars , 25

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(A) Representative GFP-labeled blastomeres that contributed to the chimeric porcine RPE cells are shown with immunofluorescence staining. Scale bars, 25 μm


( B ) Representative GFP - labeled blastomeres that contributed to chimeric porcine corneal epithelial . Almost RPE corneal epithelial of chimera stained with anti - GFP antibody . Scale bars , 75

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(B) Representative GFP-labeled blastomeres that contributed to the chimeric porcine corneal epithelial cells. Almost all RPE cells and corneal epithelial cells of the chimera stained with anti-GFP antibody. Scale bars, 75 μm


( C ) Representative immunofluorescence showed expression of an RPE marker ( BEST ) in GFP - labeled . Scale bars , 50

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(C) Representative immunofluorescence images showed the expression of an RPE marker (BEST) in GFP-labeled cells. Scale bars, 50 μm


( D ) Representative immunofluorescence showed expression of a corneal epithelial marker ( K12 ) in GFP - labeled . WT , Bama WT piglet . Mut , Bama MITFL247S / L247S piglet . CH , chimeric piglet . Scale bars , 50

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(D) Representative immunofluorescence images showed the expression of a corneal epithelial cell marker (K12) in GFP-labeled cells. WT, Bama WT piglet. Mut, Bama MITFL247S/L247S piglet. CH, chimeric piglet. Scale bars, 50 μm


A Statistical of NFATc1 expression with respect to grading in KPCmice tissues .

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A Statistical analysis of NFATc1 expression with respect to tumor grading in KPCmice tissues.


B Immunohistochemistry detection of NFATc1 p53 , illustrating G1 ‐ G2 G3 in human PDAC tissues . Scale bars , 100 μm .

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B Immunohistochemistry detection of NFATc1 and p53, illustrating G1‐G2 and G3 in human PDAC tissues. Scale bars, 100 μm.


C Statistical illustration of TMA demonstrating NFATc1 strong positive tumors ( n = 161 ) with respect to functional status of p53 expression in human PDAC tissues .

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C Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors (n = 161 patients) with respect to functional status of p53 expression in human PDAC tissues.


D Representative H & amp ; amp ; E ‐ stained sections as well as immunohistochemistry detection of HA ‐ NFATc1 p53 in well ‐ poorly differentiated pancreatic tissues of KNCmice . Scale bars , 100 μm . E of p53 expression with respect to grading by evaluating both of immunostaining number of with overexpression by means of an immunoreactivity score ( IRS ) in KNCmice ( every dot represents one mouse ) .

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D Representative H&amp;E‐stained sections as well as immunohistochemistry detection of HA‐NFATc1 and p53 in well‐ and poorly differentiated pancreatic tissues of KNCmice. Scale bars, 100 μm.E Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score (IRS) in KNCmice (every dot represents one mouse).


F Incidence of livermetastases in KNCmice ( mean values ± SD ) .

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F Incidence of livermetastases in KNCmice (mean values ± SD).


A Genome ‐ wide expression GSEA show p53 ‐ enriched signatures with " EMT " " transcript " in KNCtumor .

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A Genome‐wide expression and GSEA analysis show p53‐enriched signatures with "EMT" and "stem cell transcript" in KNCtumor cells.


B Heat map showing p53 ‐ dependent regulation of " EMT " ‐ " stemness " ‐ related genes in KNCtumor . Fold change relative to control is displayed in a green red color scheme for selected genes with FClog2 1 . 0 or FClog2 > − 1 . 0 .

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B Heat map showing p53‐dependent regulation of "EMT"‐ and "stemness"‐related genes in KNCtumor cells. Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1.0 or FClog2 > −1.0.


C Statistical of wound healing performed in KNCtumor following p53 depletion . Time ‐ lapse analyzer software was used to quantify wound closure rate . Means ± SD are shown from one out of three independent experiments .

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C Statistical analysis of wound healing assay performed in primary KNCtumor cells following p53 depletion. Time‐lapse analyzer software was used to quantify the wound closure rate. Means ± SD are shown from one out of three independent experiments.


D Western blot revealing expression upon p53 depletion in KNCtumor .

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D Western blot analysis revealing protein expression levels upon p53 depletion in KNCtumor cells.


