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 .

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

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 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 .

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

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 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 .

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 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

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 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 .

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 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

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 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 .

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

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 + ) cells in blood , using CD48 and CD150 to distinguish HSC ( hematopoietic stem cells ) , MPP ( multipotent progenitors ) and HPC ( hematopoietic progenitors ) .

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

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 - ) and HPC - 2 ( KLS CD48 + CD150 + ) cells than G - CSF to the PB .

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

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

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

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


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 - ) .

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

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 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 .

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

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 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

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 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 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

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 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 ) .

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 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 B-CONTROLLED_VAR 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 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 ) .

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

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 B-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 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 .

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 B-CONTROLLED_VAR 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. 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 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 ) .

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

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 B-CONTROLLED_VAR 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 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 ) .

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

O O O O O O 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 B-CONTROLLED_VAR 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. 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 ; 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 .

O O O O O O 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 B-CONTROLLED_VAR 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 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 .

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 B-CONTROLLED_VAR 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 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 .

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 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

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 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 .

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

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 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

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


IL - 1β ELISA of supernatants from LPS - primed ( 1 μg / mL , 4hrs . ) MDMs pre - incubated with 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) 15 mins . before treatment with either nigericin ( 10 μM , 45 mins . ) or CPPD crystals ( 250 μg / mL , 2 hrs . ) . Bars represent the mean , ± S . D . n = 13 and 11 independent blood donors for nigericin and CPPD , respectively . IL - 18 ELISA of supernatants from MDMs treated as in A . Bars represent the mean , ± S . D . n = 11 independent blood donors .

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

IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors.


LDH - release from MDMs treated as in A . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 10 and 11 independent blood donors for nigericin and CPPD , respectively .

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

LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively.


Western blots of supernatants and cell lysates from MDMs treated as in A . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) , pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) and mature caspase - 1 ( mCasp - 1 ; 20 kDa ) . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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Western blots of supernatants and cell lysates from MDMs treated as in A. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa), pro-caspase-1 (pro-Casp-1; 45 kDa) and mature caspase-1 (mCasp-1; 20 kDa). β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


Quantitative RT - PCR showing the mRNA expression levels of IL - 1β , IL - 18 , ASC , CASP1 and NLRP3 in MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ; 4 hrs . ) , as indicated . Bars represent the mean fold increase from an untreated ( UT )

O O O 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 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

Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) control


Western blots showing the relative abundance of NLRP3 , ASC , pro - caspase - 1 , pro - IL - 1β , pro - IL - 18 , USP7 and USP47 in cell lysates of MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ) for 4 hrs . , as indicated . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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

Western blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


IL - 18 ELISA of supernatants from LPS - primed ( 1 μg / mL , 10 mins . ) MDMs pre - incubated with either vehicle ( 0 . 1 % DMSO ) or P22077 ( 2 . 5 µM ) for 15 mins . before treatment with nigericin ( 10 μM , 45 mins . ) . Bars represent the mean , ± S . D . n = 9 independent blood donors .

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IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors.


Western blots of supernatants and cell lysates from MDMs as treated in C . Bands in the figure represent : Pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) and β - actin as a loading control . Blots are representative of at least 3 independent blood donors .

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Western blots of supernatants and cell lysates from MDMs as treated in C. Bands in the figure represent: Pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa) and β-actin as a loading control. Blots are representative of at least 3 independent blood donors.


LDH - release from MDMs as treated in C . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 9 independent blood donors .

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LDH-release from MDMs as treated in C. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 9 independent blood donors.


Western blots showing ASC - oligomerisation after DSS - mediated crosslinking in insoluble fractions from THP1 cell lysates . PMA differentiated THP1 cells were LPS - primed ( 1 μg / mL , 4 hrs . ) and pre - incubated for 15 min with either 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) . followed by treatment with nigericin ( 10 μM , 45 mins . ) . The ' input ' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction . Blots show membranes that were probed with an anti - ASC antibody . Data is representative of at least three independent experiments .

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Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in insoluble fractions from THP1 cell lysates. PMA differentiated THP1 cells were LPS-primed (1 μg/mL, 4 hrs.) and pre-incubated for 15 min with either 0.1% DMSO or P22077 (2.5 µM). followed by treatment with nigericin (10 μM, 45 mins.). The 'input' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction. Blots show membranes that were probed with an anti-ASC antibody. Data is representative of at least three independent experiments.


Western blots showing ASC - oligomerisation after DSS - mediated crosslinking in supernatants from THP1s treated as in A . Blots show membranes that were probed with an anti - ASC antibody . The input represents non - crosslinked supernatant . Data is representative of at least three independent experiments .

