( D ) HEK293A transiently transfected with HA ‐ p38IP RFP ‐ mAtg9 were treated with anisomycin for 30 min mAtg9 was immunoprecipitated . 5 % of lysates or immunoprecipitates were probed with anti ‐ HA anti ‐ mAtg9 antibodies ( data is representative of 3 experiments , * P = 0 . 0332 ) .

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(D) HEK293A cells transiently transfected with HA‐p38IP and RFP‐mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated. 5% of the lysates or the immunoprecipitates were probed with anti‐HA and anti‐mAtg9 antibodies (data is representative of 3 experiments, *P=0.0332).


( A ) HEK293A were transfected with RFP - mAtg9 Flag - p38 ( lanes 2 7 ) , RFP - mAtg9 , Flag - p38 HA - p38IP ( lanes 3 8 ) , or RFP - mAtg9 HA - p38IP ( lanes 4 9 ) . RFP - mAtg9 was immunoprecipitated with an anti ‐ mAtg9 antibody , co ‐ immunoprecipitated HA - p38IP was detected with an anti ‐ HA antibody , co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ Flag antibody . RFP - mAtg9 was detected with an anti ‐ Atg9 antibody .

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(A) HEK293A cells were transfected with RFP-mAtg9 and Flag-p38 (lanes 2 and 7), RFP-mAtg9, Flag-p38 and HA-p38IP (lanes 3 and 8), or RFP-mAtg9 and HA-p38IP (lanes 4 and 9). RFP-mAtg9 was immunoprecipitated with an anti‐mAtg9 antibody, the co‐immunoprecipitated HA-p38IP was detected with an anti‐HA antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐Flag antibody. RFP-mAtg9 was detected with an anti‐Atg9 antibody.


( B ) HEK293A were transfected with RFP - Atg9 HA - p38IP ( lanes 1 5 ) , Flag - Flag , Flag - p38 , HA - p38IP ( lanes 2 6 ) , or Flag - p38 HA - p38IP ( lanes 3 7 ) . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody , co ‐ immunoprecipitated RFP - Atg9 was detected with an anti ‐ Atg9 antibody , co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ p38α antibody . HA - p38IP was detected with an anti ‐ HA antibody .

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(B) HEK293A cells were transfected with RFP-Atg9 and HA-p38IP (lanes 1 and 5), Flag-Flag, Flag-p38, and HA-p38IP (lanes 2 and 6), or Flag-p38 and HA-p38IP (lanes 3 and 7). HA-p38IP was immunoprecipitated with an anti‐HA antibody, the co‐immunoprecipitated RFP-Atg9 was detected with an anti‐Atg9 antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐p38α antibody. HA-p38IP was detected with an anti‐HA antibody.


( C ) HEK293A were transfected with Flag - p38α for 24 h . were incubated in either full medium , EBSS , or EBSS plus leupeptin for 2 h , lysed , analysed for endogenous LC3 lipidation using an anti ‐ LC3 antibody , immunoblotted with anti ‐ Flag antibody . LC3 lipidation was quantified as amount of LC3II / LC3I ( data are represented as mean ± s . e . m . of triplicates , n = 2 experiments , EBSS control versus EBSS with Flag ‐ p38α , * * * P = 0 . 0026 ) .

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(C) HEK293A cells were transfected with Flag-p38α for 24 h. Cells were incubated in either full medium, EBSS, or EBSS plus leupeptin for 2 h, lysed, and analysed for endogenous LC3 lipidation using an anti‐LC3 antibody, and immunoblotted with anti‐Flag antibody. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean±s.e.m. of triplicates, n=2 experiments, EBSS control versus EBSS with Flag‐p38α, ***P=0.0026).


( A ) 293 / GFP - LC3 were transfected with control or p38α siRNA . At 72 h after transfection , were incubated in either full medium , EBSS , or EBSS with leupeptin for 2 h , then fixed visualized by confocal microscopy . Bars = 5 μm ( data are represented as mean ± s . e . m . n = 60 , EBSS control versus p38α siRNA ( * * * P = 0 . 0001 ) ; EBSS with leupeptin control versus p38α siRNA ( * * * P = 0 . 0001 ) , Student ' s t ‐ ) .

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(A) 293/GFP-LC3 cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS with leupeptin for 2 h, then fixed and visualized by confocal microscopy. Bars=5 μm (data are represented as mean±s.e.m. n=60 cells, EBSS control versus p38α siRNA (***P=0.0001); EBSS with leupeptin control versus p38α siRNA (***P=0.0001), Student's t‐test).


( B ) HEK293A were transfected with control or p38α siRNA . At 72 h after transfection , were incubated in either full medium , EBSS , or EBSS containing leupeptin for 2 h . lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . was also probed with anti ‐ actin anti ‐ p38α antibodies . LC3 lipidation was quantified as amount of LC3II / LC3I ( data are represented as mean ± s . e . m . n = 5 , full medium control versus p38α siRNA ( * * P = 0 . 0056 ) ; EBSS control versus p38α siRNA ( * * P = 0 . 048 ) ; EBSS with Leu control versus p38α siRNA ( * * P = 0 . 04 ) , Student ' s t ‐ ) .

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(B) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS containing leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐actin and anti‐p38α antibodies. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean ±s.e.m. n=5, full medium control versus p38α siRNA (**P=0.0056); EBSS control versus p38α siRNA (**P=0.048); EBSS with Leu control versus p38α siRNA (**P=0.04), Student's t‐test).


( C ) HEK293A were transfected with control or p38α siRNA . At 72 h after transfection , were incubated in either full medium or EBSS for 2 h , then fixed immunostained to detect endogenous mAtg9 localization . of with dispersed mAtg9 was quantified as in Figure 2E ( data are represented as mean ± s . e . m . n = 200 , full medium control versus p38α siRNA ( * * P = 0 . 0027 ) ; EBSS control versus p38α siRNA ( * P = 0 . 0459 ) ; Student ' s t ‐ ) . Bars = 5

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(C) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. The percentage of cells with dispersed mAtg9 was quantified as in Figure 2E (data are represented as mean±s.e.m. n=200 cells, full medium control versus p38α siRNA (**P=0.0027); EBSS control versus p38α siRNA (*P=0.0459); Student's t‐test). Bars=5 μm


( D ) HEK293A were transfected with siRNA for p38α , homogenized subjected to centrifugation , resulting post ‐ supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into pellet cytosol as in Figure 2A . Equal amounts were resolved by SDS - PAGE immunoblotted with anti ‐ MPR , anti ‐ HA , anti ‐ p38α antibodies . Data are representative of four experiments .