E X ‐ fold mRNA expression of EMT ‐ related marker genes after p53 depletion in KNCtumor . Means ± SD from at least three independent experiments are shown . Asterisks display significance ( * P 0 . 05 ) .

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E X‐fold mRNA expression of EMT‐related marker genes after p53 depletion in KNCtumor cells. Means ± SD from at least three independent experiments are shown. Asterisks display significance (*P 0.05).


F Representative expression of EMT inducers in pancreatic lysates prepared from KNCmice with different gradings .

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F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings.


A Kaplan - Meier curves showing survival of KNC , KPNC , KPΔNC mice under control of p48 ‐ Cre pancreas ‐ specific promoters ( P 0 . 0001 for KPNC or KPΔNC cohorts versus KNC , log ‐ rank , for pairwise combination ) .

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A Kaplan-Meier curves showing survival of KNC, KPNC, and KPΔNC mice under the control of p48‐Cre pancreas‐specific promoters (P 0.0001 for KPNC or KPΔNC cohorts versus KNC, log‐rank test, for pairwise combination).


B Pathological features of KPNC KP ∆ NC mice before extraction . Pancreatic tissue is encircled .

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B Pathological features of KPNC and KP∆NC mice before tumor extraction. Pancreatic tissue is encircled.


C , D HE corresponding immunohistochemistry stainings of ( C ) KPNC ( D ) KP ∆ NCmouse tumors . Scale bars , 100 μm .

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C, D HE and corresponding immunohistochemistry stainings of (C) KPNC and (D) KP∆NCmouse tumors. Scale bars, 100 μm.


E grading of PDAC from KPNC mice ( n = 10 ) .

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E Tumor grading of PDAC from KPNC mice (n = 10).


A Quantitative RT ‐ PCR of EMT ‐ related markers obtained from tumors derived from mice of indicated genotypes ( n = 3 , means ± SD ) .

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A Quantitative RT‐PCR analysis of EMT‐related markers obtained from tumors derived from mice of the indicated genotypes (n = 3, means ± SD).


B Relative mRNA expression of NFATc1 ‐ EMT ‐ related marker genes on administration of CsA ( 1 μM ) in KPNC . Data represents means ± SD from at least three independent experiments . Asterisks show significance ( * P 0 . 05 ) .

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B Relative mRNA expression of NFATc1‐ and EMT‐related marker genes on administration of CsA (1 μM) in KPNC cells. Data represents means ± SD from at least three independent experiments. Asterisks show significance (*P 0.05).


C Western blot revealing expression in KPNC from indicated following transfection of siRNA control or siRNA toward NFATc1 , respectively .

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C Western blot analysis revealing protein expression levels in KPNC cells from the indicated proteins following transfection of siRNA control or siRNA toward NFATc1, respectively.


A Sox2 mRNA expression of pancreatic lysates from KNC , KPNC , KPC mice , analyzed by real ‐ time PCR in three independent experiments ( means ± SD ) .

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A Sox2 mRNA expression of pancreatic lysates from KNC, KPNC, and KPC mice, analyzed by real‐time PCR in three independent experiments (means ± SD).


B , C Western blot shows Sox2 expression in pancreatic lysates of ( B ) KNC mice with indicated grading in ( C ) KPNC KPC mice .

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B, C Western blot analysis shows Sox2 expression in pancreatic lysates of (B) KNC mice with indicated tumor grading and in (C) KPNC and KPC mice.


D expression after siRNA ‐ mediated p53 depletion in KNC .

D Protein expression levels after siRNA‐mediated p53 depletion in KNC tumor cells.


E , F NFATc1 Sox2 expression after ( E ) NFATc1 depletion or ( F ) upon CsA ( 1 μM , 24 h ) treatment in Panc1 .

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E, F NFATc1 and Sox2 protein expression after (E) NFATc1 depletion or (F) upon CsA (1 μM, 24 h) treatment in Panc1 cells.


G Sox2 E ‐ cadherin mRNA expression after NFATc1 depletion in L3 . 6 .

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G Sox2 and E‐cadherin mRNA expression after NFATc1 depletion in L3.6 cells.


