A . Imaging of SPBs ( Cnm67 - RFP ) Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control spo13 mutants . Top , time - lapse series . Bottom , meiotic events were quantified in synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP MPC70 - GFP strains . Data information : data are representative of two independent experiments .

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

A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of two independent experiments.


C . Imaging of SPBs ( Cnm67 - RFP ) Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control containing clb1 ∆ or cdc28 - as1 , which have been treated with 1NM - PP1 at metaphase I ( 8 h in SPM ) . Meiotic events ( bottom ) were analyzed as in ( A ) . Data information : data are representative of two independent experiments .

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

C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.


A . Imaging of SPBs ( Cnm67 - RFP ) Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ containing SPC72 , spc72 - 7 , or spc72 - 7 plus hrr25 - as . were shifted from 24 to 36 ° C at 4 h in SPM treated with 1NM - PP1 to inhibit Hrr25 - as . Top , time - lapse series . Bottom , Mpc70 loading to SPBs was quantified in synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Data information : data are representative of three independent experiments .

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

A. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus hrr25-as. Cells were shifted from 24 to 36°C at 4 h in SPM and treated with 1NM-PP1 to inhibit Hrr25-as. Top, time-lapse series. Bottom, Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Data information: data are representative of three independent experiments.


C . Imaging of SPBs ( Cnm67 - RFP ) Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ spo13 ∆ control expressing an additional copy of SPC72 from PEST promoter at 4 h in SPM . Mpc70 loading ( bottom ) was analyzed as in ( A ) . PEST - SPC72 expression delays time of Mpc70 loading by 98 min ( 95 % CI , 67 - 129 ; P < 0 . 0001 ; Welch ' s t - ) . Data information : data are representative of three independent experiments .

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

C. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70 loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the time of Mpc70 loading by 98 min (95% CI, 67-129; P < 0.0001; Welch's t-test). Data information: data are representative of three independent experiments.


( B ) of containing wild - type Spo13 , Spo13 - mD , or Spo13 - mD - m2 . Meiotic events were quantified in synchronized in silico to SPB reduplication at entry into metaphase II ( t = 0 ) . Graphs show overlays of SPC72 - GFP MPC70 - GFP strains . spo13 - mD delays Spc72 removal Mpc70 loading by 30 min ( 95 % CI , 26 - 34 ; P < 0 . 0001 ) 60 min ( 95 % CI , 55 - 66 ; P < 0 . 0001 ; Welch ' s t - ) , respectively . ( C ) of expressing Clb1 - mDK different versions of Spo13 . Meiotic events were quantified as in ( B ) . spo13 - mD plus PEST - clb1 - mDK delays Spc72 removal Mpc70 loading by 72 min ( 95 % CI , 63 - 80 ; P < 0 . 0001 ) 114 min ( 95 % CI , 99 - 129 ; P < 0 . 0001 ; Welch ' s t - ) , respectively .

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

(B) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively.


C . Cdc5 interacts with Ime2 . Cdc5 - ha3 , Ime2 , Spo13 were detected by immunoblotting in whole - extracts α - Ha immunoprecipitates from indicated strains . Data information : data are representative of two independent experiments .

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

C. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains. Data information: data are representative of two independent experiments.


D . Cdc5 ' s polo - box domain ( PBD ) binds to Ime2 . Ha3 - tagged versions of wild - type PBD or phosphopeptide - binding mutant PBD - FAA were expressed at t = 5 . 5 h in SPM in depleted of Cdc20 Cdc5 . PBD - ha3 Ime2 were detected by immunoblotting in whole - extracts α - Ha immunoprecipitates . Data information : data are representative of two independent experiments .

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

D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates. Data information: data are representative of two independent experiments.


E . Cdc5 - dependent modification of Ime2 . PHSL1 - CDC20 ama1 ∆ control containing spo13 ∆ or cdc5 - as were treated with CMK ( to inhibit Cdc5 - as ) at t = 8 h in SPM ( arrow heads ) . panel shows immunoblot of whole - extracts . C , sample from proliferating . Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5 - as . arrow marks modified form of Ime2 . Data information : data are representative of two independent experiments .

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

E. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2. Data information: data are representative of two independent experiments.


C , D . Immunoblot of whole - extracts . C , sample from proliferating . ( C ) in cultures shown in ( A ) . ( D ) in cultures shown in ( B ) .

O O O O O O O O O O O O O O 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, D. Immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. (C) Protein levels in the cultures shown in (A). (D) Protein levels in the cultures shown in (B).


A , B . Mitotic cdc20 - 3 were shifted to 36 ° C for 80 min . At t = 0 , were treated with estradiol to induce expression from PEST promoter . Top , time - lapse series from imaging of SPBs ( Cnm67 - RFP ) Spc72 - GFP . Frame width , 19 μm . Bottom , of with Spc72 - GFP at SPBs . ( A ) Expression of Ime2 - ∆ C induces removal of Spc72 from SPBs , which is inhibited by co - expression of Spo13 . ( B ) Expression of Cdc5 plus Ime2 - ∆ C causes rapid removal of Spc72 , which is inhibited by co - expression of Spo13 . Data information : data are representative of three independent experiments .

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

A, B. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with estradiol to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with Spc72-GFP at SPBs. (A) Expression of Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5 plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13. Data information: data are representative of three independent experiments.


C . Mitotic cdc20 - 3 containing PEST - CDC5 plus PEST - IME2 - ∆ C / or PEST - NDT80 were shifted to 36 ° C for 30 min subsequently treated with estradiol ( t = 0 ) . Top , time - lapse series from imaging of SPBs ( Cnm67 - RFP ) Mpc70 - GFP . Frame width , 19 μm . Bottom , of with Mpc70 - GFP at SPBs . Data information : data are representative of two independent experiments .

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

C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with estradiol (t = 0). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs. Data information: data are representative of two independent experiments.


C . PHSL1 - CDC20 ama1 ∆ cdc28 - as2 control containing spo13 ∆ or cdc5 - as were treated with 1Na - PP1 at t = 3 h in SPM to inhibit Cdc28 - as2 . At t = 9 h in SPM , Cdc28 - as2 was activated by washing with cSPM lacking 1Na - PP1 ( washout ) . cdc5 - as were washed with cSPM plus CMK to activate Cdc28 - as2 inhibit Cdc5 - as . Top , time - lapse series from imaging of Mpc70 - GFP RFP - tubulin . weak , signal originates from Ndt80 - GFP . Bottom , of spindle formation Mpc70 loading to SPBs . D . PHSL1 - CDC20 ama1 ∆ containing SPC72 , spc72 - 7 , or spc72 - 7 plus cdc5 - as were shifted from 24 to 36 ° C at t = 4 . 2 h in SPM ( to inactivate Spc72 - 7 , arrow heads ) treated with CMK at t = 6 h in SPM ( to inhibit Cdc5 - as ) . Top , time - lapse series from imaging of SPBs ( Cnm67 - RFP ) Mpc70 - GFP . Bottom , of SPB separation Mpc70 loading to SPBs . Inhibition of Cdc5 - as in spc72 - 7 advances time of Mpc70 loading by 119 min ( 95 % CI , 76 - 162 ; P < 0 . 0001 ; Welch ' s t - ) . Data are representative of two independent experiments .

