( 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 .

O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE O O O B-TISSUE 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 B-SUBCELLULAR I-SUBCELLULAR 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-SUBCELLULAR I-SUBCELLULAR O O O 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) 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 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 .

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

(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 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 ) .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

O O O B-CELL_LINE 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 O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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 O O O O O O 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) 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 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 ) .

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

(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 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 .

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

(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 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 .

O O O B-CELL_LINE O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O 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) 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 and NW - 4 ) with different eye morphology were retrieved at E44 by caesarean

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 B-TISSUE B-EXP_ASSAY O O O O O O

(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 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

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

(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 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

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

(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 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

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

(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 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

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY 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

(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 images showed the expression of RPE - specific markers , including RPE65 ( B in the cultured RPE cells . 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 images showed the expression of RPE - specific markers , includin ZO - 1 ( C ) and Bestrophin ( D ) , respectively , in the cultured RPE cells . Scale bars , 50

O B-EXP_ASSAY O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O

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 analysis of RPE functional markers in cultured porcine RPE cells . GAPDH was used to confirm the cDNA quality of all the

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

(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 cells . 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 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

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

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

O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O

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


( 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

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

(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 the corneal epithelial cells of the chimeric piglet staining 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 the retina cells of the chimeric piglet staining 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 the chimeric porcine RPE cells are shown with immunofluorescence staining . 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 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

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

(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 images showed the expression of an RPE marker ( BEST ) in GFP - labeled cells . 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 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

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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 analysis of NFATc1 expression with respect to tumor 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 and p53 , illustrating G1 ‐ G2 and 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 analysis demonstrating NFATc1 strong nuclear positive tumors ( n = 161 patients ) 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 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 ) .

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

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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 " ‐ 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 .

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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 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 .

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

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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 cells . 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 protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor 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 , 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 ) .

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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 and KP ∆ NC mice before tumor 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 and corresponding immunohistochemistry stainings of ( C ) KPNC and ( 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 Tumor 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 analysis of EMT ‐ related markers obtained from tumors derived from mice of the 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 ‐ 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 ) .

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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 analysis revealing protein expression levels in KPNC cells from the indicated proteins 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 , and 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 analysis shows Sox2 expression in pancreatic lysates of ( B ) KNC mice with indicated tumor grading and in ( C ) KPNC and 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 Protein expression levels after siRNA ‐ mediated p53 depletion in KNC tumor cells .

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


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

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

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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 and promoter in KNC tumor cells ( 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 RNA polymerase II binding to Sox2 promoter upon CsA ( 1 μM , 24 h ) treatment in L3 . 6 cells ( 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 and Sox2 was performed in KPNC cells .

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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 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 ) .

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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 levels of EMT ‐ related marker genes upon Sox2 depletion in KPNC tumor cells ( 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 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 ) .

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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 and Sox2 protein expression in L3 . 6 cells ( 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 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 ) .

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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 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 ) .

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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 cells ( 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 light microscopy images as well as quantification 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 analysis of indicated miRNAs upon adenoviral re ‐ expression of p53 in KPΔNC tumor cells , 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 Protein expression of EMT marker genes after adenoviral re ‐ expression of GFP ‐ tagged p53 in KPΔNC tumor cells .

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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 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 ) .

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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 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 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 analysis of EMT marker expression in KNC cells 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 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 .

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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 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 .

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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 Quantification of KP ∆ NC tumour cell - 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 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 ) .

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(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 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 .

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(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-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 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 ) .

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 O B-SMALL_MOLECULE O 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 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 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

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 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 ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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 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 O B-SMALL_MOLECULE O 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 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 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 .

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

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 O B-ORGANISM I-ORGANISM I-ORGANISM O 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 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 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 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 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 ) .

O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O 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 O O O B-TISSUE O O 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 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 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 ) .

O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM 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 B-TISSUE I-TISSUE I-TISSUE I-TISSUE 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 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 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 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 ) .

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 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 O O B-CELL_TYPE I-CELL_TYPE 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 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 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 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 ) .

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

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 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 ) .

O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O 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 I-TISSUE I-TISSUE I-TISSUE 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

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 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 ) .