I , J ChIP assays show NFATc1 binding to Sox2 enhancer promoter in KNC ( I )

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I, J ChIP assays show NFATc1 binding to Sox2 enhancer and promoter in KNC tumor cells (I)


I , J ChIP assays show polymerase II binding to Sox2 promoter upon CsA ( 1 μM , 24 h ) treatment in L3 . 6 ( J ) .

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I, J ChIP assays show RNA polymerase II binding to Sox2 promoter upon CsA (1 μM, 24 h) treatment in L3.6 cells (J).


K Co ‐ immunoprecipitation of endogenous HA ‐ NFATc1 Sox2 was performed in KPNC .

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K Co‐immunoprecipitation of endogenous HA‐NFATc1 and Sox2 was performed in KPNC cells.


L ChIP experiments show NFATc1 binding along with H3K27 acetylation ( H3K27ac ) mark at selected Snai1 enhancer region polymerase II binding at Snai1 promoter in presence or absence of Sox2 in KPNC . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) .

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L ChIP experiments show NFATc1 binding along with H3K27 acetylation (H3K27ac) mark at the selected Snai1 enhancer region and RNA polymerase II binding at Snai1 promoter in the presence or absence of Sox2 in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).


M Relative mRNA expression of EMT ‐ related marker genes upon Sox2 depletion in KPNC ( P 0 . 05 ) .

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M Relative mRNA expression levels of EMT‐related marker genes upon Sox2 depletion in KPNC tumor cells (P 0.05).


N Zeb1 Snail mRNA in L3 . 6 after shRNA ‐ mediated depletion of Sox2 transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1 ‐ mediated induction of EMT genes ( * P 0 . 05 ) .

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N Zeb1 and Snail mRNA levels in L3.6 cells after shRNA‐mediated depletion of Sox2 and transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1‐mediated induction of EMT genes (*P 0.05).


A NFATc1 Sox2 expression in L3 . 6 ( adherent versus spheres ) .

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A NFATc1 and Sox2 protein expression in L3.6 cells (adherent versus spheres).


B Relative mRNA expression of NFATc1 Sox2 in L3 . 6 as well as in KPNCmicetumor ( adherent versus spheres ) . Graph represents data from three independent experiments ( means ± SD ) . Asterisks display significance ( * P 0 . 05 ) .

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B Relative mRNA expression of NFATc1 and Sox2 in L3.6 cells as well as in KPNCmicetumor cells (adherent versus spheres). Graph represents data from three independent experiments (means ± SD). Asterisks display significance (*P 0.05).


C sorting was performed to collect CD44 high low populations in KPNC spheres ( left panel ) , which were analyzed for NFATc1 Sox2 mRNA expression ( right panel ) .

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C Cell sorting was performed to collect CD44 high and low populations in KPNC spheres (left panel), which were analyzed for NFATc1 and Sox2 mRNA expression (right panel).


D Sox2 mRNA expression upon CsA ( 1 μM ) treatment in L3 . 6 ( adherent versus spheres ) ( * P 0 . 05 ) .

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D Sox2 mRNA expression upon CsA (1 μM) treatment in L3.6 cells (adherent versus spheres) (*P 0.05).


E Bright ‐ field microscopy as well as of KPNC spheres after transient NFATc1 depletion .

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E Bright‐field light microscopy images as well as quantification of KPNC spheres after transient NFATc1 depletion.


A Expression of indicated miRNAs upon adenoviral re ‐ expression of p53 in KPΔNC , detected by real ‐ time PCR ( P 0 . 05 ) .

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A Expression analysis of indicated miRNAs upon adenoviral re‐expression of p53 in KPΔNC tumor cells, detected by real‐time PCR (P 0.05).


B expression of EMT marker genes after adenoviral re ‐ expression of GFP ‐ tagged p53 in KPΔNC .

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B Protein expression of EMT marker genes after adenoviral re‐expression of GFP‐tagged p53 in KPΔNC tumor cells.


C , D ( C ) expression of EMT stemness marker genes upon miRNA ‐ 34a miRNA ‐ 200c overexpression using specific mimics in KPNC . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) .

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C, D Protein (C) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).


C , D mRNA ( D ) expression of EMT stemness marker genes upon miRNA ‐ 34a miRNA ‐ 200c overexpression using specific mimics in KPNC . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) .

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C, D mRNA (D) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).