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Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in supernatants from THP1s treated as in A. Blots show membranes that were probed with an anti-ASC antibody. The input represents non-crosslinked supernatant. Data is representative of at least three independent experiments.


Images from an immunofluorescence experiment showing ASC - speck formation in THP1 cells treated as in A . All images show cells that were stained with an anti - ASC antibody . Images are representative of 3 independent experiments . Scale bar 20µm . Quantification of ASC - speck formation in images depicted in C . The number of ASC specks was quantified and expressed as the percentage of cells containing specks .

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Images from an immunofluorescence experiment showing ASC-speck formation in THP1 cells treated as in A. All images show cells that were stained with an anti-ASC antibody. Images are representative of 3 independent experiments. Scale bar 20µm. Quantification of ASC-speck formation in images depicted in C. The number of ASC specks was quantified and expressed as the percentage of cells containing specks.


NLRP3 oligomerisation analysed by native PAGE . LPS - primed ( 1 μg / mL , 4 hrs . ) murine BMDMs pre - incubated with either 0 . 1 % DMSO or P22077 ( at the indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 min . ) . Digitonin - solubilised cell lysates were analysed by native - PAGE or SDS - PAGE and NLRP3 western - blotting . Western blots of supernatants and cell lysates for IL - 1β are also shown as a control for P22077 inflammasome inhibition . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) and NLRP3 ( 120 kDa ) . β - actin is shown as a loading control . Blots are representative of 4 independent murine donors .

O O O O O O O O O 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 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 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

NLRP3 oligomerisation analysed by native PAGE. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 min.). Digitonin-solubilised cell lysates were analysed by native-PAGE or SDS-PAGE and NLRP3 western-blotting. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120 kDa). β-actin is shown as a loading control. Blots are representative of 4 independent murine donors.


NLRP3 ubiquitination analysed by western blot . LPS - primed ( 1 μg / mL , 4 hrs . ) murine BMDMs pre - incubated with either 0 . 1 % DMSO or P22077 ( at the indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 mins . ) . Endogenous NLRP3 was immunoprecipitated ( IP ) with anti - NLRP3 and probed with either anti - ubiquitin ( Poly - Ub ) , anti - K63 - ubiquitin ( Poly - K63 - Ub ) or anti - NLRP3 . As a negative control , cell lysate made from LPS - primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG . Total levels of NLRP3 in cell lysates ( input ) were also probed . Western blots of supernatants and cell lysates for IL - 1β are also shown as a control for P22077 inhibition . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) and NLRP3 ( 120kDa ) . β - actin is shown as a loading control . Blots are representative of 2 independent murine donors for 2 . 5µM P22077 and 4 independent murine donors for 10 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 O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR O O O O O O O O O

NLRP3 ubiquitination analysed by western blot. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 mins.). Endogenous NLRP3 was immunoprecipitated (IP) with anti-NLRP3 and probed with either anti-ubiquitin (Poly-Ub), anti-K63-ubiquitin (Poly-K63-Ub) or anti-NLRP3. As a negative control, cell lysate made from LPS-primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120kDa). β-actin is shown as a loading control. Blots are representative of 2 independent murine donors for 2.5µM P22077 and 4 independent murine donors for 10 µM.


Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and then treated with nigericin ( 10 μM ) for differing lengths of time , as indicated . Relative , activity - based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs . Higher panels show lysates incubated with ABPs ( labelled ) , with the active DUB bound to ABP indicated by the suffix ' - VME ' . Lower panels , without probe labelling , show total USP7 or USP47 levels . ( Unlabelled ) . Western blots are representative of 3 independent MDM donors .

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Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors.


Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and then pre - incubated with either 0 . 1 % DMSO or P22077 ( 2 . 5 μM ) prior to incubation with CPPD crystals ( 250 µg / mL , 3 hrs . ) . Blots are labelled as in B and are representative of 3 independent MDM donors .

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Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then pre-incubated with either 0.1% DMSO or P22077 (2.5 μM) prior to incubation with CPPD crystals (250 µg/mL, 3 hrs.). Blots are labelled as in B and are representative of 3 independent MDM donors.


Western blots showing activity changes in both USP7 and USP47 in LPS - primed ( 1 μg / mL , 4 hrs . ) MDMs pre - incubated with different P22077 concentrations , as indicated , prior to activation by nigericin treatment ( 10 μM ; 45 mins . ) . Blots are labelled as in B and are representative of 3 independent MDM donors .

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Western blots showing activity changes in both USP7 and USP47 in LPS-primed (1 μg/mL, 4 hrs.) MDMs pre-incubated with different P22077 concentrations, as indicated, prior to activation by nigericin treatment (10 μM; 45 mins.). Blots are labelled as in B and are representative of 3 independent MDM donors.


Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium ( High K + ) conditions . MDMs were LPS - primed ( 1 μg / mL , 4 hrs . ) and then changed to media that either had normal or high concentrations of K + , and subsequently treated with nigericin ( 10 μM ; 45 mins . ) . Blots are labelled as in B and are representative of at least 2 independent MDM donors . Pro - IL - 1β ( 31 kDa ) and mature IL - 1β ( mIL - 1β , 17 kDa ) in the supernatants of these cells are shown as a control . Non - specific bands are highlighted with an asterisk .

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Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium (High K+) conditions. MDMs were LPS-primed (1 μg/mL, 4 hrs.) and then changed to media that either had normal or high concentrations of K+, and subsequently treated with nigericin (10 μM; 45 mins.). Blots are labelled as in B and are representative of at least 2 independent MDM donors. Pro-IL-1β (31 kDa) and mature IL-1β (mIL-1β, 17 kDa) in the supernatants of these cells are shown as a control. Non-specific bands are highlighted with an asterisk.


Western blots of supernatants and cell lysates from THP - 1 cells engineered by CRISPR / Cas9 to induce USP7 and / or USP47 KOs . Deficiency of both USP7 and USP47 was induced by doxycycline ( Doxy ) treatment ( 1 µg / mL ; 3 days ) , as indicated . All cells were differentiated with PMA and either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and treated with nigericin ( 10 μM , 45 mins . ) , as indicated . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) ; mature caspase - 1 ( mCasp - 1 ; 20 kDa ) ; USP7 and USP47 . β - actin is shown as a loading control . Blots are representative of at least 3 independent experiments .

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

Western blots of supernatants and cell lysates from THP-1 cells engineered by CRISPR/Cas9 to induce USP7 and/or USP47 KOs. Deficiency of both USP7 and USP47 was induced by doxycycline (Doxy) treatment (1 µg/mL; 3 days), as indicated. All cells were differentiated with PMA and either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and treated with nigericin (10 μM, 45 mins.), as indicated. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-caspase-1 (pro-Casp-1; 45 kDa); mature caspase-1 (mCasp-1; 20 kDa); USP7 and USP47. β-actin is shown as a loading control. Blots are representative of at least 3 independent experiments.


IL - 1β release measured from doxycycline treated ( 1 µg / mL ; 3 days ) PMA - differentiated and LPS - primed ( 1 μg / mL , 4 hrs . ) , nigericin activated ( 10 μM , 45 mins . ) THP - 1 cells , which contain the indicated sgRNA . Bars represent the mean percentage of IL - 1β release relative to their respective non - doxycycline treated control cells , ± S . D . LDH - release measured from cells as treated in B . Bars represent the mean percentage of LDH release relative to their respective non - doxycycline treated control cells , ± S . D .

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IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D.


( B ) Representative immunohistochemical analysis with anti - pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan - keratin positive areas ( mm2 ) of individual lesions ( dots ) formed by 6 HKCs strains of the two ancestries . n ( tumors per ancestry ) = 8 . * p < 0 . 05 . Horizontal line : median . Two - tailed unpaired t - test . Scale bar : 500μm .

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(B) Representative immunohistochemical analysis with anti-pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan-keratin positive areas (mm2) of individual lesions (dots) formed by 6 HKCs strains of the two ancestries. n(tumors per ancestry) = 8. *p<0.05. Horizontal line: median. Two-tailed unpaired t-test. Scale bar: 500μm.


Double immunofluorescence analysis of tumors formed by six HKC strains , as in the previous panels , with antibodies against Ki67 ( C ) together with anti - pan - keratin antibodies for tumor cells identification . Shown are representative images and quantification of results of individual lesions ( dots ) . Digitally acquired images were used to determine the % of Ki67 positive cells ( C ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 and K1 staining : n ( tumors per ancestry ) = 24 , 22 and 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal line : median , two - tailed unpaired t test . Scale bars : 100μm .

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Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against Ki67 (C) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of Ki67 positive cells (C) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


Double immunofluorescence analysis of tumors formed by six HKC strains , as in the previous panels , with antibodies against keratin 10 and 1 ( K10 , K1 ) ( D ) together with anti - pan - keratin antibodies for tumor cells identification . Shown are representative images and quantification of results of individual lesions ( dots ) . Digitally acquired images were used to determine the % of K1 / K10 positivity ( D ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 and K1 staining : n ( tumors per ancestry ) = 24 , 22 and 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal line : median , two - tailed unpaired t test . Scale bars : 100μm .

