C , D PBMC - derived macrophages primed with 100 ng / ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release from pigment - or ΔcylE extract - treated cells was measured by Luminex assay ( D ) . ( C ) n = 4 , * * * * P < 0 . 0001 , * * P = 0 . 002 . ( D ) n = 4 , * * P = 0 . 005 .

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

C, D PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.


A , B WT THP - 1macrophages were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by Luminex assay ( B ) . Data information : Data from three independent experiments performed in triplicate are shown , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA . ( A ) n = 3 , * * P = 0 . 0080 for WT versus ΔcovR ; * * P = 0 . 0016 for ΔcovR versus ΔcovRΔcylE . ( B ) n = 3 , * P = 0 . 03 for WT versus ΔcovR ; * P = 0 . 02 for ΔcovR versus ΔcovRΔcylE . ( C ) n = 3 , * * * * P < 0 . 0001 . ( D ) n = 3 , * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B WT THP-1macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B).Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.


C , D WT THP - 1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release from pigment - or ΔcylE extract - treated cells was measured by Luminex assay ( D ) . ( C ) n = 3 , * * * * P < 0 . 0001 . ( D ) n = 3 , * 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 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

C, D WT THP-1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).(C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.


A , B THP - 1macrophages transfected with empty vector , scrambled control , shASC , or shNLRP3 were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by Luminex assay ( B ) . Data from three independent experiments performed in triplicate are shown . Data information : Data were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM . ( A ) n = 3 , for WT : * * P = 0 . 001 ( vector versus shNLRP3 ) , * * P = 0 . 0016 ( vector versus shASC ) ; for ΔcovR : * * * P = 0 . 0008 ( vector versus shNLRP3 ) , * * * P = 0 . 0001 ( vector versus shASC ) , * P = 0 . 047 ( scrambled versus shNLRP3 ) , * * P = 0 . 0054 ( scrambled versus shASC ) . Data obtained from THP - 1 / vector was not significantly different from THP - 1 / scrambled , P = 0 . 18 . ( B ) n = 3 , * P = 0 . 025 . Data obtained from THP - 1 / vector were not significantly different from THP - 1 / scrambled , P = 0 . 89 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 THP-1macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). Data from three independent experiments performed in triplicate are shown.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (A) n = 3, for WT: **P = 0.001 (vector versus shNLRP3), **P = 0.0016 (vector versus shASC); for ΔcovR: ***P = 0.0008 (vector versus shNLRP3), ***P = 0.0001 (vector versus shASC), *P = 0.047 (scrambled versus shNLRP3), **P = 0.0054 (scrambled versus shASC). Data obtained from THP-1/vector was not significantly different from THP-1/scrambled, P = 0.18. (B) n = 3, *P = 0.025. Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.89.


C , D The shRNA THP - 1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release in supernatants was measured by Luminex assay ( D ) . Pigment - mediated cytotoxicity is dependent on the NLRP3 inflammasome components , suggesting that pigment is inducing a programmed cell death . Data shown are the average of at least three independent experiments performed in triplicate . Data information : Data were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM . ( C ) n = 3 , for 4 μM pigment : * * P = 0 . 0063 ( vector versus shNLRP3 ) , * * P = 0 . 0035 ( vector versus shASC ) , * P = 0 . 01 ( scrambled versus shNLRP3 ) , * * P = 0 . 009 ( scrambled versus shASC ) ; for 2 μM pigment : * * P = 0 . 0088 ( vector versus shNLRP3 ) , * * P = 0 . 0066 ( vector versus shASC ) , * P = 0 . 01 ( scrambled versus shNLRP3 or shASC ) ; for 1 μM pigment : * P = 0 . 02 ( vector versus shNLRP3 ) , * P = 0 . 01 ( vector versus shASC ) . Data obtained from THP - 1 / vector were not significantly different from THP - 1 / scrambled , P = 0 . 99 . ( D ) n = 3 , * * * * P < 0 . 0001 , * P = 0 . 015 .

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

C, D The shRNA THP-1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.


A , B THP - 1macrophages proficient for NLRP3 ( transfected with scrambled control , A ) or deficient for NLRP3 ( shNLRP3 , B ) were treated with 1 μM pigment or ΔcylE extract for 20 min , and propidium iodide ( PI ) was added during the final 10 min . PI uptake was measured by flow cytometry , and data shown are representative of two 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 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

A, B THP-1macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments.


C , D Intracellular potassium concentration was measured by ICP - AES . THP - 1macrophages transfected with the scrambled control ( C ) or shNLRP3 ( D ) were treated with GBSpigment ( 1 μM ) or an equivalent amount of the ΔcylE extract . At various time points , cells were lysed and intracellular [ K + ] was measured relative to untreated cells ( see bars and left y - axis ) , and percent cell death was quantified by alamar blue ( see squares , dotted connecting lines and right y - axis ) . Both NLRP3 - proficient and NLRP3 - deficient macrophages initially lose intracellular K + due to GBSpigment ( compare t = 0 min to t = 30 min ) , but the NLRP3 - deficient cells ( shNLRP3 ) are able to recover , while the scrambled control do not , demonstrating that initial K + loss occurs independently of NLRP3 . Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett ' s multiple comparison test following ANOVA ; all data were compared to control at t = 0 , error bars ± SEM . ( C ) n = 3 , * * * P = 0 . 0002 , * P = 0 . 019 , * * P = 0 . 0032 for 120 min , * * P = 0 . 0072 for 180 min . ( D ) n = 3 , * * P = 0 . 0043 , * P = 0 . 031 .

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

C, D Intracellular potassium concentration was measured by ICP-AES. THP-1macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBSpigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBSpigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031.


( E ) WT THP - 1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride , and cytotoxicity was measured by alamar blue assay . The addition of potassium chloride is able to protect the macrophages from cytolysis , demonstrating that K + efflux is essential for this process . Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM ( n = 3 , * * * * P < 0 . 0001 , * * P = 0 . 0036 ) .

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

(E) WT THP-1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).


THP - 1macrophages were treated with GBSpigment , and caspase 1 activation was measured by flow cytometry using a FLICA reagent . Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line , demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome . Results are representative of three independent experiments .

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

THP-1macrophages were treated with GBSpigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments.


WT THP - 1macrophages were treated with the caspase 1 inhibitor Z - YVAD - FMK , the caspase 3 / 7 inhibitor Z - DEVD - FMK , or DMSO only prior to treatment with the GBSpigment . YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment , demonstrating that caspase 1 is required for GBSpigment - mediated cell death , characteristic of pyroptosis , while DEVD had no effect . Data are average of three independent experiments , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA ( n = 3 , * * * * P < 0 . 0001 , * * * P = 0 . 0002 , * P = 0 . 014 ) .