O O O O B-GENEPROD O O O B-TISSUE O O O B-CELL_TYPE O B-CELL_TYPE 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 B-GENEPROD O O O O O B-ORGANISM 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 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 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 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 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 ) .

O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O 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 O O O B-TISSUE O O 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 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 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 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 ) .

O O O O O O B-EXP_ASSAY O B-TISSUE 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 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 B-TISSUE I-TISSUE I-TISSUE I-TISSUE 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 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 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 ) 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 .

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

(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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O 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 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 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 .

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

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 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 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

O O O O O O O O O O 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 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


( e ; HA - GluN2Bct - CTMm ; P = 0 . 785 compared to control ( white bar ) ; F ( 4 , 35 ) = 0 . 432 , 8 independent experiments from 8 separate cell cultures and transfections ) . Levels of cDAPK1 or wtDAPK1 cotransfected with pcDNA3 . 0 vector ( 0 : 1 , white bar ) represent the control values , arbitrarily set as 1 . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , compared with the control ; n . s . , not significant . Bars represent relative means ± s . e . m . Full - length blots are presented in Supplementary Figure 9 . Membranes reprobed for β - actin were used as loading controls . One - way ANOVA with Fisher least significant difference test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

(e; HA-GluN2Bct-CTMm; P = 0.785 compared to control (white bar); F(4,35) = 0.432, 8 independent experiments from 8 separate cell cultures and transfections). Levels of cDAPK1 or wtDAPK1 cotransfected with pcDNA3.0 vector (0:1, white bar) represent the control values, arbitrarily set as 1. *P 0.05, **P 0.01, ***P 0.001, compared with the control; n.s., not significant. Bars represent relative means ± s.e.m. Full-length blots are presented in Supplementary Figure 9. Membranes reprobed for β-actin were used as loading controls. One-way ANOVA with Fisher least significant difference test.


( a ) Left , NMDA ( 50 μM ; 30 min ) activated DAPK1 , resulting in a time - dependent decrease in its phosphorylation levels ( pDAPK1 , n = 4 ) . Right , coimmunoprecipitation ( IP ) with anti - GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA - induced association between DAPK1 and GluN2B ( n = 3 ) .

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

(a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4). Right, coimmunoprecipitation (IP) with anti-GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA-induced association between DAPK1 and GluN2B (n = 3).


( b ) Design and production of TAT - GluN2Bct - CTM and TAT - GluN2Bct peptides ( left ) using an E . coli expression system . Coomassie blue staining after SDS - PAGE assessed their purity ( right ) ; left lane , size marker .

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

(b) Design and production of TAT-GluN2Bct-CTM and TAT-GluN2Bct peptides (left) using an E. coli expression system. Coomassie blue staining after SDS-PAGE assessed their purity (right); left lane, size marker.


( c ) Bath application of TAT - GluN2Bct - CTM ( 200 μM ; n = 9 ) , but not TAT - GluN2Bct ( 200 μM ; n = 6 ) , knocked down activated DAPK1 , and this was prevented by NH4Cl ( 20 mM ; n = 5 ; one - way ANOVA ; P 0 . 001 , F ( 5 , 36 ) = 10 . 891 ) and dose - ( d , doses in μM ; n = 4 ; P 0 . 001 ; F ( 6 , 21 ) = 18 . 14 ) and time - dependent ( e ; P 0 . 001 ; F ( 8 , 44 ) = 12 . 074 ) . ( f ) A single pretreatment of TAT - GluN2Bct - CTM ( sing ; 200 μM , 60 min before and during the 30 - min NMDA stimulation ) produced a transient reduction of DAPK1 , returning to baseline within 7 h ( n = 4 ; P = 0 . 888 ) , and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h ( mult ; n = 4 ; ΔΔP = 0 . 002 ) . One - way ANOVA ; P 0 . 001 , F ( 4 , 15 ) = 10 . 389 .

