( F ) Expressing Tau - k18 ( - ) abolished Tau - induced upregulation of mRNA levels of survival genes ( bcl2 and survivin ) and dkk1 ( downstream of Wnt signaling ) in N2a cells measured by Q - PCR ( n = 6 ~ 9 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau ( F Data were analyzed by one - way

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(F) Expressing Tau-k18(-) abolished Tau-induced upregulation of mRNA levels of survival genes (bcl2 and survivin) and dkk1 (downstream of Wnt signaling) in N2a cells measured by Q-PCR (n=6~9 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau (F Data were analyzed by one-way ANOVA


( G ) Expressing Tau - k18 ( - ) or Tau - 2CA abolished Tau - induced Wnt / β - catenin transcriptional activation measured by dual reporter luciferase activity assay in HEK293 cells ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs vs Tau Data were analyzed by one - way

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(G) Expressing Tau-k18(-) or Tau-2CA abolished Tau-induced Wnt/β-catenin transcriptional activation measured by dual reporter luciferase activity assay in HEK293 cells (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vs Tau Data were analyzed by one-way ANOVA


( H ) Expressing phosphor - mimic TauS199E ( TauS199E ) further increased the transcription activity of Wnt / TCF / LEF1 by Tau in HEK293 cells ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec ; # # , P < 0 . 01 , # # # , P < 0 . 001 vs Tau ( H ) . Data were analyzed by , one - way

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(H) Expressing phosphor-mimic TauS199E (TauS199E) further increased the transcription activity of Wnt/TCF/LEF1 by Tau in HEK293 cells (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec ; ##, P<0.01, ###, P<0.001 vs Tau (H). Data were analyzed by , one-way ANOVA


The increased mRNA levels of bcl2 , survivin and dkk1 in the AD brains compared with the age - / sex - matched controls detected by Q - PCR ( n = 7 each group ) Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Ctrl ( I ) Data were analyzed by Student ' s t

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The increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group) Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Ctrl (I) Data were analyzed by Student's t test


The increased mRNA levels of bcl2 , survivin and dkk1 in the AD brains compared with the age - / sex - matched controls detected by Q - PCR ( n = 7 each group ) , and the positive correlations with the increased K49 - acetylated β - catenin ( see Fig . 1I , J ) analyzed by Pearson analysis .

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The increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group), and the positive correlations with the increased K49-acetylated β-catenin (see Fig. 1I, J) analyzed by Pearson analysis.


( H , I ) . Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation , with or without pre - incubation with 400 nM Compound 7 . Scale bars : ( H , I ) , 150 nm . Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs . PM represents plasma membrane . ( J ) . Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7 . PM represents plasma membrane . Data information : Data were analyzed using one - way ANOVA and displayed as the mean ± s . e . m . # , P < 0 . 05 ; # # , P < 0 . 01 ; # # # , P < 0 . 001 : cells pre - incubated with PTP - MEG2 inhibitors compared with control vehicles . N . S . means no significant difference . Data were from 6 independent experiments . # indicates P < 0 . 05 and # # # indicates P < 0 . 001 compared with control group .

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(H, I). Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation, with or without pre-incubation with 400 nM Compound 7. Scale bars: (H, I), 150 nm. Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs. PM represents plasma membrane. (J). Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7. PM represents plasma membrane. Data information: Data were analyzed using one-way ANOVA and displayed as the mean ± s.e.m. #, P<0.05; ##, P<0.01; ###, P<0.001: cells pre-incubated with PTP-MEG2 inhibitors compared with control vehicles. N.S. means no significant difference. Data were from 6 independent experiments. # indicates P<0.05 and ### indicates P<0.001 compared with control group.


( D ) . Phosphorylated NSF interacted with PTP - MEG2 . PC12 cells were transfected with FLAG - NSF . After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes ( H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins ) , the potential PTP - MEG2 substrate in cell lysates was pulled down with GST - PTP - MEG2 - D470A or GST control .

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(D). Phosphorylated NSF interacted with PTP-MEG2. PC12 cells were transfected with FLAG-NSF. After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes (H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins), the potential PTP-MEG2 substrate in cell lysates was pulled down with GST-PTP-MEG2-D470A or GST control.


( B ) . Relative values of the decreased phosphatase activities of different PTP - MEG2 mutants towards pNPP ( p - Nitrophenyl phosphate ) compared with wild - type PTP - MEG2 . Data information : * * * P < 0 . 001 : PTP - MEG2 mutants compared with the control group . Data were obtained from 3 independent experiments . N . S . means no significant difference . All the data were analyzed using one - way ANOVA , and displayed as the mean ± s . e . m . In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho - NSF segement than the pNPP . The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho - segement but not pNPP . The white column stands for the mutants that have similar effects on pNPP and NSF phospho - segement .

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(B). Relative values of the decreased phosphatase activities of different PTP-MEG2 mutants towards pNPP (p-Nitrophenyl phosphate) compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.


( C ) . Fold - change decreases in the phosphatase activities of different PTP - MEG2 mutants towards the NSF - pY83 phospho - segment compared with wild - type PTP - MEG2 . Data information : * * * P < 0 . 001 : PTP - MEG2 mutants compared with the control group . Data were obtained from 3 independent experiments . N . S . means no significant difference . All the data were analyzed using one - way ANOVA , and displayed as the mean ± s . e . m . In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho - NSF segement than the pNPP . The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho - segement but not pNPP . The white column stands for the mutants that have similar effects on pNPP and NSF phospho - segement .

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(C). Fold-change decreases in the phosphatase activities of different PTP-MEG2 mutants towards the NSF-pY83 phospho-segment compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.