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

C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.


A . Inhibition of Cdk1 at anaphase I . CDC20 - mAR CDC20 - mAR cdc28 - as1 were released from metaphase I - arrest with CuSO4 at 7 h in SPM ( t = 0 ) treated with 1NM - PP1 at t = 40 min to inhibit Cdc28 - as1 ( arrows ) . Top , time - lapse series from imaging of Mpc70 - GFP , SPBs ( Cnm67 - RFP ) , nuclei ( TetR - RFP ) . Bottom , of meiotic events .

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

A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events.


( D , E ) representative confocal of LC3 - II accumulation in CTNSWT CTNS - / - in presence absence of 25 nM bafilomycin ( BafA1 ) for 4 hrs , respectively ( n = 3 ) . Scale bars are 20 µm .

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

(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.


( F , G , H ) Western blotting densitometric analyses for LC3 - II / LC3 - I SQSTM1 in CTNSWT , CTNS - / - , CTNSPatient cultured in presence or in absence of 25 nM BafA1 for 4 h , respectively ( n = 3 ) .

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

(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).


( I , J ) Representative confocal of DQ - BSA BSA in CTNSWT , CTNS - / - , CTNSPatient , respectively ( n = 7 - 14 quantified ) . Scale bars are 20 µm .

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

(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.


( D ) List of metabolites that were shared significantly altered in both CTNS - / - CTNSPatient compared to control ( n = 6 ) .

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

(D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6).


( I ) List of that were significantly upregulated downregulated in CTNS - / - compared to control ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , LIPA ; Lysosomal acid lipase , ACP2 ; Lysosomal acid phosphatase , CTSS ; Cathepsin S , CTSC ; Dipeptidyl peptidase , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , SORT1 ; Sortilin , CAT ; Catalase , CTSA ; Lysosomal protective , SOD ; Superoxide dismutase .

(I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.


( B , C , D ) ROS production ( ROS / mg ) in CTNSWT , CTNS ‑ / - , CTNSPatient upon starvation for 4 hrs in presence absence of DMKG ( 4 hrs ) , respectively ( n = 3 ) .

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

(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).


Representative confocal ( scale bars are 20 µm ) of caspase 3 / 7 activation in DMKG ( 2 mM ) - treated CTNSWT , CTNS ‑ / - , CTNSPatient for 24 hrs ( n = 3 ) .

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

Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).


( H , I ) Western blotting densitometric analyses for LC3 - II / LC3 - I in CTNSWT , CTNS ‑ / - , CTNSPatient cultured in presence or in absence of BafA1 ( 25 nM ) DMKG ( 2 mM ) for 4 hrs , respectively ( n = 3 ) .

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

(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).


( E ) , Proteomic of CTNS - / - treated with cysteamine , bicalutamide , a combination of cysteamine bicalutamide , respectively ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , GLUD1 ; GLUD2 ; Mitochondrial glutamate dehydrogenase 1 / 2 , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , GSTK1 ; Glutathione S - transferase kappa 1 , COX6B1 ; Cytochrome c oxidase subunit 6B1 , ACACA ; Acetyl - CoA carboxylase 1 - Biotin carboxylase , GLS ; Glutaminase kidney isoform , mitochondrial , CASP3 ; Caspase - 3 , NPC1 ; Niemann - Pick C1 .

(E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.


( D , E ) Representative confocal of DQ - BSA in CTNSWT , CTNS - / - upon treatment with bicalutamide . Scale bars are 20 µm ( n = 8 - 14 quantified ) .

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

(D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images).


( F ) of cystine ( nmol / mg ) by HPLC - MS / MS in CTNS - / - in absence of drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 6 ) , bicalutamide ( n = 6 ) , a combination of cysteamine bicalutamide ( n = 6 ) . ( G ) of cystine ( nmol / mg ) by HPLC - MS / MS in CTNSPatient in absence of drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 4 ) , bicalutamide ( n = 4 ) , a combination of cysteamine bicalutamide ( n = 6 ) .

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(F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6). (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).


( A ) Immunocytochemistry of - derived cystinotic tubuloids ( CNTSPatient - 1 CTNSPatient - 2 ) healthy kidney tissue - derived control tubuloids ( CNTSWT - 1 CTNSWT - 2 ) for PAX8 , TP63 F - actin . Scale bar = 100 µm .

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(A) Immunocytochemistry of patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) and healthy kidney tissue-derived control tubuloids (CNTSWT-1 and CTNSWT-2) for PAX8, TP63 and F-actin. Scale bar = 100 µm.


( C , D ) of cystine ( nmol / mg ) by HPLC - MS / MS in two different - derived cystinotic tubuloids in absence of drugs ( NT ) or upon treatment with cysteamine ( 100 μM ) , bicalutamide ( 35 μM ) or cysteamine ( 100 μM ) - bicalutamide ( 35 μM ) combination treatment ( n = 3 ) . ( E ) αKG measured in - derived cystinotic tubuloids ( CNTSPatient - 1 CTNSPatient - 2 ) in absence of drugs ( NT ) or upon treatment with cysteamine , bicalutamide or cysteamine - bicalutamide combination treatment using metabolomics ( n = 3 ) .

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(C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3). (E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3).


( I ) Survival rates in ctns - / - zebrafish upon treatment with cysteamine , bicalutamide , a combination of cysteamine bicalutamide ( n = 40 per group ) .

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(I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).


( K ) Hatching rates in surviving ctns - / - zebrafish evaluated at 72 96 hrs post fertilization ( hpf ) with cysteamine , bicalutamide , a combination of cysteamine bicalutamide ( n = 40 per group ) .

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(K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).


A Time course of hippocampal CA1 region of Siglechdtr / dtr mice after DT administration . Sections were stained with an anti - Iba1 antibody ( green ) DAPI ( cyan ) . Arrow : live microglia with normal nucleus . Double arrow : apoptotic microglia with pyknotic nucleus . Arrowhead : microglial debris with no nucleus . Scale bar , 50 µm . B Number of apoptotic microglia with pyknotic or fragmented nucleus ( n = 5 per group , Kruskal - Wallis with post hoc Dunn ' s ) . C Number of live microglia with normal nucleus ( n = 5 per group , one - way ANOVA with post hoc Tukey ' s ) . D Number of pieces of microglial debris ( Iba1 + spheres with a diameter > 2 µm no nucleus ) ( n = 5 per group , Kruskal - Wallis with post hoc Dunn ' s ) .

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A Time course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 µm. B Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). C Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test). D Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 µm and no nucleus) (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test).