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

WT THP-1macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBSpigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment, demonstrating that caspase 1 is required for GBSpigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).


In utero fetal death in wild - type CD - 1mice due to infection with GBS WT , hyperhemolytic ΔcovR , and non - hemolytic ΔcovRΔcylE . Fetal death is represented by the number of dead fetuses / total number of fetuses obtained from six pregnant mice per group . ' n ' indicates total number of pups ( both live and dead ) ; * * * * P < 0 . 0001 , Fisher ' s exact 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

In utero fetal death in wild-type CD-1mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. 'n' indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.


H & amp ; amp ; E staining of uterine tissue . Open arrow indicates the presence of few mononuclear cells , whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris .

O O O O O O O O O O O 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&amp;E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.


Fetal death due to infection with hyperhemolytic GBS ΔcovR and non - hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome - deficient mice ; fetal death is represented by the number of dead fetuses / total number of fetuses obtained from 6 pregnant mice per group . ' n ' indicates total number of pups ( both live and dead ) ; * P = 0 . 011 , * * P = 0 . 0015 , * * * * P < 0 . 0001 , Fisher ' s exact test . Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS ; P = 0 . 31 , Fisher ' s exact 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 O O O O O O O O O O O O O O O O O O O O O O O O O

Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. 'n' indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.


Female pregnant wild - type ( CD - 1 , C57BL6 ) or NLRP3 - deficient mice were injected in utero with 106 - 7CFU of GBS WT , ΔcovR , or ΔcovRΔcylE and monitored for preterm birth . Surgery and GBS inoculation for each pregnant mouse were performed independently . Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls . Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains ( n = 6 / pup ; of note , pups that were delivered preterm were excluded from CFU enumeration . Scheme of pup numbering is shown in ( A ) . RUH and LUH indicate right uterine horn and left uterine horn , respectively . CFUs are not significantly different between any of the groups ( ANOVA , P = 0 . 6 , error bars ± SEM ) .

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

Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).


Female pregnant wild - type ( CD - 1 , C57BL6 ) or NLRP3 - deficient mice were injected in utero with 106 - 7CFU of GBS WT , ΔcovR , or ΔcovRΔcylE and monitored for preterm birth . Surgery and GBS inoculation for each pregnant mouse were performed independently . Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls . IL - 1β levels in GBS - infected tissues ( placenta and fetus , n = 6 / group ) was measured by Luminex assay ( * P = 0 . 025 , * * * P = 0 . 0002 , * * * * P < 0 . 0001 , Bonferroni ' s multiple comparison test following ANOVA ) . IL - 1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS ; P = 0 . 99 .

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

Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS; P = 0.99.


Quantification of NANOG - positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non - target or Kdm3b siRNA in FBS media . Values represent fold change over non - target control providing relative quantitation . Quantification of NANOG - positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non - targeting or Kdm3b siRNA in KSR media . Values represent fold change over non - target control providing relative quantitation .

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

Quantification of NANOG-positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non-target or Kdm3b siRNA in FBS media. Values represent fold change over non-target control providing relative quantitation. Quantification of NANOG-positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non-targeting or Kdm3b siRNA in KSR media. Values represent fold change over non-target control providing relative quantitation.


Quantification of NANOG positive colonies after retroviral expression of OSKM ( Left ) or OSK ( Right ) in MEF treated with non - targeting or siRNA targeting Kdm3 . Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming . Values represent fold change over non - target control providing relative quantitation .

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

Quantification of NANOG positive colonies after retroviral expression of OSKM (Left) or OSK (Right) in MEF treated with non-targeting or siRNA targeting Kdm3. Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming. Values represent fold change over non-target control providing relative quantitation.


Immunoblot of H3K9me1 / 2 histone demethylase in WT or Kdm3b CRISPR targeted pre - iPSC clones . These pre - iPSCs carry a Nanog - GFP reporter . Parent line ( WT ) , and two Kdm3b - KO clones using gRNA targeting either exon 1 ( KO # 1 ) or exon 2 ( KO # 2 ) are shown . RNA Polymerase II levels are the loading control .

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Immunoblot of H3K9me1/2 histone demethylase in WT or Kdm3b CRISPR targeted pre-iPSC clones. These pre-iPSCs carry a Nanog-GFP reporter. Parent line (WT), and two Kdm3b-KO clones using gRNA targeting either exon 1 (KO#1) or exon 2 (KO#2) are shown. RNA Polymerase II levels are the loading control.


Quantification of Nanog - GFP positive cells after 10 days of AA + 2i treatment of WT or Kdm3b - deleted pre - iPSCs .

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Quantification of Nanog-GFP positive cells after 10 days of AA+2i treatment of WT or Kdm3b-deleted pre-iPSCs.


Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b - KO pre - iPSCs .

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Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b-KO pre-iPSCs.


Quantification of percent Nanog - GFP positive cells after 10 days of AA + 2i or AA + 2i with 5µM UNC0638 in WT or Kdm3b deleted pre - iPSCs .

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Quantification of percent Nanog-GFP positive cells after 10 days of AA+2i or AA+2i with 5µM UNC0638 in WT or Kdm3b deleted pre-iPSCs.


Pearson correlation of transcripts per million ( TPM ) of WT and Kdm3b - KO pre - iPSC upon DMSO or AA + 2i treatment for 2 or 10 days and mESC , reprogramming populations from Chronis et . al ( 2017 ) , ( labelled as CC ) .

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Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC).


k - means clustering of the union of DEG ( FDR > 0 . 99 ) between day 2 and DMSO in WT and Kdm3b - KO pre - iPSCs . Values represent a log2 - fold change of day 2 over DMSO . Key pluripotency genes are shown on the right . k - means clustering of the union of DEG ( FDR > 0 . 99 ) between day 10 and DMSO in WT and Kdm3b - KO pre - iPSCs . Values represent a log2 - fold change of day 10 over DMSO . Key pluripotency genes or epigenetic regulators are shown on the right .

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k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right.


Expression of Tet enzymes and Tdg during AA + 2i treatment in WT or Kdm3b - KO pre - iPSCs . Error bars represent the standard deviation of two biological replicates from the RNA - Seq sample .

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Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample.


Quantification of percent Nanog - GFP cells of WT , Kdm3b - KO and Kdm3b - KO pre - iPSC clones expressing Tet1 catalytic domain ( Tet1CD ) , Nanog , or both .