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(D) HEK293A cells were transfected with siRNA for p38α, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol as in Figure 2A. Equal protein amounts were resolved by SDS-PAGE and immunoblotted with anti‐MPR, anti‐HA, and anti‐p38α antibodies. Data are representative of four experiments.


( E ) 72 h were transfected with control , p38α siRNA or both p38α siRNA , p38IP siRNA . At 72 h after transfection , were incubated in either full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . was also probed with anti ‐ p38α . LC3 lipidation was quantified as amount of LC3II / LC3I . Data are representative of two experiments .

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(E) 72 h cells were transfected with control, p38α siRNA or both p38α siRNA, and p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐p38α. LC3 lipidation was quantified as the amount of LC3II/LC3I. Data are representative of two experiments.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after last injection . Heme oxygenase activity is increased by CoPP decreased by SnPP in liver as measured by chromatography .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after last injection . Total leukocyte red blood in PB . CoPP increases number of WBC populations . Granulocyte monocyte are increasing , whereas lymphocyte is decreasing among PB leukocytes after CoPP .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after last injection . Heatplot of cytokine growth factor concentrations in plasma measured by Luminex . CoPP increases concentrations of a group of cytokines ( red box ) .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box).


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after last injection . Selected cytokine growth factor concentrations in plasma measured by Luminex . CoPP increases concentrations of G - CSF , MCP - 1 , IP - 10 , IL - 6 IL - 5 . Results are shown as mean + , one - way Anova with Bonferroni post - , n = 5 mice per group .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after last injection . ( A ) numbers of main leukocyte populations in PB measured by flow cytometry . Both G - CSF CoPP increase numbers of CD45 + in blood . G - CSF CoPP similarly increase monocyte numbers , but increase of granulocytes is higher after G - CSF . Lymphocytes , including T , are increased only by G - CSF not by CoPP .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after last injection . Cytokine growth factor concentrations in plasma measured by Luminex . CoPP induces a group of cytokines ( red box ) which are not induced by G - CSF .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after last injection . Plasma concentrations of selected cytokines growth factors . CoPP highly increases of endogenous G - CSF IL - 6 that are not increased by G - CSF .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after last injection . Treatment with G - CSF CoPP decreases of Ly6G + macrophages in

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BM


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after last injection . Treatment with G - CSF CoPP increases relative spleen .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP increases relative spleen weight.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after last injection . viSNE maps of CD11b + CD11c - blood , colored by SSC value . Three independent experiments were performed results were pooled . Results are shown as mean + , one - way Anova with Bonferroni post - , n = 7 mice per group .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. viSNE maps of CD11b+ CD11c- blood cells, colored by SSC value. Three independent experiments were performed and the results were pooled. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after last injection . ( A ) Representative flow cytometry plots show higher granularity ( SSC ) Ly6G expression in CoPP - mobilized granulocytes than in G - CSF mobilized granulocytes .

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C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. (A) Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after last injection . Relative abundance of different granulocyte in blood of mice treated with G - CSF CoPP . Mice treated with CoPP have higher proportion of granulocytes with mature phenotype , than mice treated with G - CSF .

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C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in blood of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after last injection . Relative abundance of different granulocyte in BM ( C ) of mice treated with G - CSF CoPP . Mice treated with CoPP have higher proportion of granulocytes with mature phenotype , than mice treated with G - CSF .

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C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in BM (C) of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after last injection . of isolated from C57BL / 6xFVB mice treated with G - CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli .

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C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli.


Gating strategy of KLS ( c - Kit + Lin - Sca - 1 + ) in blood , using CD48 CD150 to distinguish HSC ( hematopoietic ) , MPP ( multipotent progenitors ) HPC ( hematopoietic progenitors ) .

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Gating strategy of KLS (c-Kit+Lin-Sca-1+) cells in blood, using CD48 and CD150 to distinguish HSC (hematopoietic stem cells), MPP (multipotent progenitors) and HPC (hematopoietic progenitors).


CoPP mobilizes higher numbers of HSC ( KLS CD48 - CD150 + ) , MPP ( KLS CD48 - CD150 - ) , HPC - 1 ( KLS CD48 + CD150 - ) HPC - 2 ( KLS CD48 + CD150 + ) than G - CSF to PB .

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CoPP mobilizes higher numbers of HSC (KLS CD48-CD150+), MPP (KLS CD48-CD150-), HPC-1 (KLS CD48+CD150-) and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB.


Treatment with CoPP increases of KLS in BM , what is related to increase in HPC populations , but not in LT - HSC fraction .

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Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction.


number of KLS - ( c - Kit + Lin - Sca - 1 - ) is higher in mice treated with CoPP than in mice treated with G - CSF . Numbers of lineage committed progenitors : GMP ( granulocyte - macrophage progenitors , KLS - CD48 + CD150 - CD34 + ) MEP ( megakaryocyte - erythroid progenitors , KLS - CD48 + CD150 + CD34 - ) were higher after CoPP administration than after G - CSF . G - CSF CoPP similarly increase EP numbers ( erythrocyte progenitors , KLS - CD48 - CD150 - CD34 - ) .

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

The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-).


Both G - CSF CoPP treatments decrease KLS - in BM , however decrease after CoPP is smaller . G - CSF CoPP decrease percentages of MEP EP , but only G - CSF decreases of GMP . Results are shown as mean + values , one - way Anova with Bonferroni post - , n = 7 mice per group .

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

Both G-CSF and CoPP treatments decrease the KLS- percentage in the BM, however the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Results are shown as mean + individual values, one-way Anova with Bonferroni post-test, n = 7 mice per group.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . ( A ) Effect of G - CSF CoPP - induced mobilization in C57BL / 6 - Tg ( UBC - GFP ) 30Sch / J donor mice ( mean + values , one - way Anova with Bonferroni post - , n = 16 mice per group ) : Donor mice treated with CoPP had highest number of mobilized granulocytes ( i ) KLS ( ii ) , compared to control or G - CSF - treated donor

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. (A) Effect of G-CSF and CoPP-induced mobilization in C57BL/6-Tg(UBC-GFP)30Sch/J donor mice (mean + individual values, one-way Anova with Bonferroni post-test, n = 16 mice per group): Donor mice treated with CoPP had the highest number of mobilized granulocytes (i) and KLS cells (ii), compared to control or G-CSF-treated donor mice