( D ) . Statistical diagram of the quantal size in Fig . 5A and Appendix Figure S6A - D . The secretion amount of each group was standardized with respect to the control group . Data information : Green color represents the amino acids which are found to modulate foot probability based on structure analysis , and red color means amino acids which does not regulate foot probability . , * indicates PTP - MEG2 mutants overexpression group compared with the WT overexpression group . # indicates the overexpression group compared with the control group . * * , P < 0 . 01 ; * * * P < 0 . 001 and # , P < 0 . 05 ; # # , P < 0 . 01 ; # # # P < 0 . 001 . N . S . means no significant difference . Data were from 6 independent experiments . All the data were analyzed using one - way ANOVA and showed as the mean ± s . e . m .

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(D). Statistical diagram of the quantal size in Fig. 5A and Appendix Figure S6A-D. The secretion amount of each group was standardized with respect to the control group. Data information: Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. , * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.


( C ) . The GST pull - down assay suggested that PACSIN1 , MUNC18 - 1 , VAMP7 , SNAP25 , DYNAMIN1 and DYNAMIN2 directly interact with PTP - MEG2 . PC12 cells were transfected with plasmids of candidate substrates , including SYN1 , MUNC18 - 3 , PACSIN1 , SCAMP1 , MUNC18 - 1 , PPP3CA , STX17 , VAMP7 , SYT7 , SYT11 , SNAP25 , DYNAMIN1 , and DYNAMIN2 stimulated with 100 nM AngII . The tyrosine phosphorylation of these proteins was verified by specific anti - pY antibodies ( Appendix Figure S8A - E ) . The potential substrates of PTP - MEG2 in cell lysates were pulled down with a GST - PTP - MEG2 - D470A trapping mutant and then detected by Western blot . White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP - MEG2 substrate .

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(C). The GST pull-down assay suggested that PACSIN1, MUNC18-1, VAMP7, SNAP25, DYNAMIN1 and DYNAMIN2 directly interact with PTP-MEG2. PC12 cells were transfected with plasmids of candidate substrates, including SYN1, MUNC18-3, PACSIN1, SCAMP1, MUNC18-1, PPP3CA, STX17, VAMP7, SYT7, SYT11, SNAP25, DYNAMIN1, and DYNAMIN2 stimulated with 100 nM AngII. The tyrosine phosphorylation of these proteins was verified by specific anti-pY antibodies (Appendix Figure S8A-E). The potential substrates of PTP-MEG2 in cell lysates were pulled down with a GST-PTP-MEG2-D470A trapping mutant and then detected by Western blot. White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP-MEG2 substrate.


( D ) . Interactions of the PTP - MEG2 - trapping mutants with the MUNC18 - 1 - Y145 mutants and DYNAMIN2 - Y125 mutants . PC12 cells were transfected with FLAG - MUNC18 - 1 - Y145 , FLAG - DYNAMIN2 - Y125 and different mutations of the FLAG - MUNC18 - 1 - Y145A , Y145H , Y145E or Y145F , FLAG - DYNAMIN2 - Y125A , Y125E , Y125F , 24 hours before stimulation with 100 nM AngII , respectively . The cell lysates were then incubated with GST - beads - PTP - MEG2 - D470A complex for 2 hours with constant rotation . The potential PTP - MEG2 substrates were pulled down by GST - beads , and their levels were examined by the FLAG antibody with Western blot .

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(D). Interactions of the PTP-MEG2-trapping mutants with the MUNC18-1-Y145 mutants and DYNAMIN2-Y125 mutants. PC12 cells were transfected with FLAG-MUNC18-1-Y145, FLAG-DYNAMIN2-Y125 and different mutations of the FLAG-MUNC18-1-Y145A, Y145H, Y145E or Y145F, FLAG-DYNAMIN2-Y125A, Y125E, Y125F, 24 hours before stimulation with 100 nM AngII, respectively. The cell lysates were then incubated with GST-beads-PTP-MEG2-D470A complex for 2 hours with constant rotation. The potential PTP-MEG2 substrates were pulled down by GST-beads, and their levels were examined by the FLAG antibody with Western blot.


( H ) . The MUNC18 - 1 - Y145 mutations decreased the interaction between MUNC18 - 1 and SYNTAXIN1 . PC12 cells were transfected with plasmid encoding SYNTAXIN - 1 . The proteins in cell lysates were pulled down with purified GST - beads - MUNC18 - 1 - Y145 complex and the GST - MUNC18 - 1 - Y145H / E / F , and detected with SYNTAXIN1 antibody . The right histogram shows the quantified protein levels . Data information * indicates MUNC18 - 1 , mutants overexpression group compared with the WT overexpression group . # indicates MUNC18 - 1 , overexpression group compared with the control group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 N . S . means no significant difference . Data were from 3 ( H ) independent experiments . All the data were analyzed using one - way ANOVA , and showed as the mean ± s . e . m .

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(H). The MUNC18-1-Y145 mutations decreased the interaction between MUNC18-1 and SYNTAXIN1. PC12 cells were transfected with plasmid encoding SYNTAXIN-1. The proteins in cell lysates were pulled down with purified GST-beads-MUNC18-1-Y145 complex and the GST-MUNC18-1-Y145H/E/F, and detected with SYNTAXIN1 antibody. The right histogram shows the quantified protein levels. Data information * indicates MUNC18-1, mutants overexpression group compared with the WT overexpression group. # indicates MUNC18-1, overexpression group compared with the control group. *, P<0.05; **, P<0.01; *** P<0.001 N.S. means no significant difference. Data were from 3 (H) independent experiments. All the data were analyzed using one-way ANOVA, and showed as the mean ± s.e.m.


