C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in control and PHSL1 - CDC20 cells . 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 four independent experiments .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_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 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 B-MEASURED_VAR 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 control and PHSL1-CDC20 cells. 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 four independent experiments.


D . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 strains containing the indicated mutations . Meiotic events were analyzed as in ( C ) . Data information : data are representative of two independent experiments .

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

D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C). Data information: data are representative of two independent experiments.


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 .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR 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 O O O O O O O O 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 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 ) 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 .

O O O O O O B-MEASURED_VAR 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-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 ) 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 .

O O O O O O B-MEASURED_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 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 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 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 ) 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 .

O O O O O O B-MEASURED_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 B-CONTROLLED_VAR 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 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 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 ) 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 .

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

(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 , 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 .

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

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 O O O B-CONTROLLED_VAR 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

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 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 .

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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 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 .

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

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 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 .

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

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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 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 ) .

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

(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 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 .

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

(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 and significantly altered in both the CTNS - / - and CTNSPatient cells compared to control cells ( n = 6 ) .

O O O O O O O O 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

(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 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 .

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

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

(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 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 ) .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O 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 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 ) .

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(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 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 .

(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 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 ) .

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(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 ) 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 ) .

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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 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 .

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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 ) 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 ) .

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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 , and a combination of cysteamine and bicalutamide ( n = 40 embryos 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 and 96 hrs post fertilization ( hpf ) with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 .

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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 , 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 ) .

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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 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 .

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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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 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

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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 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

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

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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 quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells 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 images ( D ) and cfu quantification ( E ) of Shigella WT or ΔipaB mutant bound to HCT - 8 cells 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 and qRT - PCR quantification of intracellular Shigella in HeLa cells pre - treated with arsenite in the presence or not of amitriptyline , and corresponding

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

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 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

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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 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

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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 analysis of p38 MAPK phosphorylation in HeLa cells 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 cells pre - treated with arsenite in the absence or presence of the p38 inhibitor SB203580 , and 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 cells pre - treated with arsenite in the absence or presence of the p38 inhibitor SB203580 , and 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 quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and 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 cells pre - treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580 , and 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 quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and 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 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

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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 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

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 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

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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 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

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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 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

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 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

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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 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

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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 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

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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 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 -

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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 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 -

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 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 -

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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 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

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


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


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


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

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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 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

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

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 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 ) ,

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

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 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 ±

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ±

O 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 O B-MEASURED_VAR 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 B-MEASURED_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 O O O O O O O O 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 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 ) .

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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 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

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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 ) 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 ) .

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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 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

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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 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

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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 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

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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 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

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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 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 ) .

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

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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 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

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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 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 ) .

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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 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

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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 ) test 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 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

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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 - 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

O O O O O 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 O O O O O O

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 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 ) .

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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 + 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

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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 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 .

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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 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 .

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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.


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 .

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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.


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 .

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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.