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . Recipient mice which were transplanted with CoPP - mobilized PBMC had highest number of WBC , PLT highest hematocrit values 2 after PBMC transplantation , compared to G - CSF - mobilized control PBMC recipients ( mean + values , one - way Anova with Bonferroni post - , NaCl : n = 8 , G - CSF : n = 7 , CoPP : n = 9 mice per group ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest number of WBC, PLT and the highest hematocrit values 2 weeks after PBMC transplantation, compared to G-CSF-mobilized and control PBMC recipients (mean + individual values, one-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . Recipient mice which were transplanted with CoPP - mobilized PBMC had highest chimerism among CD45 + , granulocytes B 2 , 4 18 after transplantation . Chimerism among T was highest in recipients of G - CSF - mobilized PBMC 2 after transplantation , but 18 after transplantation it was highest in CoPP - mobilized PBMC recipients ( mean + , two - way Anova with Bonferroni post - , NaCl : n = 8 , G - CSF : n = 7 , CoPP : n = 9 mice per group ) .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest chimerism among CD45+ cells, granulocytes and B cells 2, 4 and 18 weeks after transplantation. Chimerism among T cells was the highest in recipients of G-CSF-mobilized PBMC 2 weeks after transplantation, but 18 weeks after transplantation it was the highest in CoPP-mobilized PBMC recipients (mean + SEM, two-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . majority of recipients transplanted with G - CSF CoPP - mobilized PBMC have detectable GFP chimerism among BM KLS .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. The majority of primary recipients transplanted with G-CSF and CoPP-mobilized PBMC have detectable GFP chimerism among BM KLS cells.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . Fraction of secondary recipient mice with chimerism in PB CD45 + exceeding 1 % tends to be higher after CoPP - mobilized PBMC transplant than G - CSF - mobilized PBMC transplant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Fraction of secondary recipient mice with chimerism in PB CD45+ cells exceeding 1% tends to be higher after CoPP-mobilized PBMC transplant than G-CSF-mobilized PBMC transplant.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . PB chimerism in secondary recipients is higher in CoPP group than in control group ( mean + values , Kruskal - Wallis with Dunn ' s post - , NaCl : n = 7 , G - CSF : n = 6 , CoPP : n = 8 mice per group ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. PB chimerism in secondary recipients is higher in CoPP group than in the control group (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . of secondary recipient mice with GFP chimerism in BM KLS ( G ) KLS - ( H ) higher that 1 % is similar in G - CSF CoPP groups .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Percentage of secondary recipient mice with GFP chimerism in BM KLS (G) and KLS-(H) cells higher that 1% is similar in G-CSF and CoPP groups.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . GFP chimerism in KLS ( I ) KLS - ( J ) in BM of secondary recipients ( mean + values , Kruskal - Wallis with Dunn ' s post - , NaCl : n = 7 , G - CSF : n = 6 , CoPP : n = 8 mice per group ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. GFP chimerism in KLS (I) and KLS- (J) cells in the BM of secondary recipients (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).


Wild type C57BL / 6 mice were injected with anti - G - CSF antibody alone or in combination with anti - IL - 6 antibody one hour prior to CoPP treatment ; injections were repeated daily for 5 days mice were sacrificed 6 hours after last CoPP injection . G - CSF IL - 6 in

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

Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. G-CSF and IL-6 concentration in plasma


Wild type C57BL / 6 mice were injected with anti - G - CSF antibody alone or in combination with anti - IL - 6 antibody one hour prior to CoPP treatment ; injections were repeated daily for 5 days mice were sacrificed 6 hours after last CoPP injection . CD45 + , granulocytes HSC numbers in PB measured by flow cytometry .

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 O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. CD45+ cells, granulocytes and HSC numbers in PB measured by flow cytometry.


HO - 1 deficient control C57BL / 6xFVB mice were injected with CoPP three times , every second day sacrificed 24 hours after last injection . Total leukocyte in PB ( HO - 1 + / + : n = 5 , HO - 1 - / - DMSO : n = 6 , HO - 1 - / - CoPP : n = 4 mice per group ) . Granulocyte among PB leukocytes .

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

HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Total leukocyte counts in PB (HO-1+/+: n = 5, HO-1-/- DMSO: n = 6, HO-1-/- CoPP: n = 4 mice per group). Granulocyte percentage among PB leukocytes.


HO - 1 deficient control C57BL / 6xFVB mice were injected with CoPP three times , every second day sacrificed 24 hours after last injection . Cytokine growth factor concentrations in plasma ( E - HO - 1 + / + n = 5 , HO - 1 - / - DMSO : n = 5 , HO - 1 - / - CoPP : n = 3 mice per group ) . Results are shown as mean + , two - way Anova with Bonferroni post - .

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

HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Cytokine and growth factor concentrations in plasma (E - HO-1+/+ n = 5, HO-1-/- DMSO: n = 5, HO-1-/- CoPP: n = 3 mice per group). Results are shown as mean + SEM, two-way Anova with Bonferroni post-test.


Nrf2 - deficient control C57BL / 6 mice were injected with CoPP daily for 5 days sacrificed 6 hours after last injection ( DMSO : n = 4 , CoPP : n = 5 mice per group ) . CD45 + numbers in PB measured by flow cytometry . Granulocyte among PB leukocytes .

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

Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). CD45+ cell numbers in PB measured by flow cytometry. Granulocyte percentage among PB leukocytes.


Nrf2 - deficient control C57BL / 6 mice were injected with CoPP daily for 5 days sacrificed 6 hours after last injection ( DMSO : n = 4 , CoPP : n = 5 mice per group ) . Cytokine growth factor concentrations in

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

Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). Cytokine and growth factor concentrations in plasma


Comparison of constitutively active rhodopsin mutants M257Y6 . 40 ( left [ 21 ] ) , T94I2 . 61 ( middle ) G90D2 . 57 ( right [ 16 ] ) . - trans ( yellow ) cis retinal isomers ( orange ) , side of retinal attachment K2967 . 43 ( blue slate ) , retinal counterion E1133 . 28 ( blue slate ) , GαCT peptides ( cyan ) constitutively activating mutations M257Y6 . 40 , T94I2 . 61 , G90D2 . 57 ( green ) are shown as spheres . Palmitoylation at C323H8 glycosylation at N15NT are shown as sticks . lower panels compare M257Y6 . 40 , T94I2 . 61 G90D2 . 57 retinal - binding pockets . constructs also contain an additional thermostabilizing disulfide bridge , depicted as ( c - c ) , to enhance stability [ 18 ] crystallizability [ 19 ] without changing rhodopsin activation pathway [ 20 ] .