E Western blot of EMT marker expression in KNC following depletion of miR ‐ 200c .

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E Western blot analysis of EMT marker expression in KNC cells following depletion of miR‐200c.


F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at selected Snai1 enhancer region as well as Sox2 polymerase II binding at Snai1 promoter after miR ‐ 200c overexpression in KPNC . Means ± SD are shown from one out of three independent experiments .

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F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR‐200c overexpression in KPNC tumor cells. Means ± SD are shown from one out of three independent experiments.


G ChIP in KP ∆ NC after adenoviral GFP ‐ tagged p53 overexpression . NFATc1 H3K27ac binding are illustrated on Snai1 enhancer , Sox2 polymerase II binding are demonstrated on Snai1 promoter . Means ± SD are shown from one out of three independent experiments .

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G ChIP analysis in KP∆NC tumor cells after adenoviral GFP‐tagged p53 overexpression. NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer, and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter. Means ± SD are shown from one out of three independent experiments.


H of KP ∆ NC tumour - derived spheres after adenoviralGFP ‐ tagged p53 overexpression .

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H Quantification of KP∆NC tumour cell-derived spheres after adenoviralGFP‐tagged p53 overexpression.


( B ) Immunoblot of pro - ( p31 ) ( p17 ) IL - 1β , pro - ( p45 ) ( p20 ) caspase - 1 in fractions of ASC deficient ( Pycard - / - ) immortalized murine macrophages that were primed with LPS ( 200 ng ml − 1 , for 3h ) . Fractions were incubated with recombinant ASC specks in presence of 2 , 20 or 200 µg ml − 1 of VHHASC or mutVHHASC . One representative of three independent experiments is shown . ( C ) Quantitative of band densitometry of three independent experiments as shown in B . Data represents fold change from conditions ( lanes 1 - 9 ) . vs Pycard − / − lysates incubated with ASC specks alone ( lane 2 ) . ns , p > 0 . 05 ; * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0002 , One - way ANOVA , multiple comparison ( Dunnet ) . Data is displayed as floating bars with max / min values mean ( thicker band ) .

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

(B) Immunoblot analysis of pro- (p31) and cleaved (p17) IL-1β, and pro- (p45) and cleaved (p20) caspase-1 in cytosolic fractions of ASC deficient (Pycard-/-) immortalized murine macrophages that were primed with LPS (200 ng ml−1, for 3h). Fractions were incubated with recombinant ASC specks in the presence of 2, 20 or 200 µg ml−1 of VHHASC or mutVHHASC. One representative of three independent experiments is shown. (C) Quantitative analysis of band densitometry of three independent experiments as shown in B. Data represents the fold change from all conditions (lanes 1- 9) .vs Pycard−/− lysates incubated with ASC specks alone (lane 2). ns, p > 0.05; *, p<0.05; **, p<0.005; ***, p<0.0002, One-way ANOVA, multiple comparison (Dunnet test). Data is displayed as floating bars with the max/min values and mean (thicker band).


( A ) Time - lapse confocal imaging of in vitro nucleation of soluble ASC - mTurquoise ( red ) by ASC - TagGFP ( ASC - GFP ) specks ( green ) , that were left untreated ( None ) , or pre - incubated with VHHASC or mutVHHASC ( 200 µg ml − 1 for 15 min ) . Scale bars : 10 μm . ( B ) Median ( MFI ) of mTurquoise graphed over time showing its polymerization seeded by ASC - TagGFP specks . Each shows mTurquoise MFI around a seeding ASC - TagGFP speck . Data is one representative of three independent experiments .

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

(A) Time-lapse confocal imaging of the in vitro nucleation of soluble ASC-mTurquoise (red) by ASC-TagGFP (ASC-GFP) specks (green), that were left untreated (None), or pre-incubated with VHHASC or mutVHHASC (200 µg ml−1 for 15 min). Scale bars: 10 μm. (B) Median fluorescence intensity (MFI) of mTurquoise graphed over time showing its polymerization seeded by ASC-TagGFP specks. Each line shows the mTurquoise MFI around a seeding ASC-TagGFP speck. Data is one representative of three independent experiments.