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Quantification of percent Nanog-GFP cells of WT, Kdm3b-KO and Kdm3b-KO pre-iPSC clones expressing Tet1 catalytic domain (Tet1CD), Nanog, or both.


Representative 5hmC profiles during AA + 2i conversion of WT pre - iPSCs . Illustration of the 5hmC pattern is shown in the bottom panel .

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Representative 5hmC profiles during AA+2i conversion of WT pre-iPSCs. Illustration of the 5hmC pattern is shown in the bottom panel.


Heatmap of k - means clustered 5hmC patterns with histone modifications in pre - iPSCs and mESCs from Chronis et al ( 2017 ) .

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Heatmap of k-means clustered 5hmC patterns with histone modifications in pre-iPSCs and mESCs from Chronis et al (2017).


Representative profiles of regions that undergo demethylation through a 5hmC intermediate . Top - 5hmC profile during AA + 2i treatment . Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs , data from Rais et al 2013 . Bottom - Percent 5hmC via Tet - assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days , data from Hu et al 2014 .

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Representative profiles of regions that undergo demethylation through a 5hmC intermediate. Top - 5hmC profile during AA+2i treatment. Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs, data from Rais et al 2013. Bottom - Percent 5hmC via Tet-assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days, data from Hu et al 2014.


Left - Quantification of percent Nanog - GFP positive colonies . Right - Total Nanog - GFP positive colonies obtained after ten days of AA + 2i treatment in Tdg - depleted pre - iPSC ( Tdg ) or control ( Empty ) .

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Left - Quantification of percent Nanog-GFP positive colonies. Right - Total Nanog-GFP positive colonies obtained after ten days of AA+2i treatment in Tdg-depleted pre-iPSC (Tdg) or control (Empty).


Quantification of percent Nanog - GFP positive cells of WT or Kdm3b - KO pre - IPSC clones expressing Nanog , Tet1CD , or Nanog + Tet1CD after 15 days of AA + 2i or AA + 2i with 4 or 5µM of UNC0638 .

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Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638.


Left - Heatmap of K - mean clustered 5hmC patterns in WT pre - iPSC with 5hmC profile of Kdm3b - KO pre - iPSC treated with AA + 2i for 0 , 2 or 10 days . Middle - Metaplot of 5hmC signal on day 10 between WT ( orange ) and Kdm3b - KO ( black ) for each cluster . Y - axis scale is in normalized coverage data ( 1x106 / Total Count ) . Right - Illustration of 5hmC pattern in WT and Kdm3b - KO during AA + 2i treatment .

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Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment.


Representative 5hmC profiles during AA + 2i conversion of WT and Kdm3b - KO pre - iPSC .

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Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC.


Scheme for identifying genes with both misregulated 5hmC and expression . Heatmap of two - fold misregulated 5hmC and two - fold differential expression between WT and Kdm3b - KO after 10 days of AA + 2i treatment ( 607 genes ) . Peaks are sorted by 5hmC signal in WT . RNA seq values represent a log2 - fold change of day 10 over DMSO for either WT or Kdm3b KO pre - iPSCs .

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Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs.


Box plot of expression for genes that are two - fold misregulated 5hmC and expression between WT and Kdm3b - KO on day 10 ( 607 genes ) .

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Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes).


Heatmap of POU5F1 bound peaks in both mESC and pre - IPSC ( Cluster 1 ) and mESC alone ( Cluster 2 ) with 5hmC levels during Day 0 , 2 and 10 of AA + 2i treatment .

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Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment.


Metaplots of POU5F1 binding for Cluster 1 ( Left ) and Cluster 2 ( Right ) regions in pre - iPSCs and mESCs .

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Metaplots of POU5F1 binding for Cluster 1 (Left) and Cluster 2 (Right) regions in pre-iPSCs and mESCs.


Metaplots of 5hmC Enrichment for Cluster 1 ( Left ) and Cluster 2 ( Right ) regions for Day 0 , 2 and 10 of AA + 2i treatment .

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Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment.


Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding . Right - Heatmap of 5hmC profile during AA + 2i treatment of WT pre - iPSC and POU5F1 binding in pre - iPSCs or mESCs at the loci identified in the left panel .

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Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel.


Representative 5hmC profiles during AA + 2i conversion of WT and Kdm3b - KO pre - iPSC with POU5F1 binding in pre - iPSCs and mESCs . Loci were assigned to the nearest gene .

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Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene.


ChIP - PCR for POU5F1 during AA + 2i reprogramming of WT and Kdm3b - KO pre - iPSCs . Values are fold change of enrichment between WT and Kdm3b KO ( WT / Kdm3bKO ) .

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ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO).


tSNE of Tet1 , Tet2 , Nanog , and Cdh1 expression in single cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b . MEF , Reprogramming ( REPROG ) , and mESCs cells are highlighted in red dotted lines . Specific reprogramming timepoints are highlighted within the reprogramming cluster in arrows . Color corresponds to log10 of transcript counts for each individual cell .

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tSNE of Tet1, Tet2, Nanog, and Cdh1 expression in single cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b. MEF, Reprogramming (REPROG), and mESCs cells are highlighted in red dotted lines. Specific reprogramming timepoints are highlighted within the reprogramming cluster in arrows. Color corresponds to log10 of transcript counts for each individual cell.


Bar graphs indicating number of cells expressing Tet1 , Tet2 , Cdh1 or Nanog from the reprograming population ( Day 3 , 6 , 9 and 12 ) . Number of cells per reprogramming population is highlighted within legend .

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Bar graphs indicating number of cells expressing Tet1, Tet2, Cdh1 or Nanog from the reprograming population (Day 3, 6, 9 and 12). Number of cells per reprogramming population is highlighted within legend.


Scheme for deriving Tet1 and Tet2 double knockout pre - iPSC lines . Tet1KO / KO Tet2fl / fl or Tet1WT / WT Tet2fl / fl MEFs were treated with retroviruses containing OSKM . Tet1KO / KO Tet2fl / fl or Tet1WT / WT Tet2fl / fl pre - iPSC clones were isolated and treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre - iPSCs with Tet1KO / KO Tet2fl / fl , Tet1KO / KO Tet2KO / KO , Tet1WT / WT Tet2fl / fl , and Tet1WT / WT , and Tet2KO / KO . Scale bar , 50 μm .

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Scheme for deriving Tet1 and Tet2 double knockout pre-iPSC lines. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl MEFs were treated with retroviruses containing OSKM. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl pre-iPSC clones were isolated and treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre-iPSCs with Tet1KO/KO Tet2fl/fl, Tet1KO/KO Tet2KO/KO, Tet1WT/WT Tet2fl/fl, and Tet1WT/WT, and Tet2KO/KO. Scale bar, 50 μm.


Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT / WT Tet2fl / fl , Tet1WT / WT Tet2KO / KO , Tet1KO / KO Tet2fl / fl , and Tet1KO / KO Tet2KO / KO after 10 days of AA + 2i treatment .

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Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT/WT Tet2fl/fl, Tet1WT/WT Tet2KO/KO, Tet1KO/KO Tet2fl/fl, and Tet1KO/KO Tet2KO/KO after 10 days of AA+2i treatment.


( C ) PCR analysis of a pool of transfected cells after treatment with Cas9 - D10A ' nickase ' and the indicated combination of gRNAs The ~ 200 bp product was centred around the start of exon 1 of the SLD5 gene . Efficient cutting of both template strands by Cas9 - nickase was indicated by the production of a smeary PCR product ( gRNAs 2 + 3 ) , reflecting DNA repair that mostly produced small deletions around the nicking site The combination of gRNAs 1 + 3 did not produce a smeary PCR , due to a polymorphism within the PAM motif of the genomic

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(C) PCR analysis of a pool of transfected cells after treatment with Cas9-D10A 'nickase' and the indicated combination of gRNAs The ~200 bp product was centred around the start of exon 1 of the SLD5 gene. Efficient cutting of both template strands by Cas9-nickase was indicated by the production of a smeary PCR product (gRNAs 2+3), reflecting DNA repair that mostly produced small deletions around the nicking site The combination of gRNAs 1+3 did not produce a smeary PCR, due to a polymorphism within the PAM motif of the genomic locus


( F ) Immunoblot indicating successful tagging of both alleles of SLD5 with TAP or GFP .

(F) Immunoblot indicating successful tagging of both alleles of SLD5 with TAP or GFP.


( G ) Asynchronous cultures of the indicated cells were used to generate cell extracts , which were then incubated with agarose beads coupled to anti - GFP nanobodies .

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(G) Asynchronous cultures of the indicated cells were used to generate cell extracts, which were then incubated with agarose beads coupled to anti-GFP nanobodies.


( H ) Extracts of TAP - SLD5 mouse ES cells were incubated plus or minus DNase for 30 minutes at 4 ° C , before immunoprecipitation of TAP - SLD5 .

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(H) Extracts of TAP-SLD5 mouse ES cells were incubated plus or minus DNase for 30 minutes at 4 °C, before immunoprecipitation of TAP-SLD5.


( I ) TAP - SLD5 cells were synchronised as indicated DNA content was monitored throughout the experiment by flow cytometry .

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(I) TAP-SLD5 cells were synchronised as indicated DNA content was monitored throughout the experiment by flow cytometry.


Samples were taken at the indicated times and used to isolate TAP - SLD5 by immunoprecipitation on IgG beads . The indicated proteins were monitored by immunoblotting . For reasons of space , samples A , S , M and 1 - 6 were resolved in a separate gel to samples 7 - 8 .

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Samples were taken at the indicated times and used to isolate TAP-SLD5 by immunoprecipitation on IgG beads. The indicated proteins were monitored by immunoblotting. For reasons of space, samples A, S, M and 1-6 were resolved in a separate gel to samples 7-8.


( A ) Cells were treated with 5 µM CB - 5083 as indicated ( p97i = p97 inhibitor ) . Extracts were then incubated with IgG beads to isolate the GINS complex via TAP - SLD5 and the indicated factors were monitored by immunoblotting . Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2 - 7 .

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(A) Cells were treated with 5 µM CB-5083 as indicated (p97i = p97 inhibitor). Extracts were then incubated with IgG beads to isolate the GINS complex via TAP-SLD5 and the indicated factors were monitored by immunoblotting. Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2-7.


( B ) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA ( examples marked by arrows ; colocalization of HP1 and condensed DNA in 83 % of cells , n = 103 ) .

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(B) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA (examples marked by arrows; colocalization of HP1 and condensed DNA in 83 % of cells, n = 103).


( C ) Time - lapse analysis of mouse ES cells expressing GFP - SLD5 and mCherry - PCNA Arrows indicate co - localisation on heterochromatic patches , which was observed in 14 % asynchronous cells ( n = 238 ) . GFP - SLD5 appeared and disappeared with similar kinetics to mCherry - PCNA ( 100 % cells , n = 101 ) .

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(C) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA Arrows indicate co-localisation on heterochromatic patches, which was observed in 14 % asynchronous cells (n = 238). GFP-SLD5 appeared and disappeared with similar kinetics to mCherry-PCNA (100 % cells, n = 101).


( D ) GFP - SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst . Arrows indicate accumulation of GFP - SLD5 on constitutive heterochromatin ( 53 % cells , n = 125 ) .

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(D) GFP-SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst. Arrows indicate accumulation of GFP-SLD5 on constitutive heterochromatin (53 % cells, n = 125).


( E ) Immunofluorescence of the replisome components CLASPIN ( i ) and CTF4 ( ii ) in GFP - SLD5 cells treated with p97i . Arrows indicate co - localisation of GFP - SLD5 patches with CLASPIN ( 95 % cells , n = 107 ) or CTF4 ( 91 % cells , n = 45 ) . Scale bars in all panels correspond to 10 µm .

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(E) Immunofluorescence of the replisome components CLASPIN (i) and CTF4 (ii) in GFP-SLD5 cells treated with p97i. Arrows indicate co-localisation of GFP-SLD5 patches with CLASPIN (95 % cells, n = 107) or CTF4 (91 % cells, n = 45). Scale bars in all panels correspond to 10 µm.


( A ) Cell extracts were generated after the indicated treatments and then incubated with anti - GFP beads . Factors co - purifying with GFP - SLD5 were monitored by immunoblotting .

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(A) Cell extracts were generated after the indicated treatments and then incubated with anti-GFP beads. Factors co-purifying with GFP-SLD5 were monitored by immunoblotting.


( B ) TAP - SLD5 cells were subjected to the indicated treatments . The level of LRR1 and VHL was assessed by immunoblotting . The asterisk denotes a non - specific band . ( C ) For the experiment described in ( B ) , TAP - SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting .

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(B) TAP-SLD5 cells were subjected to the indicated treatments. The level of LRR1 and VHL was assessed by immunoblotting. The asterisk denotes a non-specific band. (C) For the experiment described in (B), TAP-SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting.