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 O O O O O O O O O O O O O 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

Comparison of constitutively active rhodopsin mutants M257Y6.40 (left [21]), T94I2.61 (middle) and G90D2.57 (right [16]). All-trans (yellow) and cis retinal isomers (orange), the side of retinal attachment K2967.43 (blue slate), the retinal counterion E1133.28 (blue slate), GαCT peptides (cyan) and the constitutively activating mutations M257Y6.40, T94I2.61, and G90D2.57 (green) are shown as spheres. Palmitoylation at C323H8 and glycosylation at N15NT are shown as sticks. The lower panels compare the M257Y6.40, T94I2.61 and G90D2.57 retinal-binding pockets. All constructs also contain an additional thermostabilizing disulfide bridge, depicted as (c-c), to enhance the stability [18] and crystallizability [19] without changing the rhodopsin activation pathway [20].


Kinetic characterization of rhodopsin state . Thermal decay of state rhodopsin : A ) WT , B ) T94I C ) G90D rhodopsin . spectra are normalized to OD280 OD500 / OD480 at t = 0 . D ) Absorption maximum plotted as a function of time fitted to a single exponential decay .

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

Kinetic characterization of the rhodopsin dark state. Thermal decay of dark state rhodopsin: A) WT, B) T94I and C) G90D rhodopsin. The spectra are normalized to the OD280 and OD500/OD480 at t=0. D) Absorption maximum plotted as a function of time and fitted to a single exponential decay.


Retinal isomerization in rhodopsin state : E ) WT , F ) T94I G ) G90D rhodopsin , H ) fraction of 11 - cis retinal is plotted as a function of time fitted to a single exponential decay .

O O O B-MEASURED_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 O O O O O O

Retinal isomerization in the rhodopsin dark state: E) WT, F) T94I and G) G90D rhodopsin, H) The fraction of 11-cis retinal is plotted as a function of time and fitted to a single exponential decay.


SB stability in state rhodopsin : I ) WT , J ) T94I K ) G90D rhodopsin . L ) OD440 is plotted as a function of time fitted to a single exponential decay . decay rates were measured at 55 C .

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 O O O O O O O O O O O O O O O O O O O O

SB stability in dark state rhodopsin: I) WT, J) T94I and K) G90D rhodopsin. L) OD440 is plotted as a function of time and fitted to a single exponential decay. All decay rates were measured at 55 C.


Accelerated molecular dynamic simulation of rhodopsin state : WT , G90D2 . 57 , T94I2 . 61 T94I2 . 61 with protonated E1133 . 28 . In absence of G90D2 . 57 T94I2 . 61 state structure , both aspartate isoleucine were placed in WT ( 1GZM [ 48 ] ) using a favorable rotamer without introducing major clashes with of . However , both isoleucine aspartate in state structures introduced clashes with several were therefore optimized using energy minimization before simulation . WT simulation was done with protonated SB while in G90D2 . 57 system , both E1133 . 28 SB were protonated [ 36 ] . protonated state of E1133 . 28 was unknown in T94I2 . 61 , therefore simulation were done for both deprotonated protonated E1133 . 28 . T94I2 . 61 state conformation with deprotonated E1133 . 28 was similar to that of WT was stable , whereas state of G90D2 . 57 T94I2 . 61 with protonated E1133 . 28 was unstable with a concomitant movement of E1133 . 28 residue away from SB , thereby , causing movement of TM3 opening of E1133 . 28 - SB activation switch . In contrast to crystal structures orientation of retinal - ionone ring in simulations was variable with respect to polyene chain . This indicates a degree of positional freedom within hydrophobic binding pocket .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Accelerated molecular dynamic simulation of the rhodopsin dark state: WT, G90D2.57, T94I2.61 and T94I2.61 with protonated E1133.28. In the absence of the G90D2.57 and T94I2.61 dark state structure, both aspartate and isoleucine were placed in the WT (1GZM [48]) using a favorable rotamer without introducing major clashes with the rest of the protein. However, both isoleucine and aspartate in the dark state structures introduced clashes with several amino acids and were therefore optimized using energy minimization before simulation. WT simulation was done with the protonated SB while in the G90D2.57 system, both E1133.28 and SB were protonated [36]. The protonated state of E1133.28 was unknown in T94I2.61, therefore simulation were done for both deprotonated and protonated E1133.28. The T94I2.61 dark state conformation with deprotonated E1133.28 was similar to that of the WT and was stable, whereas the dark state of G90D2.57 and T94I2.61 with protonated E1133.28 was unstable with a concomitant movement of E1133.28 residue away from the SB, thereby, causing movement of TM3 and opening of the E1133.28-SB activation switch.In contrast to the crystal structures the orientation of the retinal -ionone ring in the simulations was variable with respect to the polyene chain. This indicates a degree of positional freedom within the hydrophobic binding pocket.


B exclusion chromatography confirms that oligomerisation tendency of FICDR371S is unperturbed relative to wild - type FICD . Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species . elution profile of a monomeric FICDL258D - H363A mutant is also shown for reference , alongside elution times of molecular standards .

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B Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild-type FICD. Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species. The elution profile of a monomeric FICDL258D-H363A mutant is also shown for reference, alongside the elution times of molecular weight standards.


C Representative immunoblot comparing ability of wild - type Arg371Ser FICD ( at a wide range of enzyme concentrations ) to catalyse accumulation of BiP - AMP in presence of physiological ATP concentrations ( 5 mM ) . At low enzyme concentrations ( early time points ) AMPylation defect imposed by Arg371Ser is manifest . At higher enzyme concentrations where deAMPylation activity of wild - type enzyme begins to dominate , residual AMPylation activity of FICDR371S results in a greater accumulation of BiP - AMP ( relative to same of wild - type enzyme ) . D of AMPylated BiP signals relative to total BiP of experiments as in C ( mean values ± , n = 4 , biological replicates ) .

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 O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_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 O O O O O O O O

C Representative immunoblot comparing the ability of wild-type and Arg371Ser FICD (at a wide range of enzyme concentrations) to catalyse the accumulation of BiP-AMP in the presence of physiological ATP concentrations (5 mM). At low enzyme concentrations (and early time points) the AMPylation defect imposed by Arg371Ser is manifest. At higher enzyme concentrations where the deAMPylation activity of the wild-type enzyme begins to dominate, the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP-AMP (relative to the same concentration of wild-type enzyme). D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C (mean values ± SEM, n = 4, biological replicates).


F Representative immunoblot comparing accumulation of BiP - AMP in reactions constituted with indicated concentrations of wild - type Arg371Ser FICD , performed as in A ( upper panel ) . lower panel is a Coomassie - stained gel of reaction constituents . An N - terminally extended version of wild - type enzyme ( residues 45 - 458was used in lanes 2 5 - 7 , to distinguish its migration on gel from mutant ( residues 104 - 445 ) . bar diagram provides of AMPylated BiP signals relative to total BiP of experiments as in F ( from n = 3 , biological replicates , mean values ± ) .