( E ) . Relative phosphatase activities of different PTP - MEG2 mutants towards the MUNC18 - 1 - pY145 phospho - segment compared with wild - type PTP - MEG2 . ( F ) . Relative phosphatase activities of different PTP - MEG2 mutants towards the DYNAMIN 2 - pY125 phospho - segment compared with wild - type PTP - MEG2 . Data information : ( E , F ) Residues are coloured according to Figure 8B . ( E , F ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 : PTP - MEG2 mutants compared with the control . N . S . means no significant difference . Data were obtained from 3 independent experiments . * * , P < 0 . 01 ; * * * P < 0 . 001 and # # P < 0 . 01 ; # # # P < 0 . 001 . N . S . means no significant difference . Data were obtained from 10 cells in each group of 6 independent experiments . . All the data were analyzed using one - way ANOVA and showed as the mean ± s . e . m .

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(E). Relative phosphatase activities of different PTP-MEG2 mutants towards the MUNC18-1-pY145 phospho-segment compared with wild-type PTP-MEG2. (F). Relative phosphatase activities of different PTP-MEG2 mutants towards the DYNAMIN 2-pY125 phospho-segment compared with wild-type PTP-MEG2.Data information: (E, F) Residues are coloured according to Figure 8B. (E, F), * P<0.05, ** P<0.01, *** P<0.001: PTP-MEG2 mutants compared with the control. N.S. means no significant difference. Data were obtained from 3 independent experiments. **, P<0.01; *** P<0.001 and ## P<0.01; ### P<0.001. N.S. means no significant difference. Data were obtained from 10 cells in each group of 6 independent experiments. . All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.


A , B : Quantitative RT - PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes ( GV and MII ) , somatic tissues , ES cells ( A ) and preimplantation embryos ( B ) . n = 3 biological

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A, B: Quantitative RT-PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes (GV and MII), somatic tissues, ES cells (A) and preimplantation embryos (B). n = 3 biological replicates


C : Cumulative numbers of pups per female showing fertility of WT and Cnot6l - / - female mice . n = 5 females for each

O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O

C: Cumulative numbers of pups per female showing fertility of WT and Cnot6l-/- female mice. n = 5 females for each genotype


D : Representative images results of oocytes collected from oviducts of WT and Cnot6l - / - mice at 16 h after hCG injection . scale bar , 100 μm . Arrows indicate polar body - 1 ( PB1 )

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D: Representative images results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 100 μm. Arrows indicate polar body-1 (PB1)


E : Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l - / - mice at 16 h after hCG injection . scale bar , 20 μm . Arrows indicate spindle

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E: Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 20 μm. Arrows indicate spindle poles


F : Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l - / - female mice . The numbers of analyzed oocytes were indicated ( n )

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F: Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l-/- female mice. The numbers of analyzed oocytes were indicated (n)


G : Quantification of preimplantation embryos derived from WT and Cnot6l - / - females that develop to the indicated stages after hCG administration and mated with adult WT males . The numbers of analyzed embryos are indicated ( n )

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G: Quantification of preimplantation embryos derived from WT and Cnot6l-/- females that develop to the indicated stages after hCG administration and mated with adult WT males. The numbers of analyzed embryos are indicated (n)


H : Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l - / - female at indicated time points after hCG administration . Scale bar , 100 μm . Data information : Error bars , s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non -

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H: Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l-/- female at indicated time points after hCG administration. Scale bar, 100 μm. Data information: Error bars, s.e.m. **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif


A : Rates of germinal vesicle breakdown ( GVBD ) and PB1 emission in oocytes cultured in vitro . Fully - grown GV oocytes were collected from PMSG - primed ( 44 h ) WT and Cnot6l - / - mice . PB1 : polar body - 1 . Error bars , s . e . m . The numbers of analyzed oocytes were indicated ( n )

O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

A: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)


B : Representative images of WT , Cnot6l - / - and Btg4 - / - oocytes showing PB1 emission at 16 h after culture . Arrows indicate PB1 . Scale bar , 100

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B: Representative images of WT, Cnot6l-/- and Btg4-/- oocytes showing PB1 emission at 16 h after culture. Arrows indicate PB1. Scale bar, 100 μm


C : Representative images of chromosome spreads made from WT and Cnot6l - / - oocytes after 16 h of in vitro maturation culture . Immunofluorescent staining of topoisomerase II ( TOP2 ) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres , respectively . Numbers of paired sister chromatids are indicated . Scale bar , 5

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C: Representative images of chromosome spreads made from WT and Cnot6l-/- oocytes after 16 h of in vitro maturation culture. Immunofluorescent staining of topoisomerase II (TOP2) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres, respectively. Numbers of paired sister chromatids are indicated. Scale bar, 5 μm


D : Percentage ( % ) of aneuploidy among in vitro cultured WT and Cnot6l - / - oocytes that have released PB1s . Error bars , s . e . m . * P < 0 . 05 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

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D: Percentage (%) of aneuploidy among in vitro cultured WT and Cnot6l-/- oocytes that have released PB1s. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


E : Rates of GVBD , PB1 emission , and normal spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes cultured in vitro . Error bars , s . e . m . * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

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E: Rates of GVBD, PB1 emission, and normal spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes cultured in vitro. Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


F : Confocal microscopy results showing spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes at metaphase I ( MI ) and metaphase II ( MII ) . Scale bar , 20

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F: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μm


G : Pericentrin immunofluorescence showing MTOCs in cultured WT , Cnot6l - / - and Btg4 - / - oocytes at MI and MII stages . Spindle and DNA were labeled by microtubule nucleation factor ( TPX2 ) and DAPI , respectively . Scale bar , 20

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G: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm


A : Live cell imaging results showing in vitro meiotic division of WT and Cnot6l - / - oocytes . Abbreviations : PM , prometaphase ; MI , metaphase I ; AI , anaphase I ; TI , anaphase I - metaphase II transition ; MII , metaphase II ; PB1 : polar body - 1 . Hours after released from GV arrest were indicated . Scale bar , 20

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O

A: Live cell imaging results showing in vitro meiotic division of WT and Cnot6l-/- oocytes. Abbreviations: PM, prometaphase; MI, metaphase I; AI, anaphase I; TI, anaphase I-metaphase II transition; MII, metaphase II; PB1: polar body-1. Hours after released from GV arrest were indicated. Scale bar, 20 μm