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Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against keratin 10 and 1 (K10, K1) (D) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of K1/K10 positivity (D) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


( B ) Kaplan Meier plots of long - term overall survival of H / NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males ( darkgreen and lightgreen lines ) and females ( red and orange lines ) , showing statistically significant association of elevated HSD17B7 expression with poorer patient survival . Statistical significance was calculated by the log - rank test between the groups . High _ female : n = 57patients . Low _ female : n = 79 patients . High _ male : n = 203 patients . Low _ male : n = 181 patients .

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(B) Kaplan Meier plots of long-term overall survival of H/NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males (darkgreen and lightgreen lines) and females (red and orange lines), showing statistically significant association of elevated HSD17B7 expression with poorer patient survival. Statistical significance was calculated by the log-rank test between the groups. High_female: n=57patients. Low_female: n= 79 patients. High_male: n= 203 patients. Low_male: n= 181 patients.


( G ) Immunofluorescence ( IF ) analysis of HSD17B7 protein expression in White and Black HKCs . Shown are representative images as well as IF signal quantification at the level of individual cells ( dots ) for the indicated strains . 19 - 46 cells were counted in each case . n ( HKC strains per ancestry ) = 5 , * * * p < 0 . 0005 , horizontal line : median , 2 - tailed unpaired t - test . Unit of Y - axis : pixels . Scale bar : 20 μm .

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(G) Immunofluorescence (IF) analysis of HSD17B7 protein expression in White and Black HKCs. Shown are representative images as well as IF signal quantification at the level of individual cells (dots) for the indicated strains. 19-46 cells were counted in each case. n(HKC strains per ancestry) = 5, ***p<0.0005, horizontal line: median, 2-tailed unpaired t-test. Unit of Y-axis: pixels. Scale bar: 20 μm.


( D - F ) Intradermal tumorigenicity assays with HKCs ( 269W ) stably infected with an HSD17B7 overexpressing lentivirus ( HSD17B7 - oe ) versus control ( Ctrl ) . Experimental conditions were the same as in Fig 2 . Mice were sacrificed 15 days after injection . Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti - pan keratin antibodies ( D , ) as well as % of Ki67 positive cells ( E ) and K1 positivity ( F ) of pan - keratin positive areas . For ( D ) , n ( tumors per condition ) = 9 ; for ( E and F ) n ( 3 fields per tumor , 5 tumors per condition ) = 15 . * p < 0 . 05 , * * * * p < 0 . 0001 . Horizontal line : median . 2 - tail unpaired t - test . Scale bar : 500μm ( D ) , 100μm ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7-oe) versus control (Ctrl). Experimental conditions were the same as in Fig 2. Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti-pan keratin antibodies (D,) as well as % of Ki67 positive cells (E) and K1 positivity (F) of pan-keratin positive areas. For (D), n(tumors per condition) = 9; for (E and F) n(3 fields per tumor, 5 tumors per condition) = 15. *p<0.05, ****p<0.0001. Horizontal line: median. 2-tail unpaired t-test. Scale bar: 500μm (D), 100μm (F).


( G , H ) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 ( HSD17B7 - oe ) versus control ( Ctrl ) . Mice were sacrificed 15 days after injection . Shown are representative images and quantification of tumor cells density , assessed by immunohistochemical staining with anti - pan keratin antibodies ( G ) as well as % of Ki67 positive cells in pan - keratin positive areas ( H ) . For ( G ) , n ( tumors per condition ) = 4 ; for ( H ) , n ( 3 - 5 fields per tumor , 4 tumors per condition ) 15 , 19 . * p < 0 . 05 ; Horizontal line : median . One - tailed paired t - test . Scale bar in ( G , H ) : 100μm .

(G, H) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7-oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti-pan keratin antibodies (G) as well as % of Ki67 positive cells in pan-keratin positive areas (H). For (G), n(tumors per condition) = 4; for (H), n(3-5 fields per tumor, 4 tumors per condition) 15, 19. *p<0.05; Horizontal line: median. One-tailed paired t-test. Scale bar in (G, H): 100μm.


( D ) Colony forming assays of the indicated SCC cell lines plus / minus HSD17B7 silencing as in the previous panels . Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in ( A ) . Data are represented as number of colonies + / - SD . n ( dishes per condition ) = 3 * p < 0 . 05 , * * p < 0 . 01 ; p * * * * < 0 . 0001 , 2 - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colony forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.01; p****<0.0001, 2-tailed unpaired t-test.