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F Representative immunoblot comparing the accumulation of BiP-AMP in reactions constituted with the indicated concentrations of wild-type and Arg371Ser FICD, performed as in A (upper panel). The lower panel is a Coomassie-stained gel of the reaction constituents. An N-terminally extended version of the wild-type enzyme (residues 45-458was used in lanes 2 and 5-7, to distinguish its migration on the gel from the mutant (residues 104-445). The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F (from n = 3, biological replicates, mean values ± SEM).


A Immunoblots of AMPylated detected with a pan - AMP antibody , BiP , FICD eIF2α ( a loading control ) in lysates of FICD ∆ CHO transiently transfected with plasmids encoding indicated derivatives of FICD . Signals corresponding to AMPylated BiP , auto - AMPylated FICDE234G , total BiP , FICD eIF2α are indicated . novel species reactive with anti - FICD antiserum in lysates of transfected with plasmids expressing FICDR371S ( * ) likely reflects a glycosylated isoform arising from creation of a new glycosylation site at Asn369 .

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

A Immunoblots of AMPylated proteins detected with a pan-AMP antibody, BiP, FICD and eIF2α (a loading control) in lysates of FICD∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD. Signals corresponding to AMPylated BiP, auto-AMPylated FICDE234G, total BiP, FICD and eIF2α are indicated. The novel species reactive with the anti-FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S (*) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369.


C of data from repeats of experiment as shown in B . upper two graphs show of with high CHOP : : GFP or high XBP : : Turq for each transfection . lower panel provides quantitation of median for each reporter . Mean ± SD , n = 4 , biological replicates .

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C Quantification of the data from repeats of the experiment as shown in B. The upper two graphs show the percent of cells with high CHOP::GFP or high XBP::Turq for each transfection. The lower panel provides quantitation of the median fluorescence intensity for each reporter. Mean ± SD, n=4, biological replicates.


B On left are immunoblots of AMPylated detected with pan - AMP , BiP eIF2α ( a loading control ) reactive antibodies in lysates of of indicated genotype . were untreated or exposed to synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) for indicated time . In panel on right are immunoblots of samples immunoprecipitated with a BiP - specific antiserum from lysates of samples shown on left .

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B On the left are immunoblots of AMPylated proteins detected with pan-AMP, BiP and eIF2α (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots of samples immunoprecipitated with a BiP-specific antiserum from the lysates of the samples shown on the left.


C Immunoblots as in B in lysates of of indicated genotype . were untreated or exposed to synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) / or thapsigargin ( Tg ) for indicated periods prior to harvest . D of of BiP - AMP to total BiP signal ( expressed in arbitrary units ) of repeats of experiment shown in C . Mean ± SD of BiP - AMP signal normalized to total BiP signal from same samples are depicted , ( P values by two t - , n = 6 , biological replicates ) .

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C Immunoblots as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) and/or thapsigargin (Tg) for the indicated periods prior to harvest. D Quantification of ratio of the BiP-AMP to total BiP signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP signal normalized to total BiP signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).


A Representative immunoblot of AMPylated in FICD ∆ CHO transiently transfected with expression plasmids encoding indicated FICD enzymes ( replicated three times ) . Note that hyperactive E234G mutant bearing K124E - H131A mutations that compromise its ability to interact with BiP , is nonetheless consistently auto - AMPylated .

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A Representative immunoblot of AMPylated proteins in FICD∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes (replicated three times). Note that the hyperactive E234G mutant bearing the K124E-H131A mutations that compromise its ability to interact with BiP, is nonetheless consistently auto-AMPylated.


C Bar diagram of amount of alkaline phosphatase [ normalised to luciferase ( a transfection marker ) ] secreted from FICD ∆ CHO co - expressing indicated FICD enzymes . K124E - H131A mutations that interfere with engagement of BiP as a substrate reverse secretion defect imposed by hyperactive FICD enzymes [ Mean ± SD , P values by ANOVA with Šídák ' s multiple comparisons , n = 8 - 16 ( biological replicates ) ] .

O O O O O O B-MEASURED_VAR I-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 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

C Bar diagram of the amount of alkaline phosphatase [normalised to cytoplasmic luciferase (a transfection marker)] secreted from FICD∆ CHO cells co-expressing the indicated FICD enzymes. The K124E-H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [Mean ± SD, P values by ANOVA with Šídák's multiple comparisons test, n = 8-16 (biological replicates)].


WT SNX10 KO Caco - 2 were treated with indicated doses of OMVs for 24h . expression of E - cadherin SNX10 was determined by immunoblots ( A ) quantified by ImageJ software ( B ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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WT and SNX10 KO Caco-2 cells were treated with indicated doses of OMVs for 24h. Protein expression of E-cadherin and SNX10 was determined by immunoblots (A) and quantified by ImageJ software (B) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


G , H Confocal of E - cadherin in Caco - 2 treated with OMVs ( 100 μg / mL ) for indicated time were captured ( G ) , of E - cadherin was quantified by ImageJ software ( H ) ( n = 3 independent experiments , n = 18 fields analysed ) . Scale bar : 20 μm . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 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

G, H Confocal images of E-cadherin staining in Caco-2 cells treated with OMVs (100 μg/mL) for the indicated time were captured (G), and the fluorescence intensity of E-cadherin staining was quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Scale bar: 20 μm. Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


A , B WT SNX10 KO Caco - 2 were treated with or without OMVs ( 100 μg / mL ) for 24 h . fold changes in mRNA of SNAI1 ( encoding Snail ) ( A ) SNAI2 ( encoding Slug ) ( B ) in Caco - 2 were determined by RT - qPCR . n = 6 independent experiments . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . ns , not significant .

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-MEASURED_VAR O O B-MEASURED_VAR 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 O O O O O O O O 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 WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. The fold changes in mRNA levels of SNAI1 (encoding Snail) (A) and SNAI2 (encoding Slug) (B) in Caco-2 cells were determined by RT-qPCR. n = 6 independent experiments. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ns, not significant.


C WT SNX10 KO Caco - 2 were treated with or without OMVs ( 100 μg / mL ) for 24 h . were extracted for Snail Slug determination by immunoblots .

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C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots.


WT SNX10 KO Caco - 2 were treated with or without OMVs ( 100 μg / mL ) for 24 h . quantiﬁed of Snail ( E ) or Slug ( F ) - positive nuclei respectively . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * * * p < 0 . 001 .