E A representative image of a survived microglial in hippocampal CA1 region of Siglechdtr / dtr mice 2 days after DT administration . Sections were stained with an anti - Iba1 antibody ( green ) DAPI ( cyan ) . A 3D image ( rightmost panel ) was reconstructed using Imaris software . Arrow : live microglia . Arrowhead : microglial debris . Scale bar , 10 µm .

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E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 µm.


A Histological of RFP + in Siglechdtr / dtr : Ccr2RFP / + mice . Lumber spinal cord after EAE induction , hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed . Iba1 immunoreactivity ( green ) RFP ( red ) are shown . were acquired using same power sensitivity , image processing was same for RFP signals ( red ) . Scale bar , 50 µm .

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A Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumber spinal cord after EAE induction, and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 µm.


B Flow cytometric of RFP + Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr / dtr : Ccr2RFP / + mice 2 days after PBS or DT administration . Representative data a graph ( n = 5 per group , two - unpaired Student ' s t - ) are shown .

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B Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown.


C Immunohistochemical localization of CD206 + perivascular macrophages ( arrows ) in hippocampal CA1 of Siglechdtr / dtr mice . Sections prepared 2 days after administration of PBS or DT were stained with anti - Iba1 ( green ) , anti - CD206 ( red ) anti - CD31 ( blue ) antibodies . Scale bar , 50 µm . D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr / dtr mice 2 days after PBS or DT administration ( n = 5 per group , two - unpaired Student ' s t - ) .

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C Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red) and anti-CD31 (blue) antibodies. Scale bar, 50 µm. D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test).


A qPCR of marker molecules for CNS types . hippocampus of WT Siglechdtr / dtr mice 2 days after PBS or DT administration was analyzed ( n = 3 per group , one - way ANOVA with post hoc Tukey ' s ) . Results are normalized to Gapdh , are shown as ratios to value of WT mice injected with PBS .

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A qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh, and are shown as ratios to the value of WT mice injected with PBS.


B Immunohistochemical detection of astrocytes in Siglechdtr / dtr mice . Hippocampal CA1 sections were prepared 2 days after PBS or DT administration , stained with an anti - GFAP antibody . were acquired using same power sensitivity , image processing was same . Scale bar , 30 µm . C Astrocyte number in hippocampal CA1 of Siglechdtr / dtr mice 2 days after PBS or DT administration ( n = 5 , two - unpaired Student ' s t - ) .

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B Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 µm. C Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test).


A Representative of microglial debris surrounded by astrocyte processes in hippocampal CA1 . Sections were prepared from Siglechdtr / dtr mice 2 days after PBS or DT administration , stained with anti - GFAP ( green ) anti - CD11b ( red ) antibodies . 3D ( lower row ) were reconstructed from confocal ( upper row ) using Imaris software . Scale bar , 10 µm .

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A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1. Sections were prepared from Siglechdtr/dtr mice 2 days after PBS or DT administration, and stained with anti-GFAP (green) and anti-CD11b (red) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Scale bar, 10 µm.


B CLEM of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr / dtr mice 2 days after DT administration . A phagocytic astrocyte , which engulfed degenerated microglial components , was identified by immunohistochemistry using anti - S100β ( green ) anti - CD11b ( red ) antibodies , DAPI ( cyan ) ( upper middle panels ) . Then , a phagocytic cup of same was analyzed by electron microscopy ( lower panel ) . Degenerated microglial cytoplasm / plasma ( arrow ) nucleus ( double arrow ) in a phagocytic cup of astrocyte , a piece of microglial debris internalized in astrocytic cytoplasm ( arrowhead ) are indicated . Scale bar , 50 µm ( upper panel ) , 10 µm ( upper panel [ inset ] middle panel ) , 2 µm ( lower panel ) .

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B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. A phagocytic astrocyte, which engulfed degenerated microglial components, was identified by immunohistochemistry using anti-S100β (green) and anti-CD11b (red) antibodies, and DAPI (cyan) (upper and middle panels). Then, a phagocytic cup of the same cell was analyzed by electron microscopy (lower panel). Degenerated microglial cytoplasm/plasma membrane (arrow) and nucleus (double arrow) in a phagocytic cup of the astrocyte, and a piece of microglial debris internalized in astrocytic cytoplasm (arrowhead) are indicated. Scale bar, 50 µm (upper panel), 10 µm (upper panel [inset] and middle panel), and 2 µm (lower panel).


C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr / dtr mice 2 days after DT administration . Sections were stained with anti - S100β ( green ) anti - CD11b ( red ) antibodies , confocal were reconstructed using Imaris software . lower two panels are higher magnification of contacted microglial debris . Arrow : microglial debris contacted by astrocyte processes ( coverage : approximately 30 or 50 % ) . Arrowhead : microglial debris internalized in astrocyte cytoplasm . Scale bar , 10 µm ( upper panel ) 2 µm ( lower panels ) . D , E of pieces of debris ( CD11b + spheres with > 0 . 5 µm diameter ) contacted with ( coverage > 30 % ) ( D ) internalized by ( E ) a single astrocyte within 30 µm - thick sections ( n = 75 from five per group ) . number of debris pieces was counted in 3D reconstructed using Imaris software . confocal were acquired using same power sensitivity , image processing was same .

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C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with anti-S100β (green) and anti-CD11b (red) antibodies, and confocal images were reconstructed using Imaris software. The lower two panels are higher magnification images of contacted microglial debris. Arrow: microglial debris contacted by astrocyte processes (coverage: approximately 30 or 50%). Arrowhead: microglial debris internalized in astrocyte cytoplasm. Scale bar, 10 µm (upper panel) and 2 µm (lower panels). D, E Quantification of the pieces of debris (CD11b+ spheres with > 0.5 µm diameter) contacted with (coverage > 30%) (D) and internalized by (E) a single astrocyte within 30 µm-thick sections (n = 75 cells from five animals per group). The number of debris pieces was counted in 3D images reconstructed using Imaris software. All confocal images were acquired using the same laser power and sensitivity, and image processing was the same.


B Expression level of phagocytic receptors their extracellular adaptor molecules ( n = 3 independent experiments ) . Values show mean ± . N . S . : no significance ; * * * q < 0 . 001 . probability values for differential expression were computed with edgeR followed by a correction of multiple testing with q - value .

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B Expression level of phagocytic receptors and their extracellular adaptor molecules (n = 3 independent experiments). Values show the mean ± SEM. N.S.: no significance; ***q < 0.001. The probability values for differential expression analysis were computed with edgeR followed by a correction of multiple testing with q-value.


Cultured astrocytes engulf microglial debris . CAG - EGFP mouse - derived astrocytes were co - cultured with or without - labelled apoptotic microglia . ( A ) of GFP ( green ) , microglial debris ( red ) GFAP immunoreactivity ( blue ) . were acquired using same power sensitivity , image processing was same in two ( red ) . Scale bar , 50 µm .

