G ) H & E , SOFG and Immunofluorescence analyses of pathological new bone in hind paws . n = 9 per group . ( H ) Quantitative analysis of CaSR + , OCN + and CaSR + OCN + cells number in pathological new bone formation sites . n = 9 per group , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New bone . Data shown as mean ± SD . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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G) H&E, SOFG and Immunofluorescence analyses of pathological new bone in hind paws. n=9 per group. (H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.


G ) H & E , SOFG and Immunofluorescence analyses of pathological new bone in hind paws . n = 9 per group .

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G) H&E, SOFG and Immunofluorescence analyses of pathological new bone in hind paws. n=9 per group.


( A ) μCT images of the spine in PGIS model ( 2D and 3D reconstruction ) . Arrow head shows spinal ankylosis .

(A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis.


( B ) H & E and SOFG staining of spine in PGIS model .

(B) H&E and SOFG staining of spine in PGIS model.


( C ) Incidence of spinal ankylosis in PGIS model . n = 5 per cage of total 6 cages per group , Fisher ' s exact test . ( D ) Quantitative analysis of structural parameters of new bone by μCT analysis . n = 5 , one ‐ way ANOVA , Bonferroni post hoc .

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(C) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 6 cages per group, Fisher's exact test.  (D) Quantitative analysis of structural parameters of new bone by μCT analysis. n=5, one‐way ANOVA, Bonferroni post hoc.


( C ) Incidence of spinal ankylosis in PGIS model . n = 5 per cage of total 6 cages per group , Fisher ' s exact test .

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(C) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 6 cages per group, Fisher's exact test.


( E ) μCT images of new bone formation ( red area ) in hind paws ( 2D and 3D reconstruction ) . ( F ) Incidence of new bone formation in DBA / 1 model . n = 9 per cage of total 3 cages per group , Fisher ' s exact test . ( G ) Quantitative analysis of structural parameters of new bone by μCT analysis . n = 9 , one ‐ way ANOVA , Bonferroni post hoc .

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(E) μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction).  (F) Incidence of new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test. (G) Quantitative analysis of structural parameters of new bone by μCT analysis. n=9, one‐way ANOVA, Bonferroni post hoc.


( E ) μCT images of new bone formation ( red area ) in hind paws ( 2D and 3D reconstruction ) .

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(E) μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction).


( H ) H & E and SOFG staining of new bone in hind paws . Data information : CB : Cortical bone ; NB : New bone

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(H) H&E and SOFG staining of new bone in hind paws. Data information: CB: Cortical bone; NB: New bone


( H ) H & E and SOFG staining of new bone in hind paws . Data information : CB : Cortical bone ; NB : New

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( I ) μCT images of the PCT in SMTS model ( 2D ) . Arrow heads shows bony projection .

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(I) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.


( J ) H & E and SOFG staining of Achilles tendon enthesis compartment in SMTS model . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage ; PCT : posterior calcaneal tuberosity .

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(J) H&E and SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity.


( K ) Quantitative analysis of area of UF . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . ( L ) Quantitative analysis of area of CF . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified

(K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc. (L) Quantitative analysis of area of CF. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage


( K ) Quantitative analysis of area of UF . n = 5 , one ‐ way ANOVA , Bonferroni post hoc .

(K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc.


( A ) Alizarin red staining of hBMSC cells for 11 days .

(A) Alizarin red staining of hBMSC cells for 11 days.


( B ) Quantification of Alizarin Red staining at 570 nm ( A570 ) . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(B) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.


( C ) RT - qPCR analysis of osteogenesis markers in hBMSC cells . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(C) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.


( D ) Alizarin red staining of hBMSC cells .

(D) Alizarin red staining of hBMSC cells.


( E ) Quantification of Alizarin Red staining at 570 nm ( A570 ) . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(E) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.


( F ) RT - qPCR analysis of osteogenesis markers in hBMSC cells . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(F) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.


( G ) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells .

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(G) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells.


( H ) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells treated with SR and NPS - 2143 .

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(H) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells treated with SR and NPS-2143.


( I ) Immunofluorescence analysis of spine in PGIS model . Quantitative analysis of CaSR + , p - PLCγ + and CaSR + p - PLCγ + cells number in spine of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; NB : New bone

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(I) Immunofluorescence analysis of spine in PGIS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in spine of PGIS model. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; NB: New bone


( I ) Immunofluorescence analysis of spine in PGIS model . Quantitative analysis of CaSR + , p - PLCγ + and CaSR + p - PLCγ + cells number in spine of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; NB : New

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( J ) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model . Quantitative analysis of CaSR + , p - PLCγ + and CaSR + p - PLCγ + cells number in Achilles tendon enthesis compartment . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage

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(J) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in Achilles tendon enthesis compartment. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage


( J ) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model . Quantitative analysis of CaSR + , p - PLCγ + and CaSR + p - PLCγ + cells number in Achilles tendon enthesis compartment . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified

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( K ) Immunofluorescence analysis of pathological new bone on dorsal surface of hind paws . Quantitative analysis of CaSR + , p - PLCγ + and CaSR + p - PLCγ + cells number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New bone

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(K) Immunofluorescence analysis of pathological new bone on dorsal surface of hind paws. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in pathological new bone formation sites. n=9, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone


( K ) Immunofluorescence analysis of pathological new bone on dorsal surface of hind paws . Quantitative analysis of CaSR + , p - PLCγ + and CaSR + p - PLCγ + cells number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New

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( L ) μCT images of the PCT in SMTS model ( 2D ) . Arrow heads shows bony projection .