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WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. quantiﬁed the percentage of Snail (E) or Slug (F)-positive nuclei respectively. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


H WT SNX10 KO Caco - 2 were transfected with Ad - vector or Ad - SNX10 , followed by treatment with or without 100 μg / mL OMVs for 24 h . from nucleus , cytoplasm total lysates were subjected to immunoblots with indicated antibodies .

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H WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies.


C , D of p - Lyn , Lyn , E - cadherin SNX10 in WT SNX10 KO Caco - 2 treated with or without OMVs ( 100 μg / mL ) for 24h were determined by immunoblots ( C ) quantified by ImageJ software ( D ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O 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 Levels of p-Lyn, Lyn, E-cadherin and SNX10 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24h were determined by immunoblots (C) and quantified by ImageJ software (D) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


E , F Caco - 2 were transfected with Ad - vector or Ad - SNX10 , followed by treatment with or without OMVs ( 100 μg / mL ) for 24h . Lysates were extracted subjected to immunoblots with indicated antibodies ( E ) quantified by ImageJ software ( F ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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E, F Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. Lysates were extracted and subjected to immunoblots with the indicated antibodies (E) and quantified by ImageJ software (F) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


I were extracted from WT SNX10 KO Caco - 2 treated with or without Tolimidone ( 10 μM ) , indicated were analyzed by immunoblots .

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I Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco-2 cells treated with or without Tolimidone (10 μM), and the indicated proteins were analyzed by immunoblots.


D WT SNX10 KO Caco - 2 were treated with or without OMVs ( 100 μg / mL ) for 24 h then subjected to IP with anti - caspase - 5 antibody .

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D WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h and then subjected to IP with anti-caspase-5 antibody.


E Representative of co - of Lyn caspase - 5 in WT SNX10 KO Caco - 2 treated with or without OMVs ( 100 μg / mL ) for 24 h . Scale bar : 20 μm . F co - localization of Lyn caspase - 5 was quantified by ImageJ software . n = 3 independent experiments , n = 18 fields analysed . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-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

E Representative images of co-staining of Lyn and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm. F The co-localization of Lyn and caspase-5 was quantified by ImageJ software. n = 3 independent experiments, n = 18 fields analysed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


H Lysates of Flag - tagged full - SNX10 or its different truncated mutants transfected Caco - 2 were subjected to immunoprecipitation .

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H Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected Caco-2 cells were subjected to immunoprecipitation.


E , WT SNX10 KO Caco - 2 were treated with OMVs ( 100 μg / mL ) for indicated time stained with antibodies against LPS Rab5 ( E ) . Scale bar : 20 μm .

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E, WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time and stained with antibodies against LPS and Rab5 (E). Scale bar: 20 μm.


G LPS in culture supernatants of WT SNX10 KO Caco - 2 stimulated by OMVs ( 100 μg / mL ) for indicated time were detected by LAL ( n = 6 independent experiments ) . H LPS in residual fractions of Caco - 2 stimulated by OMVs ( 100 μg / mL ) with or without chloroquine ( CQ ) ( 200 nM ) or bafilomycin A1 ( Baf A1 ) ( 50 nM ) for indicated time were detected by LAL ( n = 6 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not significant .

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G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco-2 cells stimulated by OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments). H LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without chloroquine (CQ) (200 nM) or bafilomycin A1 (Baf A1) (50 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.


C WT SNX10 KO Caco - 2 were treated with or without OMVs ( 100 μg / mL ) for 24h then subjected to IP with anti - PIKfyve antibody .

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C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24h and then subjected to IP with anti-PIKfyve antibody.


D , E Co - of PIKfyve caspase - 5 in WT SNX10 KO Caco - 2 treated with or without OMVs ( 100 μg / mL ) for 24 h ( D ) . Anti - PIKfyve anti - caspase - 5 antibodies were used to detect endogenous PIKfyve caspase - 5 respectively . Scale bar : 20 μm . Colocalized PIKfyve caspase - 5 were quantified by ImageJ software ( E ) ( n = 3 independent experiments , n = 18 fields analysed ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-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

D, E Co-staining of PIKfyve and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (D). Anti-PIKfyve and anti-caspase-5 antibodies were used to detect endogenous PIKfyve and caspase-5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (E) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


F Caco - 2 were stimulated by OMVs ( 100 μg / mL ) with or without apilimod ( 30 nM ) for 24h . of lysates extracted from Caco - 2 were subjected to immunoblots with indicated antibodies .

O O O O O O O O O O O O O O O O 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 Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for 24h. Proteins of lysates extracted from Caco-2 cells were subjected to immunoblots with the indicated antibodies.


G , H Co - of PIKfyve Rab5 in WT SNX10 KO Caco - 2 treated with or without OMVs ( 100 μg / mL ) for 24 h ( G ) . Anti - PIKfyve anti - Rab5 antibodies were used to detect endogenous PIKfyve Rab5 respectively . Scale bar : 20 μm . Colocalized PIKfyve caspase - 5 were quantified by ImageJ software ( H ) ( n = 3 independent experiments , n = 18 fields analysed ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-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

G, H Co-staining of PIKfyve and Rab5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (G). Anti-PIKfyve and anti-Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


I LPS in residual fractions of Caco - 2 stimulated by OMVs ( 100 μg / mL ) with or without apilimod ( 30 nM ) for indicated time were detected by LAL ( n = 6 independent experiments ) . J Caco - 2 transfected with control siRNA or CASP5 siRNA were treated with OMVs ( 100 μg / mL ) for indicated time . LPS in residual fractions of Caco - 2 were detected by LAL ( n = 6 independent experiments ) . K LPS in residual fractions of Caco - 2 stimulated by OMVs ( 100μg / mL ) with or without bafetinib ( 1μM ) for indicated time were detected by LAL ( n = 6 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). J Caco-2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs (100 μg/mL) for the indicated time. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected by LAL assay (n = 6 independent experiments). K LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100μg/mL) with or without bafetinib (1μM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 , each group ) . B , C of colons from WT , SNX10 cKO , IL - 10 KO SNX10 cKO IL - 10 KO mice was measured ( B ) analysed ( C ) at 16th week . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). B, C Length of colons from WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice was measured (B) and analysed (C) at the 16th week. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 , each group ) . E , F Representative H & E of colon tissues of WT , SNX10 cKO , IL - 10 KO SNX10 cKO IL - 10 KO mice at 16th week were shown ( E ) analyzed ( F ) . Scale bar : 100 μm . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). E, F Representative H&E images of colon tissues of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were shown (E) and analyzed (F). Scale bar: 100 μm. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 , each group ) . G content of TNF - α , IL - 6 , IL - 12 / IL - 23 p40 IL - 17A in serum of WT , SNX10 cKO , IL - 10 KO SNX10 cKO IL - 10 KO mice at 16th week were measured by ELISA . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). G The content of TNF-α, IL-6, IL-12/IL-23 p40 and IL-17A in the serum of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were measured by ELISA. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 , each group ) . J expression of E - cadherin , p - Lyn Lyn in colon epithelial tissues was determined by immunoblots .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). J Protein expression of E-cadherin, p-Lyn and Lyn in colon epithelial tissues was determined by immunoblots.