B : Data represent the mean and standard deviations of mCherry - Securin fluorescence intensity in WT and Cnot6l - / - oocytes at each time point . Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest . The numbers of analyzed oocytes were indicated ( n )

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O

B: Data represent the mean and standard deviations of mCherry-Securin fluorescence intensity in WT and Cnot6l-/- oocytes at each time point. Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest. The numbers of analyzed oocytes were indicated (n)


C : SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l - / - oocytes at 16 h after culture . Centromeres and DNA were labeled by CREST and DAPI , respectively . Scale bar , 5

O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O

C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μm


D : BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l - / - oocytes at 9 . 5 h after culture . More than 8 oocytes were observed for each genotype with similar results . Scale bar , 5

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D: BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l-/- oocytes at 9.5 h after culture. More than 8 oocytes were observed for each genotype with similar results. Scale bar, 5 μm


E : PB1 emission rates in WT and Cnot6l - / - oocytes cultured with or without MPS1 inhibitor reversin ( 5 μm ) . Reversin is added at 9 hours after culture . Error bars , s . e . m . The numbers of analyzed oocytes were indicated ( n )

O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

E: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)


A : Relative mRNA copy number dynamics in WT and Cnot6l - / - samples at the indicated stages . Error bars indicate values of the two biological

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

A: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicates


B : Box plot showing gene expression levels of WT , Cnot6l - / - and Btg4 - / - oocytes at the MII stage . Genes were divided into 10 bins according to their expression levels in the WT MII oocytes . The box indicates upper and lower quantiles , the thick line in the box indicate the median , and the whiskers represent 2 . 5th and 97 . 5th percentiles . * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non -

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Box plot showing gene expression levels of WT, Cnot6l-/- and Btg4-/- oocytes at the MII stage. Genes were divided into 10 bins according to their expression levels in the WT MII oocytes. The box indicates upper and lower quantiles, the thick line in the box indicate the median, and the whiskers represent 2.5th and 97.5th percentiles. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif


C : qRT - PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l - / - mice ( Cnot6l - / - / WT ) . Error bars , s . e . m . * * * P < 0 . 001 by two - tailed Student " s t test . n = 3 biological

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O 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: qRT-PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l-/- mice (Cnot6l-/-/WT). Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n = 3 biological repli


D : Scatter plot comparing transcripts between WT and Cnot6l - / - oocytes ( at GV , MI and MII stages ) and zygotes derived from these oocytes . Transcripts decreased or increased more than five folds in Cnot6l - / - oocyte samples were high - lighted with blue or red ,

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D: Scatter plot comparing transcripts between WT and Cnot6l-/- oocytes (at GV, MI and MII stages) and zygotes derived from these oocytes. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively


E : Heatmap showing transcripts that were degraded during meiotic maturation ( GV / Zygote > 10 ) in WT oocytes . The definition of cluster I - III is described in the

O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O

E: Heatmap showing transcripts that were degraded during meiotic maturation (GV/Zygote > 10) in WT oocytes. The definition of cluster I-III is described in the text


F : Relative mRNA copy number dynamics of the three gene clusters in WT , Cnot6l - / - and Btg4 - / -

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O

F: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples


Western blot results showing the levels of CPEB1 in WT and Cnot6l - / - oocytes at the indicated time points after meiotic resumption . Total proteins from 50 ( A oocytes are loaded in each lane . DDB1 ( A is blotted as a loading control . Experiments were performed three times with reproducible results ; a representative result is

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins from 50 (A oocytes are loaded in each lane. DDB1 (A is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shown


Western blot results showing the levels of CPEB1 in WT and Cnot6l - / - oocytes at the indicated time points after meiotic resumption . Total proteins fro 100 ( B ) oocytes are loaded in each lane α - tubulin ( B ) is blotted as a loading control . Experiments were performed three times with reproducible results ; a representative result is

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins fro 100 (B) oocytes are loaded in each lane α-tubulin (B) is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shown


Immunofluorescenc of L - homopropargylglycine ( HPG ) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l - / - mice . Scale bar , 20 μm . Error bars , standard deviations ( n = 6 oocytes for each genotype ) . * * P < 0 . 01 , * * * P < 0 .

B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

Immunofluorescenc of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001


quantification ( D ) of L - homopropargylglycine ( HPG ) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l - / - mice . Scale bar , 20 μm . Error bars , standard deviations ( n = 6 oocytes for each genotype ) . * * P < 0 . 01 , * * * P < 0 .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

quantification (D) of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001


E : Relative mRNA copy numbers of polysome - bound transcripts in WT and Cnot6l - / - oocytes at indicated stages . Error bars indicate values of the

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O

E: Relative mRNA copy numbers of polysome-bound transcripts in WT and Cnot6l-/- oocytes at indicated stages. Error bars indicate values of the replicates


F : Scatter plot comparing polysome - bound transcripts between WT and Cnot6l - / - oocytes at GV , MI and MII stages , respectively . Transcripts decreased or increased more than five folds in Cnot6l - / - oocyte samples were high - lighted with blue or red ,

O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O

F: Scatter plot comparing polysome-bound transcripts between WT and Cnot6l-/- oocytes at GV, MI and MII stages, respectively. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively


I : Quantitative RT - PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l - / - oocytes at GV , MI and MII stages . Error bars , standard deviations ( n = 3 biologica repeats )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

I: Quantitative RT-PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l-/- oocytes at GV, MI and MII stages. Error bars, standard deviations (n = 3 biologica repeats)


A : Presence of putative AREs ( AUUUA ) in transcripts of WT oocyte . Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change ( MII / GV ) in WT

O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O

A: Presence of putative AREs (AUUUA) in transcripts of WT oocyte. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (MII/GV) in WT oocytes