(L) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.


( M ) SOFG staining of Achilles tendon enthesis compartment in SMTS model . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage ; PCT : posterior calcaneal tuberosity .

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(M) SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity.


( N ) Quantitative analysis of area of UF and CF . n = 5 , Student ' s t -

(N) Quantitative analysis of area of UF and CF. n=5, Student's t-test


( A ) RT - qPCR and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells treated with IL - 1β . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

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(A) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-1β. n=3, one‐way ANOVA, Bonferroni post hoc.


( B ) RT - qPCR and Western Blot analyss of CaSR expressions in MC3T3 - E1 cells treated with TNFα . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

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(B) RT-qPCR and Western Blot analyss of CaSR expressions in MC3T3-E1 cells treated with TNFα. n=3, one‐way ANOVA, Bonferroni post hoc.


( C ) RT - qPCR and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells treated with IL - 17A . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(C) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-17A. n=3, one‐way ANOVA, Bonferroni post hoc.


( D ) RT - qPCR and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells treated with IL - 22 . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(D) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-22. n=3, one‐way ANOVA, Bonferroni post hoc.


( E ) RT - qPCR and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells treated with IL - 23 . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(E) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-23. n=3, one‐way ANOVA, Bonferroni post hoc.


( A ) Immunofluorescence analysis of spine in PGIS mice . Quantitative analysis of CaSR + p - p65 + cells and CaSR + p - Stat3 + cells number in pathological new bone formation of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; GP : Growth plate ; NB : New bone

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(A) Immunofluorescence analysis of spine in PGIS mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation of PGIS model. n = 5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; GP: Growth plate; NB: New bone


( A ) Immunofluorescence analysis of spine in PGIS mice . Quantitative analysis of CaSR + p - p65 + cells and CaSR + p - Stat3 + cells number in pathological new bone formation of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; GP : Growth plate ; NB : New

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( B ) Immunofluorescence analysis of pathological new bone of hind paws in DBA / 1 mice . Quantitative analysis of CaSR + p - p65 + cells and CaSR + p - Stat3 + cells number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . CB : Cortical bone ; NB : New bone

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(B) Immunofluorescence analysis of pathological new bone of hind paws in DBA/1 mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation sites. n = 9, one‐way ANOVA, Bonferroni post hoc. CB: Cortical bone; NB: New bone


( B ) Immunofluorescence analysis of pathological new bone of hind paws in DBA / 1 mice . Quantitative analysis of CaSR + p - p65 + cells and CaSR + p - Stat3 + cells number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . CB : Cortical bone ; NB : New

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( C ) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model . Quantitative analysis of CaSR + p - p65 + cells and CaSR + p - stat3 + cells number in PCT . n = 5 , Student ' s t - test . UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage

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(C) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-stat3+ cells number in PCT. n=5, Student's t-test. UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage


( C ) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model . Quantitative analysis of CaSR + p - p65 + cells and CaSR + p - stat3 + cells number in PCT . n = 5 , Student ' s t - test . UF : Uncalcified fibrocartilage ; CF : Calcified

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Three sex chromosome axes could be detected in the 2 - month - old XUsp26 + XUsp26 - Y spermatocytes . Immunofluorescence analysis of SYCP3 ( red ) and SYCP1 ( green ) in ( J ) , ATR ( red ) and SYCP3 ( green ) in ( K ) was performed in 2 - month - old WT and XUsp26 + XUsp26 - Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome . The arrowheads indicate the X chromosome .

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Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.


D Histological analysis of the seminiferous tubules and caudal epididymis of the 6 - month - old Usp26 + / Y and Usp26 - / Y mice .

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D Histological analysis of the seminiferous tubules and caudal epididymis of the 6-month-old Usp26+/Y and Usp26-/Y mice.


F The deletion of Usp26 causes pachytene and meiotic division arrest . Representative PAS - hematoxylin staining in 6 - month - old Usp26 + / Y and Usp26 - / Y seminiferous tubules . Parafﬁn sections from Usp26 + / Y and Usp26 - / Y testes were stained with PAS - hematoxylin . P : pachytene spermatocyte , D : diplotene spermatocyte , rST : round spermatid , eST : elongating spermatid , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions .

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F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Parafﬁn sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.