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

(c) Bath application of TAT-GluN2Bct-CTM (200 μM; n = 9), but not TAT-GluN2Bct (200 μM; n = 6), knocked down activated DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; one-way ANOVA; P 0.001, F(5,36) = 10.891) and dose- (d, doses in μM; n = 4; P 0.001; F(6,21) = 18.14) and time-dependent (e; P 0.001; F(8,44) = 12.074). (f) A single pretreatment of TAT-GluN2Bct-CTM (sing; 200 μM, 60 min before and during the 30-min NMDA stimulation) produced a transient reduction of DAPK1, returning to baseline within 7 h (n = 4; P = 0.888), and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h (mult; n = 4; ΔΔP = 0.002). One-way ANOVA; P 0.001, F(4,15) = 10.389.


( g ) Synthetic peptides TAT - GluN2B and TAT - GluN2BCTM .

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

(g) Synthetic peptides TAT-GluN2B and TAT-GluN2BCTM.


( h ) TAT - GluN2BCTM ( 25 μM ; n = 5 ; P = 0 . 001 ) , but not control TAT - GluN2B ( 25 μM ; n = 4 ; P = 0 . 223 ) , decreased native DAPK1 , and this was prevented by NH4Cl ( 20 mM ; n = 5 ; P = 0 . 302 ) . One - way ANOVA , P 0 . 001 , F ( 5 , 24 ) = 13 . 591 . Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control ( white bar ; * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ) or NMDA - treated group ( gray bar ; ΔP 0 . 05 , ΔΔP 0 . 01 and ΔΔΔP 0 . 001 ) . Membranes reprobed for β - actin were used as a loading control . Bars represent relative means ± s . e . m . Sample sizes represent number of individual experiments . Full - length blots are presented in Supplementary Figure 9 .

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

(h) TAT-GluN2BCTM (25 μM; n = 5; P = 0.001), but not control TAT-GluN2B (25 μM; n = 4; P = 0.223), decreased native DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; P = 0.302). One-way ANOVA, P 0.001, F(5,24) = 13.591. Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control (white bar; *P 0.05, **P 0.01 and ***P 0.001) or NMDA-treated group (gray bar; ΔP 0.05, ΔΔP 0.01 and ΔΔΔP 0.001). Membranes reprobed for β-actin were used as a loading control. Bars represent relative means ± s.e.m. Sample sizes represent number of individual experiments. Full-length blots are presented in Supplementary Figure 9.


( a ) Top , the synthetic cell - penetrating α - synuclein targeting peptide TAT - βsynCTM and its control TAT - βsyn . Middle , immunoblots demonstrate that TAT - βsynCTM ( 25 μM ; n = 5 ) , but not the CTM - lacking control peptide TAT - βsyn ( 25 μM ; n = 5 ) , specifically decreased the targeted endogenous α - synuclein ( one - way ANOVA , Tukey post hoc , P 0 . 001 , F ( 3 , 16 ) = 12 . 435 ) , without affecting the level of unrelated control protein PSD - 95 at 4 h ( bottom ) , and this reduction was prevented in the presence of NH4Cl ( 20 mM ; n = 5 ) . Sample sizes represent individual experiments from at least 3 separate primary cultures . (

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

(a) Top, the synthetic cell-penetrating α-synuclein targeting peptide TAT-βsynCTM and its control TAT-βsyn. Middle, immunoblots demonstrate that TAT-βsynCTM (25 μM; n = 5), but not the CTM-lacking control peptide TAT-βsyn (25 μM; n = 5), specifically decreased the targeted endogenous α-synuclein (one-way ANOVA, Tukey post hoc, P 0.001, F(3,16) = 12.435), without affecting the level of unrelated control protein PSD-95 at 4 h (bottom), and this reduction was prevented in the presence of NH4Cl (20 mM; n = 5). Sample sizes represent individual experiments from at least 3 separate primary cultures. (


( b ) Top , PSD - 95 targeting peptide TAT - GluN2B9cCTM and control TAT - GluN2B9c . Middle , TAT - GluN2B9cCTM ( 25 μM ; n = 4 ) , but not Tat - GluN2B9c ( 25 μM ; n = 4 ) , effectively degraded endogenous PSD - 95 ( one - way ANOVA , Tukey post hoc , P 0 . 001 , F ( 3 , 12 ) = 18 . 154 ) without perturbing untargeted protein α - synuclein ( bottom ) . NH4Cl rescued PSD - 95 degradation . Sample sizes represent individual experiments from at least 2 separate primary cultures . Membrane reprobing for β - actin was used as an additional specificity and loading control . ΔP 0 . 05 , * * , ΔΔP 0 . 01 and * * * P 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to the saline control ( arbitrarily set as 1 ) . Full - length blots are presented in Supplementary Figure 9 .