( C ) WES capillary electrophoresis immunoblotting of DSS cross - linked in - vitro - generated human ASC - mTurquoise ( ASC ) specks that were incubated for 1h ( RT ) with 2 , 20 or 200 µg ml − 1 of VHHASC , mutVHHASC or with 20 µg ml − 1 of a polyclonal anti - ASC Ab ( a - ASC pAb ) . Data is from one representative of four independent experiments .

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

(C) WES capillary electrophoresis and immunoblotting of DSS cross-linked in-vitro-generated human ASC-mTurquoise (ASC) specks that were incubated for 1h (RT) with 2, 20 or 200 µg ml−1 of VHHASC, mutVHHASC or with 20 µg ml−1 of a polyclonal anti-ASC Ab (a-ASC pAb). Data is from one representative of four independent experiments.


A - D ( A - B ) Human IL - 1β ( hIL - 1β ) concentrations in - free supernatants of LPS - primed ( 10 ng ml − 1 , 150 min ) human macrophages that were left untreated , or pre - incubated with VHHASC or mutVHHASC ( 100 µg ml − 1 ) , CRID3 ( 50 µM ) or VX - 765 ( 50 µM ) for 30 min before stimulation with ( A ) nigericin ( 10 µM ) , or ( B ) PFO ( 30 ng ml − 1 ) for 2h . ( C - D ) Mouse IL - 1β ( mIL - 1β ) concentrations in - free supernatants of LPS - primed mouse BMDMs ( 200 ng ml − 1 , 150 min ) , incubated with VHHs , CRID3 or VX765 , before activation with nigericin ( 10 µM ) , or PFO ( 250 ng ml − 1 ) . Data is combined from two independent experiments , each performed with two donors ( A - B ) or mice ( C - D ) , represented with symbols ( 4 donors or mice in total ) . Data is displayed as floating bars with max / min values mean ( thicker band ) .

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

A-D (A-B) Human IL-1β (hIL-1β) concentrations in cell-free supernatants of LPS-primed (10 ng ml−1, 150 min) primary human macrophages that were left untreated, or pre-incubated with VHHASC or mutVHHASC (100 µg ml−1), CRID3 (50 µM) or VX-765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml−1) for 2h. (C-D) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed mouse BMDMs (200 ng ml−1, 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml−1). Data is combined from two independent experiments, each performed with two donors (A-B) or mice (C-D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band).


E , F ( E ) Epifluorescence microscopy imaging ( F ) of ASC speck formation in BMDMs from ASC - mCitrine ( Green ) transgenic mice . were primed with LPS ( 200 ng ml − 1 , 150 min ) , pre - treated with VX - 765 ( 50 µM , 30 min ) , then treated with VHHASC , VHHmASC ( 100 µg ml − 1 ) or CRID3 ( 50 µM ) for another 30 min before stimulation with nigericin ( top ) , or PFO ( bottom ) for 2h finally fixed with 4 % PFA nuclei stained with DRAQ5 ( Blue ) . Scale bars : 100 μm . in ( E ) are representative of one out of three independent experiments that were quantified in F . Data is displayed as floating bars with max / min values mean ( thicker band ) .

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

E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC-mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml−1, 150 min), pre-treated with VX-765 (50 µM, 30 min), then treated with VHHASC, VHHmASC (100 µg ml−1) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2h and finally fixed with 4% PFA and nuclei stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are representative of one out of three independent experiments that were quantified in F. Data is displayed as floating bars with the max/min values and mean (thicker band).


THP - 1 expressing a Dox - inducible CRISPR - Cas9 cassette targeting GSDMD gene were treated with 1 µg ml - 1 Dox for one or two cycles of 72 h ( 1x , or 2x respectively ) . As control , were left untreated cultured in parallel ( - ) . ( A ) Immunoblot of GSDMD expression following indicated course of Dox treatment PMA - differentiation , where indicated . Data is from one representative of two independent experiments .

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

THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-). (A) Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA-differentiation, where indicated. Data is from one representative of two independent experiments.


GFP + specks detected in culture supernatants by ( D ) immunoblotting of ASC - GFP oligomeric state ( DSS cross - linked ) of extracellular specks recovered from supernatants of treated similarly to ( A - B ) . ( D ) is from one out of at least two independent experiments .