( C , D ) SCC13 cells stably infected with an HSD17B7 - expressing lentivirus ( HSD - oe ) versus empty vector control ( Ctrl ) ( C ) or two HSD17B7 - silencing lentivirus ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) ( D ) were analyzed as in the previous panels . Shown are individual values for 3 parallel cultures per condition together with mean + / - SD . * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 . 2 - tailed unpaired t - test . Additional metabolic measurement experiments of SCC cells plus / minus HSD17B7 overexpression and silencing , in the context of additional manipulations are shown in Fig 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SCC13 cells stably infected with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) (C) or two HSD17B7-silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean +/- SD. **p<0.005; ***p<0.0005; ****p<0.0001. 2-tailed unpaired t-test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations are shown in Fig 9.


( C ) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type ( HSD - wt ) and Y193G and K197G mutated ( HSD - mut ) forms versus empty vector control ( Ctrl ) as in ( B ) were assayed for levels of Electron Transport Chain activity , ATP and mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * p < 0 . 005 ; * * * p < 0 . 0005 . 2 - tailed unpaired t - test . Individual experimental values are provided in Dataset EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


( D ) SCC13 cells were infected with two HSD17B7 silencing lentiviruses ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) followed , 3 days after infection and antibiotic selection , by treatment with Zymosterol ( 2 . 5uM ) or ethanol vehicle alone for 48hours . Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity , ATP and mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * * * p < 0 . 0001 , 2 - tailed unpaired t - test . Individual experimental values are provided in Dataset EV3 .

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

(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( A ) left untreated or treated with increasing concentrations of LLO for 15 min . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (A) left untreated or treated with increasing concentrations of LLO for 15 min. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).


( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( B ) left uninfected ( U ) or infected with wt or Δhly Lm for 1 h . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (B) left uninfected (U) or infected with wt or Δhly Lm for 1 h. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).


( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( C ) infected with wt Lm for the indicated time points . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (C) infected with wt Lm for the indicated time points. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).


( D ) Confocal microscopy images of HeLa cells : ( D ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) , immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) and stained with DAPI ( blue ) . Arrows point to various NMHCIIA - Gp96 positive cortical bundles in different cell . Insets show high magnification image ( E ) Quantification of Pearson ' s correlation coefficient ( PCC ) within : uninfected cells ( U ) , LLO - treated cells , cortical NMHCIIA bundles or equivalent size control cellular ROI . ( Ctrl ROI ) Data are mean ± SEM ( n = 6 ) ; p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of HeLa cells: (D) left untreated or treated with LLO (0.5 nM, 15 min), immunolabelled for NMHCIIA (green), Gp96 (red) and stained with DAPI (blue). Arrows point to various NMHCIIA-Gp96 positive cortical bundles in different cell. Insets show high magnification image (E) Quantification of Pearson's correlation coefficient (PCC) within: uninfected cells (U), LLO-treated cells, cortical NMHCIIA bundles or equivalent size control cellular ROI. (Ctrl ROI) Data are mean±SEM (n=6); p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.05, **p<0.01, ***p<0.001


( F ) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) Lm ( blue ) and stained with DAPI ( white ) . Arrows indicate NMHCIIA - Gp96 cortical bundles at cortical sites close to wt Lm . ( D and F ) Scale bar - 10 μm . See also Figs EV1 and EV2 .

(F) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA (green), Gp96 (red) Lm (blue) and stained with DAPI (white). Arrows indicate NMHCIIA-Gp96 cortical bundles at cortical sites close to wt Lm. (D and F) Scale bar - 10 μm. See also Figs EV1 and EV2.


( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( A ) GFP - NMHCIIA . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( A ) Arrows indicate NMHCIIA bundles at PM blebbing sites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (A) GFP-NMHCIIA . LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (A) Arrows indicate NMHCIIA bundles at PM blebbing sites.


( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( B ) simultaneously GFP - NMHCIIA and mcherry - KDEL . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( B ) Highlights depicting ER structures within NMHCIIA bundles and PM blebs . Arrows indicate cortical ER surrounding NMHCIIA accumulations , arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (B) simultaneously GFP-NMHCIIA and mcherry-KDEL. LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (B) Highlights depicting ER structures within NMHCIIA bundles and PM blebs. Arrows indicate cortical ER surrounding NMHCIIA accumulations, arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs


( C ) Confocal microscopy images of HeLa cells left untreated ( Control ) or treated with LLO ( 0 . 5 nM 15 min ) . Cells were stained with FITC - WGA ( Plasma membrane , PM - red ) and immunolabelled for NMHCIIA ( green ) . Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated ( 1 ) or detached ( 2 ) from the cell body .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of HeLa cells left untreated (Control) or treated with LLO (0.5 nM 15 min). Cells were stained with FITC-WGA (Plasma membrane, PM-red) and immunolabelled for NMHCIIA (green). Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated (1) or detached (2) from the cell body.


( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( D ) LLO pre - incubated with cholesterol ( LLOCHT )

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

(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT)


( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( E ) LLO in medium supplemented with 140 mM K + ( HighK + ) and LLO in Ca2 + - free medium Ca2 + ( Ca2 + free )

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

(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (E) LLO in medium supplemented with 140 mM K+(HighK+) and LLO in Ca2+-free medium Ca2+ (Ca2+free)


( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( F ) LLO in the presence of 25 μM blebbistatin ( BB ) .

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

(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (F) LLO in the presence of 25 μM blebbistatin (BB).


( G ) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER - KDEL ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) .

O O O O O O O O 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) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER-KDEL (blue), NMHCIIA (green) and Gp96 (red).


( H ) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA ( green ) and Gp96 ( red ) . Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA .

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

(H) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA (green) and Gp96 (red). Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA.


( I ) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) , SLO ( 1 . 5 μg / ml , 30 min ) ( SLO ) or aerolysin ( 0 . 2 nM , 40 min ) ( AL ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL).


( A ) Confocal microscopy images of shCtrl , shGp96 or shNMHCIIA HeLa cells treated with LLO ( 0 . 5 nM , 15 min ) and immunolabelled for KDEL - proteins ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) . Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells . Scale bar - 10 μm . ( B - D ) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with ( B ) LLO ( 0 . 5 nM , 15 min ) , ( C ) aerolysin ( 0 . 2 nM , 40 min ) or ( D ) with wt Lm . Values are the mean ± SEM ( n ≥ 3 ) , p - values were calculated using ( B - C ) one - way - ANOVA with Dunnett post hoc analyses and ( D ) two - tailed un - paired Student ' s t - test , * p < 0 . 5 , * * * p < 0 . 001 . For shNMHCIIA , bundles were detected following actin staining ( Fig EV3C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with LLO (0.5 nM, 15 min) and immunolabelled for KDEL-proteins (blue), NMHCIIA (green) and Gp96 (red). Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells. Scale bar - 10 μm. (B-D) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with (B) LLO (0.5 nM, 15 min), (C) aerolysin (0.2 nM, 40 min) or (D) with wt Lm. Values are the mean ±SEM (n≥3), p-values were calculated using (B-C) one-way-ANOVA with Dunnett post hoc analyses and (D) two-tailed un-paired Student's t-test, *p<0.5, ***p<0.001. For shNMHCIIA, bundles were detected following actin staining (Fig EV3C).


( E - F ) Number of ( E ) blebs per cell or ( F ) retracting blebs per cell evaluated by time - lapse microscopy analysis of LLO - treated shCtrl or shGp96 cells . shCtrl n = 32 cells and shGp96 n = 40 cells , p - values were calculated using two - tailed un - paired Student ' s t - test * p < 0 . 5 . ( G ) Sequential frames of time - lapse microscopy analysis of LLO - treated HeLa cells expressing GFP - NMHCIIA ( shCtrl , shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl - BB ) . LLO was added to culture medium 10 seconds before t0 .

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

(E-F) Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n=32 cells and shGp96 n=40 cells, p-values were calculated using two-tailed un-paired Student's t-test *p<0.5. (G) Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFP-NMHCIIA (shCtrl, shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl-BB). LLO was added to culture medium 10 seconds before t0.


( H ) Immunoblots of Ser19 - phosphorylated MRLC ( pMRLC ) , MRLC and actin levels from shCtrl or shGp96 cells left untreated ( U ) or treated with 0 . 1 nM LLO for the indicated time points . Quantification of pMRLC levels corresponds to the mean ± SEM ( n ≥ 3 ) , p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 , * * * p < 0 . 001 . See also Appendix Fig S2 and Fig EV3 .

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

(H) Immunoblots of Ser19-phosphorylated MRLC (pMRLC), MRLC and actin levels from shCtrl or shGp96 cells left untreated (U) or treated with 0.1 nM LLO for the indicated time points. Quantification of pMRLC levels corresponds to the mean ± SEM (n≥3), p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01, ***p<0.001. See also Appendix Fig S2 and Fig EV3.


( Ai ) Confocal microscopy images of LLO - treated HeLa cells immunolabelled for the indicated proteins . Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles . ( Aii ) Quantification of Pearson ' s correlation coefficient ( PCC ) between NMHCIIA and the indicated proteins within entire LLO - treated cells , cortical bundles or equivalent size control cellular ROI ( Ctrl ROI ) . For Calpain 2 cortical bundles were defined by actin staining . Data are representative of one experiment repeated 3 independent times with similar results . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 ; * * * p < 0 . 001 , ns - non

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

(Ai) Confocal microscopy images of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles. (Aii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent size control cellular ROI (Ctrl ROI). For Calpain 2 cortical bundles were defined by actin staining. Data are representative of one experiment repeated 3 independent times with similar results. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses ** p<0.01; *** p<0.001, ns - non significant


( B ) Immunoblots of NMHCIIA , Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) ( LLO ) .