E The X and Y chromosomes were unpaired in Usp26 - / Y spermatocytes . Immunofluorescence analysis of Chr X - FISH ( green ) , Chr Y - FISH ( red ) , and SYCP3 ( white ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . The arrowheads indicate the X chromosome . F Quantification of unpaired X and Y chromosomes in 6 - month - old Usp26 + / Y and Usp26 - / Y mice ( n = 5 independent experiments ) . Red dots indicate Usp26 + / Y mice and green dots indicate Usp26 - / Y mice .

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E The X and Y chromosomes were unpaired in Usp26-/Y spermatocytes. Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. The arrowheads indicate the X chromosome. F Quantification of unpaired X and Y chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.


Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of SYCP3 ( green ) , MRE11 ( red ) ( G ) ; was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the sex chromosomes .

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Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.


Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of ; SYCP3 ( green ) , γH2AX ( red ) ( H ) ; SYCP3 ( green ) , ATR ( red ) , p - ATM ( pink ) ( I ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the sex chromosomes .

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Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of ; SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.


L Crossover on sex chromosomes was impaired in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of SYCP3 ( green ) , MLH1 ( red ) , and ATR ( white ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) .

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L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).


D Representative TUNEL results in Usp26 + / Y and Usp26 - / Y testes . Parafﬁn sections from 6 - month - old Usp26 + / Y and Usp26 - / Y testes were stained with TUNEL ( green ) and DAPI ( blue ) to show dead cells in stage IV , VIII and XII tubules with pachytene and metaphase spermatocytes , respectively . The arrowheads indicate the laggard chromosomes . P : pachytene spermatocytes , rSt : round spermatid , spz : spermatozoa , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions , arSt : apoptotic round spermatid .

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D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Parafﬁn sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.


E The protein level of three known SAC proteins was reduced in 2 - month - old and 6 - month - old Usp26 − / Y mouse testes . Immunoblotting of MAD2 , BUBR1 , PLK1 and USP26 was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 - / Y testes . H3Ser10p and Tub served as loading control .

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

E The protein level of three known SAC proteins was reduced in 2-month-old and 6-month-old Usp26−/Y mouse testes. Immunoblotting of MAD2, BUBR1, PLK1 and USP26 was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26-/Y testes. H3Ser10p and Tub served as loading control.


F Usp26 - deficient mice produced XY aneuploid round spermatids . FISH analysis of Chr X ( green ) and Chr Y ( red ) was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 − / Y round spermatids . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome , and the arrowheads indicate the X chromosome . G Quantification of different types of round spermatids in 2 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 3 independent experiments ) and 6 - month - old Usp26 + / Y ( n = 3 independent experiments ) , Usp26 - / Y mice ( n = 4 independent experiments ) . P = 0 . 0026 for XY spermatozoa in 6 - month - old Usp26 + / Y and Usp26 − / Y mice . P = 0 . 0216 for O spermatozoa in 6 - month - old Usp26 + / Y and Usp26 − / Y mice . H Usp26 - deficient mice produced XY aneuploid spermatozoa . FISH assay of Chr X ( green ) and Chr Y ( red ) was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 − / Y spermatozoa . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome , and the arrowheads indicate the X chromosome . I Quantification of different types of spermatozoa in 2 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 3 independent experiments ) and 6 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 5 independent ex experiments ) . P = 0 . 0195 for XY spermatozoa in 6 - month - old Usp26 + / Y and Usp26 − / Y mice . Data are presented as means ± SD .

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

F Usp26-deficient mice produced XY aneuploid round spermatids. FISH analysis of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y round spermatids. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. G Quantification of different types of round spermatids in 2-month-old Usp26 +/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y (n= 3 independent experiments), Usp26-/Y mice (n= 4 independent experiments). P=0.0026 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. P=0.0216 for O spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. H Usp26-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. I Quantification of different types of spermatozoa in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n= 5 independent ex experiments). P=0.0195 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. Data are presented as means ± SD.


The X and Y chromosomes were unpaired in Tex11 - / Y spermatocytes . Immunofluorescence analysis of SYCP3 ( green ) , TRF1 ( red ) , ATR ( white ) ( D ) ; SYCP3 ( green ) , MLH1 ( red ) , ATR ( white ) ( E ) was performed in in Tex11 + / Y and Tex11 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrowheads indicate the X chromosome .

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

The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome.


A , B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations . The spermatozoa from fertile men without USP26 mutations ( n = 10 independent experiments ) ; fertile men contained the USP26 c . 463C > T mutation ( The sample was analyzed in three repeated experiments ) , the USP26 c . 125T > C mutation ( n = 2 independent experiments , one sample was analyzed in two repeated experiments ) , the USP26 c . 1044T > A mutation ( n = 5 independent experiments ) , and the USP26 c . 370 _ 371insACA / 494T > C / 1423C > T mutation ( n = 10 independent experiments ) were collected to perform Chr X and Chr Y FISH . P - values are shown for comparison to fertile men without USP26 mutations .