( A ) of in 293 cells24 h after transfection with luciferase , p55 / TNFR1 , DRP - 1 Δ73 or DAPk ΔCaM , GFP . Graphs show of GFP - positive with altered . ( B ) of GFP - positive cotransfected with luciferase ( 1 ) , p55 / TNFR1 ( 2 ) , DRP - 1 Δ73 ( 3 ) , or DAPk ΔCaM ( 4 ) .

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

(A) Quantification of cell death in 293 cells24 h after transfection with luciferase, p55/TNFR1, DRP-1 Δ73 or DAPk ΔCaM, and GFP. Graphs show the percentage of GFP-positive cells with altered cell morphology. (B) Photographs of GFP-positive cells cotransfected with luciferase (1), p55/TNFR1 (2), DRP-1 Δ73 (3), or DAPk ΔCaM (4).


( C ) showing overlay of GFP phase - contrast of 293 cells24 h after transfection with GFP luciferase ( 1 ) or with GFP - DAPk ( 2 3 ) ; both a blebbing GFP - DAPk - expressing ( 2 , arrows indicate blebs ) spread nontransfected are shown ( 3 ) .

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

(C) Photographs showing the overlay of GFP and phase-contrast images of 293 cells24 h after transfection with GFP and luciferase (1) or with GFP- DAPk (2 and 3); both a blebbing GFP-DAPk-expressing cell (2, arrows indicate blebs) and spread nontransfected cells are shown (3).


( D ) Hoechst ( 4 - 6 ) of GFP - positive 293 ( 1 - 3 ) 60 h after cotransfection with GFP DAPk ΔCaM ( 1 4 ) , DRP - 1 Δ73 ( 2 5 ) , or p55 / TNFR1 ( 3 6 ) . Arrows point to condensed nuclei resulting from DRP - 1 Δ73 or DAPk ΔCaM transfections ( 4 5 ) .

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

(D) Hoechst staining (4-6) of GFP-positive 293 cells (1-3) 60 h after cotransfection with GFP and DAPk ΔCaM (1 and 4), DRP-1 Δ73 (2 and 5), or p55/TNFR1 (3 and 6). Arrows point to condensed nuclei resulting from DRP-1 Δ73 or DAPk ΔCaM transfections (4 and 5).


( A ) Scoring loss of mitochondrial potential ( ΔΨm ) . 293 expressing luciferase ( black ) , DRP - 1 Δ73 ( pink ) , DAPk ΔCaM ( purple ) , Bax ( green ) were assayed for loss of mitochondrial potential 24 h after transfection using flow cytometric of TMRM . fraction of TMRM - negative calculated from three experiments is shown in graph below . small increase in TMRM in kinase - transfected is not statistically significant .

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 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 O O O O O O O O O O O O O O O O O O O O O O O

(A) Scoring loss of mitochondrial membrane potential (ΔΨm). 293 cells expressing luciferase (black), DRP-1 Δ73 (pink), DAPk ΔCaM (purple), and Bax (green) were assayed for loss of mitochondrial membrane potential 24 h after transfection using flow cytometric analysis of TMRM fluorescence. The fraction of TMRM-negative cells calculated from three experiments is shown in the graph below. The small increase in TMRM fluorescence in the kinase-transfected cells is not statistically significant.


( B ) Scoring release of cytochrome C from mitochondria . 293 cotransfected with luciferase ( 1 2 ) , Bax ( 3 6 ) , DRP - 1 Δ73 ( 7 8 ) , or DAPk ΔCaM ( 9 10 ) GFP constructs were incubated for 24 h immunostained using anti - cytochrome C antibodies . were visualized for cytochrome C ( left ) or GFP ( right ) . Bax - transfected spread ( 3 4 ) or blebbed ( 5 6 ) are shown . In fields that also contain nontransfected , arrows point to GFP - positive cytochrome C - stained .

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

(B) Scoring the release of cytochrome C from mitochondria. 293 cells cotransfected with luciferase (1 and 2), Bax (3 and 6), DRP-1 Δ73 (7 and 8), or DAPk ΔCaM (9 and 10) and GFP constructs were incubated for 24 h and immunostained using anti-cytochrome C antibodies. Cells were visualized for cytochrome C (left) or GFP (right) fluorescence. Bax-transfected spread (3 and 4) or blebbed (5 and 6) cells are shown. In fields that also contain nontransfected cells, arrows point to GFP-positive cytochrome C-stained cells.


( C ) Assessing effects of Bcl - 2 Bcl - XL . 293 were cotransfected with luciferase , DRP - 1 Δ73 , DAPk ΔCaM , or Bax , either Bcl - 2 or Bcl - XL together with GFP . GFP - positive were visualized by microscopy scored for appearance of 24 h after transfection . Total refers to sum of morphological changes observed .

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

(C) Assessing the effects of Bcl-2 and Bcl-XL. 293 cells were cotransfected with luciferase, DRP-1 Δ73, DAPk ΔCaM, or Bax, and either Bcl-2 or Bcl-XL together with GFP. GFP-positive cells were visualized by fluorescent microscopy and scored for the appearance of cell death morphology 24 h after transfection. Total cell death refers to the sum of all morphological changes observed.


( A ) Transmission electron of 293 transfected with luciferase ( 1 ) p55 / TNFR1 ( 2a - 2c ) , DRP - 1 Δ73 ( 3a 3b ; part of fig . 3b is magnified on its right ) , DAPk ΔCaM ( 4 ) . different stages of autophagic development are depicted as follows : immature autophagic vesicles ( double arrow ) , mature autophagic vesicles autolysosomes ( black arrow ) , residual bodies ( dashed arrow ) . Empty vacuoles ( arrow ) are also shown . Arrowheads show condensed fragmented . m , mitochondria ; g , Golgi apparatus . Bar , 1 μm .