B : Presence of putative AREs in oocyte transcripts stabilized by Cnot6l - / - knockout . Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change ( Cnot6l - / - / WT ) at MII

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B: Presence of putative AREs in oocyte transcripts stabilized by Cnot6l-/- knockout. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (Cnot6l-/-/WT) at MII stage


C : Co - IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7 . HeLa cells were co - transfected with plasmids expressing HA - ZFP36L2 and FLAG - CNOT6L / 7 for 48 h before immunoprecipitation . Experiments were performed three times with reproducible results ; a representative result is

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

C: Co-IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7. HeLa cells were co-transfected with plasmids expressing HA-ZFP36L2 and FLAG-CNOT6L/7 for 48 h before immunoprecipitation. Experiments were performed three times with reproducible results; a representative result is shown


D : RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts , with or without the presence of ZFP36L2 . HeLa cells were co - transfected with plasmids expressing FLAG - ZFP36L2 and HA - CNOT6L for 48 h before immunoprecipitation using an anti - HA antibody . mRNAs recovered from the immunoprecipitates were subjected to qRT - PCR . Error bars , standard deviations ( n = 3 biological repeats ) . * * * P < 0 . 001 by two - tailed Student " s

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D: RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts, with or without the presence of ZFP36L2. HeLa cells were co-transfected with plasmids expressing FLAG-ZFP36L2 and HA-CNOT6L for 48 h before immunoprecipitation using an anti-HA antibody. mRNAs recovered from the immunoprecipitates were subjected to qRT-PCR. Error bars, standard deviations (n = 3 biological repeats). ***P < 0.001 by two-tailed Student"s t


fluorescence microscopy results ( B ) showing the translation activities of the Cnot6l 3 " - UTR ( WT and CPE mutated ) in GV - ( maintained by 2 μM milrinone ) or MII - ( released from milrinone ) arrested oocytes . GFP signal indicated translational activation of Cnot6l 3 " - UTR . An in vitro transcribed and polyadenylated mCherry mRNA was co - microinjected as a positive control . Scale

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

fluorescence microscopy results (B) showing the translation activities of the Cnot6l 3"-UTR (WT and CPE mutated) in GV- (maintained by 2 μM milrinone) or MII- (released from milrinone) arrested oocytes. GFP signal indicated translational activation of Cnot6l 3"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive control. Scale b


GFP signal indicated translational activation of Cnot6l 3 " - UTR . An in vitro transcribed and polyadenylated mCherry mRNA was co - microinjected as a positive co Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte . Error bars , s . e . m . * P < 0 . 05 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O

GFP signal indicated translational activation of Cnot6l 3"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive co Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


fluorescence microscopy results ( E ) showing the expression of GFP - fused Cnot6l 3 " - UTR in GV and MII oocytes with or without U0126 treatment ( 20 μM ) . Scale bar ,

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fluorescence microscopy results (E) showing the expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U0126 treatment (20 μM). Scale bar, 1


e expression of GFP - fused Cnot6l 3 " - UTR in GV and MII oocytes with or without U01 F : Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte The numbers of analyzed oocytes were indicated ( n )

O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O

e expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U01 F: Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte The numbers of analyzed oocytes were indicated (n)


G : Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126 . Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L , CNOT7 and / or BTG4 and are released from milrinone at 12 h after microinjection . Then the oocytes were further culture for 24 h with or without adding U0126 to the medium . Error bars , s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O

G: Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126. Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L, CNOT7 and/or BTG4 and are released from milrinone at 12 h after microinjection. Then the oocytes were further culture for 24 h with or without adding U0126 to the medium. Error bars, s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm Cell pellets collected at the indicated time points after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for caspase - 1 ( E , F ) ( E Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) bands ( E ) ( F , Quantification of band intensities for caspase - 1 p20 normalized to pro - caspase - 1 and actin ( E ) from three independent experiments are shown as fold change ( F Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 (E, F) (E Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20) bands (E) (F, Quantification of band intensities for caspase-1 p20 normalized to pro-caspase-1 and actin (E) from three independent experiments are shown as fold change (F Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm Cell pellets collected at the indicated time points after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for GSDMD or LC - 3 G ) . Representative immunoblots showing GSDMD full length ( GSDMD FL ) , cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) , LC3 - II and LC3 - I bands in cell lysates ( G ) . H , Quantification of band intensities for GSDMD N - ter normalized to actin ( G ) and LC3 - II normalized to LC3 - I and actin ( H ) from three independent experiments are shown as fold change , H ) Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for GSDMD or LC-3 G). Representative immunoblots showing GSDMD full length (GSDMD FL), cleaved GSDMD N-terminal fragment (GSDMD N-ter), LC3-II and LC3-I bands in cell lysates (G). H, Quantification of band intensities for GSDMD N-ter normalized to actin (G) and LC3-II normalized to LC3-I and actin (H) from three independent experiments are shown as fold change , H) Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.


Intestinal CD11c + DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight . B . Cell pellets from three independent experiments were immunoblotted for LC3 and actin . Upper panel . Representative immunoblot . Lower panel . Quantification of band intensities for LC3 - II normalized to LC3 - I and actin are shown as fold induction relative to samples from untreated mice . Note that LC3 - I signal from ex vivo samples is almost undetectable . Data information : Mean is indicated . * * p < 0 . 01 . Mann - Whitney non - parametric statistical test .

B-TISSUE B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Intestinal CD11c+ DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight. B. Cell pellets from three independent experiments were immunoblotted for LC3 and actin. Upper panel. Representative immunoblot. Lower panel. Quantification of band intensities for LC3-II normalized to LC3-I and actin are shown as fold induction relative to samples from untreated mice. Note that LC3-I signal from ex vivo samples is almost undetectable. Data information: Mean is indicated. **p<0.01. Mann-Whitney non-parametric statistical test.