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Cultured astrocytes engulf microglial debris. CAG-EGFP mouse-derived primary astrocytes were co-cultured with or without fluorescence-labelled apoptotic microglia. (A) Fluorescence images of GFP (green), microglial debris (red) and GFAP immunoreactivity (blue). Images were acquired using the same laser power and sensitivity, and image processing was the same in the two images (red). Scale bar, 50 µm.


( B ) of GFP ( green ) , microglial debris ( red ) LAMP - 1 immunoreactivity ( cyan ) . Scale bar , 30 µm 5 µm ( inset ) .

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(B) Fluorescence images of GFP (green), microglial debris (red) and LAMP-1 immunoreactivity (cyan). Scale bar, 30 µm and 5 µm (inset).


C Flow cytometric of uptake of microglial debris by cultured astrocytes . of internalized microglial debris was measured in GLAST + astrocytes co - cultured with or without - labelled apoptotic microglia .

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C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes. Fluorescence of internalized microglial debris was measured in GLAST+ astrocytes co-cultured with or without fluorescence-labelled apoptotic microglia.


D Expression of TAM family members their knockdown by siRNA in cultured astrocytes . Tyro3 , Axl Metk in cultured astrocytes or hippocampal tissue were examined by western blotting . Arrows indicate corresponding band ( s ) . secondary antibody against goat IgG generates a ~ 115 kDa non - specific band . ~ 115 kDa band in Axl image is Axl - specific band overlapping non - specific band . ~ 115 kDa band in Mertk image is non - specific band . Axl Mertk expression was knocked down by two siRNAs . GAPDH expression is shown as an internal control . E of siRNA knockdown in cultured astrocytes ( n = 3 independent experiments , one - way ANOVA with repeated measures ) . Knockdown of Tyro3 mRNA was examined by qPCR because of very low expression level even in control astrocytes . Knockdown efficiency of Axl Mertk was quantified from western blots . Results are normalized to Gapdh or GAPDH , are shown as ratios to value of non - transfected astrocytes .

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D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes. Tyro3, Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting. Arrows indicate the corresponding protein band(s). The secondary antibody against goat IgG generates a ~115 kDa non-specific band. The ~115 kDa band in the Axl image is the Axl-specific band overlapping the non-specific band. The ~115 kDa band in the Mertk image is the non-specific band. Axl and Mertk expression was knocked down by two siRNAs. GAPDH expression is shown as an internal control. E Quantification of siRNA knockdown in cultured astrocytes (n = 3 independent experiments, one-way ANOVA with repeated measures). Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes. Knockdown efficiency of Axl and Mertk was quantified from western blots. Results are normalized to Gapdh or GAPDH, and are shown as ratios to the value of non-transfected astrocytes.


F , G Flow cytometric of microglial debris uptake by cultured astrocytes . After knockdown of TAM family members , astrocytes were co - cultured with - labelled apoptotic microglia . of internalized microglial debris in GLAST + astrocytes was quantified by flow cytometry ( n = 4 independent experiments , one - way ANOVA with repeated measures ) . Results are shown as ratios to value of non - transfected astrocytes .

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F, G Flow cytometric analysis of microglial debris uptake by cultured astrocytes. After knockdown of TAM family members, astrocytes were co-cultured with fluorescence-labelled apoptotic microglia. Fluorescence of internalized microglial debris in GLAST+ astrocytes was quantified by flow cytometry (n = 4 independent experiments, one-way ANOVA with repeated measures). Results are shown as ratios to the value of non-transfected astrocytes.


B Representative of microglial distribution in cerebral cortex of WT mutant mice at 3 w . Sections were stained with anti - Iba1 or anti - CD11b antibodies ( red ) . Scale bar , 100 µm . C of microglial number ( n = 5 per group , one - way ANOVA ) . Note that Irf8 - / - mice were analyzed by CD11b immunostaining , while other strains were analyzed by Iba1 .

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B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w. Sections were stained with anti-Iba1 or anti-CD11b antibodies (red). Scale bar, 100 µm. C Quantification of microglial number (n = 5 animals per group, one-way ANOVA). Note that Irf8-/- mice were analyzed by CD11b immunostaining, while other strains were analyzed by Iba1 staining.


D Representative of a spontaneous apoptotic surrounding glial in cerebral cortex of WT Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) anti - caspase - 3 ( blue ) antibodies . 3D ( lower row ) were reconstructed from confocal ( upper row ) using Imaris software . Arrows indicate caspase - 3 + apoptotic . Insets are higher magnification around apoptotic . Scale bar , 20 µm 5 µm ( insets ) . E of spontaneous apoptotic surrounded by microglia in WT mutant mice . 3D reconstructed from confocal were analyzed ( n = 36 - 50 from five per group , Kruskal - Wallis with post hoc Dunn ' s ) . F of spontaneous apoptotic surrounded by astrocyte processes in WT mutant mice ( n = 36 - 50 from five per group , one - way ANOVA with post hoc Tukey ' s ) .

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D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets). E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice. 3D images reconstructed from confocal images were analyzed (n = 36-50 cells from five animals per group, Kruskal-Wallis test with post hoc Dunn's test). F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice (n = 36-50 cells from five animals per group, one-way ANOVA with post hoc Tukey's test).


D Representative of a spontaneous apoptotic surrounding glial in cerebral cortex of WT Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) anti - caspase - 3 ( blue ) antibodies . 3D ( lower row ) were reconstructed from confocal ( upper row ) using Imaris software . Arrows indicate caspase - 3 + apoptotic . Insets are higher magnification around apoptotic . Scale bar , 20 µm 5 µm ( insets ) .

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D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets).


Representative of HeLa ( B ) or HCT - 8 ( C ) infected with Shigella WT pre - treated or not with arsenite , analyzed at indicated times post - infection Cfu of intracellular in HeLa HCT - 8 pre - treated or not with arsenite infected with

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Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella


Representative ( E ) cfu ( F ) of intracellular in HeLa infected with Shigella WT after pre - treatment with TNF - α , H2O2 , anisomycin , hypoxia corresponding controls , analyzed at 0 . 5

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Representative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia and corresponding controls, analyzed at 0.5 hpi


Representative ( G ) cfu ( H ) of intracellular Shigella in HeLa pre - treated with arsenite , anisomycin , stressors plus NAC , corresponding

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Representative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls


Representative ( A ) cfu ( B ) of Shigella WT or ΔipaB mutant bound to HeLa pre - treated or not with arsenite . Ruffle formation induced by Shigella WT in panel ( A ) is indicated by

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Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads


Cfu of Shigella WT or ΔipaB mutant bound to HeLa pre - treated with anisomycin or DMSO ( control )

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Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control)


Representative ( D ) cfu ( E ) of Shigella WT or ΔipaB mutant bound to HCT - 8 pre - treated or not with

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Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite


Representative image ) , cf qRT - PCR of intracellular Shigella in HeLa pre - treated with arsenite in presence or not of amitriptyline , corresponding

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Representative image ), cf and qRT-PCR quantification of intracellular Shigella in HeLa cells pre-treated with arsenite in the presence or not of amitriptyline, and corresponding controls


Representative image cf qRT - PC of intracellular Shigella in HeLa pre - treated wit anisomycin in presence or not of amitriptyline , corresponding

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Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells pre-treated wit anisomycin in the presence or not of amitriptyline, and corresponding controls


Representative image cf qRT - PC of intracellular Shigella in HeLa transfected with ASM siRNA or control siRNA pre - treated or not with arsenite prior to

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Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection


3D reconstruction of representative of ASM ( J , K ) or ceramide ( L , M ) in HeLa treated with arsenite or anisomycin corresponding controls . ASM , ceramide Hoechst were surface converted by voxel

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3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide and Hoechst staining were surface converted by voxel distance


Western blot of p38 MAPK phosphorylation in HeLa treated with arsenite , TNF - α , H2O2 or anisomycin ; β - actin was used as loading

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Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite, TNF-α, H2O2 or anisomycin; β-actin was used as loading control


Representative image of intracellular Shigella in HeLa pre - treated with arsenite in absence or presence of p38 inhibitor SB203580 , corresponding

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Representative image of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


cfu quantificatio of intracellular Shigella in HeLa pre - treated with arsenite in absence or presence of p38 inhibitor SB203580 , corresponding

cfu quantificatio of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


Cfu of intracellular Shigella in HeLa transfected with p38 siRNA or control siRNA pre - treated or not with arsenite prior to

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Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not with arsenite prior to infection


cfu quantificatio of intracellular Shigella in HeLa pre - treated wit anysomycin in absence or presence of p38 inhibitor SB203580 , corresponding

cfu quantificatio of intracellular Shigella in HeLa cells pre-treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


Cfu of intracellular Shigella in HeLa transfected with p38 siRNA or control siRNA pre - treated or not wit anisomycin prior to

Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not wit anisomycin prior to infection


3D reconstruction of representative of ceramide in HeLa treated with arsenite ) , in presence or absence of SB203580 , corresponding controls . Ceramide Hoechst were surface converted by voxel

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3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance


3D reconstruction of representative of ceramide in HeLa treated wit anisomyci ) , in presence or absence of SB203580 , corresponding controls . Ceramide Hoechst were surface converted by voxel

3D reconstruction of representative images of ceramide staining in HeLa cells treated wit anisomyci ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance


Representative ( A ) , cfu ( B qRT - PCR of intracellular Salmonella in HeLa pre - treated with arsenite or anisomycin corresponding controls Infection was performed with Salmonella WT analyzed at 0 . 5

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Representative images (A), cfu (B and qRT-PCR quantification of intracellular Salmonella in HeLa cells pre-treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0.5 hpi


Representative of bound to HeLa pre - treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant for 10

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Representative images of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10 min


Violin plots showing distribution of number of bound per infected in HeLa pre - treated or not with arsenite followed by incubation with Salmonella WT ( E ) or Δ4 mutant ( F ) for 10

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Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT (E) or Δ4 mutant strain (F) for 10 min


Representative ( G ) cfu ( H ) of bound to HeLa pre - treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC , ΔfliC / pFliC or ΔflhC for 10

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Representative images (G) and cfu quantification (H) of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC, ΔfliC/pFliC or ΔflhC for 10 min


Representative ( I ) cfu ( J ) of Listeria bound to HeLa pre - treated or not with arsenite followed by incubation with for 20

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Representative images (I) and cfu quantification (J) of Listeria bound to HeLa cells pre-treated or not with arsenite followed by incubation with the bacteria for 20 min


Representative ( K ) cfu ( L ) of Yersinia bound / internalized by HeLa pre - treated or not with arsenite followed by 70 min incubation with

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Representative images (K) and cfu quantification (L) of Yersinia bound/internalized by HeLa cells pre-treated or not with arsenite followed by 70 min incubation with the bacteria


Schematic representation of experimental design for reinfection experiments . were infected with Shigella - mCherry ( infection ) or mock - treated . At 3 hpi , were re - infected with Shigella expressing GFP ( secondary infection ) . Secondary infection was analyzed at 0 . 5 hpi ( i . e . 1 h after addition of to ) Representative image cfu of re - infection assays with Shigella W of HeLa primarily infected with Shigella WT or ΔipaB / Inv mutant , or mock -

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Schematic representation of the experimental design for reinfection experiments. Cells were infected with Shigella-mCherry (primary infection) or mock-treated. At 3 hpi, cells were re-infected with Shigella expressing GFP (secondary infection). Secondary infection was analyzed at 0.5 hpi (i.e. 1 h after addition of bacteria to cells) Representative image and cfu quantification of re-infection assays with Shigella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated


Representative image cfu quantificatio of re - infection assays wit Salmonella W of HeLa primarily infected with Shigella WT or ΔipaB / Inv mutant , or mock -

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Representative image and cfu quantificatio of re-infection assays wit Salmonella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated


Representative ( F ) cfu ( G ) of re - infection assays with Salmonella ΔfliC , ΔfliC / pFliC or ΔflhC mutant strains in HeLa primarily infected with Shigella WT or mock -

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Representative images (F) and cfu quantification (G) of the re-infection assays with Salmonella ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock-treated


Schematic representation of experimental design for reinfections upon knockdown or inhibitor treatment . I J . Representative ( I ) cfu ( J ) of secondary infection with Shigella WT , in transfected with ASM siRNA or control

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Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment. I and J. Representative images (I) and cfu quantification (J) of the secondary infection with Shigella WT, in cells transfected with ASM siRNA or control siRNA


Cfu of secondary infection with Shigella WT in HeLa following

Cfu quantification of the secondary infection with Shigella WT in HeLa cells following amitriptyline


Cfu of secondary infection with Shigella WT in HeLa followin transfectio with MAPK p38

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Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin transfectio with MAPK p38 siRN


Cfu of secondary infection with Shigella WT in HeLa followin SB203580

Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin SB203580 treatmen


3D reconstruction of representative of ASM ( A ) or ceramide ( B ) in HeLa infected with Shigella WTor mock - treated , analyzed at 3 hpi . ASM , ceramide , Hoechst Shigella were surface converted by voxel

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3D reconstruction of representative images of ASM (A) or ceramide (B) staining in HeLa cells infected with Shigella WTor mock-treated, analyzed at 3 hpi. ASM, ceramide, Hoechst and Shigella staining were surface converted by voxel distance


ASM enzymatic activity measurement in HeLa infected with Shigella - WT or mock - treated , analyzed at 3 hpi . ASM enzymatic activity was determined in fraction corresponding to 3 . 0 × 105 per