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

A, B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations. The spermatozoa from fertile men without USP26 mutations (n = 10 independent experiments); fertile men contained the USP26 c.463C>T mutation (The sample was analyzed in three repeated experiments), the USP26 c.125T>C mutation (n = 2 independent experiments, one sample was analyzed in two repeated experiments), the USP26 c.1044T>A mutation (n = 5 independent experiments), and the USP26 c.370_371insACA/494T>C/1423C>T mutation (n = 10 independent experiments) were collected to perform Chr X and Chr Y FISH. P-values are shown for comparison to fertile men without USP26 mutations.


C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa . FISH assay of Chr X ( green ) and Chr Y ( red ) was performed in fertile men with or without the USP26 mutated haplotype . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome , and the arrowheads indicate the X chromosome .

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

C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.


Survival rates of control and Cdc50a cKO mice after oil or 4 - OHT injection ( n = 5 for each group , P * * * = 0 . 0003 ) .

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

Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).


Bar graphs showing audiogenic seizure scores following auditory stimuli ( 5 , 10 , 15 , 20 , 40 kHz or white noise ( WN ) at 70 , 80 , 90 , or 100 dB ) in control ( E ) and Cdc50a cKO ( F ) mice ( n = 8 for each group ) .

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

Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group).


Representative confocal z - stack images showing cFos ( magenta ) - positive neurons from control and Cdc50a cKO mice ( CTX : cortex , HP : hippocampus , and IC : inferior colliculus ) . Scale bar , 20 μm . Bar graphs showing the relative area of cFos - positive cells in the brains of control and Cdc50a cKO mice ( n = 5 for each group , * * P = 0 . 0030 , n . s . , not significant ) .

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

Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant).


Schematic diagram of the experiment . Representative confocal z - stack images of PSD95 ( green ) - and VGLUT1 ( red ) - positive excitatory ( A ) and Gephyrin ( green ) - and VGAT ( magenta ) - positive inhibitory ( B ) synapses in the CTX , HP , and IC ( CTX : cortex , HP : hippocampus , and IC : inferior colliculus ) of control and Cdc50a cKO mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm .

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

Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Representative current clamp traces from the IC of control and Cdc50a cKO mice ; + 100 pA ( upper ) and + 400 pA ( lower ) currents were injected into control mice ( left ) and Cdc50a cKO mice ( right ) for 500 ms .

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

Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms.


The bar graphs show the relative percentages of neurons showing PS exposure only in the soma ( left ) and overall PS exposure ( right ) ( n = 30 - 40 cells for each group ) in control and Cdc50a cKO mice .

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

The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.


Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP . Representative confocal z - stack images showing Gephyrin - positive inhibitory post - synapses in the somas ( red ) and proximal ( green ) and distal dendrites ( cyan ) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing the number of Gephyrin - positive inhibitory post - synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice ( n = 4 for each group , P * * * = 0 . 0002 , P * * * * < 0 . 0001 ) .

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

Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).


Representative confocal z - stack and colocalization images of Gephyrin ( green ) , IBA1 ( blue ) , and CD68 ( red ) in the IC of control and Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice ( n = 3 for each group , * * P = 0 . 0024 ) .

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

Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024).


Representative confocal z - stack images of microglia ( IBA1 , green , upper ) and inhibitory synapses ( Gephyrin : green , VGAT : red , lower ) in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm . Bar graphs showing the relative area of IBA1 - positive cells in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , control ; DMSO versus control ; PLX3397 , * P = 0 . 0428 , control ; DMSO versus Cdc50a cKO ; DMSO , * * * P = 0 . 0002 , Cdc50a cKO ; DMSO versus Cdc50a cKO ; PLX3397 , * * * P = 0 . 0005 ) . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , P * * * * < 0 . 0001 ) .

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

Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).


Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the IC ( inferior colliculus ) of control ; C3 + / + , control ; C3 - / - , Cdc50a cKO ; C3 + / + and Cdc50a cKO ; C3 - / - mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 10 μm .

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

Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm.


Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the IC of control ; Mertk + / + , control ; Mertk - / - , Cdc50a cKO ; Mertk + / + and Cdc50a cKO ; Mertk - / - mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm .

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

Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Schematic diagram of the experiment in which PS exposure was measured in the brains of 1 - month - and 3 - month - old wild - type mice by injecting AAV9 - GFAP - secA5 - mCherry in the neonatal stage ( P0 ) . Representative confocal z - stack colocalization images of Gephyrin ( green ) , VGAT ( blue ) , and secA5 ( red ) in the HP ( hippocampus ) of 1 - month - and 3 - month - old wild - type mice . The white circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the expression of secA5 ( B ) and IBA1 ( C ) in inhibitory post - synapses ( Gephyrin - positive , green ) and pre - synapses ( VGAT - positive , blue ) in the HP of 1 - month - and 3 - month - old wild - type mice ( n = 4 for each group , * * * P = 0 . 0009 , * P = 0 . 0248 , n . s . , not significant ) .

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

Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).