WT or SLC15A4feeble BMDCs expressing ASC - GFP and mCherry - LC3 ( A , B were infected with flagellin - expressing STm and fixed A . Cells were analyzed by fluorescence microscopy . Representative images showing ASC speck ( green ) relative to mCherry - LC3 ( red ) in two infected WT and SLC15A4feeble DCs each . B . Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test .

O O B-GENEPROD B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O 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 or SLC15A4feeble BMDCs expressing ASC-GFP and mCherry-LC3 (A, B were infected with flagellin-expressing STm and fixed A. Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry-LC3 (red) in two infected WT and SLC15A4feeble DCs each. B. Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs non - transduced ( C ) were infected with flagellin - expressing STm and fixed C . Cells were stained for endogenous ASC and p62 , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( red ) relative to p62 ( green ) in two infected WT and SLC15A4feeble DCs each . Note p62 staining surrounding ASC specks in SLC15A4feeble DCs . D . Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) ASC specks in at least 30 cells per cell type in each of 3 independent experiments . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test .

O O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-CELL_TYPE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O 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 or SLC15A4feeble BMDCs non-transduced (C) were infected with flagellin-expressing STm and fixed C. Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4feeble DCs. D. Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP ( E were infected with flagellin - expressing STm and fixed ( E , Cells were stained for endogenous ASC , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( red ) and SLC15A4 - GFP ( green ) in SLC15A4feeble DCs together with non - transduced SLC15A4feeble DCs . Note the perinuclear positioning of ASC specks in the transduced cells ( E ) . N , nucleus . Dotted white lines , cell outlines . Corresponding DIC images show nuclear position . Scale bar , 8 μm .

B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O B-GENEPROD B-CELL_TYPE O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

SLC15A4feeble BMDCs transduced with SLC15A4-GFP (E were infected with flagellin-expressing STm and fixed (E, Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4-GFP (green) in SLC15A4feeble DCs together with non-transduced SLC15A4feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP F , were infected with flagellin - expressing STm and fixed Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) and non - perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments ( F ) . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test .

B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SLC15A4feeble BMDCs transduced with SLC15A4-GFP F, were infected with flagellin-expressing STm and fixed Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non-perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP G ) were infected with flagellin - expressing STm and lysed ( G ) 1 h after infection . G . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , LC3 - II and LC3 - I , and actin bands .

B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O

SLC15A4feeble BMDCs transduced with SLC15A4-GFP G) were infected with flagellin-expressing STm and lysed (G) 1 h after infection. G. Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), LC3-II and LC3-I, and actin bands.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) were infected with flagellin - expressing STm . ( A , B ) . Cell pellets collected 1 h after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for phospho ( P ) and total mTOR , ULK1 kinase , p70 kinase and S6 , FLAG and actin . Representative immunoblots . Non - specific band right above FLAG - RagBQ99L is indicated with an asterisk ( A ) . Quantification of band intensities for P - mTOR normalized to total mTOR , P - ULK1 normalized to total ULK1 , P - p70 normalized to total p70 and P - S6 normalized to S6 , in transduced cells from 3 independent experiments are shown as fold change relative to WT control ( B ) . Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) were infected with flagellin-expressing STm. (A, B). Cell pellets collected 1 h after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for phospho (P) and total mTOR, ULK1 kinase, p70 kinase and S6, FLAG and actin. Representative immunoblots. Non-specific band right above FLAG-RagBQ99L is indicated with an asterisk (A). Quantification of band intensities for P-mTOR normalized to total mTOR, P-ULK1 normalized to total ULK1, P-p70 normalized to total p70 and P-S6 normalized to S6, in transduced cells from 3 independent experiments are shown as fold change relative to WT control (B). Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) ( E , Cell pellets were probed for caspase - 1 , GSDMD , actin ( E , Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , GSDMD full length ( GSDMD FL ) and cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) bands .

O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) (E, Cell pellets were probed for caspase-1, GSDMD, actin (E, Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.


WT or SLC15A4feeble BMDCs transduced with non - target control , ATG5 or ATG7 shRNAs G ) G ) . Cell pellets were probed for caspase - 1 , GSDMD , actin , ATG5 and ATG 7 ( G ) . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , GSDMD full length ( GSDMD FL ) and cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) bands .

O O B-GENEPROD B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

WT or SLC15A4feeble BMDCs transduced with non-target control, ATG5 or ATG7 shRNAs G) G). Cell pellets were probed for caspase-1, GSDMD, actin , ATG5 and ATG 7 (G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) H , I ) and mcherry - LC3 ( H , I ) , ( H , I ) . Cells were stained for endogenous ASC , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( green ) and LC3 ( red ) in SLC15A4feeble DCs ( H ) . Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) in at least 30 cells per cell type in each of three independent experiments ( I ) . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μ Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) H, I) and mcherry-LC3 (H, I), (H, I). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (green) and LC3 (red) in SLC15A4feeble DCs (H). Quantification of perinuclear (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μ Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom left inset . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.


B . Quantification of the number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage .

O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O

B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.


C . Representative images of unseparated ( < 1 μm apart ) , initial separation ( 1 - 5 μm apart ) , intermediate separation ( 5 - 11 μm apart ) , and pole separation ( > 11 μm apart ) of centrosomes . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom and top left insets . Scale bars = 5 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 B-SUBCELLULAR O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.


. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal - Wallis one - way ANOVA . * * P < 0 . 01 .

O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal-Wallis one-way ANOVA. **P<0.01.


E . Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT ( purple ) , CETN3 ( green ) , and SYCP3 ( red ) , and stained with DAPI ( blue ) . Zoomed images of centrosomes below each panel . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O

E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm.


A . Hematoxylin and eosin staining of 5 - micron thick testis sections of adult control , Plk1 cHet , and Plk1 cKO mice . Stars indicate examples of spermatocytes with condensed chromosomes . B . Percent testis to body weight ratios from control ( n = 8 ) , Plk1 cHet ( n = 4 ) , and Plk1 cKO ( n = 7 ) adult mice .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O

A. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. B. Percent testis to body weight ratios from control (n = 8), Plk1 cHet (n = 4), and Plk1 cKO (n = 7) adult mice.