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(B) Immunoblots of NMHCIIA, Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO).


( C ) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with 25 μg / ml of Wnt5a for the indicated time .

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(C) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time.


( D ) Confocal microscopy images of HeLa cells untreated ( Mock ) or treated with 25 μg / ml of Wnt5a for 30 min . Cells were immunolabelled for ER - KDEL ( blue ) , Gp96 ( red ) and NMHCIIA ( green ) . Arrows indicate polarised localization of NMHCIIA , ER - KDEL and Gp96 in Wnt5a - treated cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarised localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells.


( E - F ) Confocal microscopy images of shCtlr , shGp96 or shNMHCIIA HeLa cells stained for actin ( red ) and DAPI ( blue ) and immunolabelled for ( E ) NMHCIIA ( green ) or ( F ) focal adhesion kinase ( FAK ) ( green ) . ( A , D - F ) Scale bar - 10 μm . Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points . Quantification of the % of cells with ( G ) stress fibers or ( H ) focal adhesion points labelled by FAK .

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

(E-F) Confocal microscopy images of shCtlr, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). (A, D-F) Scale bar - 10 μm. Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points. Quantification of the % of cells with (G) stress fibers or (H) focal adhesion points labelled by FAK.


( I - J ) Wound closure assay . ( I ) Sequential frames of time - lapse microscopy of HeLa cells grown to confluence separated by a stopper . Stopper was removed at t0 and cellular migration was imaged for the indicated times . ( J ) Quantification of the % of wound area occupied by migrating cells over time stopper removal ( upper panel ) and respective rate of closure ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the % of wound area occupied by migrating cells over time stopper removal (upper panel) and respective rate of closure (lower panel).


( A ) Epifluorescence microscopy images of supernatants from LLO - treated ( 0 . 5 nM 15 min ) shCtlr , shGp96 or shNMHCIIA HeLa cells collected into poly - lysine - coated cover slips , fixed and stained with FITC - WGA ( green ) and DAPI ( blue ) . Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei . Scale bar - 10 μm . ( B ) Quantification of released PM blebs ( total FITCWGA / field ) . Values are the means ± SEM ( n ≥ 3 ) . * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epifluorescence microscopy images of supernatants from LLO-treated (0.5 nM 15 min) shCtlr, shGp96 or shNMHCIIA HeLa cells collected into poly-lysine-coated cover slips, fixed and stained with FITC-WGA (green) and DAPI (blue). Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei. Scale bar - 10 μm. (B) Quantification of released PM blebs (total FITCWGA/field). Values are the means ± SEM (n≥3). ***p<0.001.


( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( C ) 0 . 5 nM LLO for the indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (C) 0.5 nM LLO for the indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( D ) 1 . 5 or 3 nM LLO for 15 min ; or ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (D) 1.5 or 3 nM LLO for 15 min; or (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


( A - B ) Epifluorecence microscopy images of HeLa cells ( A ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) . Cells were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) and stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable bacteria .

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

(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.


( A - B ) Epifluorecence microscopy images of HeLa cells ( B ) infected with GFP - expressing wt Lm for 6 h . Cells were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) and stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable bacteria .

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

(A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.


( C ) Quantification of the % of infected cells with exofacial - PS sites in HeLa cells infected with wt Lm for 6 h . Data are the mean ± SEM ( n = 3 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 .

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(C) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5.


( D - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa and RAW264 . 7 cells , infected with GFP - expressing wt or Δhly Lm for 6 h . The levels of PI incorporation by uninfected cells were subtracted from infected samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria ( Fig Appendix Fig S3 ) .

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(D-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa and RAW264.7 cells, infected with GFP-expressing wt or Δhly Lm for 6 h. The levels of PI incorporation by uninfected cells were subtracted from infected samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria (Fig Appendix Fig S3).


( A ) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates ( WCL ) and IP fractions ( IP Gp96 ) of extracts of zebrafish larvae 3 dpf uninfected ( U ) or infected with wt or ∆ hly Lm ( low dose 200 - 1500 CFU ) for 24h .

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(A) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates (WCL) and IP fractions (IP Gp96) of extracts of zebrafish larvae 3 dpf uninfected (U) or infected with wt or ∆hly Lm (low dose 200-1500 CFU) for 24h.


( B ) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control ( Ctrl - mo ) or Gp96 morpholino oligonucleotides ( Gp96 mo ) .

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(B) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control (Ctrl-mo) or Gp96 morpholino oligonucleotides (Gp96 mo).