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

GFP+ specks detected in culture supernatants by (D) immunoblotting analysis of ASC-GFP oligomeric state (DSS cross-linked) of extracellular specks recovered from supernatants of cells treated similarly to (A-B). (D) is from one out of at least two independent experiments.


B , C Representative Iba1 stainings of retinal flat mounts detecting microglia / macrophages in spots 3 days after coagulation in C57BL6 / J controls ( B ) Ifnar1 - / - ( C ) mice . Scale bar : 20 µm . D of amoeboid - shaped mononuclear phagocytes in spots . Values show mean ± SD . ( n = 11 - 12 retinas ; unpaired Student ' s t - : * * * p = 0 . 0004 ) . E of immune in spots using a grid image system . Values show mean ± SD . ( n = 42 - 62 ; unpaired Student ' s t - : * * * p < 0 . 0001 ) .

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

B, C Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in C57BL6/J controls (B) and Ifnar1-/- (C) mice. Scale bar: 20 µm.D Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 11-12 retinas; unpaired Student's t-test: ***p = 0.0004).E Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 42-62 cells; unpaired Student's t-test: ***p < 0.0001).


F - K Representative fundus fluorescein angiography of C57BL6 / J ( F - H ) Ifnar1 - / - ( I - K ) mice 3 , 7 14 days after - induced damage . L of vascular leakage by analyzing pixel intensities at 3 , 7 14 days after - induced retinal damage in C57BL6 / J controls versus Ifnar1 - / - mice . Values show mean ± SD . ( n = 14 - 22 eyes ; One - way ANOVA followed by Tukey ' s post - : * * * p < 0 . 0001 ) .

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

F-K Representative fundus fluorescein angiography images of C57BL6/J (F-H) and Ifnar1-/- (I-K) mice 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in C57BL6/J controls versus Ifnar1-/- mice. Values show mean ± SD. (n = 14-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p < 0.0001).


M - P Representative of lectin stained choroidal flat mounts 7 14 days after coagulation in C57BL6 / J control mice ( M , N ) Ifnar1 - / - ( O , P ) . Dashed lines indicate CNV areas asterisk marks central optic nerve head . Scale bar : 200 µm . Q of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 4 - 11 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - : * p = 0 . 0281 ) .

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

M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in C57BL6/J control mice (M, N) and Ifnar1-/- animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µm.Q Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 4-11 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: *p = 0.0281).


B , C Representative Iba1 stainings of retinal flat mounts detecting microglia / macrophages in spots 3 days after coagulation in control mice ( B ) or IFN - b treated ( C ) . Scale bar : 20 µm . D of amoeboid - shaped mononuclear phagocytes in spots . Values show mean ± SD . ( n = 7 - 10 retinas ; unpaired Student ' s t - : * * * p < 0 . 0001 ) . E of immune in spots using a grid image system . Values show mean ± SD . ( n = 41 - 62 ; unpaired Student ' s t - : * * * p < 0 . 0001 ) .

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

B, C Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in control mice (B) or IFN-b treated animals (C).Scale bar: 20 µm.D Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 7-10 retinas; unpaired Student's t-test: ***p < 0.0001).E Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 41-62 cells; unpaired Student's t-test: ***p < 0.0001).


F - K Representative fundus fluorescein angiography of control mice ( F - H ) or IFN - b treated ( I - K ) 3 , 7 14 days after - induced damage . L of vascular leakage by analyzing pixel intensities at 3 , 7 14 days after - induced retinal damage in control mice IFN - b treated . Values show mean ± SD . ( n = 15 - 22 eyes ; One - way ANOVA followed by Tukey ' s post - : * * * p = 0 . 0004 ) .

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

F-K Representative fundus fluorescein angiography images of control mice (F-H) or IFN-b treated animals (I-K) 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in control mice and IFN-b treated animals. Values show mean ± SD. (n = 15-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p = 0.0004).


M - P Representative of lectin stained choroidal flat mounts 7 14 days after coagulation in control mice ( M , N ) IFN - b treated ( O , P ) . Dashed lines indicate CNV areas asterisk marks central optic nerve head . Scale bar : 200 µmQ of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 7 - 12 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - : * * p = 0 . 0038 ) .

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

M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in control mice (M, N) and IFN-b treated animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µmQ Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 7-12 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: **p = 0.0038).