C . TUNEL staining of paraffin embedded testis sections of adult control , Plk1 cHet , and Plk1 cKO mice . D . The number of TUNEL positive cells per seminiferous tubule cross - section was quantified in control Plk1 cHet , and Plk1 cKO mice . Two technical replicates were performed per genotype , and 100 seminiferous tubule cross sections were analyzed per replicate .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O

C. TUNEL staining of paraffin embedded testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. D. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Plk1 cHet, and Plk1 cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.


E . Hematoxylin and eosin staining of 5 - micron thick testis sections of adult control , Aurka cHet , and Aurka cKO mice . Stars indicate examples of spermatocytes with condensed chromosomes . F . Percent testis to body weight ratios from control ( n = 5 ) , Aurka cHet ( n = 3 ) , and Aurka cKO ( n = 5 ) adult mice .

E. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Aurka cHet, and Aurka cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. F. Percent testis to body weight ratios from control (n = 5), Aurka cHet (n = 3), and Aurka cKO (n = 5) adult mice.


G . TUNEL staining of paraffin embedded testis sections of adult control , Aurka cHet , and Aurka cKO mice . H . The number of TUNEL positive cells per seminiferous tubule cross - section was quantified in control Aurka cHet , and Aurka cKO mice . Two technical replicates were performed per genotype , and 100 seminiferous tubule cross sections were analyzed per replicate .

G. TUNEL staining of paraffin embedded testis sections of adult control, Aurka cHet, and Aurka cKO mice. H. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Aurka cHet, and Aurka cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.


I . Representative images of spermatocytes containing monopolar , bipolar , and multipolar spindles immunolabeled against alpha - tubulin ( red ) , centromere ( green ) , and stained with DAPI . J . Spindle pole number per primary spermatocyte was quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO . n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment .

O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O O

I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.


B . Quantification of the number of CETN3 foci per spermatocyte . n = 30 for each meiotic prophase substage .

O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O O

B. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.


C . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom and top left insets . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

C. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.


D . Quantification of the number of CETN3 foci per spermatocyte . n = 30 for each meiotic prophase substage .

D. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.


E . Centrosome ( PCNT ) separation was scored per spermatocyte . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications P values obtained from two - tailed Student ' s t - test comparing the percent of unseparated centrosomes in each mouse mutant to control . n . s . ( not significant ) , * * * P < 0 . 001 .

O O B-SUBCELLULAR O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

E. Centrosome (PCNT) separation was scored per spermatocyte. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications P values obtained from two-tailed Student's t-test comparing the percent of unseparated centrosomes in each mouse mutant to control. n.s. (not significant), ***P<0.001 .


A . Spermatocytes were immunolabeled against CETN3 ( green ) , CEP164 ( red ) , SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom and top left insets . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

A. Spermatocytes were immunolabeled against CETN3 (green), CEP164 (red), SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.


B . The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates ( n = 30 total cells ) in control , Aurka cKO , Plk1 cHet , and Plk1 cKO spermatocytes at the pachytene - stage of meiotic prophase and metaphase I .

O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O O O O

B. The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates (n = 30 total cells) in control, Aurka cKO, Plk1 cHet, and Plk1 cKO spermatocytes at the pachytene-stage of meiotic prophase and metaphase I.


C . Quantification of gamma - tubulin positive centrosomes per metaphase I spermatocyte . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 137 , 169 , 198 , and 198 , respectively .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O

C. Quantification of gamma-tubulin positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 137, 169, 198, and 198, respectively.


D . Spermatocytes were immunolabeled against gamma - tubulin ( green ) , alpha - tubulin ( red ) , and stained with DAPI ( blue ) . Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O

D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.


Western blots of gamma - tubulin from protein extracts obtained from 19dpp and 22dpp control , Aurka cKO , and Plk1 cKO spermatocytes ( E ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading .

B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

Western blots of gamma-tubulin from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (E). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


Western blots of gamma - tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16 , 19 and 22 dpp ( F ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading .

B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

Western blots of gamma-tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (F). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


A . Spermatocytes were immunolabeled against TACC3 ( green ) , SYCP3 ( red ) , CETN3 ( magenta ) , and stained with DAPI ( blue ) . Zoom images of the centrosomes are presented to the right . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

A. Spermatocytes were immunolabeled against TACC3 (green), SYCP3 (red), CETN3 (magenta), and stained with DAPI (blue). Zoom images of the centrosomes are presented to the right. Scale bars = 5 μm.


B . Quantification of TACC3 positive centrosomes per metaphase I spermatocyte . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 97 , 103 , 96 , and 105 , respectively .

O O O O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O

B. Quantification of TACC3 positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 97, 103, 96, and 105, respectively.


Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control , Aurka cKO , and Plk1 cKO spermatocytes ( D ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (D). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16 , 19 and 22 dpp ( C ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (C). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


E , F . Spermatocytes were immunolabeled against beta - catenin ( green ) , SYCP3 ( red ) , PCNT ( magenta ) , and stained with DAPI ( blue ) . Middle columns emphasize beta - catenin signal in diplonema and diakinesis spermatocytes . Scale bars = 5 μm . G . Quantification of beta - catenin positive centrosomes per spermatocyte at diakinesis . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 89 , 106 , 122 , and 93 , respectively .

O O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O

E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm. G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively.


A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom left inset . Scale bars = 5 μm .

O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.


B . Quantification of the number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage .

O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O

B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.