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ASM enzymatic activity measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi. The ASM enzymatic activity was determined in the membrane fraction corresponding to 3.0×105 cells per condition


ceramide measurement in HeLa infected with Shigella - WT or mock - treated , analyzed at 3 hpi Ceramide is shown normalized to mock - treated

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ceramide measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi Ceramide is shown normalized to mock-treated cells


Representative spinning - disk confocal of colon tissue sections of non - infected ( E F ) Shigella infected ( G H ) guinea pigs , collected at 4 hpi . E G are montages of multiple image fields ; dashed boxes in ( E G ) are shown enlarged in ( F H ) ,

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Representative spinning-disk confocal images of colon tissue sections of non-infected (E and F) and Shigella infected (G and H) guinea pigs, collected at 4 hpi. E and G are montages of multiple image fields; dashed boxes in (E and G) are shown enlarged in (F and H), respectively


Mean of ceramide signal in surface epithelial ( outermost tissue layer ) of non - infected or Shigella - infected guinea pig colon tissue

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Mean fluorescence intensity of ceramide signal in surface epithelial cells (outermost tissue layer) of non-infected or Shigella-infected guinea pig colon tissue sections


A mRNA value of AKT2 was analyzed in GEO Profiles from liver explant of Hepatitis B ( HBV ) - associated acute liver failure ( ALF ) ( GDS4387 / 225471 _ s _ at ) , from bronchial epithelial with pandemic seasonal H1N1 influenza infections in vitro ( GDS4855 / 203808 _ at ) . normal , n = 10 ; HBV , n = 17 ; control , n = 3 ; influenza A , n = 9 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using unpaired t - Error bars mean ±

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A The mRNA value of AKT2 was analyzed in the GEO Profiles from the liver explant of Hepatitis B virus (HBV)-associated acute liver failure (ALF) patients (GDS4387/225471_s_at), and from bronchial epithelial cells with pandemic and seasonal H1N1 influenza virus infections in vitro (GDS4855/203808_at). normal, n=10; HBV, n=17; control, n=3; influenza A, n=9. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using unpaired t-test Error bars mean ± SD


C qRT - PCR of Akt2 mRNA in PEMs stimulated with lipo - poly ( I : C ) ( 1 μg / mL ) , lipo - poly ( A : T ) ( 1 μg / mL ) , lipo - ISD ( 3 μg / mL ) or HSV - 1 ( MOI , 1 ) for 6 hours . n = 3 , respectively . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using one - way ANOVA Data are from three independent biological replicates Error bars mean ± ) .

O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The qRT-PCR analysis of Akt2 mRNA in PEMs stimulated with lipo-poly(I:C) (1 μg/mL), lipo-poly(A:T) (1 μg/mL), lipo-ISD (3 μg/mL) or HSV-1 (MOI, 1) for 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using one-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).


D mRNA of Akt2 ( red ) Ifnb1 ( black ) were analyzed by qRT - PC in mock VSV ( MOI , 1 ) or LM ( MOI , 1 ) - stimulated PEMs or BMDMs at indicated time . Mock VSV , n = 4 ; mock LM , n ≥ 3 .

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D The mRNA of Akt2 (red line) and Ifnb1 (black line) were analyzed by qRT-PC in the mock and VSV (MOI, 1) or LM (MOI, 1)-stimulated PEMs or BMDMs at the indicated time. Mock and VSV, n=4; mock and LM, n≥3.


G - H PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 ( 10 μM ) ( G ) for 2 hours , or knocked down of Akt2 with siAkt2 - 1 ( 20 nM ) for 48 hours ( H ) , then mRNA level of Ifnb1 was measured by qRT - PCR after lipo - poly ( I : C ) , lipo - poly ( A : T ) , lipo - ISD , VSV , or HSV - 1 stimulation for another 6 hours . n = 3 , respectively . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA Data are from three independent biological replicates Error bars mean ± ) .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 (10 μM) (G) for 2 hours, or knocked down of Akt2 with siAkt2-1 (20 nM) for 48 hours (H), then the mRNA level of Ifnb1 was measured by qRT-PCR after lipo-poly(I:C), lipo-poly(A:T), lipo-ISD, VSV, or HSV-1 stimulation for another 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).


A - B qRT - PCR detection for mRNA expression of Ifnb1 ( A ) , Ifna4 , Cxcl10 Ccl5 ( B ) in WT ( n = 3 ) Akt2 KO ( n = 3 ) PEMs stimulated with lipo - poly ( I : C ) , VSV , lipo - poly ( A : T ) , lipo - ISD or HSV - 1 for 6 hours . C expression of Ifnb1 mRNA level by qRT - PCR in WT ( n = 3 ) Akt2 KO ( n = 3 ) PEMs treated with poly ( I : C ) ( 10 μg / mL ) for 6 hours ( left panel ) or LPS ( 1 μg / mL ) for 2 hours ( right panel ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA Data are from three independent experiments Error bars mean ±

O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B qRT-PCR detection for the mRNA expression of Ifnb1 (A), Ifna4, Cxcl10 and Ccl5 (B) in WT (n=3) and Akt2 KO (n=3) PEMs stimulated with lipo-poly(I:C), VSV, lipo-poly(A:T), lipo-ISD or HSV-1 for 6 hours. C The expression of Ifnb1 mRNA level by qRT-PCR in WT (n=3) and Akt2 KO (n=3) PEMs treated with poly(I:C) (10 μg/mL) for 6 hours (left panel) or LPS (1 μg/mL) for 2 hours (right panel). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM


H Zebrafish larvae were overexpressed indicated for 48 hours challenged with VSV for another 6 hours , then mRNA of ifn1 in zebrafish larvae were measured by qRT - PCR ( left panel ) . Every dot represents three zebrafish . Horizontal square bracket shows statistical of comparison with ' PBS mock ' , shows comparison with ' PBS VSV ' . PBS mock , n = 5 ; PBS VSV , n = 13 ; AKT2 VSV , n = 7 ; AKT2 - T309A / S474A VSV , n = 9 . H & E ( middle panel ) survival rates ( Kaplan - Meier curve ) ( right panel ) were collected from zebrafish larvae after VSV micro - injection for 18 hours or longer . arrows indicated VSV - infected eye skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA or log - rank ( Mantel - Cox ) Data are from three independent biological replicates Error bars mean ± SD ) .

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H Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours, then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT-PCR (left panel). Every dot represents three zebrafish embryos. Horizontal square bracket shows the statistical analysis of the comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=5; PBS VSV, n=13; AKT2 VSV, n=7; AKT2-T309A/S474A VSV, n=9. H&E staining (middle panel) and survival rates (Kaplan-Meier curve) (right panel) were collected from zebrafish larvae after VSV micro-injection for 18 hours or longer. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test or log-rank (Mantel-Cox) test Data are from three independent biological replicates Error bars mean ± SD).