Schematic diagram of the experiment in which Annexin V was injected intravenously ( every two days ) to block PS in the brains of 1 - month - and 3 - month - old wild - type mice . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the HP of 1 - month - old wild - type mice . The white circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the HP of 1 - month - old mice after PBS or Annexin V injection ( n = 4 for each group , * * * P = 0 . 0013 , * * P = 0 . 0090 ) .

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Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).


Schematic diagram of the experiment in which microglial Mertk KO ( CX3CR1 - CreERT2 ; Mertkfl / fl ) and control ( Mertkfl / fl ) mice were generated . Both 1 - month - old microglial Mertk KO mice and 1 - month - old control mice were injected with 4 - OHT for 5 consecutive days . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( magenta ) - positive inhibitory synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice . The white circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * * P = 0 . 0027 , * P = 0 . 0062 ) . Representative confocal z - stack colocalization images of Gephyrin ( green ) , VGAT ( blue ) and secA5 ( red ) in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice . The white circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * P = 0 . 0015 ) .

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Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).


( A ) 293T cells were transfected with Flag - ULK1 or control empty vector and subjected to immunoprecipitation with anti - Flag antibody , followed by immunoblot analysis with the indicated antibodies .

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(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies.


( B ) 293T cells were co - transfected with Flag - AMPKα2 and HA - ULK1 . Cell lysates were subjected to immunoprecipitation using anti - Flag antibody followed by SDS - PAGE / immunoblot analysis with anti - HA and anti - Flag antibodies .

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(B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies.


( C ) 293T cells were transiently transfected with Flag - AMPKα2 or Flag - AMPKβ1 . After 24 h , immunoprecipitation was performed using anti - Flag or control anti - HA antibodies and analyzed by immunoblotting with anti - ULK1 and anti - Flag polyclonal antibodies .

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(C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies.


( D ) 293T cell lysates were subjected to immunoprecipitation with anti - ULK1 polyclonal antibody or control preimmune rabbit serum ( NRS ) , followed by immunoblot analysis with anti - ULK1 and anti - AMPKα antibodies .

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(D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies.


( E ) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti - AMPKα antibody followed by immunoblotting with anti - ULK1 and anti - AMPKα antibodies .

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(E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies.


( F ) Purified His6 - AMPKα1 / β1 / γ1 fusion proteins ( 250 ng ) were mixed with purified His6 - tagged ULK1 ( 1 µg ) or Bcl - XL ( 200 ng ) proteins in 1 % Triton X - 100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti - ULK1 antibody . The resulting protein complexes and 10 % of the input proteins were analyzed by immunoblotting with anti - His - Tag polyclonal antibody .

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(F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.


( B , C ) 293T cells were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti - HA antibody . The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR , raptor , AMPKα or HA - tag .

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(B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.


( A ) 293T cells were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutant ( Δ654 - 828 ) ULK1 and subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies .

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(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654-828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies.


( B ) U - 2OS cells stably expressing GFP - LC3 were infected with lentivirus expressing human Ulk1 shRNA ( shhUlk1 ) or scrambled shRNA ( shScr ) and subjected to selection with 1 µg / ml puromycin for 5 days . The cells were then transfected with wild type mouse Ulk1 ( mUlk1 ) , mUlk1 Δ654 - 828 or control empty vector for 24 h , incubated in serum - free DMEM overnight , and cultured in complete medium with or without 10 mM metformin for 20 h . The images were obtained using a fluorescence microscope . The scale bars represent 10 µm .

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(B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654-828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm.


( C ) The number of GFP - LC3 dots per GFP - positive cell in ( B ) was quantified ( mean ± s . d . ; n = 81 ) .

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(C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81).


( D ) U - 2OS cells stably expressing GFP - LC3 were transfected with 0 , 2 or 4 µg of pcDNA3 encoding HA - tagged ULK1 deletion mutant ( 554 - 828 ) . The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector . After 24 h transfection , the cells were starved in nutrient - free medium for 1 . 5 h , fixed in 4 % paraformaldehyde , and analyzed by fluorescence microscopy . The number of GFP - LC3 dots per GFP - positive cell was quantified ( mean ± s . d . ; n = 100 ) . Statistical significance was determined by Student ' s t - test and the asterisks indicate P < 0 . 0001 .

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(D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554-828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's t-test and the asterisks indicate P<0.0001.


293 cells stably expressing HA - ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti - HA or control anti - FLAG antibodies . The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies .

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293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies.


( A ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were transfected with GFP - LC3 , treated with 2 mM AICAR or control vehicle for 4 h , and then analyzed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was counted ( mean ± s . d . ; n = 60 ) . The scale bars represent 10 µm .

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(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm.


( B ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were treated with 2 mM AICAR for the indicated times . Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies . The levels of p62 are listed relative to those of untreated WT raptor cells , which were set as 1 .

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(B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1.