C . Centrosome ( PCNT ) separation was assessed in Aurka ; Plk1 dKO spermatocytes . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation

O O B-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O

C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications


( A , B ) B - ALL progression measured as ( A ) absolute number or ( B ) percentage of OFP + cells in peripheral blood of transplanted mice ( mean ± SD , each dot represents an individual mouse , CTRL = 6 mice , IFNγ = 10 mice ; * * * * p ≤ 0 . 001 , Two - Way ANOVA ; mice in blue and yellow ( B ) were also analyzed by single - cell RNA

O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY

(A, B) B-ALL progression measured as (A) absolute number or (B) percentage of OFP+ cells in peripheral blood of transplanted mice (mean ± SD, each dot represents an individual mouse, CTRL = 6 mice, IFNγ = 10 mice; **** p ≤ 0.001, Two-Way ANOVA; mice in blue and yellow (B) were also analyzed by single-cell RNA sequencing


( F , G ) Percentage of CD4 + and CD8 + cells within OFP - BM cells ( F ) and maturation state ( G ) of CD8 + lymphocytes ( CD62L - CD44 - double negative , CD62L - CD44 + effector memory , CD62L + CD44 - naive and CD62L + CD44 + central memory ) in the BM immune infiltrate at 12 - and 17 - days after B - ALL ( mean ± SD , each dot represents an individual mouse ; * * p = 0 . 005 , * * * * p ≤ 0 . 001 , Two - Way ANOVA ) .

O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) Percentage of CD4+ and CD8+ cells within OFP- BM cells (F) and maturation state (G) of CD8+ lymphocytes (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) in the BM immune infiltrate at 12- and 17-days after B-ALL (mean ± SD, each dot represents an individual mouse; ** p = 0.005, **** p ≤ 0.001, Two-Way ANOVA).


( A ) Engraftment of CD45 . 2 donor cells and lineage composition following transplantation of wild - type ( WT ) or IFNγ receptor 1 knock out ( KO ) HSPCs transduced with either the control Tie2 . NGFR ( CTRL ) or Tie2 . IFNγ ( IFNγ ) LV ( mean ± SD , each dot represents an individual mouse , WT CTRL = 7 mice , WT IFNγ = 7 mice , KO CTRL = 7 mice , KO IFNγ = 7 mice ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O

(A) Engraftment of CD45.2 donor cells and lineage composition following transplantation of wild-type (WT) or IFNγ receptor 1 knock out (KO) HSPCs transduced with either the control Tie2.NGFR (CTRL) or Tie2.IFNγ (IFNγ) LV (mean ± SD, each dot represents an individual mouse, WT CTRL = 7 mice, WT IFNγ = 7 mice, KO CTRL = 7 mice, KO IFNγ = 7 mice).


( C ) B - ALL progression measured as absolute number of OFP + cells in the peripheral blood ( mean ± SD , each dot represents an individual mouse ; * p = 0 . 0286 at day 10 ; p = 0 . 0174 at day 12 ; * * * p = 0 . 0002 ; * * * * p ≤ 0 . 0001 ; Ordinary Two - Way ANOVA ) .

O O O B-DISEASE I-DISEASE I-DISEASE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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) B-ALL progression measured as absolute number of OFP+ cells in the peripheral blood (mean ± SD, each dot represents an individual mouse; * p = 0.0286 at day 10; p = 0.0174 at day 12; *** p = 0.0002; **** p ≤ 0.0001; Ordinary Two-Way ANOVA).


( E ) Percentage of CD8 + T lymphocytes within OFP - CD45 + BM cells ( mean ± SD , each dot represents an individual mouse ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM O O

(E) Percentage of CD8+ T lymphocytes within OFP- CD45+ BM cells (mean ± SD, each dot represents an individual mouse).


( B , C ) Violin plots show the distribution of IFNγ ( B ) and MHC - II gene ( C ) signatures for each experimental condition , within each custom cell - type . Wilcox Rank Sum Test for IFNγ signature , condition IFNγ d12 vs . CTR d12 : B - ALL p = 2 . 68x10 - 19 , B cells p = 0 . 5 , plasma cells p = 0 . 78 ; Wilcox Rank Sum Test for MHC - II signature , condition IFNγ d12 vs . CTRL d12 : B - ALL p = 4 . 26x10 - 46 , B cells p = 1x10 - 9 , plasma cells p = 0 . 32 .

O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O

(B, C) Violin plots show the distribution of IFNγ (B) and MHC-II gene (C) signatures for each experimental condition, within each custom cell-type. Wilcox Rank Sum Test for IFNγ signature, condition IFNγ d12 vs. CTR d12: B-ALL p= 2.68x10-19, B cells p=0.5, plasma cells p= 0.78; Wilcox Rank Sum Test for MHC-II signature, condition IFNγ d12 vs. CTRL d12: B-ALL p= 4.26x10-46, B cells p=1x10-9, plasma cells p= 0.32.


( F ) Expression of Mki67 transcripts in B - ALL cells from CTRL d12 and IFNγ d12 conditions , extracted from the scRNAseq dataset ( * * * * p ≤ 0 . 0001 , Welch ' s test ) .

O O O O O B-GENEPROD O O B-DISEASE I-DISEASE I-DISEASE O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(F) Expression of Mki67 transcripts in B-ALL cells from CTRL d12 and IFNγ d12 conditions, extracted from the scRNAseq dataset (**** p ≤ 0.0001, Welch's test).


( I ) Loss of IFNγ response over time in B - ALL cells from the IFNγ group , potentially facilitated by reduced expression of Ifngr1 , Ifngr2 , Jak1 , Stat1 and Irf1 ( data extracted from the scRNAseq dataset statistical analysis by Two - Way ANOVA ) .

O O O O O B-GENEPROD O O O O B-DISEASE I-DISEASE I-DISEASE O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O

(I) Loss of IFNγ response over time in B-ALL cells from the IFNγ group, potentially facilitated by reduced expression of Ifngr1, Ifngr2, Jak1, Stat1 and Irf1 (data extracted from the scRNAseq dataset statistical analysis by Two-Way ANOVA).