B - D Representative Iba1 results of retinal flat mounts detecting microglia / macrophages in spot 3 days after coagulation in Cx3cr1CreER ( B ) , Ifnar1fl / fl ( C ) Cx3cr1CreER : Ifnar1fl / fl ( D ) mice . Scale bar : 20 µm . E of amoeboid - shaped mononuclear phagocytes in spots . Values show mean ± SD . ( n = 17 - 29 retinas ; unpaired Student ' s t - : * * * p = 0 . 0003 , * * p = 0 . 0044 ) . F of immune in spots using a grid image system . Values show mean ± SD . ( n = 44 - 52 ; unpaired Student ' s t - : * * * p < 0 . 0001 ) .

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

B-D Representative Iba1 staining results of retinal flat mounts detecting microglia/macrophages in laser spot 3 days after laser coagulation in Cx3cr1CreER (B), Ifnar1fl/fl (C) and Cx3cr1CreER:Ifnar1fl/fl (D) mice. Scale bar: 20 µm.E Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 17-29 retinas; unpaired Student's t-test: ***p = 0.0003, **p = 0.0044).F Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 44-52 cells; unpaired Student's t-test: ***p < 0.0001).


G - O Representative fundus fluorescein angiography of Cx3cr1CreER ( G - I ) , Ifnar1fl / fl ( J - L ) Cx3cr1CreER : Ifnar1fl / fl ( M - O ) mice 3 , 7 14 days after - induced damage . P of vascular leakage by analyzing pixel intensities at 3 , 7 14 days after - induced retinal damage in Cx3cr1CreER , Ifnar1fl / fl Cx3cr1CreER : Ifnar1fl / fl mice . Values show mean ± SD . ( n = 5 - 12 eyes ; One - way ANOVA followed by Tukey ' s post - : * * p = 0 . 0032 , * p = 0 . 0247 ) .

O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-O Representative fundus fluorescein angiography images of Cx3cr1CreER (G-I), Ifnar1fl/fl (J-L) and Cx3cr1CreER:Ifnar1fl/fl (M-O) mice 3, 7 and 14 days after laser-induced damage.P Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in Cx3cr1CreER, Ifnar1fl/fl and Cx3cr1CreER:Ifnar1fl/fl mice. Values show mean ± SD. (n = 5-12 eyes; One-way ANOVA followed by Tukey's post-test: **p = 0.0032, *p = 0.0247).


Q - V Representative of lectin stained choroidal flat mounts 7 14 days after coagulation in Cx3cr1CreER ( Q , R ) , Ifnar1fl / fl ( S , T ) Cx3cr1CreER : Ifnar1fl / fl ( U , V ) mice . Dashed lines indicate CNV areas asterisk marks central optic nerve head . Scale bar : 200 µm . W of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 4 - 11 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - : * p = 0 . 043 , * * * p = 0 . 0007 ) .

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

Q-V Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in Cx3cr1CreER (Q, R), Ifnar1fl/fl (S, T) and Cx3cr1CreER:Ifnar1fl/fl (U, V) mice. Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µm.W Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 4-11 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: *p = 0.043, ***p = 0.0007).


( b ) of CTM - GFP mCTM - GFP relative to WT - GFP in HEK 24 h after transfections with WT - GFP , mCTM - GFP ( n = 6 ) or CTM - GFP . transfected with WT - GFP or CTM - GFP were treated without ( no treatment ; n = 13 ) or with serum deprivation ( + SD ; n = 5 ; to enhance CMA activity ) or with macroautophagy inhibitor 3 - methyladenine ( + 3 - MA ; 10 mM ; n = 5 ) , proteasome inhibitor ( + MG132 ; 5 μM ; n = 6 ) , lysosome inhibitor ( + NH4Cl ; 20 mM ; n = 6 ) or pepstatin A ( + Pep A ; 10 μM ; n = 5 ) . Top , representative immunoblots of lysates for GFP . Membranes reprobed for β - actin were used as a loading control . Bottom , means ± s . e . m . of relative , normalized to WT - GFP ( arbitrarily set as 1 ; bar ) ; n represents number of independent experiments from at least 4 separate cultures . One - way ANOVA with Fisher least significant difference , F ( 12 , 73 ) = 20 . 939 . * * * P 0 . 001 versus WT - GFP ( bar ) ; ΔΔΔP 0 . 001 relative to untreated CTM - GFP ( gray bar ) . Full - blots are presented in Supplementary Figure 9 .