Representative images for the TIF analysis shown in ( C ) . Shown are POT1Y223C / - hESCs and an unedited wild - type hESC clone ( WT / WT ) isolated in parallel , as well as a POT1 WT / Q623H hESC and a contemporaneously isolated hECS clones ( WT / WT ) . Scale bars = 10µm . Box and whisker plot ( showing min , max and average ) quantifying telomere - dysfunction induced foci ( TIF ) in two POT1 Y223C / - hESC lines , one POT1 Q623H / Q623H , and one POT1WT / Q623H hESC lines compared to wild - type unedited control hESCs . Cells were analyzed 3 months post targeting . Cells were stained for TRF1 and γ - H2Ax . POT1Y223C / - cells have increased TIFs compared to an unedited wild - type cell clone , isolated and cultured in parallel ( P < 0 . 01 , two - tailed Mann - Whitney test , one biological replicate ) .

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Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.   Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).


Images of telomere and centromere FISH on metaphase spreads of cell lines as in ( B ) . Cy3 Tel - C PNA probes were used . Arrows indicate chromosomal abnormalities . E ) Quantification of chromosomal abnormalities as determined by FISH analysis in ( D ) .

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Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).


B ) Quantification of telomere length data from two STELA experiments using the same genomic DNA , shown in ( A ) and ( Appendix Figure 2C ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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B) Quantification of telomere length data from two STELA experiments using the same genomic DNA, shown in (A) and (Appendix Figure 2C). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


B ) TIF analysis of wild - type , POT1WT / Y223C hESCs , and the indicated controls using antibodies against TRF1 and γ - H2AX . Scale bars = 10µm . Lentiviral expression of TPP1 ∆ PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response . TPP1 ∆ PBD infected cells were analyzed 72h post infection and without selection .

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B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection.


E ) Quantification of two STELA experiments , including that shown in ( D ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


D ) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in ( C ) WT : wild - type , MT : mutant .

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D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant.


C ) Representative genotyping outcomes of hCD34 + cells nucleofected sgRNA - 1 , xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS . Data information : The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion . A dash inside a gray box indicates a deleted base pair .

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C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS. Data information: The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.


B ) Telomere restriction fragment assay for the five subjects shown in ( A ) . Mean telomere length is indicated .

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B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.


( A ) Histogram ( mean , 95 % CI ) of fold induction of PXR activity reporter assay ( luc , luciferase ) in LS180 cells transiently transfected with wild - type PXR and p3A4 - luc reporter plasmids . Chemical structures of FKK5 , FKK6 and FKK999 compounds are overlaid . The bar graph ( s ) depicts one representative experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of cells . * p < 0 . 05 , one - way ANOVA with Dunnett ' s post hoc test . * significant over vehicle ( DMSO ) control .

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(A) Histogram (mean, 95 %CI) of fold induction of PXR activity reporter assay (luc, luciferase) in LS180 cells transiently transfected with wild-type PXR and p3A4-luc reporter plasmids. Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.


( B ) , same as ( A ) , in HepG2 cells stably expressing pCYP1A1 luciferase plasmid ( AhR reporter ) . Chemical structures of FKK5 , FKK6 and FKK999 compounds are overlaid . Indole is colored blue . The bar graph ( s ) depicts one representative experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of cells . * p < 0 . 05 , one - way ANOVA with Dunnett ' s post hoc test . * significant over vehicle ( DMSO ) control .

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(B), same as (A), in HepG2 cells stably expressing pCYP1A1 luciferase plasmid (AhR reporter). Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. Indole is colored blue. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.


C , Histogram ( mean , 95 % CI ) of fold mRNA expression , CYP1A1 ( top panel ) , CYP3A4 ( middle panel ) and MDR1 ( bottom panel ) in LS180 cells with or without ( mock ) transfected PXR plasmid , HepaRG hepatic progenitor cells ( PXR - knockout , PXR - KO ; AhR - knockout , AhR - KO ; parental control 5F clone ) and primary human hepatocytes ( HEP ) from four donors is shown . The bar graph represents one experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of LS180 cells ; n = 2 independent experiments with one well / compound and RT - PCR performed in triplicate for each HepaRG genotype ; for each donor hepatocyte , n = 1 well / compound and RT - PCR performed in triplicate . * , # p < 0 . 05 , two - way ANOVA with Tukey ' s post hoc test . * significant over vehicle control . # significant over the same treatment in corresponding mock transfected or knock - out cells .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Histogram (mean, 95 %CI) of fold mRNA expression, CYP1A1 (top panel), CYP3A4 (middle panel) and MDR1 (bottom panel) in LS180 cells with or without (mock) transfected PXR plasmid, HepaRG hepatic progenitor cells (PXR-knockout, PXR-KO; AhR-knockout, AhR-KO; parental control 5F clone) and primary human hepatocytes (HEP) from four donors is shown. The bar graph represents one experiment of a series of experiments (n > 3) performed in four consecutive passages of LS180 cells; n = 2 independent experiments with one well/compound and RT-PCR performed in triplicate for each HepaRG genotype; for each donor hepatocyte, n = 1 well/compound and RT-PCR performed in triplicate. *, #p < 0.05, two-way ANOVA with Tukey's post hoc test. *significant over vehicle control. #significant over the same treatment in corresponding mock transfected or knock-out cells.