( D ) The activity of CUL2LRR1 was inhibited in GFP - SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP - SLD5 on constitutive heterochromatin ( 49 % cells , n = 125 ) . ( E ) Cells were treated as in ( D ) and the localisation of GFP - SLD5 was compared with CLASPIN by immunofluorescence . Arrows indicate the co - localisation of GFP - SLD5 and CLASPIN on constitutive heterochromatin ( in 90 % cells with GFP - SLD5 patches , n = 103 ) .

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(D) The activity of CUL2LRR1 was inhibited in GFP-SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP-SLD5 on constitutive heterochromatin (49 % cells, n = 125). (E) Cells were treated as in (D) and the localisation of GFP-SLD5 was compared with CLASPIN by immunofluorescence. Arrows indicate the co-localisation of GFP-SLD5 and CLASPIN on constitutive heterochromatin (in 90 % cells with GFP-SLD5 patches, n = 103).


( A ) Time - lapse analysis of mouse ES cells expressing GFP - SLD5 and mCherry - PCNA , following inhibition of CUL2LRR1 . Arrows illustrate the persistence of GFP - SLD5 on heterochromatin after disappearance of mCherry - PCNA .

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(A) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA, following inhibition of CUL2LRR1. Arrows illustrate the persistence of GFP-SLD5 on heterochromatin after disappearance of mCherry-PCNA.


( B ) time - lapse analysis of mouse ES cells expressing GFP - SLD5 and mCherry - Histone H2B ( from the CAG promoter at the ROSA26 locus after inhibition of CUL2LRR1 . Arrows indicate heterochromatic patches of GFP - SLD5 that disappear during entry into mitosis ( 98 % cells , n = 97 ) . ( C ) Equivalent data to ( B ) but for untreated cells ( 100 % of untreated cells lacked GFP - SLD5 on mitotic chromatin , n = 149 ) .

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(B) time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-Histone H2B (from the CAG promoter at the ROSA26 locus after inhibition of CUL2LRR1. Arrows indicate heterochromatic patches of GFP-SLD5 that disappear during entry into mitosis (98 % cells, n = 97). (C) Equivalent data to (B) but for untreated cells (100 % of untreated cells lacked GFP-SLD5 on mitotic chromatin, n = 149).


( A ) TRAIP co - purifies with the CMG helicase from extracts of mouse ES cells . The asterisk indicates a non - specific band .

O O O 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) TRAIP co-purifies with the CMG helicase from extracts of mouse ES cells. The asterisk indicates a non-specific band.


( B ) Mouse ES cells were processed Arrows in ( i ) indicate accumulation of GFP - SLD5 on heterochromatin during interphase , whereas arrows in ( ii ) denote GFP - SLD5 on chromatin during mitotic metaphase . Scale bars correspond to 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 O O O O O O O O O O O O O O

(B) Mouse ES cells were processed Arrows in (i) indicate accumulation of GFP-SLD5 on heterochromatin during interphase, whereas arrows in (ii) denote GFP-SLD5 on chromatin during mitotic metaphase. Scale bars correspond to 10 µm.


( C ) Immunoblot illustrating the stable expression from the CAG promoter of the indicated forms of TRAIP at the ROSA26 locus in TRAIP - / - mouse ES cells . Asterisks indicate non - specific bands . ( D ) Quantification of the percentage of mitotic cells with GFP - SLD5 on mitotic chromosomes , after inhibition of CUL2LRR1 in cells with the indicated genotypes . Cells were fixed before analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot illustrating the stable expression from the CAG promoter of the indicated forms of TRAIP at the ROSA26 locus in TRAIP -/- mouse ES cells. Asterisks indicate non-specific bands. (D) Quantification of the percentage of mitotic cells with GFP-SLD5 on mitotic chromosomes, after inhibition of CUL2LRR1 in cells with the indicated genotypes. Cells were fixed before analysis.


( A ) Top hit selected candidates are listed . The plot shows relative S9 . 6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs .

(A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs.


( B ) Representative images of HeLa cells immunostained with S9 . 6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool .

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(B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool.


( C ) Relative S9 . 6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs . More than 250 total cells from three independent experiments were considered . The median of each population is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * * * p < 0 . 001 ( Mann - Whitney U 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

(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).


( A ) Relative DRIP - qPCR signal values at RPL13A , APOE , MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre - immunoprecipitation where indicated . The mean ± SEM from at least three independent experiments is shown . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative images of immunostaining with S9 . 6 and anti - nucleolin antibodies in HeLa cells transfected with the indicated

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

(B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs


( C ) Relative S9 . 6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre - extraction ( CE ) and treated in vitro with RNaseIII and RNase H where indicated . More than 500 total cells from three independent experiments were considered . The median of each population is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) .

(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).


( A ) Representative images of single - cell alkaline gel electrophoresis ( comet assay ) of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) .

(A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+).


( B ) Comet tail moment from single - cell alkaline gel electrophoresis ( comet assay ) of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 250 total cells were considered . The mean ± SEM of the median from at least three independent experiments is shown , except for RAD1 , RAD17 and UBE2B ( n = 2 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed unpaired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Representative images and percentage of HeLa cells with more than 5 H3S10 - P foci after transfection with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . Mitotic cells were excluded for the analysis by DAPI staining . More than 300 total cells were considered . Data represent mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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

(A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . At least three experiments were performed . The median of each population in a representative experiment with at least 100 cells per condition is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). At least three experiments were performed. The median of each population in a representative experiment with at least 100 cells per condition is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 200 tracks were considered except for RAD1 + RNH1 ( n = 125 ) , ATR + RNH1 ( n = 182 ) and RAD18 + RNH1 ( n = 146 ) . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 200 tracks were considered except for RAD1 +RNH1 (n=125), ATR +RNH1 (n=182) and RAD18 +RNH1 (n=146). Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . From 40 to 200 total measurements were considered for each candidate . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

(B) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). From 40 to 200 total measurements were considered for each candidate. Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Left column : Flow cytometry profiles showing DNA content of the indicated RNase III - treated cells . G1 ( red ) , S ( blue ) and G2 ( green ) phases were calculated from the profile . Right column : Flow cytometry histograms depicting intensity of S9 . 6 signals in each phase of the cell cycle for the indicated cells . ( B ) Quantification of panel B . The mean ± SD of the S9 . 6 mean intensity of five experiments is shown . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( repeated measures ANOVA test with Bonferroni ' s post - test ) . ( C ) Graph represents fold change of S9 . 6 mean signal in G2 with respect to G1 cells . Values for five independent experiments together with the mean are shown . * p < 0 . 05 ( one - tailed paired t - test ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left column: Flow cytometry profiles showing DNA content of the indicated RNase III-treated cells. G1 (red), S (blue) and G2 (green) phases were calculated from the profile. Right column: Flow cytometry histograms depicting intensity of S9.6 signals in each phase of the cell cycle for the indicated cells. (B) Quantification of panel B. The mean ± SD of the S9.6 mean intensity of five experiments is shown. ***p<0.001, **p<0.01, *p<0.05 (repeated measures ANOVA test with Bonferroni's post-test). (C) Graph represents fold change of S9.6 mean signal in G2 with respect to G1 cells. Values for five independent experiments together with the mean are shown. *p<0.05 (one-tailed paired t-test).