( C ) Confocal microscopy images of zebrafish larvae ( Ctrl mo or Gp96 mo ) infected ( low dose ) in the tail muscle , with the GFP - expressing wt or Δhly Lm for 24 h , immunolabelled for KDEL - proteins ( green ) , and stained with phalloidin ( actin , red ) and DAPI ( white ) . Images show actin rich structures at the cell cortex ( arrows ) and ER - KDEL puncta ( arrowheads ) in larvae infected with wt strain .

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(C) Confocal microscopy images of zebrafish larvae (Ctrl mo or Gp96 mo) infected (low dose) in the tail muscle, with the GFP-expressing wt or Δhly Lm for 24 h, immunolabelled for KDEL-proteins (green), and stained with phalloidin (actin, red) and DAPI (white). Images show actin rich structures at the cell cortex (arrows) and ER-KDEL puncta (arrowheads) in larvae infected with wt strain.


( D ) CFU counts per zebrafish larvae ( Ctrl or Gp96 mo ) infected with wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm and analysed at 0 , 24 , 48 hpi . Results are mean ± SEM ( n ≥ 3 ) ( horizontal bars ) and each circle represents 1 larvae . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CFU counts per zebrafish larvae (Ctrl or Gp96 mo) infected with wt (low dose) or Δhly (high dose > 10,000 CFU) Lm and analysed at 0, 24, 48 hpi. Results are mean ± SEM (n≥3) (horizontal bars) and each circle represents 1 larvae. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01.


( E ) Survival curves of zebrafish larvae ( Ctrl - mo or Gp96 - mo ) infected with a wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm . Results are the mean ± SEM ( n ≥ 3 ) . wt infection of Ctrl and Gp96 mo , n = 28 larvae ; Δhly infection of Gp96 mo n = 13 . p - values were calculated using Log Rank test , * * p < 0 . 01 .

(E) Survival curves of zebrafish larvae (Ctrl-mo or Gp96-mo) infected with a wt (low dose) or Δhly (high dose > 10,000 CFU) Lm. Results are the mean ± SEM (n≥3). wt infection of Ctrl and Gp96 mo, n=28 larvae; Δhly infection of Gp96 mo n=13. p-values were calculated using Log Rank test, **p<0.01.


( F ) CFU counts per zebrafish larvae infected with a high dose of ∆ hly Lm at different times post - infection . Results are mean ± SEM ( n ≥ 3 ) ( horizontal bars ) ; each point represents 1 larvae . See also Fig EV5 .

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(F) CFU counts per zebrafish larvae infected with a high dose of ∆hly Lm at different times post-infection. Results are mean ± SEM (n≥3) (horizontal bars); each point represents 1 larvae. See also Fig EV5.


A ) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation ( 15 Gy ) followed by analysis by SDS - PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin . ( NT , no treatment ) . The lysate from the 24 hours time point was treated with phosphatase ( Ppase ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS-PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin. (NT, no treatment). The lysate from the 24 hours time point was treated with phosphatase (Ppase).


B ) U2OS cells were left untreated or treated with ATM ( KU 55933 , 10 μM ) , ATR ( ETP - 464 , 1 μM ) , or DNA - PK ( NU7441 , 1 μM ) inhibitors 30 minutes prior to exposure to IR ( 15 Gy , 2h recovery ) . Immunoblots were performed with indicated antibodies .

O O O O O O O O O O O O B-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 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) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies.


C ) U2OS cells were left untreated ( NT ) or exposed to HU ( 2 mM ) for the indicated times . The lysate from the 24 hour time point was treated with phosphatase ( Ppase ) . Immunoblots were performed with indicated antibodies .

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

C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies.


D ) Untreated U2OS cells or cells exposed to either HU ( 2 mM , 24h ) or IR ( 15 Gy , 2h recovery ) alone or together with ATM inhibitor ( KU55933 , 10 μM ) or inhibitors to ATR ( ATR # 1 : ETP - 464 , 1 μM ; ATR # 2 : AZ - 20 , 3 μM ) were harvested and lysates were prepared . The inhibitors were added 30 minutes prior to HU or IR treatment . Immunoblots were performed with indicated antibodies .

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D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies.


E ) ATR kinase assay was performed with N - terminal PALB2 ( aa 1 - 560 ; GST - PALB2 - N ) as a substrate and HA - tagged ATR as kinase and visualized by Fujifilm Phosphoimager . 293T cells transfected with empty vector ( EV ) , GST , and ATR inhibition ( AZ - 20 , 30 μM ) are controls . HA - ATR and GST - PALB2 - N was controlled by immunoblotting with HA and GST antibodies .

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E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.