D WT Akt2 KO PEMs were infected with or without VSV for 6 hours . Then , lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti - AKT2 antibody , followed by immunoblotting using anti - IRF3 antibody ( left panel ) . Alternatively , lysates were prepared for immunoprecipitation using anti - IRF3 antibody , followed by immunoblotting using anti - AKT2 antibody ( right panel ) to detect endogenous interaction between AKT2 IRF3 . Data information : * Data are representative of two or three independent biological

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D WT and Akt2 KO PEMs were infected with or without VSV for 6 hours. Then, cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti-AKT2 antibody, followed by immunoblotting using anti-IRF3 antibody (left panel). Alternatively, cell lysates were prepared for immunoprecipitation using anti-IRF3 antibody, followed by immunoblotting using anti-AKT2 antibody (right panel) to detect the endogenous interaction between AKT2 and IRF3. Data information: * Data are representative of two or three independent biological replicates


G Immunofluorescent microscopic imaging for ( left panel ) statistics ( right panel ) for IRF3 translocation in WT Akt2 KO PEMs at 6 hour post - VSV infection . IRF3 ( red ) , Nuclei ( Hoechst , green ) . arrows indicate nuclei with IRF3 translocation . Mock , n = 2 ; VSV , n = 3 . Bar , 20 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA Data are representative of two or three independent biological replicates Error bars mean ± ) .

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G Immunofluorescent microscopic imaging for (left panel) and statistics analysis (right panel) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post-VSV infection. IRF3 (red), Nuclei (Hoechst, green). The white arrows indicate the nuclei with IRF3 translocation. Mock, n=2; VSV, n=3. Bar, 20 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM).


H Immunoassay of IRF3 , AKT2 , LaminB1 GAPDH in fractions from WT Akt2 KO PEMs after VSV treatment for 6 hours . Tubulin LaminB1 were used as nucleic loading control respectively . Data information : * Data are representative of two or three independent biological

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H Immunoassay of IRF3, AKT2, LaminB1 and GAPDH in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 hours. Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates


I Immunoassay of IRF3 , SP1 Tubulin in fractions of HEK293T overexpressed with indicated constructs for 24 hours stimulated with VSV for 6 hours . Tubulin SP1 served as nucleic loading control respectively . Data information : * Data are representative of two or three independent biological

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I Immunoassay of IRF3, SP1 and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 hours and stimulated with VSV for 6 hours. Tubulin and SP1 served as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates


D Immunofluorescence microscopy ( top panel ) of AKT2 - , Flag - IRF3 - or Flag - IRF3 - T207A - overexpressed MEF after poly ( A : T ) stimulation for 6 hours . Flag ( IRF3 , red ) Hoechst ( nuclei , blue ) . Immunoassay of - cytoplasm extractions ( bottom panel ) from HEK293T - IRF3 KO with overexpression of indicated plasmids for 24 hours followed by VSV infection for 6 hours . GAPDH SP1 were used as nucleic loading control respectively . Bar , 20 μm . Data information : * Data are representative of three independent

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D Immunofluorescence microscopy (top panel) of AKT2-, Flag-IRF3- or Flag-IRF3-T207A-overexpressed MEF cells after poly(A:T) stimulation for 6 hours. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay of nuclear-cytoplasm extractions (bottom panel) from HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours. GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively. Bar, 20 μm. Data information: * Data are representative of three independent experiments


F Survival rates ( left panel ) until 72 hours H & E ( right panel ) at 18 hours of zebrafish larvae with indicated expression VSV challenge . arrows indicated VSV - infected eye skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) Data are representative of three independent

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F Survival rates (left panel) until 72 hours and H&E staining (right panel) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


G qRT - PCR of VSV copies in overexpressed zebrafish with VSV challenge at 18 hours later . Every dot represents three zebrafish . Horizontal vertical square bracket shows statistical of comparison with ' PBS mock ' , shows comparison with ' PBS VSV ' . PBS mock , n = 15 ; PBS VSV , n = 9 ; IRF3 VSV , n = 16 ; IRF3 - T207A VSV , n = 11 ; AKT2 VSV , n = 14 ; AKT2 - T309A / S474A VSV , n = 9 ; IRF3 + AKT2 VSV , n = 9 ; IRF3 + AKT2 - T309A / S474A VSV , n = 13 ; IRF3 - T207A + AKT2 VSV , n = 10 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA Data are representative of three independent experiments Error bars mean ± SD ) .

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G qRT-PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=15; PBS VSV, n=9; IRF3 VSV, n=16; IRF3-T207A VSV, n=11; AKT2 VSV, n=14; AKT2-T309A/S474A VSV, n=9; IRF3 + AKT2 VSV, n=9; IRF3 + AKT2-T309A/S474A VSV, n=13; IRF3-T207A + AKT2 VSV, n=10. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are representative of three independent experiments Error bars mean ± SD).


H survival rates of over - expressed zebrafish larvae as long as 72 hours after VSV challenge . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) Data are representative of three independent

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H The survival rates of over-expressed zebrafish larvae as long as 72 hours after the VSV challenge. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


B Survival rates of WT Akt2 KO mice after lethal dose injection of VSV during 120 hours ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) Data are representative of three independent experiments

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B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


E WT Akt2 KO mice were injected with VSV for 24 hours ( 1 × 106 PFU / g , i . v . ) . Representative of VSV infected ( GFP ) were collected by microscope ( left panel ) VSV copies ( WT , n = 6 ; Akt2 KO n = 7 ) were measured by qRT - PCR ( right panel ) in liver of mice . Bar , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a unpaired t - Data are representative of three independent experiments respectively in which different groups of mice were sacrificed quantified for indicated experimental purposes Error bars mean ± SD ) .

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E WT and Akt2 KO mice were injected with VSV for 24 hours (1 × 106 PFU/g, i.v.). Representative images of the VSV infected cells (GFP) were collected by fluorescence microscope (left panel) and the VSV copies (WT, n=6; Akt2 KO n=7) were measured by qRT-PCR (right panel) in the liver of mice. Bar, 100 μm. Data information: *P < 0.05, **P < 0.01; using a unpaired t-test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD).