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

(b) Top, PSD-95 targeting peptide TAT-GluN2B9cCTM and control TAT-GluN2B9c. Middle, TAT-GluN2B9cCTM (25 μM; n = 4), but not Tat-GluN2B9c (25 μM; n = 4), effectively degraded endogenous PSD-95 (one-way ANOVA, Tukey post hoc, P 0.001, F(3,12) = 18.154) without perturbing untargeted protein α-synuclein (bottom). NH4Cl rescued PSD-95 degradation. Sample sizes represent individual experiments from at least 2 separate primary cultures. Membrane reprobing for β-actin was used as an additional specificity and loading control. ΔP 0.05, **,ΔΔP 0.01 and ***P 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (arbitrarily set as 1). Full-length blots are presented in Supplementary Figure 9.


( a ) Immunoblotting for phosphorylated DAPK1 ( pDAPK1 ) revealed a time - dependent activation ( dephosphorylation ) of DAPK1 by H2O2 treatment ( 300 μM ; 30 min ; n = 4 ) .

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

(a) Immunoblotting for phosphorylated DAPK1 (pDAPK1) revealed a time-dependent activation (dephosphorylation) of DAPK1 by H2O2 treatment (300 μM; 30 min; n = 4).


( b ) Bath application of 100 μM TAT - GluN2Bct - CTM ( 36 . 54 ± 7 . 1 % of control ; n = 8 ; P = 0 . 001 ) , but not TAT - GluN2Bct ( 89 . 13 ± 10 . 78 % ; n = 7 ; P = 0 . 311 ) , 60 min before and during H2O2 treatment ( 300 μM ; 30 min ) knocked down DAPK1 at 2 h after washout , and this effect was rescued by NH4Cl ( 20 mM ; n = 8 ; * * P = 0 . 003 to control ; P = 0 . 106 to H2O2 - treated ) . One - way ANOVA , F ( 4 , 34 ) = 11 . 628 , P 0 . 001 . Bars represent DAPK1 levels relative to saline control group . β - actin was used as a loading control .

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

(b) Bath application of 100 μM TAT-GluN2Bct-CTM (36.54 ± 7.1% of control; n = 8; P = 0.001), but not TAT-GluN2Bct (89.13 ± 10.78%; n = 7; P = 0.311), 60 min before and during H2O2 treatment (300 μM; 30 min) knocked down DAPK1 at 2 h after washout, and this effect was rescued by NH4Cl (20 mM; n = 8; **P = 0.003 to control; P = 0.106 to H2O2-treated). One-way ANOVA, F(4,34) = 11.628, P 0.001. Bars represent DAPK1 levels relative to saline control group. β-actin was used as a loading control.


( c ) LDH assay revealed that H2O2 treatment ( 300 μM ; 30 min ) resulted in a significant increase in neuronal death 12 h after treatment ( n = 8 ; 2 . 50 ± 0 . 12 ; P 0 . 001 to control ) , which was rescued by breaking down H2O2 with catalase ( 100 U in 10 μl phosphate - buffered saline ; n = 4 ; 1 . 17 ± 0 . 02 ; P = 0 . 001 to H2O2 group ) . H2O2 - induced neurotoxicity was significantly reduced by TAT - GluN2Bct - CTM ( 50 μM ; applied 60 min before and maintained throughout the experiments ; n = 9 ; 1 . 56 ± 0 . 08 ; P = 0 . 001 to H2O2 group ) , but not by TAT - GluN2Bct ( 50 μM ; n = 9 ; 2 . 63 ± 0 . 10 ; P = 0 . 105 to H2O2 group ) or the NMDAR antagonist APV ( 1 μM ; n = 4 ; 2 . 16 ± 0 . 14 ; P = 0 . 169 to H2O2 group ) . NH4Cl abolished the neuroprotective effect of TAT - GluN2Bct - CTM ( n = 4 ; 2 . 39 ± 0 . 27 ; P = 0 . 538 compared to H2O2 group ) . One - way ANOVA , P 0 . 001 , F ( 6 , 41 ) = 26 . 842 . * * * , ΔΔΔP 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to the saline control ( white bar , arbitrarily set as 1 ) . n represents individual experiments from at least 3 separate primary cultures . Full - length blots are presented in Supplementary Figure 9 .