D , Chromatin Immunoprecipitation ( ChIP ) assay in LS174T cells . Top panel , PCR products cells exposed to vehicle or FKK6 and run on a 2 % agarose gel . DNA 100 base pair marker ; Vehicle , 10 % DMSO ; FKK6 ( 10 μM ) ; 1 / 10 Input - 0 . 2 million cells before IP ; IgG - IP with polyclonal rabbit IgG ; PXR - IP with PXR antibody . Bottom panel , quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH ( fold expression ) . Dash line , 2 - fold expression . The data is one representative experiment of two independent experiments ( each n = 3 biological replicates , n = 4 technical replicates ) . Mean fold expression is expressed above each histogram ( mean + SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Chromatin Immunoprecipitation (ChIP) assay in LS174T cells. Top panel, PCR products cells exposed to vehicle or FKK6 and run on a 2% agarose gel. DNA 100 base pair marker; Vehicle,10% DMSO; FKK6 (10 μM); 1/10 Input - 0.2 million cells before IP; IgG - IP with polyclonal rabbit IgG; PXR - IP with PXR antibody. Bottom panel, quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH (fold expression). Dash line, 2-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biological replicates, n = 4 technical replicates). Mean fold expression is expressed above each histogram (mean + SD).


E , PXR TR - FRET assay . TR - FRET ratio 520 / 495 nm are plotted against concentration of compound ( s ) . Half - maximal inhibitory concentrations IC50 were obtained from interpolated standard curves ( sigmoidal , 4PL , variable slope ) ; error bars show standard deviation of n = 2 independent experiments each with four technical replicates .

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E, PXR TR-FRET assay. TR-FRET ratio 520/495 nm are plotted against concentration of compound(s). Half-maximal inhibitory concentrations IC50 were obtained from interpolated standard curves (sigmoidal, 4PL, variable slope); error bars show standard deviation of n = 2 independent experiments each with four technical replicates.


F , Microcalorimetric titrations of PXR ligand binding domain ( LBD ) with FKK5 and FKK6 . A single - site binding model was used for fitting the data . The upper panel shows the output signal , dQ / dt , as a function of time . The middle panel shows the integrated heats as a function of the ligand / PXR molar ratio in the cell . The solid line represents the best non - linear least - squares fit of the data . The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain .

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F, Microcalorimetric titrations of PXR ligand binding domain (LBD) with FKK5 and FKK6. A single-site binding model was used for fitting the data. The upper panel shows the output signal, dQ/dt, as a function of time. The middle panel shows the integrated heats as a function of the ligand/PXR molar ratio in the cell. The solid line represents the best non-linear least-squares fit of the data. The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain.


A , PXR ( luciferase ) reporter assay in HEK293T cells transiently transfected with PXR plasmids ( wild - type and ligand binding domain mutant C285I / C301A ) . RLU , relative light units are shown normalized to beta - galactosidase ( β - Gal ) expression . The histogram represents one experimental data ( of n > 2 independent experiments each performed in quadruplicate ) mean ( 95 % CI ) ; n . s . not significant ; * p < 0 . 05 , two - way ANOVA .

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A, PXR (luciferase) reporter assay in HEK293T cells transiently transfected with PXR plasmids (wild-type and ligand binding domain mutant C285I/C301A). RLU, relative light units are shown normalized to beta-galactosidase (β-Gal) expression. The histogram represents one experimental data (of n > 2 independent experiments each performed in quadruplicate) mean (95 %CI); n.s. not significant; * p < 0.05, two-way ANOVA.


B , Histogram represents fold change in mRNA expression , normalized to GAPDH , by RT - qPCR from Caco - 2 cells exposed to compounds . DMSO , 0 . 1 % DMSO vehicle ; 9 . 3 is 9 . 3 - fold expression . The data is one representative experiment of two independent experiments ( each n = 3 biologic replicates , n = 4 technical replicates ) .

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B, Histogram represents fold change in mRNA expression, normalized to GAPDH, by RT-qPCR from Caco-2 cells exposed to compounds. DMSO, 0.1% DMSO vehicle; 9.3 is 9.3-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biologic replicates, n = 4 technical replicates).


Points and error represent mean ( 95 % CI ) values for control ( TNFα 5 ng / ml ) normalized NF - κB ( luciferase ) reporter activity in LS180 cells after C , Passage 13 ( P13 ) and D , Passage 8 ( P8 ) and C , D , Line graph depicts one representative experiment of a series of experiments ( n > 4 ) performed in four consecutive passages of cells . Data was expressed as mean with 95 % CI of four technical replicates .

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Points and error represent mean (95 %CI) values for control (TNFα 5 ng/ml) normalized NF-κB (luciferase) reporter activity in LS180 cells after C, Passage 13 (P13) and D, Passage 8 (P8) and C, D, Line graph depicts one representative experiment of a series of experiments (n > 4) performed in four consecutive passages of cells. Data was expressed as mean with 95% CI of four technical replicates.