Bone marrow from WT Akt2 KO mice ( CD45 . 2 ) were adoptively transferred to lethally irradiated CD45 . 1 mice infected with VSV . representative were analyzed Data information : * Data are representative of three independent experiments

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Bone marrow cells from WT and Akt2 KO mice (CD45.2) were adoptively transferred to lethally irradiated CD45.1 mice and infected with VSV. representative images were analyzed Data information: * Data are representative of three independent experiments respectively


I Survival rates of WT mice that were intraperitoneal injected with CCT128930 ( 20 mg / kg ) twice every other day before VSV infection ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) Data are representative of three independent experiments

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I Survival rates of WT mice that were intraperitoneal injected with CCT128930 (20 mg/kg) twice every other day before VSV infection (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


immunoblot of IRF3 , LaminB1 GAPDH in PIC fractions ( C ) from WT Akt2 KO mice treated with TMPD for 2 . GAPDH LaminB1 were used as nucleic loading control respectively . Data information : * Data are representative of three independent

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immunoblot analysis of IRF3, LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions (C) from WT and Akt2 KO mice treated with TMPD for 2 weeks. GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of three independent experiments


H & E - ( E left panel ) immunofluorescence of IgG deposits ( E right panel ) in kidney sections were collected from WT Akt2 KO bone marrow - reconstituted CD45 . 1 mice Bar , 50 μm . Data information : * Data are representative of three independent

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H&E-staining (E left panel) and immunofluorescence of IgG deposits (E right panel) in the kidney sections were collected from WT and Akt2 KO bone marrow-reconstituted CD45.1 mice Bar, 50 μm. Data information: * Data are representative of three independent experiments


J . qRT - PCR of relative mRNA expression of AKT2 ( left panel ) IFNA ( middle panel ) or IFNβ1 ( right panel ) in CD14 positive monocytes isolated from SLE ( n = 9 ) or healthy donors ( n = 9 ) . Data information : * P < 0 . 05 , * * P < 0 . 01 * * * * P < 0 . 0001 ; using unpaired t - or correlation analyses Data are pooled from different individuals Error bars mean ± SD ) .

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J. qRT-PCR analysis of the relative mRNA expression levels of AKT2 (left panel) and IFNA (middle panel) or IFNβ1 (right panel) in CD14 positive monocytes isolated from SLE patients (n=9) or healthy donors (n=9). Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using unpaired t-test or correlation analyses Data are pooled from different individuals Error bars mean ± SD).


K Immunofluorescence of IRF3 ( Red ) , AKT2 ( Green ) , DAPI ( Blue , nucleus ) in CD14 + from SLE followed by of about mean amount of AKT2 in this as well as mean amount of IRF3 in same nucleus with Image J software . ( n = 39 ) were collected 3 . Bar , 2 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 * * * * P < 0 . 0001 ; using correlation analyses Data are pooled from different individuals or representative of three independent

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K Immunofluorescence of IRF3 (Red), AKT2 (Green), DAPI (Blue, nucleus) in CD14+ cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software. Cells (n=39) were collected 3 patients. Bar, 2 μm. Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using correlation analyses Data are pooled from different individuals or representative of three independent experiments


Ex vivo generation of CAR T . Activated human PBMC were left untransduced or incubated with CD8 - LVCD19CAR at an MOI of 2 . Five days later , expression of CD19 - CAR CD8 were determined on CD3 + . Numbers indicate of in respective gate .

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Ex vivo generation of CAR T cells. Activated human PBMC were left untransduced or incubated with CD8-LVCD19CAR at an MOI of 2. Five days later, expression of CD19-CAR and CD8 were determined on CD3+ cells. Numbers indicate the percentage of cells in the respective gate.


Detection of CAR T by vector copy numbers ( VCN ) . Genomic was isolated from peritoneal cavity , spleen blood . VCN were determined in technical duplicates by qPCR for two mice of each group . Presence of B in transplanted PBMC is indicated below .

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Detection of CAR T cells by vector copy numbers (VCN). Genomic DNA was isolated from peritoneal cavity, spleen and blood cells. VCN were determined in technical duplicates by qPCR for two individual mice of each group. Presence of B cells in the transplanted PBMC is indicated below.


isolated from peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for percentages of human CD8 + in CD3 + ( D ) , within fraction of human CD45 + . Representative density plots are shown for peritoneal .

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Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD8+ in CD3+ cells (D), within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.


isolated from peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for percentages of CAR + or RFP + in CD8 + CD8 - fractions , respectively ( E ) within fraction of human CD45 + . Representative density plots are shown for peritoneal .

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Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of CAR+ or RFP+ cells in the CD8+ and CD8- fractions, respectively (E) within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.


isolated from peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for percentages of human CD19 + within fraction of human CD45 + . Representative density plots are shown for peritoneal .

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Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD19+ cells within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.


Mice were transplanted with B - depleted human PBMC then received CD8 - LVCD19CAR ( filled circle ) or PBS ( open circle ) . As control , CD8 - LVCD19CAR or PBS were injected into mice transplanted with non - depleted PBMC . Data represent mean ± SD for groups ( CD8 - LVCD19CAR : n = 6 in D , n = 4 in G ; CD8 - LVRFP : n = 4 ; PBS : n = 4 in G , others n = 3 ) .

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Mice were transplanted with B-cell depleted human PBMC and then received CD8-LVCD19CAR (filled circle) or PBS (open circle). As control, CD8-LVCD19CAR or PBS were injected into mice transplanted with non-depleted PBMC. Data represent mean ± SD for all groups (CD8-LVCD19CAR: n=6 in D, n=4 in G; CD8-LVRFP: n=4; PBS: n=4 in G, all others n=3).


CAR T in CD8 + CD8 - T harvested from blood , spleen bone marrow of PBS ( PBS ) or vector - injected ( CAR ) mice determined by flow cytometry .

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CAR T cell levels in the CD8+ and CD8- T cells harvested from blood, spleen and bone marrow of PBS (PBS) or vector-injected (CAR) mice determined by flow cytometry.


Detection of CAR T by determining vector copy numbers ( VCN ) in genomic isolated from CD8 + enriched harvested from bone marrow .

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Detection of CAR T cells by determining vector copy numbers (VCN) in genomic DNA isolated from CD8+ enriched cells harvested from bone marrow.


Detection of CAR T in bone marrow quantified by flow cytometry .

Detection of CAR T cells in bone marrow quantified by flow cytometry.


CD19 + B in blood , spleen bone marrow determined by flow cytometry .

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CD19+ B cell levels in blood, spleen and bone marrow determined by flow cytometry.


Relative human CD19 + B level in blood calculated by normalizing at day were sacrificed to those before vector administration .

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Relative human CD19+ B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration.


Cytokine in plasma of mice obtained from blood at 7 after vector injection . distinct symbols used for each mouse are identical to ones used in Fig . 2 . Mean values ± SD of n = 2 technical replicates .

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Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig. 2. Mean values ± SD of n=2 technical replicates.


Immunohistochemistry of paraffin embedded sections from lungs , brain meninges , brain striatum , spleen liver of vector - injected mouse M16 ( CAR ) a control mouse injected with PBS ( PBS ) stained against CD8 ( αCD8 ) , CAR ( αCAR ) , CD4 ( αCD4 ) , or CD19 ( αCD19 ) . Black arrowheads point at infiltrated lymphocytes , red arrowheads at histiocytes , yellow indicates B - lymphocyte rich zones .

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Immunohistochemistry of paraffin embedded sections from the lungs, brain meninges, brain striatum, spleen and liver of the vector-injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads at histiocytes, and the yellow line indicates B-lymphocyte rich zones.