( A ) DNA - RNA hybrids , DNA damage and fork stalling in 9 - 1 - 1 / ATR / CHK1 , ATM / CHK2 and PRR deficient cells . DNA - RNA hybrids represent the mean ± SEM of all the DRIP data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Paired t - test ) . DNA damage represents the mean of medians ± SEM of all the comet tail moment data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( paired t - test ) . Fork stalling represents the median ± SEM of all the fork asymmetry data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DNA-RNA hybrids, DNA damage and fork stalling in 9-1-1/ATR/CHK1, ATM/CHK2 and PRR deficient cells. DNA-RNA hybrids represent the mean ± SEM of all the DRIP data ***p<0.001, **p<0.01, *p<0.05 (Paired t-test). DNA damage represents the mean of medians ± SEM of all the comet tail moment data ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


B ) VEGF neutralizing antibody ( B20 ) failed to suppress BCE cell growth induced by LN - 229 CM , n = 3 . Data information : Bars and error bars represent mean ± SD . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test . ns , not statistically significant .

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B) VEGF neutralizing antibody (B20) failed to suppress BCE cell growth induced by LN-229 CM, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.


G ) LIF and VEGF synergistically stimulated BCE cell growth . Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods , n = 3 . Data information : Bars and error bars represent mean ± SD . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test . ns , not statistically significant .

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G) LIF and VEGF synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.


A ) The JAK inhibitor baricitinib ( Ba ) blocked LIF - induced STAT3 phosphorylation . BCE cells were pre - incubated with DMSO , baricitinib ( 2 μM ) , cobimetinib ( Co ) ( 150 nM ) or BEZ235 ( BE ) ( 5 nM ) for 1 hour and were then treated with vehicle or LIF ( 10 ng / ml , Sigma ) for 15 minutes . Ctrl , no pre - incubation with inhibitors .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O 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) The JAK inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were pre-incubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM) or BEZ235 (BE) (5 nM) for 1 hour and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 minutes. Ctrl, no pre-incubation with inhibitors.


C ) and D ) STAT3 knockdown in BCE cells . BCE cells were transfected with siNegative and siRNAs targeting STAT3 . qRT - PCR were performed to examine STAT3 mRNA levels . STAT3 level in siNegative was set as 1 . Data from three independent experiments were averaged and are presented in C . In D , cells transfected with siRNAs were treated with LIF ( 10 ng / ml ) or vehicle for 15 minutes . Whole - cell lysates were subjected to Western blotting with the indicated antibodies . Data information : Bars and error bars represent mean ± SD . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test .

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C) and D) STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT-PCR were performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with the indicated antibodies. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


E ) LIF - induced BCE cell growth was abolished by STAT3 knockdown . BCE cells with STAT3 knockdown were cultured with LIF ( 10 ng / ml , Sigma ) or vehicle . Cell proliferation was analyzed after 3 days . Fluorescence reading at 590 nm for each vehicle group was set as 1 , n = 3 . siNegative , negative control siRNA not targeting any known genes . Data information : Bars and error bars represent mean ± SD . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test .

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E) LIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n=3. siNegative, negative control siRNA not targeting any known genes. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


B ) . JAK inhibitor baricitinib blocked activation of STAT3 by LIF . BAE cells pre - incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF ( 10 ng / ml ) for 15 minutes . Whole - cell lysates were subjected to Western blotting with indicated antibodies . Ctrl , no pre - incubation with inhibitors ; Ba , baricitinib ( 2 μM ) ; Co , cobimetinib ( 150 nM ) ; BE , BEZ235 ( 5 nM ) .

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

B). JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells pre-incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml) for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no pre-incubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM).


E ) . Knockdown of STAT3 in BAE cells . BAE cells were transfected with siRNAs targeting STAT3 . In E , cells transfected with siRNAs were treated with LIF ( 10 ng / ml , Sigma ) and vehicle for 15 minutes . Whole - cell lysates were subjected to Western blotting with indicated antibodies .

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

E). Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies.


F ) . LIF - induced BAE cell growth inhibition was abolished by knockdown of STAT3 . BAE cells with STAT3 knockdown were cultured with LIF ( 10 ng / ml , Sigma ) and vehicle . Cell proliferation was analyzed after 3 days . Fluorescence reading for each vehicle group was set as 1 , n = 3 . Data information : Bars and error bars represent mean ± SD . All experiments were carried out in three independent studies . siNegative , negative control siRNA not targeting any known genes . Two - way ANOVA was used as statistical test .

F). LIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n=3. Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test.


C , D ) OCT - A imaging of LIF - treated mouse retina . Adult mice were intravitreally injected with 1 μl of LIF ( 50 ng , Sigma ) or vehicle solution ( PBS ) . Retinal OCT - A images were obtained 7 days after the injection and representative images are shown . Blood vessel density was determined as percentage of vessel - covered area / total area surface using ImageJ software and shown in D ) , n = 7 - 8 . Data information : Bars and error bars represent mean ± SEM . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical 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 O O O O O O O O O O O O O O O O O O O

C, D) OCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n=7-8. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


E ) . STAT3 was phosphorylated in retinal ECs after LIF treatment . 50 ng of our in - house LIF with low - endotoxin levels was injected . Phospho - Stat3 was examined on cryosection of retina after 2 hour by IHC . Magnification in the dashed box is shown in the lower panel . n = 5 . Scale bar = 100 µm .

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E). STAT3 was phosphorylated in retinal ECs after LIF treatment. 50 ng of our in-house LIF with low-endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 hour by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm.