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

(c) LDH assay revealed that H2O2 treatment (300 μM; 30 min) resulted in a significant increase in neuronal death 12 h after treatment (n = 8; 2.50 ± 0.12; P 0.001 to control), which was rescued by breaking down H2O2 with catalase (100 U in 10 μl phosphate-buffered saline; n = 4; 1.17 ± 0.02; P = 0.001 to H2O2 group). H2O2-induced neurotoxicity was significantly reduced by TAT-GluN2Bct-CTM (50 μM; applied 60 min before and maintained throughout the experiments; n = 9; 1.56 ± 0.08; P = 0.001 to H2O2 group), but not by TAT-GluN2Bct (50 μM; n = 9; 2.63 ± 0.10; P = 0.105 to H2O2 group) or the NMDAR antagonist APV (1 μM; n = 4; 2.16 ± 0.14; P = 0.169 to H2O2 group). NH4Cl abolished the neuroprotective effect of TAT-GluN2Bct-CTM (n = 4; 2.39 ± 0.27; P = 0.538 compared to H2O2 group). One-way ANOVA, P 0.001, F(6,41) = 26.842. ***,ΔΔΔP 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). n represents individual experiments from at least 3 separate primary cultures. Full-length blots are presented in Supplementary Figure 9.


( b ) 2 , 3 , 5 - Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo . ( c ) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE 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 B-TISSUE O O O B-EXP_ASSAY O

(b) 2,3,5-Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo. (c) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting.


( d ) Immunoblots demonstrated specific DAPK1 knockdown in the infarct ( Ipsi ) but not contralateral ( Contra ) side following application of TAT - GluN2BCTM ( 10 mg per kilogram , i . v . ; n = 3 ; t ( 4 ) = 14 . 459 , P 0 . 001 ) but not TAT - GluN2B ( 10 mg per kilogram , i . v . ; n = 3 ; t ( 4 ) = 0 . 739 , P = 0 . 501 ) . β - actin was used as a loading control ; two - tailed Student ' s t - test * * * P 0 . 001 ; n . s . , not significant .

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

(d) Immunoblots demonstrated specific DAPK1 knockdown in the infarct (Ipsi) but not contralateral (Contra) side following application of TAT-GluN2BCTM (10 mg per kilogram, i.v.; n = 3; t(4) = 14.459, P 0.001) but not TAT-GluN2B (10 mg per kilogram, i.v.; n = 3; t(4) = 0.739, P = 0.501). β-actin was used as a loading control; two-tailed Student's t-test ***P 0.001; n.s., not significant.


( e ) HE ( left ) and immunohistochemical DAPK1 ( right ) staining of adjacent brain sections . Compared with those in saline ( top ) and TAT - GluN2B - treated ( middle ) controls , TAT - GluN2BCTM treatment ( bottom ) selectively reduced infarct area ( left ) and DAPK1 levels ( right ) ipsilaterally . Black outlining delineates infarct areas as visualized with HE staining .

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

(e) HE (left) and immunohistochemical DAPK1 (right) staining of adjacent brain sections. Compared with those in saline (top) and TAT-GluN2B-treated (middle) controls, TAT-GluN2BCTM treatment (bottom) selectively reduced infarct area (left) and DAPK1 levels (right) ipsilaterally. Black outlining delineates infarct areas as visualized with HE staining.