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 O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O 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) Transmission electron micrographs of 293 cells transfected with luciferase (1) p55/TNFR1 (2a-2c), DRP-1 Δ73 (3a and 3b; part of fig. 3b is magnified on its right), and DAPk ΔCaM (4). The different stages of autophagic development are depicted as follows: immature autophagic vesicles (double arrow), mature autophagic vesicles and autolysosomes (black arrow), and residual bodies (dashed arrow). Empty vacuoles (white arrow) are also shown. Arrowheads show condensed and fragmented chromatin. m, mitochondria; g, Golgi apparatus. Bar, 1 μm.


( B ) Transmission electron of MCF - 7 transfected with luciferase ( 1 ) , p55 / TNFR1 ( 2a - 2c ) , DRP - 1 Δ73 ( 3a 3b ) , DAPk ΔCaM ( 4 ) . Immature autophagic vesicles ( double arrow ) , autophagic vesicles ( black arrow ) , residual bodies ( dashed arrow ) are shown . Insets in B , 2b 2c , correspond to empty vacuoles , whereas those in B , 3a 4 , correspond to autophagic vesicles . Arrowheads show condensed fragmented . m , mitochondria ; dm , darkened mitochondria ; n , nucleus ; g , Golgi apparatus ; v , vacuoles ; ly , lysosomes ; er , endoplasmic reticulum .

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 O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transmission electron micrographs of MCF-7 cells transfected with luciferase (1), p55/TNFR1 (2a-2c), DRP-1 Δ73 (3a and 3b), and DAPk ΔCaM (4). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and residual bodies (dashed arrow) are shown. Insets in B, 2b and 2c, correspond to empty vacuoles, whereas those in B, 3a and 4, correspond to autophagic vesicles. Arrowheads show condensed and fragmented chromatin. m, mitochondria; dm, darkened mitochondria; n, nucleus; g, Golgi apparatus; v, vacuoles; ly, lysosomes; er, endoplasmic reticulum.


( A ) of autophagy in 293 MCF - 7 . Graphs show of MDC - positive resulting from luciferase or DRP - 1 Δ73 transfections ( mean ± SD calculated from triplicates of 100 each ; note that total number comprises transfected nontransfected ) . These experiments were repeated three times with reproducible results .

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 O O O O 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) Quantification of autophagy in 293 and MCF-7 cells. Graphs show the percentage of MDC-positive cells resulting from luciferase or DRP-1 Δ73 transfections (mean ± SD calculated from triplicates of 100 cells each; note that total cell number comprises transfected and nontransfected cells). These experiments were repeated three times with reproducible results.


( B ) of MDC - stained MCF - 7 from same experiment shown in A . Arrows point to MDC - positive . Transfections : luciferase ( 1a 1b ) DRP - 1 Δ73 ( 2a 2b ) at low ( a ) high ( b ) magnifications .

O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(B) Photographs of MDC-stained MCF-7 cells from the same experiment shown in A. Arrows point to MDC-positive cells. Transfections: luciferase (1a and 1b) and DRP-1 Δ73 (2a and 2b) at low (a) and high (b) magnifications.


( A ) of - associated morphologies in 293 . Graphs show of harboring spectrum of morphological changes occurring as a result of luciferase , p55 / TNFR1 , DRP - 1 Δ73 , or DAPk ΔCaM transfections in presence or absence of caspase inhibitors BD - fmk or z - VAD - fmk ( 100 μM ; mean ± SD calculated from triplicates of 100 each ) . This experiment was repeated three times with reproducible results .

O 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 B-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

(A) Quantification of cell death-associated morphologies in 293 cells. Graphs show the percentage of cells harboring all spectrum of morphological changes occurring as a result of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM transfections in the presence or absence of the caspase inhibitors BD-fmk or z-VAD-fmk (100 μM; mean ± SD calculated from triplicates of 100 cells each). This experiment was repeated three times with reproducible results.


( B ) of - associated morphologies in MCF - 7 . Experimental conditions were performed as in A in presence or absence of BD - fmk ( 100 μM ) .

O O O O O O O O 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) Quantification of cell death-associated morphologies in MCF-7 cells. Experimental conditions were performed as in A in the presence or absence of BD-fmk (100 μM).


( C ) Graphs show of harboring complete spectrum of - associated morphological changes occurring as a result of cotransfections of luciferase , p55 / TNFR1 , DRP - 1 Δ73 , or DAPk ΔCaM with caspase inhibitor crmA or control luciferase vector ( mean ± SD calculated from triplicates of 100 each ) . Immunoblot shows expression of transfected HA - tagged DAPk DRP - 1 Δ73 , confirming that cotransfection with crmA did not alter expression of - inducing .

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

(C) Graphs show the percentage of cells harboring the complete spectrum of death-associated morphological changes occurring as a result of cotransfections of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM with the caspase inhibitor crmA or control luciferase vector (mean ± SD calculated from triplicates of 100 cells each). Immunoblot shows the expression levels of transfected HA-tagged DAPk and DRP-1 Δ73, confirming that cotransfection with crmA did not alter expression of the death-inducing proteins.


( D ) Transmission electron of 293 transfected with p55 / TNFR1 ( 1 ) or DRP - 1 Δ73 ( 2a 2b ; parts of these figures are magnified at right ) treated with caspase inhibitor BD - fmk ( 100 μM ) . Immature autophagic vesicles ( double arrow ) , autophagic vesicles ( black arrow ) , autolysosomes ( arrow ) are shown . m , mitochondria ; g , Golgi apparatus .

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

(D) Transmission electron micrographs of 293 cells transfected with p55/TNFR1 (1) or DRP-1 Δ73 (2a and 2b; parts of these figures are magnified at the right) and treated with the caspase inhibitor BD-fmk (100 μM). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and autolysosomes (white arrow) are shown. m, mitochondria; g, Golgi apparatus.


( E ) Western blots of lysates prepared from HeLa transfected with luciferase , p55 / TNFR1 , DRP - 1 Δ73 , or DAPk ΔCaM GFP incubated for 24 h . Blots were reacted with anti - PARP antibodies , anti - caspase - 8 antibodies , anti - caspase - 3 antibodies , anti - HA antibodies for DRP - 1 Δ73 DAPk ΔCaM detection , anti - β - tubulin antibodies to quantitate loading .

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

(E) Western blots of cell lysates prepared from HeLa cells transfected with luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM and GFP and incubated for 24 h. Blots were reacted with anti-PARP antibodies, anti-caspase-8 antibodies, anti-caspase-3 antibodies, anti-HA antibodies for DRP-1 Δ73 and DAPk ΔCaM detection, and anti-β-tubulin antibodies to quantitate protein loading.