F ) . Five - day old neonatal mice were intravitreally injected with LIF ( 50 ng ) or vehicle solution ( PBS ) . After 3 days , retinas were subjected to IF staining with Dyight - 488 - labeled lectin . Representative images for similar ocular loci and quantification of lectin - labeled area using ImageJ software were shown , n = 4 . Scale bar = 100 µm . Data information : Bars and error bars represent mean ± SEM . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical 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 O O O O

F). Five-day old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar ocular loci and quantification of lectin-labeled area using ImageJ software were shown, n=4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


A ) . LIF enhanced laser - induced CNV . Intravitreal administration of LIF ( 10 ng and 100 ng , Sigma ) and OSM ( 10 ng ) after laser - induced CNV . PBS was used as vehicle control . OCT - A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction . Quantification of CNV area is shown . Dashed line circles indicate the CNV in OCT - A images . n = 5 . Dot plot shows all data points from three independent experiments . Scale bar = 100 µm .

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A). LIF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm.


D ) . Avascular area in choroid in sodium iodate model was determined and quantified using Image J . E ) . Thickness of retina in H & E staining was quantified and bar graph was shown using ImageJ . Data information : Bars and error bars represent mean ± SEM . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test .

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D). Avascular area in choroid in sodium iodate model was determined and quantified using Image J. E). Thickness of retina in H&E staining was quantified and bar graph was shown using ImageJ. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


( G ) Human choroidal EC ( HCEC ) and mouse retinal EC ( MREC ) were treated with LIF 10 ng , LIF 100 ng for 30 min . ( PBS was used as control ) . STAT3 phosphorylation was detected by western blot .

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(G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot.


( H ) H & E staining of LIFR and CD31 in mouse eye sections . The magnified images of the dashed line boxes , a and b , are shown on the right panel . Box a shows LIFR staining in retina and box b showed LIFR staining in RPE / choroid layer .

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(H) H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer.


( I ) Whole mount staining of LIFR and CD31 in mouse retina . The fluorescent staining for CD31 ( green ) and LIFR ( red ) is shown . White arrows indicate the co - staining of CD31 and LIFR on retinal ECs .

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(I) Whole mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs.


Violin plots of the log scale normalized expression of LIF receptor subunits , IL6ST ( A ) , LIFR ( B ) , LIF ( C ) , IL6R ( D ) in all cell types in the human choroid Note the wide distribution of IL6ST ( gp130 ) , the common subunit to all receptors of the IL - 6 family ( A ) . In contrast , LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes ( B ) . LIF is expressed in fibroblasts , macrophages / monocytes and pericytes and minimally in ECs ( C ) . The specific IL - 6 receptor ( IL6R ) is highly expressed in macrophage / monocytes ( D ) , while LIFR has little expression in these cells ( B ) .

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Violin plots of the log scale normalized expression of LIF receptor subunits, IL6ST (A), LIFR (B), LIF (C), IL6R (D) in all cell types in the human choroid Note the wide distribution of IL6ST (gp130), the common subunit to all receptors of the IL-6 family (A). In contrast, LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes (B). LIF is expressed in fibroblasts, macrophages/monocytes and pericytes and minimally in ECs (C). The specific IL-6 receptor (IL6R) is highly expressed in macrophage/monocytes (D), while LIFR has little expression in these cells (B).


Violin plots of the log scale normalized expression of LIF receptor subunits KDR ( E ) in all cell types in the human choroid KDR shows , as expected , high expression selectively in EC ( E ) .

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Violin plots of the log scale normalized expression of LIF receptor subunits KDR (E) in all cell types in the human choroid KDR shows, as expected, high expression selectively in EC (E).


A . Total lipids were extracted from PfCDPK7 - KO or 3D7 parasites and separated by HPTLC . After quantification by GC - MS content relative to total fatty acids was determined . Relative abundance of individual phospholipids was quantified . Disruption of PfCDPK7 caused a significant decrease in PC ( mean ± SEM , n = 3 ; biological replicates , * * * p < 0 . 001 , t - test ) .

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A. Total lipids were extracted from PfCDPK7-KO or 3D7 parasites and separated by HPTLC. After quantification by GC-MS content relative to total fatty acids was determined. Relative abundance of individual phospholipids was quantified. Disruption of PfCDPK7 caused a significant decrease in PC (mean± SEM, n=3; biological replicates, ***p<0.001, t-test).


E . 3D7 or PfCDPK7 - KO parasites were cultured in the presence or absence of 200 μM choline in culture medium . After 3 days , parasitemia was determined by counting parasites on thin blood smears ( mean ± SEM , n = 6 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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E. 3D7 or PfCDPK7-KO parasites were cultured in the presence or absence of 200 μM choline in culture medium. After 3 days, parasitemia was determined by counting parasites on thin blood smears (mean± SEM, n=6; biological replicates, *p< 0.05, ***p<0.001, ANOVA).


A - B . 3D7 ( A ) or parasites overexpressing EK - GFP ( B ) were used to prepare protein lysates followed by immunoprecipitation using anti - PfCDPK7 or anti - GFP antibody , respectively . Subsequently , protein lysate or indicated IP were subjected to Western blotting using anti - PMT ( A ) , anti - GFP ( B ) . PMT was found to co - IP with PfCDPK7 ( A , lane 2 ) and EK - GFP with PfCDPK7 ( B , lane 3 ) .

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A-B. 3D7 (A) or parasites overexpressing EK-GFP (B) were used to prepare protein lysates followed by immunoprecipitation using anti-PfCDPK7 or anti-GFP antibody, respectively. Subsequently, protein lysate or indicated IP were subjected to Western blotting using anti-PMT (A), anti-GFP (B). PMT was found to co-IP with PfCDPK7 (A, lane 2) and EK-GFP with PfCDPK7 (B, lane 3).


C . 3D7 and PfCDPK7 - KO parasites at schizont stage ( ~ 40 h ) were used for Western blotting using anti - PMT antisera . Actin served as a loading control . Right panel : Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin . Fold change in PMT was determined with respect to 3D7 ( mean ± SEM , n = 4 ; biological replicates , * * p < 0 . 01 , t - test ) .

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C. 3D7 and PfCDPK7-KO parasites at schizont stage (~40 h) were used for Western blotting using anti-PMT antisera. Actin served as a loading control. Right panel: Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin. Fold change in PMT was determined with respect to 3D7 (mean± SEM, n=4; biological replicates, **p<0.01, t-test).


A . Parasites overexpressing EK - GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first ( 48 h . p . i ) and second cycle ( 96 h . p . i ) . Fold change in parasitemia of EK _ S37A parasites with respect to WT EK overexpressing parasites was compared , which was significantly reduced ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , ANOVA ) .

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A. Parasites overexpressing EK-GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first (48 h.p.i) and second cycle (96 h.p.i). Fold change in parasitemia of EK_S37A parasites with respect to WT EK overexpressing parasites was compared, which was significantly reduced (mean± SEM, n=3; biological replicates, *p<0.05, ANOVA).