( f ) Left , brain sections stained with Fluorojade B in rats injected with saline ( n = 6 ) , TAT - GluN2B ( n = 5 ) or TAT - GluN2BCTM ( n = 5 ) after treatment as shown in a . Right , quantification of cellular damage by counting the number of Fluorojade B - positive cells in each 640 × 640 pixel image at 10 × magnification . TAT - GluN2BCTM ( 10 mg per kilogram ) displayed more prominent neuroprotection in the cortex ( P 0 . 001 ) and striatum ( P 0 . 001 ) as compared to TAT - GluN2B ( 10 mg per kilogram ) . Cortex : H ( 2 ) = 41 . 235 ; P 0 . 001 ; striatum : H ( 2 ) = 38 . 808 ; P 0 . 001 . Kruskal - Wallis ANOVA on ranks with Dunn ' s post hoc ; bars represent relative means ± s . e . m . , * * * , ΔΔΔP 0 . 001 . n values represent tissue from 3 animals collected from at least 2 litters . Scale bars 1 mm in b , c , e , 20 μm in f . Full - length blots are presented in Supplementary Figure 9 .

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

(f) Left, brain sections stained with Fluorojade B in rats injected with saline (n = 6), TAT-GluN2B (n = 5) or TAT-GluN2BCTM (n = 5) after treatment as shown in a. Right, quantification of cellular damage by counting the number of Fluorojade B-positive cells in each 640 × 640 pixel image at 10× magnification. TAT-GluN2BCTM (10 mg per kilogram) displayed more prominent neuroprotection in the cortex (P 0.001) and striatum (P 0.001) as compared to TAT-GluN2B (10 mg per kilogram). Cortex: H(2) = 41.235; P 0.001; striatum: H(2) = 38.808; P 0.001. Kruskal-Wallis ANOVA on ranks with Dunn's post hoc; bars represent relative means ± s.e.m., ***,ΔΔΔP 0.001. n values represent tissue from 3 animals collected from at least 2 litters. Scale bars 1 mm in b,c,e, 20 μm in f. Full-length blots are presented in Supplementary Figure 9.


( L ) Larval NSCs of control ( ctrl ; yw ) , pr - set7716 / Df , and UAS - pr - set7 induced with insc - Gal4 at 72h ALH were stained for Pr - set7 , Mira and DNA . ( M , N ) Quantifications of NSC diameters ( M ) at 24h ALH and EdU incorporation ( N ) for genotypes in ( O ) . In ( M ) , n = 152 for control ; n = 129 for pr - set7716 / Df . In ( N ) , for control , n = 601 , 13 BL at 24h ALH ; n = 988 , 14 BL at 48h ALH ; n = 813 , 9 BL at 72h ALH and n = 829 , 10 BL at 96h ALH . For pr - set7716 / Df , n = 566 , 12 BL at 24h ALH ; n = 808 , 13 BL at 48h ALH ; n = 1032 , 10 BL at 72h ALH and n = 825 , 9 BL at 96h ALH . Data information : Quantification data are presented in as mean ± SD M , N ) . n numbers in N ) were total NSCs counted with total brain lobes measured , while each brain lobe considered as one biological replicate . White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann - Whitney test M , N ) . * * * P < 0 . 001 . Scale bars , 5 μm L ) ,

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

(L) Larval NSCs of control (ctrl; yw), pr-set7716/Df, and UAS-pr-set7 induced with insc-Gal4 at 72h ALH were stained for Pr-set7, Mira and DNA. (M, N) Quantifications of NSC diameters (M) at 24h ALH and EdU incorporation (N) for genotypes in (O). In (M), n=152 for control; n=129 for pr-set7716/Df. In (N), for control, n=601, 13 BL at 24h ALH; n=988, 14 BL at 48h ALH; n=813, 9 BL at 72h ALH and n=829, 10 BL at 96h ALH. For pr-set7716/Df, n=566, 12 BL at 24h ALH; n=808, 13 BL at 48h ALH; n=1032, 10 BL at 72h ALH and n=825, 9 BL at 96h ALH. Data information: Quantification data are presented in as mean ± SD M, N). n numbers in N) were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann-Whitney test M, N). ***P < 0.001. Scale bars, 5 μm L),