O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(b) Quantification of CTM-GFP and mCTM-GFP relative to WT-GFP in HEK cells 24 h after individual transfections with WT-GFP, mCTM-GFP (n = 6) or CTM-GFP. Cells transfected with WT-GFP or CTM-GFP were treated without (no treatment; n = 13) or with serum deprivation (+ SD; n = 5; to enhance CMA activity) or with macroautophagy inhibitor 3-methyladenine (+ 3-MA; 10 mM; n = 5), proteasome inhibitor (+ MG132; 5 μM; n = 6), lysosome inhibitor (+ NH4Cl; 20 mM; n = 6) or pepstatin A (+ Pep A; 10 μM; n = 5). Top, representative immunoblots of cell lysates for GFP. Membranes reprobed for β-actin were used as a loading control. Bottom, means ± s.e.m. of relative protein levels, normalized to WT-GFP (arbitrarily set as 1; white bar); n represents the number of independent experiments from at least 4 separate cultures. One-way ANOVA with Fisher least significant difference test, F(12,73) = 20.939. ***P 0.001 versus WT-GFP (white bar); ΔΔΔP 0.001 relative to untreated CTM-GFP levels (gray bar). Full-length blots are presented in Supplementary Figure 9.


( c ) Representative confocal of colocalization of GFP with lysosome marker LAMP - 1 in COS - 7 transfected with either WT - GFP ( n = 17 ) or CTM - GFP ( n = 16 ) . Scale bar , 20 μm .

O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O

(c) Representative confocal images of colocalization of GFP with the lysosome marker LAMP-1 in COS-7 cells transfected with either WT-GFP (n = 17) or CTM-GFP (n = 16). Scale bar, 20 μm.


b ) Reciprocal coimmunoprecipitation ( IP ) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1 . Flag - tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA - GluN2Bct - CTM or HA - GluN2Bct - CTMm at various ratios in HEK , coimmunoprecipitation / or immunoblotting were performed 24 h thereafter . Anti - HA was used to detect HA - GluN2Bct - CTM HA - GluN2Bct - CTMm , anti - FLAG to detect wtDAPK1 cDAPK1 .

O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-CELL_LINE O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O B-GENEPROD O O O O O

b) Reciprocal coimmunoprecipitation (IP) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1. Flag-tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA-GluN2Bct-CTM or HA-GluN2Bct-CTMm at various ratios in HEK cells, and coimmunoprecipitation and/or immunoblotting were performed 24 h thereafter. Anti-HA was used to detect HA-GluN2Bct-CTM and HA-GluN2Bct-CTMm, anti-FLAG to detect wtDAPK1 and cDAPK1.


( c ) HA - GluN2Bct - CTM specifically dose - dependently decreased level of cDAPK1 ( n = 4 independent experiments from 4 separate cultures transfections ; P 0 . 001 ; F ( 5 , 18 ) = 18 . 27 ) but not

O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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) HA-GluN2Bct-CTM specifically and dose-dependently decreased the level of cDAPK1 (n = 4 independent experiments from 4 separate cell cultures and transfections; P 0.001; F(5,18) = 18.27) but not wtDAPK1


( d ; n = 3 independent experiments from 3 separate cultures transfections ; P = 0 . 933 ; F ( 5 , 12 ) = 0 . 249 ) . HA - GluN2Bct - CTM - mediated cDAPK1 knockdown was significantly reduced by NH4Cl ( c ; 20 mM ; n = 4 ; ΔΔΔP 0 . 001 compared to HA - GluN2Bct - CTM : cDAPK1 8 : 1 group ) by mutational inactivation of

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

(d; n = 3 independent experiments from 3 separate cell cultures and transfections; P = 0.933; F(5,12) = 0.249). HA-GluN2Bct-CTM-mediated cDAPK1 knockdown was significantly reduced by NH4Cl (c; 20 mM; n = 4; ΔΔΔP 0.001 compared to HA-GluN2Bct-CTM:cDAPK1 8:1 group) and by mutational inactivation of CTM