C . IFA was performed on parasites expressing WT EK - GFP or its S37A mutant 6 - 12h post - invasion to detect ( i ) RAP1 , which is transferred to PV post invasion , and ( ii ) MSP1 which is located at the PVM . Parasite expressing WT EK - GFP exhibited typical round ring like shape . In contrast , several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV ( Scale bar - 1 μm ) .

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C. IFA was performed on parasites expressing WT EK-GFP or its S37A mutant 6-12h post-invasion to detect (i) RAP1, which is transferred to PV post invasion, and (ii) MSP1 which is located at the PVM. Parasite expressing WT EK-GFP exhibited typical round ring like shape. In contrast, several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV (Scale bar-1 μm).


E . Parasites EK - GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts / segmentors . The graph shows average number of merozoites per schizont from three biological replicates . Each replicate represents an average of merozoites from 30 - 40 schizonts . ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ) .

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E. Parasites EK-GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts/segmentors. The graph shows average number of merozoites per schizont from three biological replicates. Each replicate represents an average of merozoites from 30-40 schizonts. (mean± SEM, n=3; biological replicates, *p<0.05, t-test).


A . 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h . Subsequently , parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7 . While PfCDPK7 was present in puncta proximal to ER ( left panel ) , inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme ( right panel ) ( Scale bar - 1 μm ) .

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A. 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h. Subsequently, parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7. While PfCDPK7 was present in puncta proximal to ER (left panel), inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme (right panel) (Scale bar-1 μm).


C . 3D7 parasites were treated with DMSO or 50nM MMV048 for 20 - 24h . Subsequently , IFA was performed ~ 24h post - invasion to detect RAP1 and MSP1 ( left panel ) Right panel , Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed ( mean ± SEM , n = 3 ; biological replicates , * * p < 0 . 01 , t - test ; Scale bar - 1 μm ) .

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C. 3D7 parasites were treated with DMSO or 50nM MMV048 for 20-24h. Subsequently, IFA was performed ~24h post-invasion to detect RAP1 and MSP1 (left panel) Right panel, Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed (mean± SEM, n=3; biological replicates, **p<0.01, t-test; Scale bar-1 μm).


A - B . 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10 - 12h . Subsequently , parasites were incubated with 14C - Cho ( A ) or 14C - Eth ( B ) and were harvested after 12h . Total lipids were extracted and separated by TLC . The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry ( upper panel ) . Bottom Panel ( A and B ) , fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined ( mean ± SEM , n = 4 ; biological replicates , * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ; ns , not significant ) .

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A-B. 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10-12h. Subsequently, parasites were incubated with 14C-Cho (A) or 14C-Eth (B) and were harvested after 12h. Total lipids were extracted and separated by TLC. The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry (upper panel). Bottom Panel (A and B), fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined (mean±SEM, n=4; biological replicates, **p<0.01, ***p<0.001, ANOVA; ns, not significant).


C - D . UCT943 and MMV048 treated 3D7 ( C ) or EK - GFP overexpressing parasites ( D ) were subjected to Western blotting using anti - PMT ( C ) and anti - GFP ( D ) antibodies ( left panels ) . Right Panels : densitometry was performed for bands corresponding to PMT ( C ) or EK - GFP ( D ) , which were normalized with respect to Actin . Fold change in PMT or EK was determined with respect to DMSO treated parasites ( Mean ± SEM , n = 4 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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C-D. UCT943 and MMV048 treated 3D7 (C) or EK-GFP overexpressing parasites (D) were subjected to Western blotting using anti-PMT (C) and anti-GFP (D) antibodies (left panels). Right Panels: densitometry was performed for bands corresponding to PMT (C) or EK-GFP (D), which were normalized with respect to Actin. Fold change in PMT or EK was determined with respect to DMSO treated parasites (Mean± SEM, n=4; biological replicates, *p<0.05, ***p<0.001, ANOVA).


B . Ring - infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4 - 64 and DAPI to assess lipid uptake . Subsequently , FM4 - 64 associated fluorescence was detected by fluorescence microscopy ( upper panel ) or quantitated by FACS ( lower panel ) and % change in FM4 - 64 uptake in inhibitor - treated parasites was determined . ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ; Scale bar - 1 μm ) .

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B. Ring-infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4-64 and DAPI to assess lipid uptake. Subsequently, FM4-64 associated fluorescence was detected by fluorescence microscopy (upper panel) or quantitated by FACS (lower panel) and % change in FM4-64 uptake in inhibitor-treated parasites was determined. (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).


D . Trophozoite stage infected erythrocytes were incubated with FM4 - 64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7 - KO parasites . Subsequently , FM4 - 64 associated fluorescence was visualized by live microscopy ( top panel ) and also analyzed by FACS and % change in FM4 - 64 uptake in KO parasites was determined ( bottom panel ) ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ; Scale bar - 1 μm ) .

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D. Trophozoite stage infected erythrocytes were incubated with FM4-64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7-KO parasites. Subsequently, FM4-64 associated fluorescence was visualized by live microscopy (top panel) and also analyzed by FACS and % change in FM4-64 uptake in KO parasites was determined (bottom panel) (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).


( C - E ) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate ( TCL ) and further increases by IFN - γ activation ( C , E ) . Equal protein amounts were loaded for phagosomal and TCL samples . Vimentin serves as loading control ; EEA1 as purity marker . ( D ) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome ( 30 min pulse , 0 , 30 , and 180 min chase , respectively ) . LAMP1 serves as purity marker . Representative images of three replicates .

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(C-E) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate (TCL) and further increases by IFN-γ activation (C, E). Equal protein amounts were loaded for phagosomal and TCL samples. Vimentin serves as loading control; EEA1 as purity marker. (D) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome (30 min pulse, 0, 30, and 180 min chase, respectively). LAMP1 serves as purity marker. Representative images of three replicates.


( H ) Ubiquitin Chain Restriction ( UbiCRest ) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs . UT = untreated .

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(H) Ubiquitin Chain Restriction (UbiCRest) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs. UT = untreated.


( B ) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264 . 7 cells and its increased abundance in response to IFN - γ activation . Rab7 serves as purity control , iNOS as activation control and vimentin as loading control . Representative image of three replicates . Relative mobilities of reference proteins ( masses in kDa ) are shown on the right of each blot .

(B) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264.7 cells and its increased abundance in response to IFN-γ activation. Rab7 serves as purity control, iNOS as activation control and vimentin as loading control. Representative image of three replicates. Relative mobilities of reference proteins (masses in kDa) are shown on the right of each blot.