E , LS174T PXR - KO ( knockout ) cells transiently transfected with pNL3 . 2 . NF - κB - RE vector . mean ( + SD ) . * , * * p < 0 . 05 , two way ANOVA with Tukey ' s multiple comparisons test .

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E, LS174T PXR-KO (knockout) cells transiently transfected with pNL3.2.NF-κB-RE vector. mean (+ SD). *, ** p < 0.05, two way ANOVA with Tukey's multiple comparisons test.


A In Caco - 2 monolayer cells , Log10 relative expression of IL8 mRNA with or without cytokines ( 50 ng / ml IL - 1 , 10 ng / ml IFNγ , 10 ng / ml TNF α ) , and cytokine cocktail plus FKK drugs ( 10 and 25 μM ) with 2 h or 12 h incubations .

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A In Caco-2 monolayer cells, Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations.


B , Salmonella invasion in colony forming units per ml ( CFU / ml ) at the different time periods of incubation .

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B, Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation.


C , confocal images of NFĸB ( red ) and the nucleus ( blue ) . Scale bars = 80 µm . Arrows represent localization of NFĸB ( red , cytoplasmic ; purple , nuclear ) . Confocal images shown are representative images from ( n = 3 ) biological replicates .

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C, confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm. Arrows represent localization of NFĸB (red, cytoplasmic; purple, nuclear). Confocal images shown are representative images from (n = 3) biological replicates.


Log10 relative expression of IL8 mRNA with or without cytokines ( 50 ng / ml IL - 1 , 10 ng / ml IFNγ , 10 ng / ml TNF α ) , and cytokine cocktail plus FKK drugs ( 10 and 25 μM ) with 2 h or 12 h incubations . In human intestinal organoids ( HIO ) ,

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 B-CONTROLLED_VAR 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

Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations. In human intestinal organoids (HIO),


Salmonella invasion in colony forming units per ml ( CFU / ml ) at the different time periods of incubation . In human intestinal organoids ( HIO ) ,

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Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation. In human intestinal organoids (HIO),


confocal images of NFĸB ( red ) and the nucleus ( blue ) . Scale bars = 80 µm . n human intestinal organoids ( HIO ) ,

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confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm. n human intestinal organoids (HIO),


Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per individual sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK5 ( n = 9 paired samples ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an individual patient . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc test .

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 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 O O O

Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK5 (n = 9 paired samples). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.


Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per individual sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK6 ( n = 3 paired samples ; one removed due to poor inflammatory response ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an individual patient . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc test .

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 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 O O O O O O O O O O O

Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK6 (n = 3 paired samples; one removed due to poor inflammatory response). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.


Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per individual sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK9 ( n = 5 ; 3 removed due to poor response and tissue integrity ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an individual patient . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc test .

Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK9 (n = 5; 3 removed due to poor response and tissue integrity). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.


Fold induction ( mRNA ) of genes in C57BL / 6 mice gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . The entire experiment was repeated two independent times and one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays and normalized to internal control , GAPDH . The histograms show mean ( 95 % CI ) values for gene expression . The dotted line marks basal fold

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Fold induction (mRNA) of genes in C57BL/6 mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expression


Fold induction ( mRNA ) of genes in pxr - / - mice gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . The entire experiment was repeated two independent times and one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays and normalized to internal control , GAPDH . The histograms show mean ( 95 % CI ) values for gene expression . The dotted line marks basal fold

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

Fold induction (mRNA) of genes in pxr -/- mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expression


Fold induction ( mRNA ) of genes in hPXR mice ( mice expressing the human PXR gene ) gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . The entire experiment was repeated two independent times and one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays and normalized to internal control , GAPDH . The histograms show mean ( 95 % CI ) values for gene expression . The dotted line marks 2 - fold gene induction .

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Fold induction (mRNA) of genes in hPXR mice (mice expressing the human PXR gene) gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks 2-fold gene induction.


After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . A , % weight loss from baseline ( day 1 vs day 10 )

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. A, % weight loss from baseline (day 1 vs day 10)


After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . B , colon length ( cm )

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. B, colon length (cm)


After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . C , Inflammation

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. C, Inflammation score


After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . D , Fecal lipocalin 2 ( pg / ml )

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. D, Fecal lipocalin 2 (pg/ml)


After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . E , serum FITC - dextran ( μg / ml ) .

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. E, serum FITC-dextran (μg/ml).


After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . F , fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS ( n = 3 / group ; each PCR performed in quadruplicate ) .

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

After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. F, fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS (n = 3/group; each PCR performed in quadruplicate).


As in Fig 5 , pxr - / - mice underwent the identical experimental procedure and analysis . The entire experiment was repeated three independent times and one representative experiment is shown .

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As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.


( 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 .

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(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.


( A - B ) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells . ( C ) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes , shows that RNF115 ligase activity is required for the increase of phagosome maturation .

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(A-B) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells. (C) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes, shows that RNF115 ligase activity is required for the increase of phagosome maturation.